PHYSICIANS POSTER SESSIONS
Poster Session 1 Cell therapy/cellular therapy I P001 Abstract Withdrawn
P002 HLA-haploidentical allografting with high-dose posttransplant cyclophosphamide: clinical outcomes and immune-reconstitution A. Busca1,*, E. Maffini1, F. Ferrando1, C. Dellacasa1, R. Pulito1, E. Saraci1, L. Giaccone1, M. Boccadoro1, P. Omede`1, B. Bruno1 1 Division of Hematology, Department of Molecular Biotechnology and Health Sciences, University of Torino, A.O.U. Citta` della Salute e della Scienza di Torino, Torino, Italy Introduction: HLA-haploidentical allografting is a potential cure in patients (pts) without a HLA-identical donor. Materials (or patients) and methods: Between April 2010 and November 2014, 41 pts (median age 50, r 21-71 years) with hematologic malignancies (33 acute leukemias; n ¼ 2 CML; n ¼ 6 lymphomas) were transplanted from a HLAhaploidentical donor. Fifty% had advanced disease at the time of transplant. Eight pts had received a previous allograft. Conditioning was either reduced-intensity (11 pts) with fludarabine (30 mg/m2/day for 5 days), cyclophosphamide (Cy) (14.5 mg/Kg/day for 2 days) and single dose 2 Gy TBI, or myeloablative (29 pts) with thiotepa (TT) (5 mg/Kg/day for 2 days), fludarabine (50 mg/m2/day for 3 days) and i.v. Busulphan (Bu) (3.2 mg/Kg/day for 3 days) or thiotepafludarabine-melphalan (140 mg/m2/day) (1 patient), followed by T-cell replete bone marrow (34 pts) or peripheral blood stem cells (7 pts). Post-transplant immunosuppression consisted of Cy, 50 mg/Kg/day on days þ 3 and þ 4, followed by mycophenolate mofetil and FK-506. G-CSF was administered from day þ 5 until engraftment. In 22 (54%) pts, T-cell reconstitution and thymic activity were studied for the first 2 years post-transplant. T-cells subsets in both the CD4 þ and CD8 þ T cell compartments were evaluated by flow-cytometry: naı¨ve (CD45RO- and CD27 þ ); central memory (CD45RO þ CD27 þ ); effector memory (CD45RO- CD27-); and revertant (CD45RA þ CD45RO þ ). Thymic function through T-cell receptor rearrangement excision circles (TRECs) was measured by RT quantitative PCR on DNA extracted from peripheral mononuclear cells and from sorted CD4 þ and CD8 þ T cells. Here we present preliminary data on thymic output and naı¨ve CD4 þ T cell recovery Results: Neutrophil recovery occurred at a median of 17 days. Incidence of acute grade II-IV and III-IV GVHD was 28,2% and 7,6%. Blood stream infections were documented in 13 pts (7 gram þ bacteria; 6 gram- bacteria). Six had probable invasive fungal infection, in 2 cases after prophylaxis with micafungin and in 4 with azoles. CMV reactivation occurred in 21 and EBV reactivation, successfully treated with rituximab, in 1. At a median follow-up of 501 days (r 18-1278 days), 19 pts (67%) are alive, 11 (58%) in complete remission. Median OS and DFS at 2 years were 46% and 34%. Overall, 14 pts (34%) died of relapse and 8 pts (19%) of transplant related mortality (TRM). The probability of TRM was 16% at 100 days and 26% at 1 year.
(CD4 þ CD27 þ CD45RA þ ) Naı¨ve T cells decreased from a median of 23.92/ul at day 28 to 1.8/ul at day 90, then gradually increased to 3.8/ul, 3.9/ul, 8.7/ul at 6, 12 and 18 months, respectively. Median TREC copies/100 ng DNA from sorted CD4 þ cells decreased from 14.5 (r 0-202.2) pre-transplant to 4.4 (r 0-49.2) at day 90, than increased to 9.5 (r 0-134.2), 24.8 (r 2.7-83), 41.5 (r 5.5-102.2) and 47.41 (r 31.5-584.3) at 6, 12, 18 and 24 months, respectively. In healthy donors, median TREC copies/100 ng DNA was 74.5 in sorted CD4 þ cells. Conclusion: Feasibility of HLA-haploidentical transplantation with high-dose post-transplant Cy in heavily pretreated pts was confirmed. Thymic activity and immune recovery of naı¨ve CD4 þ T cells were slow and less robust when compared with transplants from HLA identical donors. Longer follow-up on a larger series of pts is needed to evaluate long-term outcomes and correlate clinical issues with immunological data. Disclosure of Interest: None declared. P003 Cost analysis of dendritic cell therapy for acute myeloid leukemia patients A. Van de Velde1,*, P. Beutels2, S. Anguille1, A. Gadisseur1, W. Schroyens1, A. Verlinden1, E. Smits3, V. Van Tendeloo4, G. Nijs3, S. Dom5, I. Cornille5, Z. Berneman1 1 Department of Hematology, Antwerp University Hospital, 2 Centre for Health Economics Research & Modeling Infectious Diseases, University of Antwerp, 3Centre for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, 4Vaccine & Infectious Disease Institute, University of Antwerp, 5Business Intelligence, Antwerp University Hospital, Antwerp, Belgium Introduction: Treatment of acute myeloid leukemia (AML) is a significant economic burden for patients, insurance companies, and society and is doubled over 10 year. Hospitalization costs are the key drivers. Most costs are associated with remission induction treatment. When relapse occurs, high costs are incurred again, particularly when another round of chemotherapy is given. Findings suggest that savings to the healthcare system could be achieved by sustaining complete remission (CR) status for longer periods. Materials (or patients) and methods: Between January 2005 and December 2010, a total of 55 adults with AML received antileukemic therapy at the Antwerp University Hospital (UZA). The analysis excluded five patients because of incomplete cost data about remission-induction therapies in their referring hospitals. A total of 50 patients were included in the analysis. Fifteen patients were treated with chemotherapy alone (group 1), 25 patients with chemotherapy followed by allogeneic hematopoietic stem cell transplantation (HSCT) (group 2) and 10 patients with chemotherapy followed by immunotherapy using dendritic cells engineered to express the Wilms’ tumor (WT1) protein (group 3). (Van Tendeloo, 2010). The Ethics Committee at UZA approved this cost study and patients in group 2 and 3 provided signed, informed consent respectively for their allogeneic HSCT and for their enrollment in the immunotherapy study. Data regarding patient characteristics were obtained from patient’s medical records. Professional and facility charges were collected using electronic billing information. Drug costs and drug administration costs were based on list prices published by the Belgian reimbursement authority (RIZIV/INAMI). Room costs and care at the hematology ward, pharmacy, intensive care unit, laboratory tests and medical imaging were analyzed. The cost of dendritic cell vaccine preparation was h 20,450 per patient.
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Results: The median cost in group 1 (15 patients) was h 32, 648 (range: h 4,759 - h 140,383). Only 2 patients in group 1 went into remission after induction therapy and received consolidation therapy. The median post-consolidation treatment cost in group 1 was h 35,581 (range: h 30,069 - h 41,093). All patients in group 1 died within 5 year after diagnosis, 13 patients died within 1 year and 5 died within 1 month. The median cost in group 2 (25 patients) was h 185,212 (range: h 87,932 - h 449,155). The median post-consolidation treatment cost in group 2 was h 117,241 (range: h 31,364 - h 304,366). Five-year survival in group 2 was 19%. Seventeen patients in group 2 died within 1 year after HSCT. The median cost in group 3 (10 patients) was h 114,505 (range: h 45,114 - h 207,731). The median post-consolidation treatment cost in group 3 was h 40,748 (range: h 26,907- h 156,870). Five-year survival in group 3 was 30%. Four patients in group 3 died within 1 year after vaccination. Conclusion: It is critical to focus on developing new therapies that can prevent relapse and maintain AML patients’ CR status to maximize their survival. Immunotherapy using dendritic cells engineered to express the Wilms’ tumor protein is one of these new therapies and in this study has 1/3 of the post consolidation costs of HSCT. Disclosure of Interest: None declared. P004 Generation of HLA*A02:01 restricted CD19 specific T cells in CD19 non-tolerant hosts A. Maurberger1,*, H. Balzer1, E. Roth2, E. Kieback3, A. Kremer1, A. Mackensen1, H.-M. Ja¨ck2, W. Uckert3, A. Gerbitz1 1 Medicine 5, Haematology/Oncology, Erlangen University Hospital, 2Division of Molecular Immunology, Department of Internal Medicine III, Friederich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Erlangen, 3Max-Delbru¨ck-Center for Molecular Medicine, Humboldt-University Berlin, Berlin, Germany Introduction: Targeting the B cell specific antigen CD19 by antibodies or CAR-modified T cells appears to be very effective in treatment of leukemias and lymphomas. However, downregulation of surface CD19 renders target cells invisible for antibody/CAR mediated therapies making it also an attractive target for TCR-transduced T cell therapy. One major obstacle for the generation of high avidity T cells with specificity for self antigens is circumvention of central tolerance. Materials (or patients) and methods: CD19 deficient mice were crossbred onto an HLA*A02:01 transgenic HHD2 background to generate CD19 non-tolerant animals. CD19-/-HHD2 mice were immunized against human CD19 using peptidepools containing HLA*A02:01 binding fragments of the huCD19 protein. Immunogenic peptides were identified by ELISA and peptide-specific T cells were enriched and expanded by restimulation in vitro. TCRs from specific T cells were identified by nested PCR and cloned into a pMP71 vector. Human T cells were retrovirally transduced to express a CD19 specific TCR. Results: Using repetitive peptide pool immunization we identified a dominant CD19 derived peptide and isolated specific HLA*A02:01 restricted CD8 þ T cells. When restimulated with the specific peptide in vitro T cells displayed proliferation and showed a strong secretion of IFNg. After four rounds of in vitro restimulation flow cytometric T-cell receptor analysis revealed a TCRva4.3 and TCRvb4 restriction which was confirmed by nested PCR. Repetitive stimulation in vitro resulted in enrichment of CD8 þ T cells binding peptide loaded HLA multimers from 2% up to 20%. CD19 specific T cells could be further enriched by flow cytometry based on peptide loaded HLA multimer. A purity of 498% was achieved. After cloning of both TCR chains into an MP71 vector backbone human T cells were retrovirally transduced to express a CD19 specific TCR. TCR transduced T cells were enriched by flow cytometry using antibodies specific for TCRvb4 (498% purity) and expanded by CD3/28 bead stimulation.
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Conclusion: Our results demonstrate that human CD19 serves as a T-cell antigen in the context of human HLA*A02:01. By utilizing a xenogenic approach central tolerance was circumvented and human CD19 specific T cells were generated and expanded. Retroviral transduction of the identified TCRs into human T cells will allow for testing this approach in preclinical models. Disclosure of Interest: None declared. P005 Optimised protocol for clinical scale isolation and expansion of invariant NKT cells for prevention of aGVHD A. Rotolo1,*, S. Loaiza1,2, S.-A. Finn2, E. Bray2, O. Pello2, S. Uddin2, A. Bradshaw2, J. F. Apperley1, A. Chaidos1, A. Karadimitris1 1 Centre for Haematology, 2John Goldman Centre for Cellular Therapy Hammersmith Hospital, Imperial College London, London, United Kingdom Introduction: Acute graft–versus-host disease (aGVHD) severely limits the wider applicability of allogeneic HSCT. iNKT cells, a rare subset of CD1d-restricted, glycolipid-specific T cells, may be crucial in the regulation of aGVHD. In line with animal models showing a role of donor iNKT cells in protecting/abrogating aGVHD, we found that a high donor CD4– iNKT cell dose in the graft is associated with a 4x reduction of the relative risk of clinically significant aGVHD. Therefore, we hypothesise that in vitro expanded donor iNKT cells adoptively transferred to allogeneic HSCT recipients receiving an iNKT-poor graft will prevent occurrence of clinically significant aGVHD. Towards this goal, we propose to develop a clinical scale GMP-compliant protocol to isolate and expand donor CD4– iNKT cells. Materials (or patients) and methods: iNKT cells were positively selected from fresh PBMC/frozen lymphapheresis products using iNKT magnetic beads (6B11mAb) and expanded with irradiated autologous PBMCs, IL2 and aGalCer (aGC) or aCD3/CD28-coated beads. For the GMP-compliant protocol, the CliniMACS device and GMP-grade TexMACS medium and IL2 were used. Expanded cells were characterized by flow cytometry-based cytokine profile. Results: First we optimised a protocol for iNKT isolation and expansion aiming to preserve the pre-selection relative frequency of CD4– cells in the post-expansion cell product. Using iNKT-specific magnetic beads, iNKT cells were effectively selected from fresh PBMCs as well as frozen lymphapheresis products with high yield and purity (Table 1). We tested 3 different post-selection expansion protocols, using aCD3/CD28 beads, autologous aGC-loaded PBMCs or aCD3/CD28 beads in the presence of autologous PBMCs (n ¼ 3). iNKT cells were expanded 4450x by all 3 methods in o3 weeks. However, the aGC-based protocol was less dependent on the initial iNKT purity and it was the only one that preserved the relative frequency of the CD4– cells. Next we adopted the aGC-based
approach for clinical scale iNKT selection and expansion from donor frozen lymphapheresis products using the CliniMACS apparatus and GMP-grade reagents (TexMACS medium and IL2). In 2 experiments, pre-selection iNKT cells were 0.13% of T cells in donor 1 (1.4x106,B0.03% of TNC) and 0.009% of T cells in donor 2 (1.6x104,o0.002% of TNC). Post CliniMACS selection, iNKT cells were 94% and 36% of T cells respectively. In both cases cells were effectively expanded, achieving B700x increase by day 18 from donor 1. Analysis of the Th1/2/ 17 cytokine profile showed that clinical scale expanded iNKT cells displayed a preferential Th1-like profile. Conclusion: We optimised a protocol for in vitro iNKT cell isolation and expansion, which ensures preservation of the relative frequency of the CD4– fraction that likely imparts protection from aGVHD. This and the 4100x expansion by 4 weeks using GMP-compliant reagents and processes provides proof-of-principle that clinical scale iNKT isolation and expansion is feasible. Notably, the process is efficient for iNKT-rich as well as iNKT-poor donors. These results show that with availability of GMP-grade iNKT selection beads and aGC the process of clinical scale iNKT selection and expansion can be easily rendered fully GMP-compliant and therefore suitable for clinical use. References: Chaidos et al, Blood 2012. Kuwatani et al, Immunol Lett 2006. Leveson-Gower et al, Blood 2011. Yang et al, Transfusion 2010. Disclosure of Interest: None declared.
HLA identical family members (n:5) or alternative donors (n:3) at Hacettepe University (n:4) or Bahc¸es¸ehir University (n:4). Of these 8 children who underwent HSCT, 4 received Busulfan (Bu) and Fludarabin (Flu) and 4 received Bu, Flu and Thiothepa as a conditioning regimen. Engraftment was achieved in 7/8 children at median day þ 14 and day þ 23 for neutrophils and platelets; respectively. The median chimerism level was 96% (82-100%) at 1 month after HSCT. Veno-occlusive disease developed in 2 children, pulmonary hypertension was seen in 2, and post-transplant hypercalcemia was seen in 1 child during HSCT. All complications related to HSCT were successfully treated. The mean follow up duration was 20.8±4.5 months (2 months-60 months). During the follow up period, 12 of 14 children are alive. Among 8 children who underwent HSCT; 7 are alive with no complications related to HSCT. However, only 1 child who received HSCT from HLA 6/10 identical mother (haploidentical HSCT) had engraftment failure and HLA identical unrelated donor search has been continued for 2nd HSCT. Conclusion: In this study; among 8 children who underwent MSC-HSC co-transplantation; 7 achieved engraftment and they are alive with no HSCT related complications. In conclusion; this pilot study suggests that MSC-HSC co-transplantation may be useful to decrease early morbidity and mortality in children with MIOP. However, MSC administration alone does not appear to change the clinical course of the disease. Disclosure of Interest: None declared.
P006 Mesenchymal Stem Cell Therapy in Malign Infantile Osteopetrosis: Hacettepe Experience From Turkey B. Tavil1,*, G. Karasu2, B. Kus¸konmaz1, F. V. Okur1, M. O. Kaynak1, M. Çetin1, D. Uc¸kan1 1 Hacettepe University Faculty of Medicine, Pediatric Hematology Unit, Ankara, 2Medicalpark Go¨ztepe Hospital, Bahc¸es¸ehir University, ˙Istanbul, Turkey
P007 Post-thaw DMSO depletion using a cGMP-compliant spinning-membrane separation device B. Calmels1,2,*, E. Guyon1, C. Wegener3, G. Bouchet1, J. Couquiaud1, L. Regimbaud1, C. Lemarie1,2, P. Houze4, K. Min3, C. Chabannon1,2 1 centre de therapie cellulaire, 2INSERM CBT-1409, institut paolicalmettes, marseille, France, 3Fresenius Kabi USA LLC, Lake Zurich, United States, 4laboratoire de biochimie, hopital saintlouis, paris, France
Introduction: Malignant infantile osteopetrosis (MIOP) is an autosomal recessive fatal disorder. Even if hematopoietic stem cell transplantation (HSCT) is performed from an HLA identical donor, morbidity and mortality of HSCT is very high in MIOP. This study aims to apply mesenchymal stem cell (MSC) to the children with MIOP to decrease the morbidity and mortality of HSCT, to decrease the complications related to MIOP, and to support engraftment. Materials (or patients) and methods: This study included 14 children with the diagnosis of MIOP referred to Hacettepe University Faculty of Medicine, Pediatric Bone Marrow Transplantation Unit between December 2012 and December 2014. Detailed laboratory and imaging studies were performed in our hospital. Ethical approval was obtained from local ethical committee of Ankara University (Number: 29-589) and from the HSCT scientific advisory committee of Turkey Ministry of Health. Mesenchymal stem cell suspensions were prepared at GMP Laboratories of Ati-gen cell in Trabzon and applied by intravenous infusion. This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK 1001 project, 112S127). Results: The mean age of 14 children (7 boys/7 girls) with MIOP was 8.0±6.7 months (1-24 months). Homozygous mutation was detected in TCIR gene for 7 children, in ClCN7 gene for 5 children, in STX gene for 1 child, and in RANK gene for 1 child. Among these 11 children; 8 received MSCs as MSCHSC co-transplantation and the other 3 children received only MSCs while waiting for HSCT, but they were not received HSCT because neuropatic form of the disease (n:2) or his parents were not give permission (n:1). A total of 18 MSC infusions were performed for 11 children with MIOP. The mean MSC dosage was 3.4±1.3 x106/kg (median: 3.5 x106/kg; range: 2-5.4 x106/kg). MSC donors were family members who were HLA 510/10 identical with the children. HSCTs were performed from
Introduction: Cryopreserved stem cell grafts are still widely infused to many patients, both in the autologous or allogeneic settings. Cryopreserved grafts can be thawed at the bedside or thawed and washed at the cell therapy laboratory. We recently published that post-thaw washing did not impair hematopoietic engraftment, in a cohort of 2,930 autologous transplanted patients receiving either unwashed or washed grafts (Calmels B et al, 2014). Post-thaw washing can be implemented using various methods such as manual centrifugation, automated centrifuge-based or spinning-membrane devices such as the Lovo (Fresenius Kabi). Materials (or patients) and methods: We here report the pre-clinical evaluation of the Lovo for washing thawed stem cell grafts. We took advantage of 15 apheresis products intended for destruction and cryopreserved in 2 identical bags; after dry-thawing (PlasmaTherm, Barkey), bags were connected to the Lovo, diluted at 20 ml/min with þ 4-81C 6% hydroxyethylstarch 130/0.4 (Voluven, Fresenius Kabi) and processed using 2- or 3-cycles reduction (a cycle referring to one pass through the spinning membrane). Results: After washing, CD34 and CD45 absolute counts and viability were evaluated (Stem-Kit, Beckman Coulter) and DMSO was quantified by capillary zone electrophoresis (P/ACE, Beckman Coulter). Lengths of procedures were 15 min and 18 min for 2- and 3-cycles, respectively. Post-wash data show consistent viability, effective DMSO depletion and relevant CD34 recovery. Conclusion: We conclude that this new spinning-membrane device enables for high efficiency DMSO depletion while preserving optimal CD34 viability and recovery. Moreover, the length of procedures does not significantly delay the process, as compared to bedside thawing. Post-thaw washing using automated cell processing systems have thus to be preferred since they provide multiple benefits such as DMSO and cell
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debris depletion, precise determination of infused CD34 cell dose, increased cellular stability as well as adjustable washing efficiency and final volume. Disclosure of Interest: B. Calmels: None declared, E. Guyon: None declared, C. Wegener Employee of: Fresenius Kabi, G. Bouchet: None declared, J. Couquiaud: None declared, L. Regimbaud: None declared, C. Lemarie: None declared, P. Houze: None declared, K. Min Employee of: Fresenius Kabi, C. Chabannon: None declared P008 Adoptive Immunotherapy of refractory systemic adenovirus infections after allogeneic umbilical cord blood (UCB) or Peripheral Blood stem cell transplantation C. Qian1,*, V. DECOT2, Y. WANG1, H. L. CAI3, V. VENARD4, H. JEULIN4, M. DE CARVALHO BITTENCOURT3, J. H. DALLE5, C. POCHON6, B. BRUNO7, C. PAILLARD8, S. VIGOUROUX9, C. JUBERT10, P. CEBALLOS11, A. MARIE CARDINE12, C. GALAMBRUN13, L. CLEMENT6, D. BENSOUSSAN1 1 UTCT, Cell Therapy Unit , 2UTCT Cell Therapy Unit , 3Plateforme Nancytomique, 4Laboratoire de Virologie , CHU de Nancy, VANDOEUVRE LES NANCY, 5Service d’he´mato-immunologie , Hoˆpital Robert Debre´, Paris, 6UTMA , CHU de Nancy, VANDOEUVRE LES NANCY, 7Service d’He´matologie pe´diatrique CHU de LILLE, CHU de LILLE, Lille, 8Service d’He´matologie Pe´diatrique, CHU de Strasbourg, STRASBOURG, 9Service de transplantation me´dullaire, 10Servide d’He´matologie Pe´diatrique, CHU de Bordeaux, BORDEAUX, 11Service de transplantation me´dullaire, CHU de Montpellier, MONTPELLIER, 12Service d’He´matologie Pe´diatrique, CHU de Rouen, ROUEN, 13Service d’He´matologie Pe´diatrique, CHU La Timone, MARSEILLE, France Introduction: Adenovirus (ADV) systemic infection refractory to antiviral treatment after allogeneic stem cell transplantation is associated with a high mortality rate up to 50%. Adoptive transfer of ADV-specific T cells (ADV-STs) is becoming an alternative treatment that has already proved feasible, safe and helpful in viral clearance and immune reconstitution related to an in vivo expansion of ADV-STs leading to clinical improvement. Materials (or patients) and methods: We previously demonstrated the feasibility of isolating ADV-STs from healthy donors using the IFNg-capture system from Miltenyi followed by immunomagnetic selection on the CliniMACS device (AissiRothe´ et al, 2010). We have now included 13 patients among 14 in a multicentric phase I/II clinical trial consisting in infusion of polyclonal ADV-STs generated by a 6-hour ex vivo stimulation with Pept-ADV5 hexon (peptide pool from the immunodominant Hexon protein, Miltenyi Biotec) of leukapheresis collected from their original stem cell donor or from third party haploidentical donors, followed by isolation of IFNg producing cells. Results: We report on the generation of 12 ADV-ST preparations and the infusion of 10 pediatric and adult patients (8/2) with ADV infection (n ¼ 5; blood and/or stool viral load) or ADV disease (n ¼ 5). Five patients had experienced grade II GVHD controlled by immunosuppressive treatments before infusion. They received a mean of 5.84 103 CD3-IFNg þ cells/ kg (range: 0,25 to 31.78 103) 1 to 9 months after HSCT. In vivo expansion of transferred ADV-STs was observed in 9 of 10 patients from day 14 to 60 following adoptive transfer infusion associated with ADV load decrease or clearance for 9 of them (90%) but one patient experienced a
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secondary increase. Neither de novo GVHD, nor side effects were observed. Reactivation of GVHD occurred in 3 patients and could be controlled by immunosuppressive treatments in 2 patients. In one patient, with stabilized GVHD grade II before infusion who experienced a modulation of immunosuppression before ADV-VSTs infusion, GVHD worsened in grade III with cutaneous, hepatic and intestinal manifestations responsible for the patient death. Two other patients died, one related to ADV disease (ADV and EBV meningitis), but none of the responders had ADV-associated mortality. Conclusion: Adoptive transfer of ADV-STs is a feasible and well-tolerated therapeutic option, representing a fast and efficient procedure to achieve reconstitution of antiviral T-cell and decrease or clearance of ADV viral load. We expect that complete analysis of all the clinical data from the trial will allow attributing GVHD reactivations either to VSTs infusion or modulation of immunosuppressive drugs. References: Aissi-Rothe´ L, Decot V, Venard V, Jeulin H, Salmon A, Clement L, Kennel A, Mathieu C, Dalle JH, Rauser G, Cambouris C, De Carvalho M, Stoltz JF, Bordigoni P, Bensoussan D. Rapid generation of full clinical grade human anti-adenovirus cytotoxic T cells for adoptive immunotherapy. J Immunother. 2010; 33: 414–424. Disclosure of Interest: None declared. P009 Mononuclear cell collection for extracorporeal photochemotherapy by using the ‘‘off-line’’ system: a study comparing an automatic and a semiautomatic apheresis device in graft versus host disease E. Gonzalez-Arias1,*, C. Pascual1, A. Perez-Corral1, L. Solan1, S. Redondo1, M. Bastos1, D. Serrano1, J. Gayoso1, M. Kwon1, J. Anguita1, J. Diez-Martin1 1 Hematologia, Hospital Gregorio Maran˜on, Madrid, Spain Introduction: Extracorporeal photochemotherapy (ECP) is an effective cell therapy employed in several diseases, including graft-versus-host disease (GVHD) after stem cell transplantation (SCT). When ECP is performed using the off-line technique, optimal mononuclear cells (MNC) collection by leukapheresis (LA) is necessary for further manipulation. The objective of our study was to compare the semiautomatic COBE Spectra (Terumo BCTs, Lakewood, CO, USA; version 7.0) and the new automated Spectra OPTIA (Terumo BCTs v.5.0) in terms of efficacy and safety in ECP. Materials (or patients) and methods: All patients with refractory acute and chronic GVHD in treatment with ECP were retrospectively studied, from June 2014 (when OPTIA device was introduced into our service) to November 2014. Patients were alternatively assigned with either apheresis device. Peripheral blood samples were obtained before and after each LA procedure, along with a sample of each product just before reinfusion. The cell blood count (CBC) was perfomed in all samples with Unicell DHX 800 (Beckman Coulters). Collection efficiency (CE) of white blood cell (WBC) and total mononuclear cells (TMNC) and platelet loss were calculated as follows: WBC CE% ¼ [productWBCx106/mL x product volume* mL/ preapheresis NC (x106/mL)x(Vol processed Ml-AC volume Ml)]x100. TMNC CE% ¼ [product MNCx106/mL x product volume* mL/ preapheresis MNC (x106/mL)x (vol processed mL-ACvolume mL)]x100.
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Platelet loss ¼ [Preapheresis PL count-Postspheresis PL count/ Preapheresis PL count] x100. *In all cases we completed with saline solution to a total volume of 300ml. We compared patient’s CBC before and after the LA, the CBC of the product and the procedure variables with both devices, using non-parametric methods (U Mann-Whitney). Results: One hundred fourteen procedures (63 in COBE and 53 in OPTIA) were carried out in 9 patients (8 with chronic GVHD and 2 with acute GVHD). The median age was 39 years old (range: 18-56). Central venous access was used in 8/9 (88,8%) patients. Both devices were safe, and there were no significant adverse events recorded. CBC results before and after LA are detailed in Table 1. The processed volume and the amount of ACD used were less in the Optia group. Hemoglobin and hematocrit product levels were higher in the Optia group, and TMNC count was similar with both devices. Notably, TMNC CE, WBC CE and platelet loss was better with OPTIA device. There was no difference in the time of the procedure. Further details are shown in Table 2. Conclusion: In our study, the Spectra Optia has proven to be more effective in collecting MNC and WBC than COBE device. The lower platelet loss is an important aspect because these patients often have low platelet counts. Fewer ACD used in the Optia group may be an advantage that may result in a lower symptoms of hypocalcemia. In our short experience, OPTIA has proven to be a safe and very effective choice for MNC collection ECP in patients with EICH after SCT. Furthermore, it has the advantage of being fully automatic and therefore independent of the technical skills. References: C. del Fante et al. Transfusion 2013; 53: 2027– 2033. Disclosure of Interest: None declared.
P010 Treatment of Secondary Graft Failure after Hematopoietic Stem Cell Transplantation with Alpha/Beta T-Cell Depleted Grafts as Booster Infusions E. Rådestad1,2,*, H. Wikell1,3, M. Engstro¨m1,2, E. Watz1,3, B. Sundberg1,2, S. Thunberg3, M. Uzunel3, J. Mattsson1,2, M. Uhlin1,2 1 Department of Oncology-Pathology, Karolinska Institute, 2Center for Allogeneic Stem Cell Transplantation, 3Clinical Immunology and Transfusion Medicine, KAROLINSKA UNIVERSITY HOSPITAL, Stockholm, Sweden Introduction: Infusions of extra lymphocytes from the original stem cell donor can be used as a treatment after hematopoietic stem cell transplantation (HSCT) for, e.g., relapse, poor immune reconstitution or secondary graft failure. In peripheral blood, 95% of T-cells express the ab T-cell receptor and the remaining T-cells express the gd T-cell receptor, which are not dependent upon human leukocyte antigen (HLA)- molecules for activation. As ab T-cells are the primary mediators of graft versus host disease (GVHD), depleting them from the graft should reduce this risk, leaving gd T-cells as primary T-cell type in the booster infusion. Materials (or patients) and methods: In this pilot study, five patients transplanted with HLA-matched related or unrelated donors were treated with ab T-cell depleted stem cell boosters, with secondary graft failure as primary indication. Secondary graft failure was in this study defined as initial signs of engraftment with subsequent development of bone marrow hyperplasia requiring frequent transfusions beyond day 60 as well as prolonged neutropenia and thrombocytopenia. All patients also suffered from infectious complications prior to the infusion, which is likely to have been associated with the poor graft function and development of secondary graft failure. The ab T-cell depletion was performed using a CliniMACS system (Miltenyi Biotech). Analysis of the booster infusion fraction as well as blood samples from the patients was performed by using flow cytometry and evaluation of chimerism as well spectratyping of gd-chains by using PCR. Monitoring of white blood cells (WBC), platelets, and granulocytes was carried out repeatedly during 30 days post infusion.
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Results: The majority of gd T-cells in the boosters expressed Vd2 and/or Vd9. Median log depletion of gd T-cells in the products was 3.72, and the mean yield of gd T-cells was 84.4%. Most patients receiving ab-depleted stem cell boosters increased their levels of WBC, platelets, and/or granulocytes 30 days post infusion. Regarding the infectious complications, the stem cell booster had a positive effect for four out of five patients. The most obvious response could be observed in the three patients with the best-combined response in WBC and granulocyte count. The cell characterization by flow cytometry on peripheral blood samples 3-7 months post infusion showed that the gd T-cell population was similar and had normalized in the four out of five patients that were characterized, regardless of the spread observed in the gd T-cell repertoire in the characterized booster infusion fractions. Conclusion: In conclusion, we describe the use of ab T-cell depleted grafts as stem cell booster in five patients suffering from infectious complications due to secondary graft failure after HSCT with encouraging results. No signs of GVHD or other side effects were detected and four out of five patients were alive six months after booster infusion. The individual contribution of gd T-cells, NK-cells, or CD34 stem cells cannot be determined based on this pilot study. A larger pool of patients with longer follow-up time is needed to confirm the data in this study and compare the treatment modality to other therapy options. Disclosure of Interest: None declared. P011 Preemptive cytokine-induced killer (CIK) cell infusions sustained remission in high-risk leukemia patients after allogeneic stem cell transplantation E. Rettinger1,*, S. Huenecke1, V. Pfirrmann1, M. Merker1, A. Willasch1, A. Jarisch1, J. Soerensen1, H. Boenig2, G. Bug3, T. Klingebiel1, P. Bader1 1 University Hospital Frankfurt, Goethe University, Department for Children and Adolescents, Division for Stem Cell Transplantation and Immunology, 2German Red Cross Blood Donor Service Baden-Wu¨rttemberg-Hessen, Division for Cell Processing, 3University Hospital Frankfurt, Goethe University, Department of Medicine II, Hematology, Oncology, Rheumatology and Infectious Diseases, Frankfurt/Main, Germany Introduction: Allogeneic stem cell transplantation (allo-SCT) is an established treatment option for high-risk leukemia patients, but relapse limits the therapeutic success. Relapse can be treated by donor lymphocyte infusion (DLI), but T cells within DLI raise the risk for acute graft versus host disease (aGvHD). Cytokine-induced killer (CIK) cells, mainly T cells with characteristics of natural killer (NK) cells, demonstrate nonmajor histocompatibility complex-restricted cytotoxicity against hematological malignancies with negligible alloreactivity and therefore were licensed as an immunotherapy. Materials (or patients) and methods: Here we report our experience of repetitive, dose-escalating CIK cell infusions in 14 leukemia patients with risk of relapse (n ¼ 11) or overt relapse (n ¼ 3) showing no signs of aGvHD 4grade I after allo-SCT. Results: Between Aug 11, 2011 and Nov 30, 2014 14 patients (o18yr, n ¼ 13, median age 9, range 1-17yr; 418yr, n ¼ 1, age 69yr) with hematological malignancies (AML, n ¼ 7; ALL, n ¼ 6; CML, n ¼ 1) received a total of 58 CIK cell infusions (median number 3, range 1-10 per patient) from matched unrelated (n ¼ 6) or haploidentical (n ¼ 8) stem cell donors at a minimum of 3 weeks after allo-SCT and an interval of 4-6 weeks between infusions (median follow up after 1st CIK cell infusion 213 days, range 56-802 days). 6 patients received CIK cell infusions based on minimal residual disease or chimerism status, 5 patients, most of whom weren’t in remission at the time of allo-SCT, were treated prophylactically, and 3 relapsed patients received CIK cell infusions with or without chemotherapy. Clinical batches of CIK cells were generated from peripheral blood mononuclear cells of the original stem cell donors under good manufacturing practiceconditions in the presence of interferong, anti-CD3 antibody,
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interleukin (IL)-2 and IL-15. Cellular composition of CIK cell infusions showed that CD19 þ B cells, CD14 þ monocytes and CD3-CD56 þ natural killer (NK) cells were quantitatively depleted while conventional CD3 þ CD56- T cells and CD3 þ CD56 þ T-NK cells made up the bulk of the CIK cell product. T cells, evenly distributed between the CD4 þ and CD8 þ compartment, were activated, which was shown by CD25 þ expression and were mostly TCRa/b type.Starting T cell dose within CIK cell infusions was 1x106 T cells/kg body weight. Regardless of donor type, dose escalation continued up to 1x108 T cells/kg body weight if no signs of aGvHD 4grade I occurred. Median doses of infused T cells were 6x107 in the matched unrelated and 1x107/kg in the haploidentical transplantation setting. Mild signs of aGvHD occurred in 2 patients (14%). Another 3 patients (21%) developed aGvHD grade III. In 6 patients (43%), 3 with overt and 3 with impending relapse, CIK cell infusions provided transient remission (median 218 days, range 56-563 days), followed by another allo-SCT in 2 cases. Deaths of 2 heavily pretreated AML-patients (14%) who remained in remission after CIK cell infusions were not CIK cell-related. Another 6 leukemia patients (43%), 3 of whom were not in remission at the time of SCT, are still in complete remission. All of them were offered CIK cell infusions from haploidentical donors. Conclusion: Allogeneic CIK cell infusions seemed to be very promising and may improve immune cell-based therapies especially in leukemia patients with impending relapse after allo-SCT. Disclosure of Interest: None declared. P012 Donor lymphocyte infusion (DLI) as salvage treatment for refractory or relapsed lymphoid malignancies after allogeneic stem cell transplantation: Experience from a single centre G. Martin Sanchez1,*, A. Insunza1, L. Yan˜ez1, A. Bermudez1, M. Colorado1, M. Lopez Duarte1, C. Amunarriz2, O. Irati1, B. Lopez Pereira1, C. Richard1, J. L. Arroyo2, E. Conde1 1 Hospital Universitario Marques de Valdecilla, 2Banco de Sangre y Tejidos de Cantabria, Santander, Spain Introduction: DLI has been used during the last decades for relapsed hematological malignancies after allogeneic stem cell transplantation, with different results. We present the outcomes and complications of patients with relapsed or refractory lymphoid malignancies treated with DLI. Materials (or patients) and methods: We retrospectively reviewed the 30 patients who received DLI between 1999 and 2014 after T cell-replete allogeneic stem cell transplantation. The median age at DLI was 42 (range: 8-69) years old. Diagnosis was multiple myeloma (MM) in 9 patients, Hodgkin´s lymphoma (HL) in 7, chronic lymphocytic leukemia (CLL) in 6, acute lymphoblastic leukemia (ALL) in 6 and non-Hodgkin´s lymphoma (NHL) in 2. All patients were off immunosuppressive treatment and with no active GVHD. Median time from progression or relapse to the first DLI was 63 (17-315) days, and 16 (53%) patients were additionally treated with chemotherapy or radiotherapy. A total of 71 DLI doses were infused, with a median of 2 (1-6) doses per patient. The maximum DLI dose varied between patients with an HLA matched donor (n ¼ 27) and mismatched (n ¼ 3, including one haploidentical) donor, with a median CD3 dose of 7,5x107/kg and 0,2x107/kg respectively. Relapses and responses were defined by disease-specific criteria based on flow cytometry, cytogenetic and imaging studies. Acute and chronic graft versus host disease (GVHD) was classified according to the Glucksberg and NIH criteria respectively. Results: Eleven (36,7%) patients achieved a complete response (CR), which lasted a median of 12 (1-61) months. Analyzing patients by diagnosis we observed a higher CR rate in ALL patients (4/6) followed by MM (3/9), CLL (2/6) and HL (2/7). All patients with MM and ALL relapsed within a median of 6 (1-12) and 8 (1-21) months, respectively, but none of the 4 patients with HL and CLL relapsed after a median follow-up of 33 (10-61) months (Table 1). Five (16,7%) patients
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(MM: 2, CLL: 1, HL: 1, NHL: 1) achieved a partial response, which was transient in all cases, with a median duration of 3 months. Overall 14 (47%) patients developed GVHD, which was acute in 4 cases (13%) and chronic in 10 (34%). Median time since the first DLI dose to GVHD onset was 33 (28-56) days in acute and 116 (89-223) days in chronic forms. Only 2 (7%) patients developed grade III-IV acute GVHD and 5 (17%) moderatesevere chronic GVHD. Skin was the most commonly affected organ in both acute and chronic GVHD. After a median follow-up of 20 (1-129) months for the entire cohort, patients who achieved CR after DLI had a significant higher overall survival at 2 years, 66% vs 44% (P ¼ 0,018). Disease progression was the main cause of death (n ¼ 16) and none of the patients died because of GVHD. Conclusion: We observed a low incidence of severe acute and chronic GVHD after DLI (7% and 17% respectively). More than half of the patients achieved partial or complete response (n ¼ 16). CR lasted more than 10 months in 7 (23%) cases, with no relapse at last follow-up in 4 of them (HL:2, CLL:2). DLI was a safe treatment and provided durable responses in a subgroup of patients with relapsed or refractory lymphoid malignancies. Disclosure of Interest: None declared. P013 Immunemodulation of blasts in AML-patients (pts) with clinically approved response modifiers to improve antileukemic T-cell reactivity: An ex vivo simulation of the clinical situation H. Schmetzer1,*, Z. Stankova1, D. Deen1, A. Hirn-Lopez1, Y. Vokac1, A. Rabe1, T. Stoschek1, T. Kroell1, C. Wendtner2, C. Schmid3, J. Tischer1 on behalf of Christoph Schmid and Johanna Tischer contributed equally 1 Dept for hematopoetic cell transplantations, University Hospital Munich Grosshadern, Munich, 2Dept for Hematology and Infectiology, Municipal Hospital Munich-Schwabing, Munich, 3 Dept for hematopoetic cell transplantations, Med2, Municipal Hospital Augsburg, Augsburg, Germany Introduction: Background: Antileukemic T-cell-reactivity has to be improved/re-established in AML-pts in vivo to prevent relapses. Ex vivo leukemia-derived DC (DCleu) are the most effective antileukemic T-cell-stimulators. Aim: To develop and test combinations of clinically approved immune modifiers that convert blasts into a ‘DCleu-vaccine‘, that induces specific antileukemic T-cell response and Tcell memory without induction of blast proliferation. Materials (or patients) and methods: Methods: A culture system using heparinised whole blood (‘WB‘, to simulate the in vivo situation) was developed to generate DCleu ex vivo from AML blasts from 52 AML-pts in active stages of the disease. 11 KITS containing selected combinations of 7 clinically approved compounds (GM-CSF, INTRON,
CALCIMYCIN, PICIBANIL, PGE1, PGE2, TNFa) were tested for their capability to generate DCleu and different DC-subtypes, as well as their potential to induce an (unwanted) blast proliferation. Further, the capability of DCleu generated from WB to stimulate antileukemic activity of T-cells from pts before or after SCT in vitro was evaluated. Results: Results: 1. Generation of DC (DCleu ) from WB: Our culture system qualified to test the convertibility from blastcontaining WB to DCleu using 11 different KITS of clinically approved immune modifiers. Average results of 7 Kits were comparable to those generated with standard methods, the remaining KITS did not produce efficient amounts of DC. Moreover patient-individuell variations were observed. 2. Some KITS induced ex vivo blast proliferation in individual pts‘ WB samples. 3. A ranking list allowed the evaluation of the best 5 of 11 blast modulating KITs. 4. Antileukemic reactivity of T-cells stimulated with KIT-treated WB could always be improved compared to controls, although with variations. 5. Blast lysis correlated with higher proportions of DC-subtypes (DC, DCleu), with proportions of CD8 þ Tcells detectable after mixed lymphocyte culture, IL-12 and IFNg concentrations and lower concentrations of IL-6 and -8 in culture supernatants. Conclusion: Conclusion and next steps: KITs can create DCsubtypes and especially DCleu from blasts in WB cultures. Their blast-proliferation-stimulating potential as well as their capacity to generate antileukemic T-cells can be evaluated. A ranking list based on DC, DCleu subtypes, antileukemic reactivity and cytokine profiles should be created to allow an individualised selection of ’best KIT‘ for in vivo applications in mouse or human. References: (Patent pending: reference number: 102014014993.5). Disclosure of Interest: None declared. P014 Expansion of Bone Marrow Derived Mesenchymal Stem Cells Using a GMP Compliant Closed System Bioreactor and Quality Analysis of the Expanded Cells I. Kozanoglu1,2,*, E. Maytalman3, P. Bilir2, G. Unver2, C. Siber2, C. Boga4, H. Ozdogu4 1 Physiology, Baskent University medical Faculty, 2Cell Processing Unit, BASKENT UNIVERSITY ADANA ADULT BONE MARROW TRANSPLANTATION CENTER, 3Cell Processing Unit, Baskent University Adult Bone Marrow Transplantation Center, 4Clinical Unit, BASKENT UNIVERSITY ADANA ADULT BONE MARROW TRANSPLANTATION CENTER, Adana, Turkey Introduction: The production of MSCs, requires extensive resources, space, labor and Good Manufacturing Practices compliance for clinical uses. In recent years, use of bioreactors have become popular as an alternative to open systems. The aim of this study is to share the quality control results of bone marrow derived MSCs expanded using a bioreactor (Quantum Cell Expansion System, Teruma BCT, Colorado, USA).
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Materials (or patients) and methods: 25 milliliters of bone marrow aspirated from a normal healthy donor, 4 liters of media and 4 liters of PBS was loaded to the Quantum machine using the closed system set. The cells were fed by media according to the results of regular lactate measurements as recommended by the manufacturer. After 14 days of culture, the cells were removed from the system using trypsin and cultured for the second passage. The samples taken from the final product were analysed for viability, cell count, microbial growth, endotoxins and telomerase enzyme activity. Flow cytometric studies, and lymphocyte proliferation inhibition tests were also performed. MSCs were differentiated to adipocytes and osteocytes. Results: Starting with a 25 ml sample of bone marrow, after 21 days of culture, 160 million cells were obtained. Cell viability was shown to be 99%. No microbial growth was observed and endotoxin analysis was negative. The cells were CD45 and CD34 negative but CD73, CD105 and CD90 positive. MSCs cultured with peripheral lymphocytes were shown to inhibit lymphocyte proliferation and the cells were shown to differentiate into adipocytes and osteocytes. Telomerase enzyme activity was determined to be below %1.5 RTA. Conclusion: Some studies have reported that immunomodulatory capacity of MSCs produced in different laboratories, although GMP compliant may not be standard. Moreover, culturing for too many passages and presence of hundreds of manipulations are important factors affecting the standardization of the cells. Bioreactors are advantageous as they provide a closed system (cells do not have any contact with the CO2 in the air), they require minimum air quality for production (GMP Class C is sufficient), more cells are obtained in less time and the product quality is high. Production with bioreactors also help decreasing the cost and time of cell expansion. Disclosure of Interest: None declared.
In T helper cells, compared to unstimulated cells, low-dose IL-2 or IL-15 alone and the combination with TKD peptide induced an up-regulation of KIR2DL2/3 and CD94. NKG2D expression increased after stimulation with IL-2 or IL-15. DNAM1 upregulation appeared only after stimulation with IL-15. NKRP1 was upregulated by IL-2± TKD and by IL-15 alone. Other NK receptors showed no or minimal expression on Th cells. In CTL cells, treatment with interleukines alone or with TKD peptide induced expression of KIR2DL2/3, CD94, DNAM1, NKRP1A. Cell number positive for NKG2D, NKp80, ILT2 and CD16 was increased by IL-2 or IL-15 and CD16 expression was also increased by the combination IL-2 ± TKD peptide. Other NK receptors showed no or minimal expression on CTL cells. In NKT cells, DNAM-1, KIR2DL2/3, KIR2DL4, NKG2D, CD94 and LAMP1 were up-regulated with IL-2 or IL-15 alone or in combination with TKD peptide. The expression of ILT-2 was increased after stimulation with IL-2 or IL-15 and also after stimulation with IL-15 plus TKD peptide. Up-regulation of NKG2C and NKp30 could be detected only after IL-15 treatment. NKRP1A and NKp80 were down-regulated by IL-2 or IL-15 and NKp80 showed even lower expression after stimulation with IL-12 plus TKD peptide. Other NK receptors showed no or minimal expression on NKT cells. Conclusion: The study showed that some activatory and inhibitory NK receptors are also expressed on CD3 þ CD4 þ T cells, CD3 þ CD8 þ T cells, B cells and NKT cells and can be regulated by the treatment with low-dose IL-2 or IL-15 themselves or in combination with TKD peptide under in vitro condition. Disclosure of Interest: I. Hromadnikova Funding from: This work was done under the framework of CellEurope project (FP7-People-2012-ITN, No. 315963) coordinated by Professor Anne Dickinson from University of Newcastle upon Tyne, UK., S. Li: None declared, A. Dickinson: None declared
P015 Influence of in vitro IL-2 or IL-15 alone or in combination with Hsp 70 derived 14-mer peptide (TKD) on the expression of NK cell activatory and inhibitory receptors on peripheral blood mononuclear cells I. Hromadnikova1,*, S. Li1, A. Dickinson2 1 Dpt. Molecular Biology and Cell Pathology, Third Faculty of Medicine, Charles University Prague, Prague, Czech Republic, 2 Institute of Cellular Medicine, Haematological Sciences, The Medical School, Newcastle University, Newcastle Upon Tyne, United Kingdom
P016 In vitro study of peripheral blood mononuclear cell cytotoxicity against Hsp70 positive human leukaemia cell lines in paediatric patients indicated to haematopoietic stem cell transplantation I. Hromadnikova1,*, S. Li1, P. Sedlacek2, A. Dickinson3 1 Dpt. Molecular Biology and Cell Pathology, Third Faculty of Medicine, Charles University Prague, 2Dpt. of Pediatric Hematology and Oncology, University Hospital Motol, Second Faculty of Medicine, Charles University Prague, Prague, Czech Republic, 3 Institute of Cellular Medicine, Haematological Sciences, The Medical School, Newcastle University, Newcastle Upon Tyne, United Kingdom
Introduction: NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. The aim of the current study was to investigate the expression of NK activatory and inhibitory receptors on T helper lymphocytes, cytotoxic T lymphocytes, B lymphocytes and NKT cells after preincubation of PBMN cells of healthy individuals with interleukines (IL-2, IL-15) alone and in combination with hsp70 derived peptide (TKD) using flow cytometry. This is the follow-up study of the expression of NK receptors on NK cells, whose results have already been reported. Materials (or patients) and methods: Flow cytometry was performed on days þ 1, þ 3, þ 5 (stimulated and unstimulated PBMN cells) using a standard direct immunofluorescence technique (anti-human monoclonal antibodies conjugated with FITC, PE, APC) on a FACSCaliburTM. NK activatory receptors (CD16, CD94,NKG2D, NKG2C, NKp46, NKp44, NKp30, NKp80, KIR2DL4, DNAM-1, and LAMP1) and NK inhibitory receptors (NKG2A, KIR2DL2/L3, ILT-2, and NKR-P1A) in healthy individuals were studied. Results were expressed as the percentage of receptor expressing cells on day þ 5. Results: In B cell population, ILT2 and LAMP were up-regulated and NKRP1A was down-regulated after stimulation with IL-2 or IL-5. NKG2D was up-regulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD peptide. Other NK receptors showed no or minimal expression on B cells.
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Introduction: NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. The aim of the study was to investigate the in vitro PBMN cell cytotoxicity after stimulation with IL-2 or IL-15 alone or in combination with hsp70 derived peptide (TKD) against human leukaemia cell lines derived from patients with CML, ALL and AML (UoC-M1, THP-1, TF-1, K562, Jurkat, CCRF-CEM) during the onset and/or relapse of the disease showing high membranebound Hsp70 positivity using ELISPOT. Materials (or patients) and methods: The first set of paediatric patients indicated to haematopoietic stem cell transplantation was analysed to see NK cell response against various leukemia cell line. 13 patients were analysed (1 cALL CR1, 1cALL CR2, 1 preBALL CR2, 1 CML CP1, 4 MDS, 1 SAA, 1 iMPAL, 1 SCID, 1 AML M5 CR1, 1 ALCL). All patients´ fresh peripheral blood samples were analysed in three different effector:target ratios (E/T 3:20, 1:10, 1:20). The constant concentration of target cells (50 000 cells/per well) and descending concentration of effector cells (7500, 5000 and 2500 cells/per well) were used. Results: Patient with SCID had no T and NK cells, which resulted in negative response in ELISPOT assay as expected. Very low response was observed in case of 2 patients with MDS-RAEBt
and MDS 7-. In case of other 2 patients with MDS (both MDS RAEB) significant number of spots was detected, however one patient did not show significant difference between responses to IL-2 or IL-15 with or without TKD peptide and the second patient showed increased response to IL-15 þ TKD compared to IL-15 alone only. The patient with SAA responded quite well in ELISPOT assay mainly to interleukines, but no significant increase of spots was observed when TKD peptided was added. The patient with iMPAL showed the significant increase in lysis of leukaemia cells derived from Jurkat, CCRF-CEM, K562 and UOCM1 leukaemia cell lines after the stimulation with the combination of IL-2 and/or IL-15 and TKD peptide compared with interleukines alone. Similarly the patient with cALL CR2 showed the highest response to leukaemia cell lines (Jurkat, CCRF-CEM, K562 and UOC-M1) after the treatment with IL-15 and TKD peptide. In case of patient (AML M5 CR1) the significant response to UoC-M1 (AML M1) and TF-1 (AML M6) cell line appeared after IL-15 þ TKD treatment compared to IL-15 alone. In case of patients with cALL CR1, CML CP1, preBALL CR2 and ALCL the immunotherapy will not probably be effective, since the combination of interleukines with TKD peptide did not lead to the increase of the PBMN cell response. Conclusion: PBMCs from different patients responded differently to various Hsp70 membrane positive leukemia cell lines after stimulation with low dose interleukines and TKD peptide. In the case of cALL CR2 and iMPAL patients the in vitro assay indicated that the immunotherapy with interleukines and Hsp70 derived peptide may be considered as a treatment option, if the patients´ own leukemic blasts will be highly Hsp70 positive. In the case of patients with cALL CR1, preBALL CR2 and CML CP1 the immunotherapy will not probaly be effective.
Disclosure of Interest: I. Hromadnikova Funding from: This work was done under the framework of CellEurope project (FP7-People-2012-ITN, No. 315963) coordinated by Professor Anne Dickinson from University of Newcastle upon Tyne, UK. , S. Li: None declared, P. Sedlacek: None declared, A. Dickinson: None declared P017 Full donor chimerism without graft-versus-host disease: the key factor for maximum benefit of pre-emptive donor lymphocyte infusions (pDLI) J. Feliu1,*, V. Potter1, J. Clay1, L. Floro1, C. Saha1, L. Barber2, G. Orti3, A. A. Alnagar1, F. Grimaldi1, M. Kenyon1, P. Krishnamurthy4, K. Raj1, D. McLornan1, H. de Lavallade1, A. Pagliuca1, G. Mufti1 1 Haematology Department, King’s College Hospital, 2Department of Haematological Medicine, King’s College London, London, United Kingdom, 3Servei d’Hematologia, Hospital Universitari de la Vall d’Hebron, Barcelona, Spain, 4Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom Introduction: Reduced-intensity conditioned (RIC) protocols and T-cell depletion (TCD) deliver low transplant-related mortality and decreased rates of graft-versus-host disease (GvHD). These advantages may however be mitigated by increased relapse rates and delayed achievement of full donor chimerism (FDC). Pre-emptive donor lymphocyte infusions (pDLI) potentially facilitate conversion of mixed donor chimerism (MDC) to FDC, with the aim of decreasing relapse
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risk. Despite many centres adopting this approach, the rutine use of pDLI remains controversial due to a lack of published data on the risk/benefit profile of this intervention. Herein we present the results of a large single centre analysis of the efficacy and toxicity of pDLI delivered via a uniform escalating dose schedule. Materials (or patients) and methods: A retrospective analysis of 119 consecutive patients (72 males/47 females) who received pDLI between Jan 2001 and Dec 2013 was performed. pDLI was delivered after TCD RIC allogeneic haematopoietic stem-cell transplantation (HSCT) for a range of haematological malignancies. Diagnoses included: AML (n ¼ 48), MDS (n ¼ 35), HL/NHL/CLL/ALL (n ¼ 24), MPN (n ¼ 8) and MDS-MPN (n ¼ 4). Donors were 81 identical siblings and 37 unrelated donors (18/37 were 1-2 antigen mismatched). FDC was defined as unfractioned and CD3 chimerism Z95%. Chimerism o95% in either fraction was considered MDC. Indications for pDLI were falling CD3 chimerism, CD3 o50% or mixed XY karyotype/FISH. Results: Median follow-up after DLI was 52.5 months. The 119 patients received 276 pDLI doses. 78 (66%) had a 2nd DLI dose, 40 (34%) a 3rd and 21 (18%) Z4 doses. Median number of pDLI doses per patient was 2 (range 1-7) and median cumulative pDLI dose was 1.56 x107 CD3 cells/Kg (range 5 x105 - 2.8 x108). Median time from HSCT to 1st pDLI was 267 days (range 83-3228). Efficacy: 71 patients (60%) converted to FDC after pDLI, of whom 1 returned to MDC. Relapse occurred in only 11/71 FDC patients (16%) compared to 16/46 MDC patients (35%) (P ¼ 0.024). The group who achieved FDC after pDLI had improved event-free survival (EFS) (P ¼ 0.0002) and overall survival (OS) (P ¼ 0.0003), compared to those with post-DLI MDC (Fig 1). These results did not differ for those with myeloid vs lymphoid malignancies. Interestingly, patients in FDC who developed DLI-induced GvHD showed a similar outcome to those with post-DLI MDC (Fig 2). Toxicity: Cumulative incidence of GvHD at 5 years after DLI was 37% (95% CI, 29% - 48%) and median time to GvHD onset after the 1st pDLI dose was 7 days (range, 1-39). The post-DLI FDC status was associated with the development of DLI-induced GvHD (P ¼ 0.034), but there was no correlation with pre-DLI GvHD, degree of HLA mismatch, T-cell dose or time from HSCT to DLI. Conclusion: A majority of patients who receive pDLI convert to FDC and impressively retain that status, potentially contributing to low rates of relapse and excellent long-term OS. The achievement of FDC after pDLI has an impact on survival, and those patients who achieve FDC without GvHD, experience maximum clinical benefit. Strategies to minimise DLI-induced GvHD should be considered to maximise the therapeutic potential of this intervention. Disclosure of Interest: None declared. P018 Immunosuppressive Properties by Activation of B7-H1 in Human Mesenchymal Stem Cells is mediated through STAT-1 signaling J. E. Park1,*, D.-H. Lee2, I. K. Jang2, K. C. Lee3 1 Pediatrics, Ajou University School of Medicine, Suwon, 2Biomedical Research Institution, Lifeliver Co. Ltd., Youngin, 3Pediatrics, College of Medicine, Korea University, Seoul, Korea, Republic Of Introduction: Mesenchymal stem cells (MSCs) derived from either bone marrow (BM) or Wharton’s jelly (WJ) inhibit proliferation of activated T cells, and interferon-gamma (IFN-g) plays an important role in this process. This IFN-g-licensed veto property is B7-H1-dependent in cell contact inhibition. However, the signaling pathway involved in B7-H1 expression of MSCs remains largely undefined. We demonstrate here a activation of B7-H1 through engagement of Signal Transducer and Activator of Transcription(STAT)-1 signaling in MSCs. Materials (or patients) and methods: Human BM- and WJ-MSCs were isolated and cultured. We compared the immunosuppressive effect of BM- and WJ-MSCs on
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phytohemagglutinin (PHA)-induced T-cell proliferation using direct and indirect co-culture system. B7-H1 expression on BMand WJ-MSC was detected by flow cytometry. The small interfering RNA method was used to knockdown the expression of STAT1. Results: The inhibitory effect of MSCs on T lymphocytes was observed in PHA-induced T cell proliferation assay. B7-H1 expression on human BM- and WJ-MSCs by IFN-g was increased time-dependently by flow cytometry. The inhibitory effect of MSCs on T cell proliferation could be restored when the anti-B7-H1 monoclonal antibody was used to block the B7H1. When the STAT-1 small interfering RNA was used to interfere with its signaling, B7-H1 expression on MSCs treated with IFN-g were decreased and T cell proliferation could be restored. Conclusion: These results show that the activation of B7-H1 by IFN-g in human BM- and WJ-MSCs is mediated through the STAT-1 signaling for immunosuppressive properties. References: 1. In Keun Jang, Hee-Hoon Yoon, Mal Sook Yang, Hyo Eun Kim, Doo-Hoon Lee, Myoung Woo Lee, Dae Seong Kim, Jun Eun Park. B7-H1 Inhibits T Cell Proliferation through MHC Class II in Human Mesenchymal Stem Cells. Transplant Proc. 2014; 46: 1638–1641 2. Chinnadurai R1, Copland IB, Patel SR, Galipeau J. IDOindependent suppression of T cell effector function by IFN-glicensed human mesenchymal stromal cells. J Immunol. 2014; 192(4): 1491–1501. 3. Tipnis S1, Viswanathan C, Majumdar AS. Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells: role of B7-H1 and IDO. Immunol Cell Biol. 2010; 88(8): 795–806. Disclosure of Interest: None declared. P019 Allogenic mesenchymal stem cell transplantation as a rescue therapy for lupus nephritis patient refractory to conventional induction therapy D. Wang1, H. Zhang1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Allogenic mesenchymal stem cells (MSCs) transplantation demonstrated significant clinical efficacy on various autoimmune diseases. Here we analyzed the role of allogenic MSCs transplantation in renal remission in patients with active and refractory lupus nephritis (LN). Materials (or patients) and methods: Eighty-one patients with active and refractory lupus nephritis in China from 2007 to 2010 were enrolled in the study. Allogenic bone marrow or umbilical cord derived MSCs were administered intravenously at the dose of one million cells per kilogram of bodyweight. Then all the patients were followed up for 12 months to evaluate renal remission as well as possible adverse events. The primary outcomes were renal complete remission (CR) and partial remission (PR) at each visit times, as well as renal flares. The secondary outcomes included renal activity score, total disease activity score, renal function and serologic index. Rates of overall survival, renal remission, as well as relapse at different visit times were calculated by Kaplan-Meier method and were statistically tested with the log-rank test. We calculated the HR and their 95% CIs using the univariate Cox proportional hazards model. Results: The overall survival rate during 12 months follow-up was 95% (77/81). The probability of renal remission was 41% (18% CR and 23% PR) at 3 months, 45% (18% CR and 27% PR) at 6 months and 44% (23% CR and 21% PR) at 12 months after allogenic MSCs transplantation. Renal remission was not correlated with age, disease duration, MSCs source and baseline SLEDAI score, but was significantly correlated with baseline proteinuria (P ¼ 0.003, 95%CI 0.336-0.794) and serum creatinine levels (P ¼ 0.047, 95%CI 0.224-0.990) by COX regression analysis. Eleven in 81 (14%) patients underwent renal flare in 12 months follow up after a prior complete or
partial remission. Renal activity evaluated by BILAG score significantly declined after MSCT, in parallel with the obvious amelioration of renal function. Total disease activity evaluation by SLEDAI score also decreased after treatment. Additionally, the doses of concomitant prednisone and immunosuppressive drugs were tapered. Four of 81 patients died of uncontrolled lupus nephritis unrelated to MSCT. Conclusion: Allogenic MSCs transplantation resulted in renal remission within 12 months visit, which could be used early as a potential induction therapy for active and refractory lupus nephritis. Disclosure of Interest: None declared. P020 Therapeutic effects of mesenchymal stem cells, anti-tumor necrosis factor and anti-CD20 On collagen induced arthritis Y. Sun1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Tremendous progress has been made in the development of non-conventional therapies for rheumatoid arthritis (RA). In this study, the effects of mesenchymal stem cells (MSCs) transplantation on established collagen-induced arthritis (CIA) were evaluated and compared with two kinds of biologic agents, anti-tumor necrosis factor (TNF) and antiCD20 antibody. Materials (or patients) and methods: CIA was induced with the immunization of type II collagen (CII) and CFA in DBA/1J mice. Human umbilical cord derived MSCs (5 106), anti-TNF antibody (100ug) and anti-CD20 antibody (200ug) were intraperitoneally injected into 3 groups of mice on day 28 after the immunization. The control group was treated with human fibroblasts (5 106). All mice were sacrificed 3 weeks later and arthritis severity was assessed by clinical and histology scoring. The frequency of CD4 þ T cell subsets, B cells and plasma cells in spleen was analyzed by flow cytometry. Serum levels of autoantibody to mouse CII were determined by ELISA. The ability of MSCs to modulate Treg/ Th17 cell percentages in CD3/CD28 stimulated DBA/1J T cells was assessed in vitro. Results: MSCs treatment significantly decreased the severity of arthritis and pathology scores, which was comparable to anti-TNF or anti-CD20 treatment. Anti-CD20 treatment depleted nearly half of B220 þ cells, and markedly reduced the frequency of plasma cells and serum levels of autoantibody compared to the control group (738±187 vs. 1817±447 U/ml, Po0.001). The decrease of autoantibody level was also detectable in those with anti-TNF treatment (663±336 U/ml) and MSCs treatment (1057±362 U/ml), but neither of these two treatments had an impact on the percentage of B cells or plasma cells. All of the three treatments resulted in a decrease in Th1 subset, but none of them altered the percentage of Th2 subset. Except anti-CD20 treatment, both MSCs and anti-TNF treatment significantly decreased the percentages of Th17 cells. Notably, only MSCs treatment increased the percentages of regulatory T cells (11.39±0.85 % vs 7.37±1.82 % in the control group, Po0.01). In vitro study confirmed that MSCs could induce the generation of Foxp3 þ T cells but reduce the percentages of pathogenic IL-17 þ Foxp3 þ T cells. Conclusion: MSCs exerted comparable therapeutic effects as biological agents on CIA through different mechanisms. MSCs may provide a promising approach for the treatment of RA. Disclosure of Interest: None declared.
Hematopoietic stem cells P021 Haploidentical T-cell replete hematopoietic transplantation conditioned with thiotepa-fludarabinebusulfan (TBF) and followed by graft versus host disease prophylaxis including high-dose cyclophosphamide and tacrolimus: Results from 4 Spanish centers A. Esquirol1,*, M. J. Pascual2, M. A. Cuesta2, J. L. Pin˜ana3, R. Rojas4, M. Alejandra2, R. Martino1, J. Sierra1 1 Hematology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, 2Hematology department, Hospital Regional Universitario Carlos Haya, Malaga, 3Hematology Department, Hospital Clı´nico Universitario de Valencia, Valencia, 4Hematology Department, Hospital Reina Sofia, Cordoba, Spain Introduction: Hematopoietic stem cell transplantation (HSCT) is an effective therapy for a variety of severe haematological diseases. Approximately 40% of patients who need a transplant and lack a related donor has an unrelated donor in due time. Cord blood transplants are another option but hematopoietic and immune recoveries are slow. Haploidentical family transplantation using T-cell repleted grafts and high-dose cyclophosphamide (HAPLO-CY) are increasingly being performed as an alternative. Materials (or patients) and methods: Thirty-four patients without a related or unrelated donor consecutively received an HAPLO-CY between 04/13 and 09/14 in 4 hospitals using the same protocol. High-dose conditioning regimen included thiotepa 5 mg/kg x 2 days (-7 and -6), fludarabine 50 mg/m2 x 3 days (-5, -4 and -3) and busulfan 1 mg/kg/6h x 3 days or its equivalent IV dose (day -5, -3 and -2). In patients older than 55 years busulfan administration was limited to two days. Graft versus host disease (GvHD) prophylaxis consisted of cyclophosphamide 50 mg/kg on day þ 3 and þ 4, and tacrolimus continuous iv infusion from day þ 5 and adjusting for blood levels 5-10 ng/L. Results: Median age of the patients was 49 years (range 5-69), 52.9% male and 47.1% female. Diagnosis distribution was: AML 10 (29.4%), MDS 12 (35.3%), ALL 6 (17.6%), Hodgkin Lymphoma 1 (2.9%), CLL 2 (5.9%) and MM 3 (8.8%). Disease status at transplant was 44.1% in complete remission (23.5% CR1, 14.7% CR2 and 5.9% CR3), 26.4% in partial or very good partial remission, 11.8% had stable disease, 17.7% had progression or refractory disease. 29.4% of patients had received a prior transplant. Median age of the donors was 33 years (range 19-74), being in 35.3% a son, 14.7% a daughter, 11.8% a brother, 14.7% a sister, 8.8% the father and 14.7% the mother. Peripheral blood and bone marrow stem cells source were used in 67.6% and 32.4% of cases, respectively. Median CD34 cell dose infused was 3.7x10e6/Kg. Median times to neutrophil (0.5x109/l) and platelet (4 50x109/ l) recoveries were þ 17 and þ 23 days, respectively (2 patients had graft primary failure, one of them recovered). Median follow up of the series is 181 days (range 40-495). Grades 3-4 gastrointestinal, mucositis and liver toxicities have been observed in 4.7%, 26.4% and 14.7% of cases (only one patient developed hepatic sinusoidal obstructive syndrome). Non-severe hemorrhagic cystitis has been observed in 35.3% of patients. The cumulative incidence of acute GvHD grade 2-4 was 26% and of grade 3-4 was 23%. 22 patients were at risk for chronic GvHD, 3 patients (8.8%) developed limited and 3 (8.8 %) moderate/severe GvHD. The cumulative incidence of non-relapse mortality (NRM) was 21% at one year and the causes were acute GvHD (2), infections (2), idiopathic encephalopathy (2) and graft failure (1). Relapse was observed in 4 patients, 2 of them remaining alive. Overall survival (OS) and progression free survival (PFS) are 71.4% and 70.1% at one year.
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Conclusion: Haploidentical T-cell repleted transplantation conditioned with TBF and followed by cyclophosphamide and tacrolimus as GVHD prophylaxis is a valid option with encouraging OS and PFS at one year. Extrahematological toxicities and NRM were limited. Disclosure of Interest: None declared.
in pediatric matched sibling donor transplants using granulocyte colony-stimulating factor-primed bone marrow and steady-state bone marrow. Pediatr Transplantation 2007: 11: 279–285. Disclosure of Interest: None declared.
P022 Clinical outcomes and graft characteristics in pediatric hematopoietic stem cell transplantation: effect of granulocyte-colony stimulating factor priming A. Fettah1,*, N. Ozbek1, F. Azik1, Z. Avci1, P. Isik1, N. Yarali1, B. Tunc1 1 Pediatric Hematology and Bone Marrow Transplantation Unit, Ankara Children’s Hematology and Oncology Research Hospital, Ankara, Turkey
P023 Important prognostic impact of early monocytes recovery after reduced intensity conditioning double umbilical cord blood allogeneic stem cell transplantation in adults A. Le Bourgeois1,*, T. Guillaume1, J. Delaunay1, P. Perterlin1, V. Dubruille1, S. Le Guill1, P. Moreau1, M. Mohty2, L. Campion3, P. Chevallier1 1 he´matologie clinique, CHU Hoˆtel Dieu, Nantes, 2he´matologie clinique, hoˆpital st Antoine, Paris, 3Institut de Cance´rologie de l’Ouest , Rene´ Gauducheau , St Herblain, France
Introduction: Hematopoietic stem cell transplantation from HLA-matched siblings significantly improved the long-term survival of the children with hematologic malignancies and bone marrow failure syndromes. Traditionally, steady-state bone marrow (S-BM) has been used as the source of stem cells. However, after obtaining that granulocyte colony-stimulating factor (G-CSF) promotes hematopoietic progenitors and increase their numbers both in the bone marrow (BM) and in peripheral blood (PB), transplantation after G-CSF mobilization has gained regard. Results of clinical trials demonstrated that G-CSF primed PB stem cells (G-PBSCs) offer the advantage of higher stem cell dose, accelerated engraftment and shorter neutropenic period compared to those with S-BM. On the other hand, it has been associated with a higher risk of chronic graft versus host disease (GvHD). Studies concerning the use of G-CSF stimulated bone marrow (G-BM) revealed that it was safe and able to produce rapid engraftment as is the G-PBSCs with a lower incidence of chronic GvHD compared to those who received G-PBSCs. In this retrospective study, we compared the clinical outcomes of children who had matched related hematopoietic stem cell transplantation (HSCT) by means of G-BM cells with those who transplanted with S-BM cells. Materials (or patients) and methods: From April 2010 to November 2013, a total of 64 pediatric patient and HLAmatched family donors were enrolled in the study at our Bone Marrow Transplantation Unit. Steady state bone marrow was harvested from 35 donors (S-BM group). Bone marrow priming was performed to 29 donors (G-BM group) when the weight of donor and recipient was discordant. Priming was performed in G-BM group with G-CSF at 10 mg/kg/day for 3 consecutive days, given as a single-dose subcutaneous injection. Results: There were 29 patients (14 male, 15 female; median age 10.5 years, range: 1.8-16.5) in the G-BM group and 35 patients (17 male, 18 female; median age 7 years, range: 1-16.3) in the S-BM group. Mean difference between weight of recipient and donor was smaller in G-BM group compared to those in S-BM group [4 kg (range: -20.5 & 38) vs. 18 kg (range: -10 & 52), P ¼ 0.001]. Granulocyte colony stimulating factor primed bone marrow (G-BM) yielded higher numbers of CD34 þ cells (180/mL vs. 125/mL, P ¼ 0,05), total nucleated cells (7,1x108/kg vs. 4,1x108/kg, P ¼ 0,0001), CFU-GM (13,82x104/kg vs. 7,95x104/kg, p:0,017) compared to those in S-BM. Neutrophil engraftment (14 vs. 15 days, p:0,699), platelet engrafment (25 vs. 24 days, p:0,994), length of stay in hospital (38 vs. 38 days, p: 0,707), overall survival and disease free survival were similiar in G-BM and S-BM groups. Also the cumulative incidence of grade II to IV acute GvHD (%10,3 vs. %11,4, P ¼ 0,809) and chronic GvHD (%20,7 vs. %17,1, p: 0,720) were similiar. It was observed that the use of G-CSF has no effect on risk of acute and chronic GvHD. Conclusion: We concluded stem cell mobilization with G-CSF is an effective and safe method in children without increasing the risk of GvHD. References: 1-Chiang KY, Haight A, Horan J, Olson E, Gartner A, Hartmen D, et al. Clinical outcomes and graft characteristics
Introduction: Little is known regarding the impact of hematopoietic and immune recoveries after double umbilical cord blood (dUCB) allogeneic stem cell transplantation (allo-SCT), especially after the TCF (low dose 2 Grays total body irradiation þ cyclophosphamide 50 mg/Kg 1 day þ fludarabine 200 mg/m2 5 days) reduced-intensity conditioning (RIC) regimen, which is considered as a standard RIC regimen for dUCB allo-SCT in adults. Materials (or patients) and methods: Here we considered a homogeneous cohort of 47 patients (males: n ¼ 24; median age: 55.5 years (range: 17.4-69.1, myeloı¨d disease 55%) who engrafted after a dUCB TCF allo-SCT performed between November 2006 and April 2013 in our department. GVHD prophylaxis consisted of cyclosporine þ mycophenolate mofetyl in all cases. All patients received G-CSF from day 1 until neutrophils recovery. The median nucleated cells dose infused was 4.17 107/kg. The aim of the study was to investigate the impact on outcomes of the recovery of the following cellular subsets: leucocytes, monocytes, lymphocytes, neutrophils at day þ 30 and day þ 42, and CD4 þ , CD8 þ T cells, B and NK cells at day þ 100. Median counts of leukocyte subpopulations were used to separate our patients into two groups of analysis. Results: Median times for neutrophils and platelets recoveries were 17 days (range: 6-59) and 37 days (range: 0-164), respectively. With a median follow-up of 30.4 months (range: 2.8-77.5), the 3-year overall and relapse-free survivals (OS, RFS), relapse incidence (RI), and non-relapse mortality (NRM) were 65.7%, 57.2%, 27.1% and 19%, respectively. The cumulative incidences of grade II-IV and grade III-IV acute GVHD were 38.3% and 10.6%, respectively, while, 3-year incidence of chronic GVHD was 53.5% (limited 42%, extensive 11.5%). In multivariate analysis, early higher monocytes and leukocytes count after transplant were the two independent factors associated with a significantly higher OS (monocytes 4615/ mm3 at day þ 30, HR: 0.07; 95%CI: 0.08-0.57, P ¼ 0.013, leukocytes 4 4250/mm3 at day þ 42, HR: 0.24; 95%CI: 0.0610.098, P ¼ 0.047). Early haematological recovery was predictive of lower NRM (leukocytes 4 2760/ mm3 at day þ 30, HR: 0.10; 95%CI: 0.01-0.91, P ¼ 0.042, lymphocytes 4 395/mm3 at day 42, HR: 0.10, 95%CI: 0.11-0.93, P ¼ 0.044). As well, higher monocytes count at day þ 42 (4830/mm3) remained independently associated with better RFS (HR: 0.18, 95%CI: 0.05-0.68, P ¼ 0.011). Older age and lymphoid disease were other independently factors predicting higher RFS (455 years, HR: 0.90; 95%CI: 0.85-0.96, P ¼ 0.001, lymphoid disease, HR 0.18; 95%CI: 0.05-0.63, P ¼ 0.007). No factor was predictive of grade II-IV or grade III-IV acute GVHD or chronic GVHD. Conclusion: These results suggest that early haematological recoveries (especially higher monocytes count) are predictive of outcome after dUCB TCF RIC allo-SCT in adults. Immune recovery seems to have no impact on survivals in this series while influence of age has to be confirmed by other studies. Thus, our results pave the way for future studies aiming to closely and prospectively monitor the kinetics of hematopoietic and immune recoveries after this type of graft. As all
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patients received G-CSF after transplant, other immunostimulatory cytokines should be tested to ensure sufficient hematopoietic recovery in the setting of adult dUCB TCF RIC allo-SCT. Disclosure of Interest: None declared. P024 Hematopoietic Stem Cell Transplantation in the elderly: nutritional and geriatric assessment A. Z. Pereira1,*, J. Silva2, A. P. Barrere2, M. Tanaka2, F. Lucio2, M. Nicastro1, S. Piovacari2, P. Rodrigues1, L. Koch1, N. Hamerschlak1 1 Oncology, Hematology and Hematopoietic Stem Cell Transplantation, 2Clinical Nutrition, Hospital Israelita Albert Einstein, S.Paulo, Brazil Introduction: Hematopoietic stem cell transplantation (HSCT) may improve outcomes of patients with hematologic malignancies not curable with conventional therapies. Being in some diseases the only curative option. HSCT in elderly patients with good performance status and no comorbidities could, in fact, not only survive the transplant with reasonable risk, but also benefit in the same measure as younger patients. Objectives: To study and correlate nutrition and geriatric assessment in elderly patients undergoing HSCT. Materials (or patients) and methods: A retrospective study of 17 elderly patients (460 years) undergoing HSCT May 2012 to January 2014 in the Hematology-Oncology and Bone Marrow Transplantation Center at Albert Einstein Hospital in Sa˜o Paulo, Brazil. All patients were evaluated approximately one month prior to HSCT. In the geriatric assessment were done hand-grip strength(HGS), questions about mobility and functional limitation. In the nutrition, we studied the Body Mass Index(BMI) (kg/m2), and serum levels of vitamin D, zinc and albumin. Results: 17 elderly patients were observed in this study, mean age was 65,5±3,8 years, BMI was 28±6,0 kg/m2, HGS was 28±8,5 kg; serum levels of albumin 3,2±0,5g/dl (normal:3,55,0); serum level of vitamin D 23,4±14 ng/ml (normal 420); serum levels of zinc 65,5±18 mg/dl (normal: 66-132,5 mg/dl). We found the negative correlation between BMI and HGS (rp ¼ 0,42). There were a significant and positive association between serum levels of zinc and albumin, and HGS and grades of mobility questions (Po0,05). The serum levels of vitamin D weren’t significantly associated with geriatric factors. Conclusion: Our study showed that the obese patients with more risks of complications in HSCT had more functional limitation. Besides low levels of zinc and albumin were associated with worst results in the geriatric assessment. In the elderly the immobility and weakness can increased the complications after HSCT. The geriatric and nutrition assessment are important to improve HSCT results. References: 1. Liu P, Zhang ZF, Cai JJ, Wang BS, Yan X. NRS2002 assesses nutritional status of leukemia patients undergoing hematopoietic stem cell transplantation. Chin J Cancer Res 2012; 24(4): 299–303. 2. Horsley P, Bauer J, Gallagher B. Poor nutritional status prior to peripheral blood stem cell transplantation is associated with increased length of hospital stay. Bone Marrow Transplant 2005; 35(11): 1113–1116. 3. Hadjibabaie M, Iravani M, Taghizadeh M, Ataie-Jafari A, Shamshiri AR, Mousavi SA, et al. Evaluation of nutritional status in patients undergoing hematopoietic SCT. Bone Marrow Transplant 2008; 42(7): 469–473. 4. Iestra JA, Fibbe WE, Zwinderman AH, van Staveren WA, Kromhout D. Body weight recovery, eating difficulties and compliance with dietary advice in the first year after stem cell transplantation: a prospective study. Bone Marrow Transplant 2002; 29(5): 417–424. 5. Kyle UG, Chalandon Y, Miralbell R, Karsegard VL, Hans D, Trombetti A, et al. Longitudinal follow-up of body composition in hematopoietic stem cell transplant patients. Bone Marrow Transplant 2005; 35(12): 1171–1177. Disclosure of Interest: None declared.
P025 Voriconazole as antifungal prophylaxis in allogeneic hematopoietic stem cell transplantation: ten years of experience in a single institution C. CHIC ACEVEDO1,*, M. J. LLAMAS POYATO1, E. GARCIA TORRES1, P. MARI1, G. DIEZ1, J. SERRANO1, J. R. MOLINA1, C. MARTIN1, F. MARTINEZ1, C. HERRERA1, R. ROJAS1 1 HEMATOLOGY, HOSPITAL UNIVERSITARIO REINA SOFIA, CORDOBA, Spain Introduction: The arrival of new active azoles against filamentous fungi has encouraged its use in the prophylaxis of invasive fungal disease (IFD) in patients undergoing allogeneic hematopoietic stem cell transplantation (AlloHSCT). The aim of this study was to analyze the incidence of IFD in patients who received Voriconazole as prophylaxis. Materials (or patients) and methods: In this retrospective study, we analyze 252 consecutive allo-HSCT patients (pediatrics and adults) undergoing this procedure in our institution. Patients were recruited from June 2004 to April 2014. All patients received antifungal prophylaxis with oral Voriconazole 200mg/ 12h (5mg/kg/12h in children) from day 0 to day þ 75. Antifungal prophylaxis was prolonged in patients requiring treatment with high doses of corticosteroids (42mg /kg /day) and/or with two immunosuppressive drugs. IFDs were classified according to the revised criteria of the EORTC/MSG 2008. Only proven and probable IFDs were considered for this study. Drug toxicity was evaluated according to CTCAEv4.1. Fungal free survival (FFS) was calculated early and late in the post-transplant period ( þ 100, þ 360 day respectively). The development of adverse drug reactions (ADRs) was analyzed as well as the most influential prognostic factors in the development of IFD and in overall survival (OS). The incidence of IFD in patients receiving Voriconazole as antifungal prophylaxis was compared to our historical cohorts (98 patients recruited from 2001 to 2004 undergoing Allo-HSCT who were administered Fluconazole as antifungal prophylaxis). Results: 157 patients (61.6%) were male and 95 (37.3%) female. The median age was 35 years (1-68). 29 patients (11.5%) had suffered IFD previously. The incidence of IFD in patients receiving Voriconazole as primary prophylaxis was 7.5% (n ¼ 19) and 3.6% in secondary prophylaxis. These results show a 50% reduction in the incidence of IFD in our historical cohorts. Considering only primary prophylaxis, the median onset of IFD was 115 days after Allo-HSCT. There were 5.9% (n ¼ 15) probable cases of IFD and 1.6% (n ¼ 4) proven cases. 9 patients developed early IFD and 10 late one: the latter group were mainly patients with cGVHD receiving long-lasting corticosteroid treatment. The most common cause of IFD was Aspergillus: (n ¼ 15), followed by Mucor (n ¼ 2), Candida (n ¼ 1) and Scedosporium (n ¼ 1). 81 patients discontinued Voriconazole: 61 (24%) temporarily and 20 (8.3%) permanently. The commonest cause for discontinuation was the development of ADRs in 66 patients (26.2%) being liver toxicity the most frequent (44 patients, 17.4%). OS was 59% with a median follow up of 30 months (1-118m). Global FFS at 100 days and 365 days after Allo-HSCT was 93.7% (88.7-98.7) and 89% (83-95) respectively. We found a relation between treatment with high doses of corticosteroids (42mg/kg/day) and incidence of IFD (P ¼ 0.011), with a FFS of 307 days vs 336 days in those receiving vs not receiving this treatment. This variable lost its significance when included in the multivariate analysis (P ¼ 0.051). We found no links between IFD and CMV infection (P ¼ 0.14) or severe mucositis (P ¼ 0.41). Conclusion: Voriconazole as primary prophylaxis in Allo-HSCT has reduced the incidence of IFD to 50% in our institution. We therefore suggest that it is an effective drug to prevent IFD in Allo-HSCT. Disclosure of Interest: None declared.
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P026 Successful bone marrow processing for transplantation with Spectra Optia cell separator Version 9 C. Wissing1,*, A. Prinsloo1, N. Besson2 1 oncology, Pretoria east Hospital, Pretoria, South Africa, 2 Scientific marketing, TerumoBCT, Brussels, Belgium Introduction: Bone marrow (BM) is one source of hematopoietic stem cells (HSC) transplantation. Before storing of both autologous and allogeneic bone marrow, it is strongly recommended to downsize bone marrow volume in order to reduce the cryogenic storage space but also to prevent DMSO toxicity during infusion. In addition, ABO-incompatible grafts may be RBC depleted to minimise the risk of ABO-induced transfusion reactions. Several methods are available. In our center, erythrocyte depletion of BM harvests has been performed on a continuous flow cell separator because of its high mononuclear cell (MNC) yield, low erythrocyte contamination and closed processing system.This study describes a single center experience in processing 11 harvested bone marrow in the blood separator Spectra Optia version 9 (TerumoBCT). Materials (or patients) and methods: 4 patients received BM transplants form ABO incompatible donor and 7 of the harvests were autologous. Directly after ending of the collection, BM was processed on the Spectra Optia version 9. Briefly, volume of the BM was determined as well as complete blood count. The CD34 þ cell counts were measured by flow cytometry and the 7-aminioactinomycin D (7-AAD) dye was used to exclude non-viable cells. 6-8 cycles were processed at an inlet of 60 ml/ min. Neutrophil engraftment was defined as the number of days after infusion until the circulating peripheral blood count reached 0.5x10E9/l for 3 consecutive days and platelet engraftment as the number of days after infusion to reach peripheral blood count Z50x10E9/L. Results are presented as mean±SD. Results: The initial bone marrow collections had a mean volume 1451±262 mL (20.8±3.7 mL/kg)(cf. Table 1). Following processing on the Spectra Optia, the mean product volume was 155.5±37.2 mL (which means a volume reduction 89.1±2.8%) and final product hematocrit was very low 3.45±0.87%. Red blood cell volume reduction was 98.85±0.45%. The mean CD34 þ cells recovery was high 106.7±32.6%. The mean CD34 þ cell dose infused was 2.3±1.4x10E6/kg; the overall CD34 þ cell viability was 98.7±1.2%. Neutrophil engraftment at 0.5x10E9/l averaged 20.8±3.7 days. Conclusion: Spectra Optia is safe and effective to reduce both the volume and red blood cell content of bone marrow collections while ensuring high CD34 þ cells recoveries. Because of the low residual red blood cell content, ABO incompatibility between donor and recipient can be successfully overcome by BM processing using Spectra Optia. Quality of the graft is not affected by BM processing. Disclosure of Interest: None declared.
P027 Prolonged Regression of Ileocolonic Crohn’s Disease following Autologous Haemopoetic Stem Cell Transplantation C. Hawkey1,* on behalf of On behalf of the ASTIC Trialists 1 NOTTIGHAM DIGESTIVE DISEASES CENTRE, UNIVERSITY OF NOTTINGHAM, Nottingham, United Kingdom Introduction: In the ECCO-EBMT ASTIC trial autologous Haemopoietic stem cell transplantation (HSCT) led to mucosal healing in Crohn’s disease. We now report prolonged regression of ileocolonic disease over 2years. Materials (or patients) and methods: Patients with impaired quality of life from active Crohn’s disease not amenable to surgery, despite treatment with Z3 immunosuppressive agents all underwent stem cell mobilisation before randomisation to immunoablation followed by unselected cyclophosphamide-based conditioning and HSCT after 1 month (Early HSCT)or 1 year (delayed HSCT). They were assessed using Crohns Disease activity index (CDAI), Simple Endoscopic Scores (SES-CD), Inflammatory Bowel Disease Questionnaire (IBDQ) and EuroQoL. Results: Forty four patients with ileocolonic involvement underwent mobilisation before randomisation to Early (n ¼ 23, all treated) or delayed (n ¼ 21) HSCT. One Early HSCT patient died 14 days after HSCT. One randomised to delayed HSCT withdrew immediately, 4 did not proceed because they required surgery (n ¼ 3) or were too well after 1 year. Sixteen underwent delayed HSCT. The CDAI fell from 321 (median, IQR 242-433) to 162 (76-294, P ¼ 0.002) one year after Early HSCT vs 353 (300-462)to 310 (236-403, P ¼ 0.124) over year 1 prior to delayed HSCT. At year 2 the CDAI was maintained at 161 (59232, P ¼ 0.555 vs Year 1) in Early HSCT patients and fell to 165 (88-298, P ¼ 0.022) following delayed HSCT. The SES-CD was 13 (8.5-24.5) at baseline, 3(1-10) after one year (p Conclusion: Improvements gained one year after HSCT are maintained into a second year. HSCT delayed by one year appears to be effective. There is a progressive improvement in quality of life. If improvements continue to be durable, the risks of HSCT might become acceptable for a wider group of patients. Disclosure of Interest: None declared. P028 Mobilization in patients over 65 years of age and patients between 55 to 65 years of age is similar in terms of safety and success in Multiple Myeloma and lymphoma patients: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, C. Bal3, I. Dora1, B. Kosan1, E. Er1, N. Baskan1, B. Saritas1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, Kocaeli, 3 Biostatistics Department, Osmangazi University, Eskisehir, Turkey Introduction: Autologous stem cell transplantation is a standard procedure in relapsed lymphoma patients and in multiple myeloma patients after first line treatment especially in patients less than 65 years old. However, autologous stem
Table 1. n ¼ 11
Mean
Standard deviation
BM volume before processing (mL) BM volume after processing (mL) BM volume reduction (%) CD34 þ cells viability (%) CD34 þ cells 10E6/kg before processing CD34 þ cells 10E6/kg after processing CD34 recovery (%) Red blood cell volume reduction (%) ANC 50 engraftment (days)
1451.5 154.9 89.1 98.7 2.4 2.3 106.7 98.9 19.5
262.6 37.3 2.7 1.2 1.4 1.4 32.6 0.5 8.0
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[P028]
cell transplantation could be applied to patients over 65 years if they fullfill certain criterias . In the present study, we aimed to compare the results of mobilization in multiple myeloma or lymphoma patients who were over 65 years and between 5565 years. Materials (or patients) and methods: There were 40 patients over 65 years and there were 81 patients between 55-65 years. The stem cells were harvested from the peripheral blood. While, 30 of the 81 patients aged between 55-65 were female, 12 of the 40 patients over 65 years were female. 75,6% of the patients aged between 55 and 65 and 72,5% of the patients over 65 years were Multiple Myeloma patients. Chi-square test (continuity correction) was used as the statistical method to compare the results. Characteristics of the patients are shown in Table 1. Results: While, median age of the group of the patients who were between 55-65 years was 60, median age of the patients over 65 years were 67(66-80). There were no significant differences between the 2 groups in terms of mobilization regimens used, febrile neutropeni incidence, transfusion requirements (erythrocyte and thrombocyte) and total CD34 þ cell count (P40,005) (Table 2). Conclusion: Mobilization in patients older than 65 years is as safe and successful as mobilization in patients between 55-65 years. Disclosure of Interest: None declared. P029 First 100 Day Results Multiple Myeloma And Lymphoma Patients Over 65 Years are Similar to Multiple Myeloma And Lymphoma Patients Who are 55-65 Years Old: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, C. Bal3, I. Dora1, B. Kosan1, E. Er1, B. Saritas1, N. Baskan1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, Kocaeli, 3 Biostatistics Department, Osmangazi University, Eskisehir, Turkey Introduction: We aimed to compare results of Autologous Hematopoietic Stem Cell Transplantation (AHSCT) patients who were over 65 years to those between 55 to 65 years. Materials (or patients) and methods: We retrospectively analyzed results of 40 patients who were over 65 years and had AHSCT and 81 patients who were 55-65 years and had AHSCT. Peripheral blood was used as the source of the stem cells. Acyclovir and fluconazole were started at the day of transplantation and piperacillin-tazobactam were started when neutropenia ensues prophilactically. We compared 100th day results. Chi-square test (continuity correction) was
used as the statistical method to compare the results. Characteristics of the patients are shown in Table 1. Results: Characteristics of the patients are shown in Table 1 and clinical results are shown in Table 2. While, median age of the group of the patients who were between 55-65 years was 60, median age of the patients over 65 years were 67(66-80). Median stem cell count infused were similar in the 2 groups (5,1 vs 5,3, P40,05). Median follow-up times of the 2 groups were not significantly different (11 months vs 13 months, P ¼ 0,35). Median time to neutrophil and thrombocyte engraftment, hospitalization durations and bacterial and viral infection rates of the 2 groups did not differ significantly (P40,05). 100th day mortality ratios of the 2 groups were similar (1,3% vs 2,7%, P ¼ 0,241). Conclusion: AHSCT in patients over 65 years is as safe as AHSCT in patients between 55 to 65 years. Disclosure of Interest: None declared.
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P030 Comparative study of two techniques for routine volume reduction of umbilical cord blood D. A. Ivolgin1,2,*, A. B. Smolyaninov3 1 Scientific research laboratory of cells Technologies North Western State Medical , 2Pokrovsky Stem Cell Bank, 3University named after I.I.Mechnicov, Saint-Petersburg, Russian Federation Introduction: The aim of this study is to compare a semiautomated versus an automated method for cord blood volume reduction in terms of efficacy, cost and time required to process units. Materials (or patients) and methods: Umbilical Cord Blood units (UCB) (n ¼ 280) were collected at the maternity clinics of St. Petersburg and Leningrad region. UCB units were processed at the Research Laboratory of cell technologies of Mechnikov NorthWestern State Medical University and Pokrovsky stem cell bank from April to December 2014 for public storage. UCB volume reduction was performed using the automated method Sepax S100 (Biosafe, Switzerland) (n ¼ 140) or the semi-automated method MacoPress Smart (Macopharma, France) (n ¼ 140). The Total Nucleated Cell (TNC) count and the hematocrit rates were analyzed pre-and post-processing using a Coulter AcT diff 2 ("Beckman Coulter", USA)hematology analyzer. The number of CD34 þ cells and cell viability were analyzed using a FC500 flow cytometer (CXP software with the laser wavelength of 488 nm, a set of reagents StemKit, "Beckman Coulter", USA). Statistical analysis was carried out using SPSS. All data are represented as Mean±s.d. The student t-test was used to compare the data between each group. The differences were considered statistically significant at Po0.05. In order to compare both techniques in similar conditions, the use of the semiautomated method was adapted to a final volume of 21 mL. Results: The TNC recovery using the semi-automated and the automated methods was: 81.9±0.7% and 80.38±0.7%, the hematocrit values were: 45.48±1.4% and 36.3±1.1%, the CD34 þ cells count were: 2.95±0.2(.106) and 2.56±0.2(.106) and the cell viability was 89.49±0.93% and 89.54±0.9%, respectively. The comparison was also done per group of volumes as shown in the table below (*Po0.01). The TNC recovery was statistically higher in group 2 when compared to group 1as shown by Student t-tests values calculated in pairs. The initial UCB volumes processed were of importance and led to higher differences in TNC recovery in favor of group 2. By contrast, the hematocrit was found to be statistically lower for group 1 when high volumes were processed; No statistical differences were noticed for lower volumes. The CD34 þ counts were similar for both groups for high volumes while a significant statistical difference was found for volumes lower than 80 mL in favor of group 1. Finally, no significant differences for cell viability levels were found between the two groups. The time required to process one UCB unit was shorter with the semi-automated method when compared to the automated one: 25±3 min and 54±2 min respectively (Po0.05). The price calculation per CBU shown that the
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automated method was 3.75 times more expensive than the semi-automated method. Conclusion: Taken together, the comparison of the two techniques show a higher TNC recovery using the semiautomated method but associated with a higher level of hematocrit in the corresponding processed UCB units. The cost of use was clearly in favor of the semi-automated method. Disclosure of Interest: None declared. P031 Intravenous busulfan in combination with thiotepa and fludarabine (TBF) as preparative regimen for patients with lymphoma submitted to allogeneic stem cell transplantation (HCT) E. Pennese1,*, S. Santarone1, P. Olioso1, P. Bavaro1, G. Papalinetti1, P. Accorsi1, T. Bonfini1, F. Papola2, P. Di Bartolomeo3 1 Department of Hematology, Transfusion Medicine and Biotechnology, Pescara, 2Ospedale San salvatore, Cntro Regionale Immunoematologia, L’Aquila, 3Department of Hematology, Transfusion Medicine and Biotecnology, Pescara, Italy Introduction: Allogeneic HCT is a potentially curative option in relapsed/refractory lymphomas due to either the preparative regimen and the graft-versus-lymphoma effect. Intravenous Busulfan (iv-BU), in association with Thiotepa and Fludarabine (TBF regimen), is an active drug against lymphoid malignancies. Our study was designed to assess the safety and efficacy of TBF regimen in patients with lymphoma undergoing alloHCT. Materials (or patients) and methods: From 2010 to 2014, 10 patients (8 males) with either advanced Hodgkin lymphoma (HL) (n ¼ 7) or non-Hodgkin lymphoma (NHL) (n ¼ 3) received an alloHCT from HLA identical sibling (n ¼ 5), or HLA haploidentical donor (n ¼ 4), or matched unrelated donor (MUD) (n ¼ 1). The median age was 41 yr (range, 23-60). The median number of previous chemotherapy lines for each patient was 5 (range, 2-6). The donor’s median age was 46 yr (range, 22-61). Conditioning regimen was myeloablative (TBFMAC: Thiotepa 5 mg/kg/day i.v. at days -7 and -6, Busulfan 3.2 mg/kg/day in a single i.v. infusion over 3 hours and Fludarabine 50 mg/m2/day i.v. in 1 hour at days -5, -4 and 3) in 6 patients or at reduced intensity (TBF-RIC) by deleting one dose of Thiotepa and Busulfan in 4 patients. Six patients were given peripheral blood stem cells (PBSC) collected from identical sibling donors or MUD and received Cyclosporine (CSA) and short course Methotrexate (MTX) as graft-versushost disease (GvHD) prophylaxis. Four patients transplanted from haploidentical donor received unmanipulated bone marrow stem cells (BMSC) and a combination of 5 drugs (CSA, MTX, Mycophenolate mofetil, anti-CD25 monoclonal antibody Basiliximab and Fresenius Anti-thymocyte globulin) for GvHD prophylaxis. Results: The median dose of CD34 þ and CD3 þ cells infused was 6.0x106/kg (range 3.5-10.7) and 245x106/kg (range 188857) for patients transplanted with PBSC and 2.3x106/kg (range 2.0-2.3) and 25.8x106/kg (range 15.8-41) for patients transplanted with BMSC, respectively. All patients engrafted with a median time of 18 days (range 13-33) and 20 days (range 13-75) to reach Z0.5x109/L neutrophil count and Z20x109/L platelet count. No primary or secondary graft failure occurred. At day 60 post-transplant all patients showed a full-donor chimerism. Grade I-II acute GvHD and limited chronic GvHD occurred in 3 and 4 patients, respectively. No patient showed veno-occlusive disease of the liver. One patient had hemorrhagic cystitis associated to BK virus reactivation. The WHO toxicity was classified as follows: slight increase of total bilirubin and SGOT/SGPT in 3 and 1 patients, respectively; mild or severe oral mucositis in 9; gastrointestinal involvement in 3; impaired renal function in 2. Toxicities resolved in all patients. No patient died of transplant-related cause. One HL patient, transplanted in active disease, showed progression of his disease and died
on day 312. With a median follow-up of 20 months (range 156), overall survival and progression-free survival were 90% and 70%, respectively. Conclusion: Our small series of 10 patients, all of them heavily pretreated and with refractory disease, shows that alloHCT is a feasible procedure in patients with lymphoma. The TBF regimen has been well tolerated with minimal secondary effects. More patients and longer follow-up are needed to assess the TBF regimen efficacy. Disclosure of Interest: None declared. P032 Comparison of different enzymatic and non-enzymatic methods on cell surface expression of CXCR4 by human CD34 þ Hematopoietic Stem Cells and Bone Marrow Mesenchymal Stem Cells G. Aydin1,2, F. Aerts-Kaya1,2,*, D. Uckan-Cetinkaya1,2,3 1 Center for Stem Cell Research and Development, 2Institute of Health Sciences, HACETTEPE UNIVERSITY, 3Department of Pediatric Hematology, Hacettepe University Medical Faculty, Ankara, Turkey Introduction: The most commonly used method to detach adherent cells is trypsinization. Trypsin is a serine protease and acts by cleaving peptide chains at the carboxyl side of the amino acids, Lysine or Arginine. As such, it may cleave cellular antigens, present at the surface of the cell. CXCR4 is an important cell surface molecule involved in homing and engraftment of CD34 þ hematopoietic stem cells (HSCs) and Mesenchymal Stem Cells (MSCs). Trypsin and other enzymatic methods, used to dissociate adherent cells/digest tissue may affect the homing ability of these cells by cleaving specific surface antigens. Here, we compared the effects of different enzymatic and nonenzymatic cell detachment methods on the expression of CXCR4 by CD34 þ HSCs and human Bone Marrow (BM) Mesenchymal Stem Cells (MSCs) and recovery of CXCR4 expression in time and after incubation with different cytokines. Materials (or patients) and methods: Peripheral blood (PB), cord blood (CB) and BM CD34 þ HSCs and BM-MSCs were treated using non-enzymatic dissociation solutions (GIBCO, Enzyme-Free Cell Dissociation Buffer and Biological Industries, Non-enzymatic Cell Dissociation Solution) and enzymatic solutions (0.25% or 0.05% Trypsin/EDTA), Mesencults enzymatic dissociation solution (Stem Cell Technologies), Gibcos TrypLEt Express (Life Technologies) and Accutase cell detachment solution (Capricorn Scientific), for 15 minutes at 371C at 5% CO2. Control cells were collected by pipetting in PBS. PBS þ 2% Fetal Calf Serum (FCS) was used to stop the reactions. Recovery of CXCR4 after 0.25% Trypsin/ EDTA treatment was measured after incubation with PBS þ 2% FCS (control) or different cytokine cocktails for 4 and 24 hours at room temperature using a FACSARIA (Becton Dickinson). Results: CXCR4 surface expression on BM-CD34 þ cells collected with PBS and the non-enzymatic solutions from GIBCO and Biological Industries was 26.7%, 28.3% and 23.2%, respectively. After Trypsin/EDTA 0.25%, Trypsin/EDTA 0.05% and Mesencults enzymatic solutions, CXCR4 levels rapidly decreased to 1.1%, 1.9% and 4.4%, respectively. CXCR4 expression by CD34 þ cells, irrespective of source, was negatively affected by enzymatic solutions. CXCR4 cell surface expression by MSCs was 29.7% in the control group and decreased to 0.9-6.9% after incubation with any solution. Spontaneous recovery of CXCR4 at 2 hours, 4 hours, 8 hours and 24 hours was not observed and incubation in presence of different cytokine cocktails resulted in minimal surface expression of CXCR4, with a maximum of 5.2% after 24 hours. Conclusion: Treatment of CD34 þ cells and MSCs with enzymatic solutions rapidly reduced CXCR4 cell surface levels and may negatively affect the homing and engraftment ability of these cells. Spontaneous recovery was not observed and incubation in presence of different cytokine cocktails did not result in re-expression of CXCR4 on the cell surface. This study
may have important implications for the choice of collection methods used in the preparation of clinical products. To maintain maximum CXCR4 levels and achieve optimal homing/engraftment of cells, the use of non-enzymatic and/or mechanical dissociation methods is considered most appropriate. Disclosure of Interest: None declared. P033 Autoimmune hemolytic anemia after allogeneic HSCT – A case report I. Bodova1,*, S. Sufliarska1, O. Fabri1, Z. Laluhova Striezencova1, P. Svec1, A. Chocholova1, J. Horakova1 1 BMT Unit, Departement of Pediatric Hematology and Oncology, Bratislava, Slovakia Introduction: Autoimmune hematologic disease following HSCT may affect one or more cell lines. Its association with GVHD, T-depletion, unrelated HSCT or serotherapy supports the assumption of its development on the basis of immune dysregulation or incomplete immune reconstitution. Materials (or patients) and methods: Between 11/1995 and 9/2014, 194 allogeneic HSCT were performed in 184 consecutive children at BMTU in Bratislava, Slovakia. 117 patients (64%) were treated for malignant disease and 67 (36%) childrens for non-malignant. There were 107 (58%) boys and 77(42%) girls with a median age 9.7 y (0.2 to 18.5 y). 106 patients (55%) had matched related donor: 100x sibling, 3x matched parent, 3x haploidentical parent). 88 childrens (45%) had matched unrelated donor. Results: Autoimmune haemolytic anaemia occurred in two patients (1,03%). First patient was 5 y girl with ALL in 2nd complete remission after allogeneic unrelated HSCT, from þ 70 D she developed signs of slight autoimmune haemolytic anaemia, after adding prednisolone 2 mg/kg/day to CS-A prophylaxis haemolysis resolved with no need of transfusion. The second patient was 16 y girl after unrelated HSCT for CML in 1st chronic phase. She was treated for diabetes mellitus type 1 and autoimmune thyroiditis. Due to major AB0 incompatibility (patient pre-HSCT 0 Rh positive, PBSC donor AB Rh positive) she was transfused with erythrocytes 0 Rh positive, platelets AB. The titre of the immune anti-A and anti-B antibodies before HSCT was slightly increased. She engrafted þ 9 D, readmitted to the hospital on þ 97 D with acute GVHD – GIT, with diarrhoea and vomiting. She responded very good to adding corticosteroids to CS-A prophylaxis. From þ 104 D developed severe anemia with signs of autoimmune haemolysis with positive free and bound polyspecific antierythrocyte autoantibodies, cross-checks before erythrocyte transfusion had been positive. Erythrocyte transfusions were administered due to vital indications (Hb 34 g/l) after corticoids premedication. Despite high doses of corticosteroids and IVIG, severe hemolysis persisted with need for erythrocytes transfusions (12x). Rituximab 375 mg/m2/day therapy started þ 112 D, first 4 doses weekly and the next doses monthly. Last transfusion was indicated after 3rd dose of Rituximab, hemolysis resolved. In total, 7 doses of Rituximab were administered. Currently, the girl is 11 months after HSCT in a good clinical condition and no signs of GVHD, good function of allogeneic graft with molecular remission of CML. Conclusion: The results of our study are similar to the published data. AIHA is the most frequent autoimmune haematological disease emerging after allogeneic HSCT. Rituximab showed to be effective treatment in AIHA after HSCT refractory to corticosteroids and IVIG. Disclosure of Interest: None declared.
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P034 Clinical relevance of anti-HLA Antibodies and DSA in unmanipulated haploidentical stem cell transplantation L. Laget1,*, J. El Cheikh2, A. Basire1, S. Simon1, P. Ladaique2, C. Lemarie´2, D. Blaise2, C. Chabannon2, C. Picard1,3 1 Laboratoire d’Immunoge´ne´tique et d’histocompatibilite´, EFS Alpes-Me´diterrane´e, 2Institut Paoli Calmettes, 3UMR 7268 ADES, Aix-Marseille Universite´/EFS/CNRS, Marseille, France Introduction: Background: The first clinical results of unmanipulated haplo-identical stem cell transplantation (haploHSCT) using cyclophosphamide after graft are promising. However, the detection in many adult recipients of preformed anti-HLA antibodies (Abs) directed against donor mismatched HLA antigens (DSA) raises the question of their clinical relevance, i.e. risks of graft failure, delayed engraftment and graft versus host disease (GVHD) all of which may contribute to reduced overall survival (OS). Objectives:The main objective of this study is to evaluate the clinical relevance of anti-HLA Abs and /or DSA recipient and donor in unmanipulated Haplo-SCT, including engraftment, the development of GVHD and overall survival. We also evaluated the effectiveness of 3 desensitization protocols for patients with DSA before transplant. Materials (or patients) and methods: The study was conducted in thirty haplotype-mismatched donor – recipient pairs taken care of at a single institution (Institute Paoli Calmettes). Patients were transplanted with T-cell replete blood cells collected by apheresis from G-CSF mobilized donors, following a non-myeloablative conditioning regimen, and received post-transplant cyclophosphamide. Anti-HLA antibodies screening and identification of donor and recipient were performed by luminex assay at Day 0 and at D3, D8, D15 and D30 post-graft and DSA were assigned in all serum samples. Results: The presence of preformed anti-HLA Abs was detected in 50% of recipients and 31 % of donors. Men were predominantly and preferably immunized against HLA class I, probably related to excessive sensitivity of the technique used (Luminex). HLA classes I DSA were detected in 3 recipients. Only one graft failure was observed in a patient with DSA at high levels (Mean Fluorescence Intensity 4 10 000) despite a desensitization protocol. Two out of three desensitization protocols (rituximab þ plasmapheresis/ rituximab þ platelets transfusions) had been effective and had significantly reduced the DSA rates to allow an engraftment. A comparison of three recipient groups (15 Abs negative, 12 Abs positive/DSA negative and 3 DSA positive) showed no significant difference on clinical end-points (table). Anti-HLA Abs donor seemed to be associated with a delayed neutrophil recovery (P ¼ 0.07). Monitoring of anti-HLA Abs after transplantation
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showed the absence of development of DSA recipient and donor, the absence of anti-HLA Abs donor detection and a loss of anti-HLA Abs recipient at D30 in many cases Conclusion: These data suggest that the preformed anti-HLA Abs in the patient and/or donor in unmanipulated haplo-HSCT are not associated with a risk of graft failure, except potentially when DSA is detected at high levels. Nevertheless the donor’s anti-HLA allo-immunization appears to be a parameter to consider when selecting the donor since there are usually several potential haploidentical donors in the patient’s family. However, the presence of DSA should no longer be a barrier to HLA haplo-HSCT, providing that further studies with new desensitization protocols are conducted and successful. Disclosure of Interest: None declared. P035 The prognostic impact of CD34 þ /CD38- cell burden at diagnosis and during disease course in acute myeloid leukemia patients undergoing allogeneic stem cell transplantation M. Jentzsch1,*, M. Bill1, S. Leiblein1, K. Schubert1, M. Ple1, U. Bergmann1, K. Wildenberger1, L. Schmalbrock1, M. Cross1, W. Po¨nisch1, G.-N. Franke1, V. Vucinic1, G. Behre1, D. Niederwieser1, S. Schwind1 1 Ha¨matologie, int. Onkologie und Ha¨mostaseologie, Universita¨tsklinikum Leipzig, Leipzig, Germany Introduction: In acute myeloid leukemia (AML) Leukemia initiating cells (LIC) are enriched within the CD34 þ /CD38bone marrow (BM) compartment. LIC may survive chemotherapy as minimal residual disease & cause relapse. Allogeneic stem cell transplantation (SCT) is a potentially curative consolidation therapy for AML patients (pts). However, relapse remains a common cause of death. Risk stratifying pts is crucial to adjust post-SCT care strategies. High LIC burden at diagnosis is associated with worse outcome in pts receiving chemotherapy, but larger studies on outcome after SCT in which graft-versus-leukemia (GvL) effects contribute to treatment success are missing. Materials (or patients) and methods: Here we analyzed 169 AML pts (median age 62 years [y]; range 19-75y) who received myeloablative (MAC, Cyclophosphamid 120 mg/kg þ 12 Gy total body irradiation [TBI], n ¼ 49) or non-myeloablative (NMA, Fludarabine 90 mg/m2 þ 2 Gy TBI, n ¼ 120) SCT in complete remission (CR). European LeukemiaNet (ELN) genetic classification was: Favorable 28%, Intermediate-1 26%, -2 20%, Adverse 26%. Using flow cytometry percentage of BM CD34 þ /CD38cells at diagnosis & in CR before & at day 28 after SCT was measured. Donors were human leucocyte antigene (HLA)-
matched related (21%) or HLA-matched (56%) or mismatched (4 ¼ 1 antigen; 23%) unrelated. 29% of pts developed acute graft-versus-host disease (GvHD; 4 ¼ grade 2) & 58% chronic GvHD. Median follow-up was 3.4y. The amount of CD34 þ / CD38- BM cells was also measured in 15 healthy donors (median 0.2 [0-0.5]). Results: A cutpoint of ¼ 45% defined a high CD34 þ /CD38burden which associated with lower platelet count (P ¼ .03) at diagnosis. Pts with high CD34 þ /CD38- burden more frequently had a complex karyotype (P ¼ .04), deletion 5 or 5p (P ¼ .009) & more secondary AML (P ¼ .006). Interestingly, no pts with high CD34 þ /CD38- burden was CEBPA mutated. The amount of CD34 þ /CD38- cells in CR was not different to levels in healthy donor BMs regardless of the burden at diagnosis (P ¼ .33 & P ¼ .14 at SCT for pts with high & low CD34 þ /CD38- burden respectively; P ¼ .51 & P ¼ .39 after SCT for pts with high & low CD34 þ /CD38- burden respectively). A high diagnostic CD34 þ /CD38- burden associated with shorter event free (EFS, Po.001, Fig. 1A) & overall survival (OS, P ¼ .004, Fig. 1B). A prognostic impact was also seen when we analysed the NMA (EFS P ¼ .02 & OS P ¼ .002) & MAC subset (EFS P ¼ .05) separately. In multivariate analysis pts with high CD34 þ /CD38-burden had shorter EFS & OS (Hazard Ratio [HR] 2.1, P ¼ .003 & HR 1.7, P ¼ .04). Conclusion: The worse outcome associated with higher CD34 þ /CD38- burden at diagnosis was likely mediated by LIC within the CD34 þ /CD38-population escaping the GvL effect of SCT. Thus LIC persistence at low levels may not easily be overcome by SCT. In the future evaluation of CD34 þ / CD38- cell burden at diagnosis may improve AML pts risk stratification & help to personalize post-SCT care. Furthermore, incorporating LIC targeting therapies may improve survival of AML pts receiving SCT. Disclosure of Interest: None declared.
EORTC/MSG 2008. Drug toxicity was evaluated according to CTCAEv4.1 criteria. Results: We found differences between the 2 groups in terms of: type of graft (bone marrow was more frecuently in fluconazole group), regimen and type of conditioning (reduced-intensity conditioning was more common in voriconazole group) and presence of cGVHD (more frecuenty in voriconazol group). The incidence of IFD was 16.3% in the Fluconazole group (n ¼ 16) vs 7.1% (n ¼ 7) in Voriconazole group (P ¼ 0.046). The median of FFS was 24.5 days and 106 days (P ¼ 0.003). According to the EORTC/MSG criteria,taking into account only patients who developed IFD, the percentage of possible IFD in the first group was 50% vs 14.8% in the second group; Probable: 37.5% vs. 71.4%; Proven: 12.5% vs. 14.28%. We found significant statistical differences at 360 days post SCT (83.7% vs 92.9%;P ¼ 0.034). Deaths caused by IFD were 10.1% in Fluconazole (n ¼ 10) and 3.1% in Voriconazole (n ¼ 3) (P ¼ 0.027). The median follow-up was 34 months. There was no significant difference in overall survival: 52.1% vs 52.6% (P ¼ 0.95). There were no differences in causes of death (P ¼ 0.326). Eleven patients (11.22%) presented adverse effects (AE)in the first group vs 18 patients (18.36%) in the second one (P ¼ 0.065). The most frequent AE for both groups was hepatic toxicity. There were not significant differences in the suspension (temporary and permanent) of the drug between the two groups. Conclusion: This study confirms that the use of Voriconazole as antifungal prophylactic treatment in patients undergoing allo-SCT decreases the incidence of IFD and increases FFS versus Fluconazole. In spite of a greater toxicity in Voriconazole group,this toxicity was non severe and rapidly recovered with the withdrawal of the drug. Disclosure of Interest: None declared.
P036 Voriconazole used as antifungal prophilaxis decreases invasive fungal disease in patients undergoing allogenic stem cell transplantation in our institution (Allo-SCT) M. J. LLAMAS POYATO1,*, C. CHIC ACEVEDO1, E. GARCIA TORRES1, J. SERRANO LOPEZ1, J. R. MOLINA HURTADO1, C. MARTIN CALVO1, F. MARTINEZ GUIBELARDE1, R. ROJAS CONTRERAS1 1 HEMATOLOGY, REINA SOFIA UNIVERSITY HOSPITAL, CORDOBA, Spain
P037 Influence of donor’s KIR2DL1-R 245 allelic snp on patient outcome after haploidentical stem cell transplant M. Bastos Oreiro1,2,*, C. Martinez Laperche1,2, D. Carbonell1, D. Champ1, E. Buces1,2, C. Pascual1, A. Perez Corral1, P. Balsalobre1, M. Kwon1, D. Serrano1, J. Gayoso1, J. Anguita1, I. Bun˜o1,2, J. L. Die´z Martı´n1 1 Hematology, Hospital General Universitario Gregorio Maran˜o´n, 2 Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n, Madrid, Spain
Introduction: Invasive fungal disease (IFD) causes significant morbidity and mortality in patients undergoing Allo-SCT. Some new triazole-drugs with anti-filamentous activity (as Voriconazole) have being used during the last years as antifungal prophylaxis for high-risk patients.The aim of our study was to compare the incidence of IFD in patients undergoing Allo-SCT in our institution using Fluconazole vs Voriconazole as prophylactic treatment. Materials (or patients) and methods: We analyse retrospectively 196 allo-SCT consecutive patients (paediatrics and adults) submitted to this procedure in our center from March 2001 to April 2008. A total of 98 patients received antifungal prophylaxis with Fluconazole and 98 with Voriconazole. All patients received antifungal prophylaxis with oral Voriconazole 200 mg/12 hours (5mg/kg/12h in children) or Fluconazole 200 mg/12 hours (5mg/kg/day in children) from day 0 to day þ 75. Antifungal prophylaxis was prolonged in patients requiring treatment with high doses of corticosteroids (42mg/kg/day) and/or with 2 immunosuppressive drugs. The temporary suspension was applied in those patients with toxicity,without reaching criteria for withdrawal,being able to continue the study drug if the suspected toxicity resolved in 7 days. AGA detection was done twice weekly from year 2004. It was considered positive when the index OD was Z 0.5 in 2 consecutive measurements or Z 0.8 on a single determination. When IFD was suspected a new determination of AGA and a high resolution CT were done as well as a bronchoalveolar lavage. IFDs were according to the revised criteria of the
Introduction: Killer-cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells play an important role in the graft versus tumour effect. KIRs are highly polymorphic. Donor KIR2DL1-R snp has been associated with stronger signalling function than others. Our aim in this analysis is to evaluate this donor polymorphism in the setting of T cellrepleted haploidentical hematopoietic stem-cell transplantation (HSCT) with post-infusion high-dose cyclophosphamide (PiCy). Materials (or patients) and methods: Forty-nine adult patients who underwent T-cell repleted haploidentical HSCT with PiCy in our institution were included. Demographic data, KIR genotype and snp are summarized in Table 1. Donor KIR2DL1 functional allele typing was retrospectively performed using single nucleotide polymorphism assay (Bari et al, Clinical Immunology 2011). KIR genotype was analyzed by PCR (KIR Typing, Miltenyi Biotec) using genomic DNA. DNA was purified by the (Maxwell 16 Blood DNA Kit, Promega) from peripheral blood examples. HLA typing was used to identify HLA class I KIR ligands. Results: We did not find progression free survival (PFS) differences between patients who received a KIR2DL1-R 245 homozygous graft or a KIR2DL1-R 245/KIR2DL1-C245 graft (76% vs. 70% P ¼ 0,7). However, patients who received a KIR2DL1-R 245 graft with HLA-C ligand-receptor mismatch had a marked tendency to better PFS compared with those who received a graft with KIR2DL1-R 245 and no HLA-C ligandreceptor mismatch or KIR2DL1-R 245/KIR2DL1-C245 graft,
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(93% vs. 74% respectively, P ¼ 0.057) one year after transplant (Figure 1). Remarkably, and despite the small number, this trend remains if we perform a sub analysis in Hodgkin lymphoma patients (PFS100%, 47%, p: 0.07). Conclusion: Donor KIR2DL1-R 245 allelic snp associated with KIR HLA-C ligand-receptor mismatch could affects recipient outcomes after T cell-repleted haploidentiacal HSCT with PiCy, especially in HL. If this information is confirmed, these findings could have significant repercussion for donor selection. Disclosure of Interest: None declared. P038 In vitro evaluation of NK cytotoxicity in the setting of T-cell-replete haploidentical transplant M. Bastos Oreiro1,2,*, J. Anguita1, C. Falero1, L. Solan1, A. Perez Corral1, C. Pascual1, C. Martinez Laperche1,2, P. Balsalobre1, C. Mun˜oz1, D. Serrano1, M. Kwon1, A. Perez3, I. Bun˜o1,2, J. Gayoso1, J. L. Die´z Martı´n1 1 Hematology , Hospital General Universitario Gregorio Maran˜o´n, 2 Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n, 3Pediatric hematology, Hospital Universitario La Paz, Madrid, Spain Introduction: NK cells has been identified as an important element for graft vs. tumor effect in allogeneic transplant. Our aim in this analysis was to evaluate, by two different methods,
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the ‘‘in vitro’’ cytotoxicity activity of NK cells and to correlate this with patient’s evolution in the setting of T-cell-replete haploidentical hematopoietic stem-cell transplantation (HSCT) with post-infusion high-dose cyclophosphamide (PiCy). Materials (or patients) and methods: Since 03-2013 to 062014, 16 consecutive patient-donor pairs with haematological malignancies who underwent T-cell-replete haploidentical HSCT with PiCy in our institution were included. Patients demographic data is included in Table 1. The cytotoxicity of NK cells was evaluated by 2 methods: 1) Conventional 2-hour europium-TDA release assay (Perkin-Elmer Wallac, Turku, Finland). K562 (erythroleukemia cell line) were used as the target cell; cytolysis was measured by fluorimetry (VictorTM, Wallac) in resting conditions and stimulated overnight with IL15, as previously described (Blomberg et al, J Immunol Methods, 1995). Percentage of degranulation was grouped in low (less than 25%), medium (25-50%) and high (up 50%). The number of NK cells in donor blood was calculated by flow cytometry, multiplying the lymphocyte counts to the percentage of CD3- CD56 þ NK cells. 2). Degranulation by flow cytometry: the percentage of NK cell degranulation was analyzed through the expression on the cell surface of CD107a. This was determined basal and after 2 hours incubation at 371 with K562 cell line. Results: We found that the correlation between tests used for the cytotoxicity assessment was good (correlation coefficient: 0.88, P ¼ 0,000) (Figure 1). Four patients relapsed (progression free survival (PFS): 86% 1 year after transplant).
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Two patients died for no transplant related causes. Median follow up was 19.8 month (r: 6-22). Five cases presented high levels of cytotoxicity by fluorometric assay, of these, four presented more than 50% degranulation by flow cytometry. One case showed more than 50% degranulation by flow cytometry but low-medium levels of cytotoxicity by fluorometric assay (Figure 1). Higher levels of cytotoxicity evaluated by fluorometric assay as well as by flow cytometry appear to be associated with lower relapse rate (HR 0.419, IC95%: 0.17-1.07, P ¼ 0.053; HR 0.88, IC95%: 0.77-1.01, P ¼ 0.088, respectively). Conclusion: Cytotoxicity evaluated by fluorometry assay as well as by flow cytometry could be a helpful tool for selecting the most alloreactive donor, in the setting of T-cell-replete haploidentical HSCT PiCy. Disclosure of Interest: None declared. P039 Outcome improvement following haploidentical stem cell transplantation in patients with high risk leukemia: A comparison of high dose post-transplant cyclophosphamide (PT-CY) versus prophylactic DLI K. alimoghaddam1, A. ghavamzadeh1, M. barkhordar1,* 1 tehran University of Medical Sciences, Hematology- oncology and SCT research center , Tehran, Iran, Islamic Republic Of Introduction: Haploidentical transplantation has become a clinical option for patients lacking an HLA-matched donor. Multiple methods have been made to improve outcome after haploidentical stem cell transplantation (SCT). High dose cyclophosphamide in the early post transplant period (PTCY) is an effective strategy for GVHD prevention and engraftment facilitation due to immunogenic tolerance. Prophylactic DLI was applied to improve both relapse rate and immunity reconstitution after haploidentical SCT. Here we report the outcome of 40 patients of Haploidentical SCT in our center that randomizeded in two arm of PT-CY versus Prophylactic DLI. Materials (or patients) and methods: From Jun 2010 to August 2014, 40 patients ( 27 male, 13 female) with high risk acute leukemia (29 AML, 11 ALL), whose have not suitable related or unrelated donor underwent haploidentical SCT from family members and randomized in two arms. Median patients age was 26.6 year (17-55 year) in PT-CY arm and27.3 year (13-
58 year) in DLI arm. Identical regimen for conditioning and GVHD prophylaxis were used in two arms . The myeloablative conditioning regimen comprised busulfan and cyclophosphamide and ATG. GVHD prophylaxis consisted of cyclosporine and methotrexate. In PT-CY arm patients undergone HSCT and received additional dose of cyclophosphamide 50mg/kg at þ 3 and in DLI arm after transplantation patients received DLI from the same donor after subcutaneous G-CSF at day þ 30 with average cell dose 1-2 107 mononuclear cells (MNC)/kg. Results: Totally 40 patients enrolled, 20 patients allocated in each arm. Median follow up was 720 days in PT-CY arm and 534 days in DLI arm. 5 patients in PT-CY arm and 7 in DLI arm were died. Cause of deaths were infection in 4 patients of DLI arm and 2 patients of PT-CY arm . disease relaps in 2 patients of PT-CY arm and 1 patient of DLI arm. Acute GVHD (grade 2-3) accur in 10 and 16 patient in PT-CY and DLI arm respectively (P-value ¼ 0.046). Chronic GVHD (moderate and severe) accur in 3 and 7 patient in PT-CY and DLI arm respectively (P-value ¼ 0.20). One year disease free survival was 79% and 84% in PT-CY and DLI arm respectively (P-value ¼ 0.97). One year overall survival was 76% and 71% in PT-CY and DLI arm respectively (P-value ¼ 0.43). Conclusion: The results of this study reveals no statistical difference between two arms in DFS and OS and chronic.GVHD, probably due to small size of patients and short time of follow up. However the acute GVHD was significantly lower in PT-CY arm. Disclosure of Interest: None declared. P040 Favourable aoutcome with donor lymphocyte infusion for secondary graft failure in X-linked Hyper Ig M Syndrome M. Torrent1,*, L. Sisinni1, N. Pardo1, L. Medina2, S. Querol2, L. Alsina3, I. Badell1 on behalf of Pediatric BMT Unit, Hospital Santa Creu I Sant PauUniversitat Auto`noma de Barcelona 1 Universitat Auto`noma de Barcelona, Pediatric BMT Unit. Hospital de la Santa Creu i Sant Pau, 2Banc de Sang i Teixits de Barcelona, 3Allergy and Clinical Immunology Department, Hospital Sant Joan de De´u, Barcelona, Spain Introduction: X-linked hyper-Ig M syndrome (XHIGM) is a rare primary immunodeficiency caused by mutations in the gene encoding CD40 ligand glycoprotein. These patients have low
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levels of IgG and IgA with high or normal levels of IgM. Patients presenting with this disease undergo monthly immunoglobulin treatment. Hematopoietic stem cell transplantation (HSCT) should be considered and performed as soon as possible. Mixed donor/recipient chimerism (MC) is increasingly common in the pediatric HSCT setting due to the increased use of reduced intensity preparative regimens for non-malignant diseases. Interventions such as rapid immunosuppression withdrawal, donor lymphocyte infusion (DLI), or stem cell boost are frequently employed to improve donor chimerism. Materials (or patients) and methods: A one year old boy with XHIGM underwent HSCT in 10/24/2013. Unmanipulated bone marrow from total HLA matched unrelated donor (MUD) was the source. Conditioning regimen was based in intravenous alemtuzumab (0.6 mg/kg), fludarabine (180 mg/m2) and busulfan (14 mg/kg with adjusted dose) HSCT complications were CMV reactivation and grade II acute GVHD (cutaneous þ intestinal) which were solved with methylprednisolone 2 mg/kg/day. Initial chimera study (11/18/2013) showed 94% donor cells in myeloid compartment and 84% in lymphocytes. Progressive decrease in donor engraftment, mainly in lymphocytes, despite of rapid withdrawal of immunosuppression was observed. Donor chimera on 2/10/2014 showed 78% in granulocytes and 11% in lymphocytes so we decided to start DLI to improve donor chimerism. On 3/20/2014 he received the first DLI at a dose of 0.1x106 CD3 þ /kg recipient weight. DLI were administered every four weeks with a progressive increase in number of infused T-cells: 0.5x106 CD3 þ /kg, 1x106 CD3 þ /kg, 5x106 CD3 þ /kg and 10x106 CD3 þ /kg from the fifth and subsequent infusions. Results: A total of 10 DLI were performed at the moment, observing progressive improvement of mixed chimerism, achieving after the 9th DLI (11/12/2014) donor cells 100% in granulocytes and 93% in lymphocytes. DLI were well tolerated. No GVHD reactivation or infection was observed. Conclusion: In our experience it is very important closed chimera monitoring to early detect loss of engraftment and to prevent with therapeutic actions as immunossuprpression withdrawal or DLI. Our patient achieved total donor chimerism after monthly DLI without toxicity. Disclosure of Interest: None declared. P041 Comparison of The Adverse Reactions Due to Hematopoietic Stem Cell Infusion Between The Patients Younger Than And Elder Than 60 Years Old N. Baskan1,*, D. Cekdemir1, E. Birtas Atesoglu1, Z. Gulbas1 1 Hematology, Anadolu Medical Center Hospital, Bone Marrow Transplantation Unite, Kocaeli, Turkey Introduction: HSCT is a therapeutic modality and number of the elderly patients undergoing HSCT has been steadily increasing as number of the elderly patients having diagnosis of hematological malignancies increases. In the present study, adverse reactions due to infusion of HSC were compared between the patients elder and younger than 60 undergoing HSCT. Materials (or patients) and methods: A total of 314 patients including 88 (28%) patients with multiple myeloma (MM), 31 (9,9%) patients with Hodgkin’s lymphoma (HL), 17 (5.4%) patients with myelodysplastic syndrome (MDS), 27 (8.6%) patients with acute lymphocytic leukemia, 47 (15%) patients with acute myeloid leukemia (AML), 53 (16.9%) patients with non-Hodgkin lymphoma, 2 (0.6%) patients with solid tumors, 21 (6.7%) patients with chronic myeloid leukemia, 21 (6,7%) patients with with chronic lymphocytic leukemia (CLL), 5 (1.6%) patients with IMF, and 16 (5.1%) patients with AA who underwent transplantation in the bone marrow transplantation (BMT) unit of Anadolu Medical Center Hospital between 2013 and 2014 were enrolled to this study. The patients underwent autologous, allogeneic, haploidentical, or
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unrelated transplantations. Mean age of the patients was 45 (range: 14 – 80) and 120 (38.2%) of the patients were female and 194 (61.8%) of them were male. Of the patients included in the present study, 262 (83.4%) were younger than 60 and 52 (16.6%) were elder than 60. Of the patients elder than 60, 34 (65.4%) had MM, 1 (1.9%) had MDS, 2 (3.8%) had AML, 12 (23.1%) had NHL, 1 (1.9%) had CML, 1 (1.9%) had CMML, and 1 (1.9%) had AA (Table 2). The patients elder than 60 received infusion of 6.11x106/kg of HSC (range: 2.10x106/kg – 21.30x106/kg) whereas those younger than 60 received infusion of 6.21x106/kg (range: 1x106/kg – 20x106/kg) of HSC. Two hundred and seventeen (69.1%) patients underwent infusion of frozen samples (FS) using DMSO while 97 (30.9%) patients underwent infusion of unfrozen samples (UFS). Results: Of 314 patients receiving infusion of HSC, 139 (44.3%) developed AR while 175 (55.7%) didn’t. ARs were observed in 21 out of 52 (40.4%) patients elder than 60 and in 118 (45%) out of 262 patients less than 60 years old. ARs were not observed in 31 out of 52 (59.6%) patients elder than 60 and in 144 (55%) out of 262 patients less than 60 years old. Hypertension (BP Z140-90 mm Hg) was found in 17.3% of the patients more than 60 years old and in 11.5% of the patients less than 60 years old. Rate of nausea was 14.1% in the patients above 60 years old and 5.8% in the patients less than 60 years old. Conclusion: When all evaluations were considered, nausea, vomiting, tachycardia, and chest pain were more common in the patients less than 60 years old while hypertension and hypoxemia were more common in those younger than 60. Disclosure of Interest: None declared. P042 Outpatient care for leucopenic patients after autologous stem cell transplantation N. Zwinkels-Barendse1,*, P. Ympa1,1 1 HagaZiekenhuis, location Leyweg, The Hague, Netherlands Introduction: Within the hematology department of HagaZiekenhuis, the Hague, Netherland, patients do not have the choice between: outpatient care several times a week or clinical care during the leucopenic phase following an autologous stem cell transplantation (ASCT). The core or central question is: ‘‘Is it feasible to treat patients in an outpatient setting during the leucopenic phase following ASCT, in both a responsible and safe way?’’. The motivation for the nurse practitioner (NP) to investigate this subject was the fact that other Dutch hospitals have already achieved such a clinic to treat their patients. It is of interest to know if this treatment option offers a solution to the shortage of hospital beds caused by the bed reduction, imposed by the government. Materials (or patients) and methods: The research design is a quantitative, not experimental research. The research took place on the hematology department of HagaZiekenhuis. The research population were 41 patients, older than 18 years who were treated for a hematologic oncological disease. They underwent an ASCT between January 2012 and February 2013. The study comprised a review of the patients clinical records and a combined questionnaire, sent to 37 patients ,fluent in the Dutch language. The questionnaire consisted of the Lastmeter (a validated tool to measure the load of all kind of aspects) and several closed and open questions. Results: The data-analysis of the medical records focused on: weight and TPN, fever and antibiotics, transfusions and leucopenia. There were significant differences between two patient groups undergoing different forms chemotherapy: HDM (high dose Melfalan) versus BEAM Carmustine, Etoposide, Cytatabine and Melfalan). The significant differences were mostly unfavorable for the BEAM patient group. The study also focused on the length of hospitalization. Patients who stayed longer in the hospital, lost more weight. Fever and use of
antibiotics lengthend hospitalization. The answers to the combined questionnaires showed that patients experienced suffering during their hospital stay for ASCT. The majority of problems were of a physical nature and some emotional. Because of the nature of the physical complaints, patients assume that hospitalization is required. Most patients were satisfied about the length of the hospitalization. The patients did not feel that they had missed out on the opportunity to choose between a clinical and outpatient treatment option. If given this choice, most of them would choose for hospitalization following ASCT. Conclusion: It is feasible in HagaZiekenhuis for patients undergoing HDM, to be treated in both a safe and responsible fashion in an outpatient setting for a period of time during the leucopenic phase following ASCT. However care plans and protocols are needed to treat patients in their leucopenic phase in an outpatient setting. Data needed to achieve such care plans and protocols are: physical condition at discharge, the home situation, hospital care and outpatient care. Attention is needed for patient education, communication between involved care providers and availability of a (emergency) hospital bed including the financial consequences. Disclosure of Interest: None declared. P043 Donor- Specific anti-HLA Antibodies: an emerging problem in Haematopoietic Stem cell transplantation P. Marenco1,*, G. Grillo1, E. Zucchetti1, B. Forno1, M. L. Pioltelli1, I. Lotesoriere1, G. Cornacchini2, G. Lando2, G. Bertani2, R. Cairoli1 1 BMT unit, 2Department of Transfusional Medicine, Niguarda Hospital, Milan, Italy Introduction: Donor-Specific anti-HLA Antibodies (DSA) are involved in graft failure after transplant (Tx). Complement Dependent Cytotocity (CDC) as a routine pretransplantation test has been debated for a long time and remains controversial. However, the increase of Unrelated Donor and, more recently, haploidentical donor transplants as well as the availability of more sensible screening tests for DSA (solid phase immunoassay Luminex) bring a new challenge to the transplant community. Materials (or patients) and methods: Our standard policy is to test every recipient of an allogeneic transplant for anti HLA Class I and II antibodies and to proceed to CDC crossmatch against the donor in all the Unrelated Donor and haplo Txs,
and for related donor Txs, only in case of positivity for anti HLA antibodies. However, regardless of the result of CDC against donor, all the antiHLA positive recipients are screened for DSA. Median Fluorescence Intensity (MFI) is recorded as titre and reported to BMT Unit physician who is responsible for strategy to reduce DSA before stem cell infusion, taking into consideration: DSA titres, class I or II of antibodies, and a possible titre reduction after conditioning. Our policy is: Plasma Exchange (PEX) (1 or 2 procedures, day -1 and day 0), i.v. Immunoglobulin 1gr/Kg day 0, and, in case of anti HLA class I Antibodies, DSA immunoadsorbtion on HLA selected Platelets day 0. Results: In the last 2 years (2013 and 2014) 63 allogeneic Txs were performed in our Centre. We report here (Table 1) the 4 cases (6,3%) with DSA positivity. 3/3 pts evaluable for engraftment regularly engrafted, one pt is not valuable yet. None experienced secondary graft failure or other DSA related problems. To be noted: in two case DSA identification led to a change in donor selection (1 UD, 1 related haploidentical) in order to avoid donor bearing Antigens or Alleles targeted by DSA at higher MFI levels. Conclusion: DSA are an emerging problem, critical levels for engraftment have still to be identified and best strategy to make transplantation safe has to be found in prospective studies. We already revised our recipient preTx evaluation SOP in order to have DSA results earlier, available for a better donor selection. Disclosure of Interest: None declared. P044 Neutrophil engraftment kinetic relationship to timing of Granulocyte Colony Stimulating Factor (G-CSF) in melphalan-conditioned autologous stem cell transplant for multiple myeloma patient cohorts from three United Kingdom centres R. Krishna1,*, H. A. Abhishekh1, R. Brown2, D. Qadir3, V. Ellis1, A.-M. Sjursen2, A. Drake3, C. Ardern1, J. Thachil1, M. Saif1, F. Dignan1, F. Willis3, A. Bloor2, M. Koh3, E. Tholouli1, M. Klammer3, J. Cavet2 1 Haematology, Manchester Royal Infirmary, 2Haematology, Christie Hospital, Manchester, 3Haematology, St Georges Hospital, London, United Kingdom Introduction: G-CSF is routinely administered after high-dose melphalan-conditioned autologous stem cell transplants
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(SCT) in patients with multiple myeloma (MM) to accelerate neutrophil engraftment. Literature on optimal timing of initiation of GCSF however remains limited. This study was performed to investigate the kinetics of neutrophil engraftment in relation to timing of GCSF in three MM SCT cohorts Materials (or patients) and methods: We retrospectively collected data of 157 MM patients who underwent autologous SCT from three U.K. hospitals with differing protocols for initiation of GCSF: GCSF was initiated per protocol on Day þ 7 (n ¼ 60), Day þ 10 (n ¼ 60), Day þ 3 (n ¼ 37) at each centre, and dates of neutrophil and platelet engraftment were recorded as per EBMT guidelines. Additionally patient characteristics including age, gender, weight, creatinine clearance, transplant characteristics incl. dose of CD34 cells, number of transplant (1st vs 2nd), melphalan dose (140mg/m2 vs 200mg/m2), use of plerixafor during harvest, peak CRP during SCT, and GCSF characteristics incl. type and dose along with day of initiation of G-CSF were extracted from patient records. Data were analysed using SPSS by univariate & forward multivariate regression Results: The mean number of days to engraftment were as follows: 13.58 ( þ 3 group), 12.26 ( þ 7 group), and 12.70 ( þ 10 group), censored by death pre-engraftment (1 patient þ 3 cohort). The median day of neutrophil engraftment was as follows: day 12 (Interquartile Range [IQR] 11-18) for þ 3 group, day 12 (IQR 12-13) for þ 7 group, day 13 (IQR 12-13) for þ 10 group. Neutrophil engraftment kinetic was significantly different across the groups [F: 4.09; P ¼ 0.019]. Although patients in the þ 3 group had delay in the overall mean neutrophil engraftment day due to some late engraftments, there were a significant majority of early neutrophil engraftments [w2 ¼ 73.8; Po0.001], with 75% engrafting by day 13. However, time to platelet engraftment was not affected significantly [F: 0.993; P ¼ 0.373]. Day of neutrophil engraftment correlated with duration of hospital stay [r ¼ 0.339: Po0.001]. There was also correlation between stem cell dose and time to neutrophil engraftment [r ¼ -0.315; Po0.001]. Conclusion: In this study, there was a significant acceleration of neutrophil engraftment in the patient group with earliest
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initiation of G-CSF (Day þ 3) which might suggest benefit; however, a proportion of later engrafting patients in this group also resulted in a longer mean overall, suggesting early GCSF cannot overcome other factors e.g. active MM or sepsis. Higher CD34 cell dose also led to earlier engraftment, which correlated with shorter admissions. Data analysis regarding pharmaco-economics & length of stay is ongoing. This study highlights the need for prospective studies in order to determine the best timing for initiation of GCSF in SCT for MM Disclosure of Interest: None declared. P045 Impact of natural-killer immune reconstitution on the development of cytomegalovirus infection, non-relapse mortality, and graft versus host disease after allogeneic stem cell transplantation R. Rodriguez-Veiga1,*, P. Montesinos2, A. Torres Gomez2, A. Muntasell3, L. Cordon2, A. Sempere2, I. Cano2, D. Martinez-Cuadron2, B. Boluda2, I. Lorenzo2, F. Lopez-Chulia2, M. J. Arilla2, A. Lancharro2, I. Jarque2, J. Sanz2, L. Larrea4, G. Sanz2, M. Lopez-Botet3, M. A. Sanz2 1 Hematology, 2Hospital Universitario La Fe, Valencia, 3IMIM: Institut Hospital del Mar d’Investigacions Me`diques, Barcelona, 4 Centro de transfusiones de la Comunidad Valenciana, Valencia, Spain Introduction: The outcome of patients receiving allogeneic stem cell transplant (alloSCT) is related with their immune recovery kinetics. Many studies showed the influence of B and Tlymphocytes in the prevention of relapse and infections after alloSCT, but the potential impact of natural-killer (NK) cell recovery and maturation has not been established. We aim to analyze prospectively the kinetics of NK and other lymphocyte subpopulations and its relation with the risk of post-alloSCT complications. We will compare the immune recovery between unrelated cord blood (UCBT) and HLA identical peripheral blood (HLA-Id). Materials (or patients) and methods: Data from all consecutive patients with hematologic malignant diseases
undergoing alloSCT in our institution were recorded (including CMV infection, grade 2-4 acute GVHD, non-relapse mortality [NRM], and relapse). From May 2013, we have analyzed prospectively by citometry the immune reconstitution after alloSCT on days þ 28, þ 100 and þ 180, including a basic immune assessment (CD45, CD3, CD8, CD4, CD19, and CD16 þ 56 þ ), and a specific NK-cell analysis (NKG2A and NKG2C). Statistical analyses, including description and comparison of variables, as well as univariate cumulative incidence (CI) method, were performed using R.14.2 version. Continuous variables were categorized using the median value. Results: We have studied 94 alloSCT, with the following main characteristics: 45 UCBT (4/6 and 5/6 HLA compatibility) and 49 HLA-id (5 unrelated and 44 sibling donor), median age 49 years (16-65), male 52 (55%), more frequent underlying disease acute myeloid leukemia 44 (47%), acute lymphoid leukemia 20 (21%), conventional conditioning regimens 54 patients (57%). We found that the absolute number (cell/mL) of NK-CD16 þ 56 þ , NKG2A, NKG2C was similar in UCBT and HLAid at day þ 28 and þ 180, but NK-CD16 þ 56 þ and NKG2A were significantly higher in UCBT vs HLA-id at day þ 100 (301 vs 148, P ¼ .006; 230 vs 73, P ¼ .0002). Additionally, the absolute numbers of T-CD4 þ , T-CD8 þ cells were significantly higher in HLA-id vs UCBT at day þ 28, þ 100, and þ 180, while the B-CD19 þ cells were higher in HLA-id vs UCBT at day þ 28, but not after. In UCBT patients, the 1 year CI of CMV was related with CD3 o31 cell/mL at þ 100 (P ¼ .02), and NKG2C 427 cell/mL at þ 180 (P ¼ .02); NRM with CD3 o63 cell/mL (P ¼ .01), CD4 o42 cell/mL (P ¼ .002), CD19 o20 cell/mL (P ¼ .03) (all at þ 180). In HLA-id patients, the 1 year CI of CMV was related with CD19 o18 cell/mL at þ 100 (P ¼ .04), and NKG2C 421 cell/mL at þ 180 (P ¼ .03); NRM with CD19 o18 cell/mL at þ 100 (P ¼ .04); and acute GVHD with CD19 o18 cell/mL at þ 100 (P ¼ .006). We found no impact of immune reconstitution on relapse. Conclusion: In this preliminary and early prospective analysis performed in a small cohort, we found differences between UCBT and HLA-id patients in the kinetics of reconstitution of NK-, T-, and B-cell subpopulations after alloSCT. As it has been described, the T-cell recovery plays a role in the prevention of CMV infection and may protect against NRM. In addition, the CD19 reconstitution could protect against CMV infection and acute GVHD in HLA-id patients, and it could be associated with less NRM in UCBT and HLA-id. As previous studies have suggested, the CMV infection was associated with NKG2C expansion in alloSCT recipients. Disclosure of Interest: None declared. P046 Peripherally inserted central catheters in stem cell transplantation. Results from a large monocentric study S. Sica1,*, S. Francia2, P. Chiusolo1, L. Laurenti1, F. Sora`1, E. Metafuni1, S. Giammarco1, G. Scoppettolo3, M. Pittiruti4 1 Hematology Department, 2Universita` Cattolica del Sacro Cuore, Rome, Italy, 3Infectious Disease Department, 4Emergy Surgery Department, Universita` Cattolica del Sacro Cuore, Rome, Italy Introduction: Peripherally inserted central catheters (PICCs) are widely used in different clinical situations and may play a role also in patients undergoing hematopoietic stem cell transplantation (HSCT). Materials (or patients) and methods: We have retrospectively reviewed our experience with silicon Groshong closedended valved PICCs (S-PICC) and polyurethane power injectable open-ended non-valved PICCs (P-PICC) used in patients undergoing autologous or allogeneic HSCT. All PICCs were positioned by a team of specifically trained physicians and nurses (adopting a standardized insertion protocol) and utilized by specifically trained nurses of our hematology unit (adopting standardized maintenance protocols for prevention of infective and non-infective complications).
Results: In our hematology unit, in the last seven years (20082014), 285 PICCs were inserted in 280 patients (115 females and 165 males; mean age 49, range 19–68 years): in particular, 185 PICCs were inserted in 183 autologous HSCT patients and 100 PICCs in 97 allogeneic HSCT patients. Both S-PICC and P-PICC were used in autologous and in allogeneic HSCT. No major insertion-related complication occurred. Mean duration of PICCs was 25 days in autologous vs. 52 days in allogeneic HSCT. Most PICCs were removed electively, while 14.6% were removed because of accidental PICC removal (1%) or proven catheter complication (3.8%) or suspected PICC infection (9.8%). Symptomatic catheter-related venous thrombosis occurred in 5.26%. Catheter-related bloodstream infection (CRBSI) was proven in 8 cases (2.8%, 0.8 episodes per 1000 PICC days); 14 PICCs were found to be colonized (4.9%, 1.4 per 1000 PICC days), with no sign of infection; there were 43 cases of non-catheter related bacteremia (15.1%). There was no significant difference between autologous and allogeneic HSCT for any of the above described complications. No significant differences were found between S-PICC and P-PICC in terms of duration or of incidence of thrombotic or infective complications. Though, S-PICC were associated with some minor complications (accidental arterial puncture during insertion, 0.5%; primary tip malposition, 2.2 %; malfunction due to lumen occlusion, 6%), which did not occur with P-PICCs. Conclusion: Our data confirm that PICCs are safe and effective in the management HSCT patients, being associated with a low incidence of both early and late complications: insertion complications are minimal and - adopting proper insertion and management protocols – also the incidence of PICC-related thrombosis and of CRBSI is very low. P-PICCs have some advantages over S-PICCs in terms of cost (being less expensive) and in terms of incidence of mechanical complications (malposition and lumen occlusion). Disclosure of Interest: None declared.
Gene therapy P047 A sensitive and rapid HPLC assay for semi-quantitative analysis of globin chain levels in blood after transplantation of autologous hematopoietic stem cells transduced by a lentiviral ?a-t87q globin vector in ?-thalassemia major and sickle cell disease A. AMIN1,*, P. BOURGET1, B. GOURMEL2, J. MAGALON3, F. TOUZOT3, A. MAGNANI3, L. CACCAVELLI3, F. J. PIERCIEY Jr.4, M. CAVAZZANA3, E. PAYEN5, P. LEBOULCH5,6,7, O. NEGRE4,5 1 Department of Clinical Pharmacy, University Hospital NeckerEnfants Malades, AP-HP, 2Department of Medical Biochemistry, Saint-Louis Hospital, AP-HP, 3Biotherapy Department, University Hospital Necker-Enfants Malades, AP-HP, Paris, France, 4Bluebird bio, Cambridge, MA, United States, 5CEA, Institute of Emerging Diseases and Innovative Therapies (CEA-U. Paris-Sud) and Inserm U. 962, Fontenay-aux-Roses, France, 6Mahidol University and Ramathibodi Hospital, Bangkok, Thailand, 7Harvard Medical School and Brigham & Women’s Hospital, Boston, MA, United States Introduction: In patients (Pts) with b-thalassemia major (b-TM) and sickle cell disease (SCD), gene therapy (GT) strategies involving ex vivo transduction of haematopoietic stem cells (HSC) have the potential: (a) to correct a defective erythropoiesis, (b) to sustainably induce high-level production of modified b-globin in the red blood cell lineage, and ultimately (c) to reduce or stop transfusion needs in Pts. To this aim, a family of human b-globin expressing lentiviral vectors was developed and extensively characterized. The amino-acid
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P048 Establishment of a T cell receptor biobank for patients with acute myeloid leukemia to be individually treated by genetically modified donor lymphocyte infusions after haploidentical stem cell transplantation R. Klar1, E. Bra¨unlein1, S. Audehm1, M. Bassani-Sternberg2, S. Mall1, D. Busch3, M. Mann2, C. Peschel1, A. M. Krackhardt1,* 1 Medizinische Klinik III, Klinikum rechts der Isar, TU Mu¨nchen, 2 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 3Institut fu¨r Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universita¨t Mu¨nchen, Mu¨nchen, Germany
(AA) substitution bA-T87Q from the g and d-globin genes1 was incorporated in the therapeutic b-globin. This AA substitution renders the expressed human b-globin protein strongly inhibitory of HbS polymerization in SCD models2 and detectable by HPLC analysis, differentially from other human b-like globin species in b-TM3. A prior trial (LG001) demonstrated clinical benefits of such a lentiviral vector (HPV569) in b-TM. A further improved bA-T87Q vector (LentiGlobin BB305) with both greater transduction efficiency and similar preclinical safety profile was developed; it is under clinical evaluation in the HGB-205 (France) and HGB-204 trials (USA). In this innovative therapeutic context, a reverse-phase HPLC (RP-HPLC) assay was setup to allow quick quantification of globin chains, including the therapeutic chain bA-T87Q. The ion exchange HPLC method that is commonly used to analyze hemoglobin (Hb) is recognized as unable to separate HbAT87Q and HbA. The challenges were: (a) to discriminate b-globin chains with a single AA difference, (b) to isolate the therapeutic globin from both the pathological and normal globins even in Pts receiving transfusions, and (c) to achieve quantification limits as low as possible. Materials (or patients) and methods: In brief, HSC from Pts with b-TM or SCD were harvested, transduced ex vivo with the lentiviral bA-T87Q-globin vector, grown in clonogenic assays, and infused after Bu-based conditioning. bA-T87Q expression was assessed by the RP-HPLC assay on progenitor cells in vitro and on red blood cells from Pts treated by GT3. Clean cell lysates were injected directly onto a C4 (250x4.6 mm) column. Chemical species were detected at 220 nm. Results: After completion of validation stage, the RP-HPLC assay was able to separate and proportionally quantify 9 fractions of Hb in less than 60 min: the heme group, and a, bE, bA, bS, bA-T87Q, gA, gG, and d subunit globin chains, obtained either from a whole blood sample (50 mL) or from in vitro clonogenic cultures with a sensitivity limit of B2,000 erythroblasts (Figure 1). Conclusion: A sensitive and rapid semi-quantitative RP-HPLC assay was developed for the separation of 9 fractions of Hb. It can be applied to: (a) early detection of b-TM or SCD, Hb variants analysis, and various hemoglobinopathies, (b) study of trends and evolution of bA-T87Q blood concentration in Pts undergoing GT, (c) study of bA-T87Q expression on erythroblasts after ex vivo transduction. New clinical and preclinical applications of the method are being explored. References: 1. Takekoshi et al. PNAS 1995. 2. Pawliuk et al. Science 2001. 3. Cavazzana-Calvo et al. Nature 2010. Disclosure of Interest: None declared.
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Introduction: Acute myeloid leukemias (AML) are highly aggressive diseases with often unfavorable prognosis. Allogeneic hematopoietic stem cell transplantation (SCT) and subsequent donor lymphocyte infusions are effective, still treatment failure is frequent. The optimization of haploidentical SCT protocols have led to an enhanced usage of this treatment procedure. The HLA mismatched situation allows redirection of T cells by T cell receptors (TCR) in an HLAmismatched setting. Such genetically modified T cells can be transferred as donor lymphocyte infusions with defined antileukemic specificity. However, target structures are limited and successful treatment will likely imply tools with diverse specificities as well as HLA restrictions. Materials (or patients) and methods: By applying the immunopeptidomic approach on samples of patients with myeloid leukemia and melanoma, we have identified novel HLA ligands that are derived from genes which are restrictedly expressed in hematopoietic cells or overexpressed in malignant cells as well as presented on different HLA molecules. We furthermore optimized a workflow that allows isolation of peptide specific T cells after stimulation of naı¨ve T cells with single HLA-mismatched dendritic cells (DC) with the aim to generate a biobank of leukemia-reactive TCR with specificity for the identified HLA ligands. Results: Using that workflow, T cells with desired specificity were sorted by HLA multimers as well as applying IFN-g capture resulting in several T cell lines and TCR with specificity for diverse hematopoietic differentiation antigens as well as cancer testis antigens as MPO, ITGA2B and PRAME. Within these specificities, a broad spectrum of peptidedependent HLA reactivities as HLA-A1, HLA-A2, HLA-B7, HLAB15, HLA-B42 is covered. Isolated T cell lines and TCR are extensively tested for peptide-dependent anti-leukemic efficacy as well as on- and off-target toxicity. Efficacy has been demonstrated for TCR2.5D6 with specificity for MPO in diverse in vivo models and this TCR is currently prepared for clinical translation. Conclusion: Taken together, we herein show that combination of immunopeptidomics and isolation of peptide specific TCR in the single HLA-mismatched setting is a powerful method to develop therapeutic tools for novel antileukemic immunotherapeutic strategies. The selected TCR build a base for a TCR biobank which can be used in a personalized way in patients undergoing haploidentical SCT for AML. Disclosure of Interest: R. Klar Conflict with: Patent, E. Bra¨unlein: None declared, S. Audehm: None declared, M. Bassani-Sternberg: None declared, S. Mall: None declared, D. Busch: None declared, M. Mann: None declared, C. Peschel: None declared, A. Krackhardt Conflict with: Patent P049 Abstract Withdrawn
P050 A clinically feasible protocol of TCR gene editing to treat Multiple Myeloma without inducing GvHD S. Mastaglio1,*, P. Genovese1, Z. Magnani1, B. Camisa1, E. Landoni1, G. Schiroli1, E. Provasi1, A. Lombardo1, A. Reik2, N. Cieri1, M. Ponzoni1, F. Ciceri1, C. Bordignon3, M. Holmes2, P. Gregory2, L. Naldini1, C. Bonini1 1 San Raffaele Scientific Institute, Milan, Italy, 2Sangamo Biosciences, Richmond - CA, United States, 3Molmed, Milan, Italy
Camisa: None declared, E. Landoni: None declared, G. Schiroli: None declared, E. Provasi: None declared, A. Lombardo: None declared, A. Reik Employee of: Employment at Sangamo BioSciences , N. Cieri: None declared, M. Ponzoni: None declared, F. Ciceri: None declared, C. Bordignon: None declared, M. Holmes Employee of: Employment at Sangamo BioSciences , P. Gregory Employee of: Employment at Sangamo BioSciences , L. Naldini: None declared, C. Bonini: None declared
Introduction: Gene transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach of adoptive immunotherapy for cancer patients. However, T cells transduced with an exogenous TCR produced suboptimal clinical results. This could be due to the dilution of the tumor specific TCR that competes with endogenous TCRs for its expression on the T cell surface. Moreover, mispairing between endogenous and exogenous TCR a and b chains could generate new TCRs with unpredictable and potentially harmful specificities. To overcome these issues, our group developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to a and b chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes by lentiviral vectors Provasi (et al, Nature Medicine 2012). Materials (or patients) and methods: The complete editing procedure requires multiple manipulation steps involving repeated cell activation cycles and 4 transduction procedures. We recently developed the ‘single TCR editing’ (SE), based on the disruption of the single endogenous TCR a chain, followed by transfer of both the a and b chains of the tumor specific TCR, that generates redirected T cells fully devoid of their natural TCR repertoire, in a single round of cell activation. The aim of this project is to determine efficacy and safety of SE T cells in vitro and in vivo. We validated the SE protocol exploiting an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by 60% of high risk multiple myeloma). Results: The SE strategy allowed the rapid production of high numbers of tumor specific T cells, enriched for cells with an early differentiated phenotype. When tested against the U266 myeloma cell line (NY-ESO-1 þ HLA-A2 þ ) in different functional assays (co-culture, g-IFN and 51Cr release), all NY-ESO-1 redirected T cells showed a very high killing potential. However, when we compared the alloreactive potential of the different NY-ESO-1 specific T cell populations in mixed lymphocyte reactions against completely mismatched allogeneic targets, we interestingly observed that the lysis of the allogeneic target by TR cells was significantly higher than that of SE T cells (P ¼ 0.05). These results were validated in NSG mice, engrafted with U266 cells and infused 5 weeks later with NY-ESO-1 specific T cells. All animals treated with tumor specific T cells were completely disease-free at the time of sacrifice, demonstrating the powerful anti-tumor potential of the NY-ESO-1 TCR. However, the overall survival of mice treated with TR vs SE cells was 26% vs 100% respectively (Po0,001), explained by the significant difference in acute and chronic GvHD occurrence (71% vs 0%, Po0,001). This suggests that the residual endogenous polyclonal TCRs and mispaired TCRs expressed on the cell surface of TR T cells can lead to offtarget recognition, while SE T cells are devoid of such reactivity. Conclusion: The single gene editing protocol enables the rapid generation of clinically relevant doses of highly performing tumor specific T cells, fully devoid of their endogenous TCR repertoire, and thus particularly appealing for a future clinical translation. Donor-derived SE T cells, with a significantly reduced alloreactive potential, could be especially suitable to treat patients with minimal residual disease after allogeneic hematopoietic stem cell transplantation. Disclosure of Interest: S. Mastaglio: None declared, P. Genovese: None declared, Z. Magnani: None declared, B.
P051 Abstract Withdrawn
Non-hematopoietic stem cells P052 Tissue-resident stem cells – natural distribution, isolation and biological characteristics A. Klimczak1,*, T. Jurek2, U. Kozlowska1, M. Czuba2, M. Rorat2, M. Paprocka1 1 Institute of Immunology and Experimental Therapy Polish Academy of Sciences, 2Department of Forensic Medicine, Medical University, Wroclaw, Poland Introduction: Natural distribution and biological characteristics of multipotent stem cells responsible for tissue regeneration in human organs is still under investigation. The aim of this study was to verify wheatear it is possible to propagate in vitro, from different human tissues obtained from deceased donors, a population of cells that represent stem cells, and to characterize biologic properties of these cells. Materials (or patients) and methods: Tissue samples were collected from 16 deceased donors (age 9 months to 48 years) with approval by the local Bioethics Committee. Samples of bone marrow (BM), skeletal muscle, heart, liver, and skin were harvested from12,5 h to 48 h after dead. BM was collected to the heparinized tube and mononuclear cells were isolated in the ficoll gradient. Tissue samples were divided and prepared for routine formalin-fixed paraffin block or placed in PBS supplemented with antibiotics for cell isolation. After tissue enzymatic digestion with trypsin, long-term cultures in standard DMEM medium (tissues) or in alpha MEM medium (bone marrow) supplemented with 10% FBS and antibiotics, at 37oC and 5% CO2 was performed. Culture medium was exchanged every 3 days until cells reached 60-70% confluence. Tissue distribution and the phenotype of the cultured cells were characterized by flow cytometry and immunocytochemistry for: CD45, CD34, CD117, CD56, PAX7, CD73, CD90, CD105. Results: Common feature of tissue localized stem cells was expression of c-kit (CD117), CD34 and CD56. Skeletal muscle progenitor cells exclusively express transcriptional factor PAX7. CD73 and CD90 were present on single stem cells of examined tissues and were localized in specific tissue compartments: between the basal lamina and sarcolemma of myofibres of the muscle, in the periportal area of the liver, in the basal layer of epidermis and in the epithelium of adnexal structure of the skin, and were connected to myocytes and fibroblasts in the cardiac niches. We obtained live adherent cells after culture of BM, skeletal muscle and skin progenitor cells. After P1 cell expansion BM-, muscle- and skin-derived live adherent cells exhibit similar phenotype (CD90 þ , CD73 þ , CD105 þ ) characteristic for naı¨ve mesenchymal stem cells (MSC). Muscle progenitor cells (but not from BM or skin) after culture additionally expressed CD56 and CD34, and this observation suggest that those population of MSC is muscle-specific. All
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cultured cells of BM-origin were negative for hematopoietic markers CD45, CD34 and CD117. We were not able to obtain live cells from liver and heart. Conclusion: Stem cells with MSC characteristics can be generated in vitro from several human tissues including BM, skeletal muscle, and skin up to 48 h after dead. The isolation and expansion of progenitor cells from human tissues is an option to obtain a population of stem cells with naı¨ve MSC phenotype. Thus, the opportunity to expand live tissue-origin MSC from deceased donor might have important clinical alternative in regenerative medicine for MSC generated from tissues from live donors. Study supported by National Science Center grant N N407 121940. Disclosure of Interest: None declared. P053 Gene expression of Notch receptors and ligands by human Mesenchymal Stem Cells from different tissues G. Aydin1,2, F. Aerts-Kaya1,2,*, A. Gunel-Ozcan1,2, D. Uckan-Cetinkaya1,2,3 1 Center for Stem Cell Research and Development, 2Institute of Health Sciences, HACETTEPE UNIVERSITY, 3Department of Pediatric Hematology, Hacettepe University Medical Faculty, Ankara, Turkey Introduction: Multipotent Mesenchymal stromal cells (MSCs) can be found in bone marrow (BM), placenta and umbilical cord. BM-MSCs also support expansion of Hematopoietic Stem Cells (HSCs) in vitro. The CD271 antigen can be used for prospective isolation of BM-MSCs. CD271bright BM cells display high clonal capacity and differentiation potential, but also support lymphoid differentiation and hematopoiesis in vivo (1). Notch signaling plays an important role in the regulation of T-cell development and stem cell maintenance. Here, we compared gene expression levels of Notch receptors and their ligands in BM-MSCs, CD271 þ MSCs, Placenta amnion (PA), Placenta chorion (PC) and Umbilical cord (UC)derived MSCs, to identify the optimal cell source of MSCs for support of in vitro expansion of HSCs, capable of lymphohemopoiesis. Materials (or patients) and methods: BM-MSCs were isolated from healthy human BM aspirates. CD271 þ MSCs were prospectively isolated by magnetic selection (Miltenyi). PA-
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MSCs, PC-MSC and UC-MSCs were isolated by mechanic dissociation and enzymatic digestions, using Trypsin/EDTA, collagenase IV and dispase I. BM-MSCs (n ¼ 6), CD271 þ MSCs (n ¼ 6), PA-MSCs (n ¼ 7), PC-MSCs (n ¼ 5), and UC-MSCs (n ¼ 4) were cultured up till passage 3. MSC immunophenotype was measured using a FACSARIA (Becton Dickinson). Total RNA was purified and 250 ng RNA was used for cDNA synthesis. Gene expression was assessed by real-time qPCR (LightCycler 480II, Roche) using RealTime ready Single Assays (Roche). All target genes were normalized against the house keeping gene, ACTB and samples were run at least in triplicate. The crossing point (Cp or Ct) for target and reference genes for each sample was calculated according to Second Derivative Maximum Method using LightCyclers 480 Software (Roche). 2 DDCt method was used for relative quantitative PCR analysis by applying a log2 transformation. Gene expression of Notch-2/3/4, Jag-1/2, DLL1/4 in PA/PC/UC/CD271 þ MSCs was determined relative to BM- MSCs. Results: BM-MSCs ane CD271 þ MSCs abundantly expressed CD29, CD44, CD73, CD90, CD105 and CD166, but lacked expression of CD14, CD45, CD34, CD31. In comparison to BMMSCs, UC-MSCs expressed lower levels of CD44 and CD73 and increased levels of CD200 (Po0.01), whereas PA and PC-MSCs expressed lower levels of CD90 (Po0.01). All groups lacked gene expression of NOTCH4. Relative NOTCH2 and NOTCH3 expression was lower in PA/PC/UC/CD271 þ MSCs compared to BM-MSCs. The lowest expression of NOTCH2 and NOTCH3 was found in PA-MSCs with 3.7 and 5.26 fold decrease, respectively. JAG1 expression was reduced in PA-MSCs (-5.99), PC-MSCs (-3.43) and UC-MSCs (-3.63). JAG2 and DLL1 were expressed at lower level in CD271 þ MSCs and PA-MSCs. PCMSCs and UC-MSCs expressed higher levels of JAG2, DLL1 and DLL4. CD271 þ MSCs and PA-MSCs were almost similar to BMMSCs for DLL4 expression. Among MSCs source types, CD271 þ MSCs showed the closest NOTCH2, NOTCH3, JAG1 and DLL4 gene profile to BM-MSCs. Conclusion: CD271 þ MSCs showed a NOTCH2, NOTCH3, JAG1, DLL4 expression profile similar to BM-MSCs. These two groups differed only in expression of JAG2 and DLL1. PC-MSCs and UC-MSCs displayed higher expression of DLL1 and DLL4, which may be important for in vitro support of lymphopoiesis, T and B cell development. References: Kuc¸i S, et al. Haematologica. 2010 Apr; 95(4): 651-659. Disclosure of Interest: None declared.
P054 Chemical inducers of Endoplasmic Reticulum (ER) Stress suppress proliferation and adipogenic differentiation of human Mesenchymal Stem Cells B. Ulum1, F. Aerts-Kaya1,2,*, H. T. Teker3, G. Balta4, D. Uckan-Cetinkaya1 1 Center for Stem Cell Research and Development, 2Institute of Health Sciences, Hacettepe University, 3Department of Biological Sciences, Middle East Technical University, 4Department of Pediatric Hematology, Hacettepe University Medical Faculty, Ankara, Turkey Introduction: The Endoplasmic Reticulum (ER) plays an important role in lipid and protein synthesis and protein folding. Accumulation of unfolded proteins in the ER lumen or protein production exceeding the capacity of ER, results in ‘‘ER stress’’, which causes a series of complex signal pathways, called the ’Unfolded Protein Response’ (UPR) and ensures ER homeostasis. Different levels of ER stress are required for cell proliferation and differentiation. Mesenchymal Stem Cells (MSCs) can be isolated from bone marrow (BM), adipose tissue, placenta, umbilical cord and differentiate into fat, bone and cartilage cells. Our aim was to investigate the effect of chemicals, that modulate ER stress and asses their effect on proliferation and differentiation of MSCs using inducers of ER stress Thapsigargin (TG) and Tunicamycine (TM) and inhibitors of ER stress TUDCA and 4-PBA. Materials (or patients) and methods: Human BM samples (3-5 mL) were obtained from BM transplantation donors, sceduled for BM harvest. Mononuclear cells were plated in DMEM-LG/MDCB201 in presence of Fetal Calf Serum. MSCs were propagated until passage 3. Cells were seeded in triplicate into e-plate wells at 2000 cells/well (xCELLigence, Roche) to assess real time proliferation using impedance measurements. Cell proliferation was followed for 10 days. For adipogenic and osteogenic differentiation MSCs were maintained in DMEM-LG/10% FCS with different concentrations of the chemicals TG, TM, TUDCA and 4-PBA in presence of adipogenic differentiation medium (1 uM Dexamethasone, 60 uM Indomethacin, 500 uM Isobutylmethylxanthine and 5 ug/mL Insulin) or osteogenic differentiation medium (100 nM dexamethasone, 10 mM b-glycerophosphate and 0.2 mM L-Ascorbic Acid). Semiquantitative analysis of differentiation was performed after three weeks of culture, using spectrophotometric comparison of Oil Red O dye inclusion as a measurement of adipogenic differentiation. Results: Treatment with ER stress inducer TG resulted in complete inhibition of proliferation at all doses tested (50-400 nM), whereas treatment with TM at 100-500 nM resulted in a dose dependent suppression of proliferation. Treatment with inhibitor of ER stress TUDCA, did not affect cell proliferation at doses from 50-200 uM, whereas 4-PBA displayed a dosedependent inhibition of proliferation with maximum inhibiton of B50% at doses of 5 mM. The inhibitory effects of TG and TM on MSC proliferation could not be counteracted by either TUDCA or PBA. TUDCA had no clear effects on MSCs, but treatment with 500 nM TM resulted in loss of cells and cell death within a week. ER stress induction using TG resulted in partial and complete inhibition of adipogenesis at 25 and 50 nM, whereas treatment with TM resulted in a 40% reduction of adipogenic differentiation. In contrast, ER stress inhibitors TUDCA and 4PBA caused a slight increase in adipogenic differentiation (120% in comparison to control). 4-PBA count not restore adipogenesis in cultures treated with TG or TM. Most, likely because 4-PBA and TG/TM affect different ER stress signaling pathways. Conclusion: These data demonstrate that ER stress inducing chemicals suppress MSC proliferation and affect its adipogenic differentiation capacities, which cannot be counteracted by chemical chaperons TUDCA and 4-PBA. Disclosure of Interest: None declared.
P055 Mir-663 impairs the effects of bone marrow-derived mesenchymal stem cells on MRL/lpr mice L. Geng1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Previously we have shown that miR-663 was increased in bone marrow derived mesenchymal stem cells (BMSCs) from SLE patients and involved in the functional deficiency of BMSCs through inhibiting transforming growth factor b1 (TGF-b1) production. This study was undertaken to explore whether the modulation of miR-663 in BMSCs could affect their therapeutic effects on MRL/lpr mice. Materials (or patients) and methods: Negative-control-miR663 (miR-663-C), mimics-miR-663 (miR-663-M) and inhibitormiR-663 (miR-663-I) eukaryotic expression vector were artificially transfected into BMSCs and intravenous injected (1 106) into 20 weeks old female MRL/lpr mice. 8 weeks later, mice were sacrificed, with kidneys, lymph node harvested and spleen weighed. Their serum and urinary samples were collected for the measurement of autoantibodies (including IgG, ds-DNA and ANA) and cytokines (TGF-b1, IL-4, IFN-g, IL-17A and so on) by ELISA, and proteinuria by coomassie blue staining assay. Immune complex deposition including IgG and complement 3 (C3) in kidney sections was performed by immunofluorescence staining. The percentages of Th1, Th2, Th17, regulatory T cells (Treg) and follicular T helper (Tfh) cells in splenic mononuclear cells were detected by flow cytometry. Results: Compared to the miR-663-C and miR-663-M group, miR-663-I transfected BMSCs displayed enhanced therapeutic effects on MRL/lpr mice, as shown by significantly declined spleens and lymph nodes size as well as reduced serum IgG and anti-dsDNA levels. Compared to miR-663-C and miR-663-I group, mice treated with miR-663-M transfected BMSCs presented enlarged glomerulus with hyper cellularity and meningeal expansion, and greater amounts of immune complex deposition including IgG and C3 in the meningeal and peripheral capillary loops. Meanwhile, Treg cell percentages were increased in miR-663-I group compared with those in miR-663-M and miR-663-C group (13.3±1.12% vs. 6.90±0.82% and 8.25±1.07%, overall Po0.05), while Tfh cell percentages were decreased (8.58±1.09% vs. 22.9±4.24% and 12.40±1.61%, overall Po0.05). Conclusion: Inhibition of miR-663 in MSCs enhanced the therapeutic effects of MSC transplantation on MRL/lpr mice. Disclosure of Interest: None declared. P056 UC-MSCs inhibit the differentiation and proliferation of T Follicular helper cell in Rheumatoid Arthritis R. Liu1, X. Li1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: The aim of this study is to elucidate whether umbilical cord derived mesenchymal stem cell (UC-MSCs) has immunomodulatory effect on T follicular helper (Tfh) cell under rheumatoid arthritis (RA) background. Materials (or patients) and methods: PBMCs that isolated from RA patients and healthy contorl (HC) were cocultured with UCMSCs at a ratio of 100:1, 10:1 and 1:1 respectively through cell-tocell contact or in a transwell system at a ratio of 1:10. After 3 days’ coculture, PBMCs were collected and the frequency of CD4 þ CXCR5 þ PD-1 þ T cell was examined by flow cytometry. Purified naı¨ve T cells isolated from PBMC of RA patients were cocultured with human UC-MSCs for 4 days at a ratio of 1:10 under Tfh cellpolarizing condition. To detect the effect of MSC on Tfh proliferation, CFSE-labeled purified CD4 þ T cells isolated from PBMC of RA patients or HC were stimulated with anti-CD3/CD28 and cocultured with UC-MSCs for 5 days. The frequency of CD4 þ CXCR5 þ PD-1 þ T or CD4 þ CXCR5 þ PD-1 þ AnnexinV þ
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T was examined by flow cytometry and the level of IL-21 in the cultured supernatant was measured by ELISA. The mRNA levels of indoleamine 2,3-dioxygenase (IDO), IL-10, PGE2, HGF, TGF-b and HLA-G in UC-MSCs were tested by RT-PCR. IDO activity of was measured by high-performance liquid chromatography (HPLC), and P-STAT-1/3/5 and p-Akt in UC-MSCs were tested by Western blot. IDO inhibitor 1-MT or anti-IL-10 antibody was added into UC-MSCs and Tfh cell differentiation coculture system for 5 days and then the frequency of CD4 þ CXCR5 þ PD-1 þ T was examined by flow cytometry. Results: UC-MSCs were able to suppress the generation of Tfh cell both in RA patients and in HC in vitro, which was dosedependent and not relied on cell-to cell contact. UC-MSCs suppressed the differentiation and proliferation of Tfh cell that was more prominent in samples from RA patients, but had no effect on Tfh cell apoptosis. The level of IL-21 in UC-MSCs and Tfh cell differentiation coculture system was significantly decreased. Dramatic increases of both IDO mRNA expression and IDO enzymatic activity were detected in UC-MSCs after coculturing with naı¨ve T cells under Tfh cell-polarizing condition. Meanwhile, pSTAT-1/3/5 and pAkt in these UCMSCs were increased. The addition of the IDO inhibitor 1-MT, but not anti-IL-10 antibody, could reverse the suppressive effect of UC-MSCs on the differentiation of Tfh cell. Conclusion: UC-MSCs suppress Tfh cell differentiation and proliferation in RA patients, which is partially mediated by the secretion of IDO. Disclosure of Interest: None declared. P057 UC-MSCs inhibit T cell autophagy and apoptosis in patients with systemic lupus erythematosus through mitochondrial transfer J. Chen1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: This study is aimed to investigate the role of umbilical cord derived mesenchymal stem cells (UC-MSCs) on autophagy and apoptosis in T cells from SLE patients, and to explore the underline mechanisms involved in this process. Materials (or patients) and methods: Peripheral blood mononuclear cells (PBMC) were isolated from SLE patients and healthy donors, and cultured under stimulation with anti-CD3/28 antibodies in the presence or absence of autophagy inhibitor 3MA (5mM for 6h) or activator rapamycin (50nM for 48h). Autophagy levels and apoptotic rates were measured by flow cytometry with the detection of LC3IIB and Annexin V respectively. To determine the effects of MSCs on T cell autophagy and apoptosis, UC-MSCs were cocultured with T cells at the ratio of 1:10 directly or in transwell system. To observe the changes of pathways upstream of autophagy after MSC treatment, an AMPK activator was added to the cocultures. Meanwhile, mitochondria transmembrane potential (DCm), which was closely related to APPK activation, was marked and measured in MSCs and T cells by MitoTracker Deep Red (MDR). Results: T cells from SLE patients had both elevated autophagy level and apoptotic rate compared with those from normal controls, which were further increased after antiCD3/CD28 stimulation. Apoptotic rate of T cells significantly correlated with autophagy level (r ¼ 0.570, Po0.0001 for CD4 þ T; r ¼ 0.508, P ¼ 0.0001 for CD8 þ T). Inhibition of autophagy with 3-MA decreased the apoptotic rate of total T cells, whereas activation of autophagy with rapamycin increased the apoptotic rate. UC-MSCs significantly inhibited T cell autophagy and T cell apoptosis after cell-to-cell contact coculturing, yet the effect was diminished in transwell system. When AMPK activator added to the cultures, the ability of UCMSCs to regulated T cell apoptosis was greatly impaired. Mitochondria in UC-MSCs could be transferred to SLE T cells when directly cocultured. Consequently, the elevation of DCm in T cells was downregulated after MSC treatment, along with the reduction of AMPK.
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Conclusion: Autophagy levels are elevated in T cells from SLE patients, leading to aberrant apoptosis. UC-MSCs may inhibit T cell autophagy and apoptosis through mitochondrial transfer. Disclosure of Interest: None declared. P058 Leptin and neutrophil activating protein 2 promote mesenchymal stem cells senescence through the activation of PI3K/Akt pathway in systemic lupus erythematosus H. Chen1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Mesenchymal stem cells (MSC) derived from patients with systemic lupus erythematosus (SLE) displays enhanced senescence. Here the aim is to explore the role and mechanism of SLE inflammatory environment on the regulation of MSC senescence. Materials (or patients) and methods: Mesenchymal stem cells were isolated from fresh umbilical cords or human bone marrow. Cellular senescence was measured by senescenceassociated b-galactosidase (SA-b-gal) staining, and the mRNA and protein levels of cell cycle inhibitors p53 and p21 were detected by real time PCR and western blot analysis. Proteins involved in senescence related signaling pathways, including Jak/stat, Ras/MEK/Erk, NF-kB and PI3K/Akt, were detected by western blot. Human serum collected from healthy controls and SLE patients were applied for the measurement of 80 human cytokines and chemokines by RayBios human cytokine antibody arrays. Then differentially expressed factors were replicated by ELISA using independent samples. Results: SLE serum significantly increased the percentage of SA-b-gal positive MSCs, and upregulated the expression of p53 and p21. Jak/stat1/5, PI3K/Akt and MEK/Erk pathways in MSCs were activated after SLE serum stimulation. Treatment of PI3K/ Akt signaling pathway inhibitor, LY294002, could reverse SLE serum induced senescence, while blocking Jak/stat with AG490 or MEK/Erk with PD98059 did not have an effect. As detected by cytokine antibody arrays and independent sample ELISA, leptin and NAP2 were both significantly elevated in SLE serum, and blocking either of them could partially inhibited MSC senescence. Leptin and NAP2 acted synergistically to promote more senescent cells than leptin or NAP2 alone, which could be completely blocked by PI3K/Akt pathway inhibitor. Conclusion: Elevation of leptin and NAP2 in SLE serum helps to accelerate the cellular senescence of MSCs. PI3K/Akt signaling transduction is important in the senescent effect. Disclosure of Interest: None declared. P059 Mesenchymal stem cells from patients with rheumatoid arthritis display impaired function in inhibiting Th17 cells Y. Sun1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Mesenchymal stem cells (MSCs) possess multipotent and immunomodulatory properties and are suggested to be involved in the pathogenesis of immune-related diseases. This study investigates the biological characteristics and immunomodulatory function of bone marrow MSCs from patients with rheumatoid arthritis (RA). Materials (or patients) and methods: In vitro cultures of bone marrow MSCs from RA patients and controls were established and characterized by their immunophenotype and differentiation potential into adipocytes and osteoblasts. We assessed the proliferation ability, cellular apoptosis and migration potency of MSCs from RA patients (RA MSCs). Using a protein array we evaluated the cytokine production of RA MSCs. The immunomodulatory capacity of RA MSCs on
suppressing proliferation of peripheral blood mononuclear cells (PBMCs) and on the distribution of CD4 þ T cell subsets (Th17, Treg and Tfh cells) was also investigated. Results: Although differentiation potential and apoptosis rate were similar to those of control MSCs, RA MSCs showed decreased proliferative activity and aberrant cell migration. No significant differences were observed in cytokine production between RA MSCs and control MSCs. RA MSCs appeared to be indistinguishable from control MSCs in terms of suppressing the proliferation of PBMCs, decreasing the proportion of Tfh cells and inducing the polarization of Treg cells. However, the ability to inhibit the polarization of Th17 cells was impaired in RA MSCs, which may be related to the low expression of CCL2 in RA MSCs after co-culture with CD4 þ T cells. Conclusion: MSCs from RA patients have abnormalities compared with their control counterparts. These aberrations may develop after the abnormal immune responses in RA. The functionally altered MSCs may then contribute to the pathogenesis of RA. Disclosure of Interest: None declared. P060 The oncometabolite R-2-hydroxyglutarate impairs the function of human mesenchymal stromal cells and induces DNA hypermethylation L. Liu1,*, K. Hu1, B. Wang1, Y. Zhang1, S. Fu1, X. Yu1, H. Huang1 1 Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Introduction: Elevated serum and myeloblast R-2-hydroxyglutarate (R-2HG) are specific features of IDH1/2-mutant acute myeloid leukemia. Recent evidence indicated that R-2HG is indeed an oncometabolite capable of blocking the differentiation of hematopoietic cells and altering epigenetic modification. Mesenchymal stromal cells (MSCs) are an integral component of the bone marrow microenvironment. Moreover, marrow aspirate R-2HG is significantly elevated in IDH-mutant patients. In this study, we therefore investigated the effect of R-2HG on the proliferation, differentiation, cytokine expression and the DNA methylation status of bone marrow MSCs. Materials (or patients) and methods: The human MSCs were obtained from bone marrow of healthy adult donors. MSCs were treated without or with R-2HG at concentrations of 0.1 to 1.5mM. The proliferation of MSCs was detected by Cell Counting Kit-8. To determine the effects of R-2HG on MSC differentiation, the cells were cultured in osteoinductive and adipogenic medium. Specific stains were performed and differentiation-related genes were quantified. The mRNA l evels of related cytokines were measured by Real-time PCR. DNA methylation status was explored by Infinium 450 K arrays. Results: Our results indicated that proliferation of MSCs was not influenced by R-2HG. R-2HG (1.0-1.5mM) inhibited the osteoblast differentiation of MSCs as measured by reduced mineralization (Alizarin-red S stain). R-2HG significantly reduced the expression of both early (Runx2 and IBSP) and late (Osterix and BGLAP) osteoblast differentiation-related genes, supporting the results obtained in the functional assays. Moreover, R-2HG (1.0-1.5mM) promoted the adipogenic differentiation of MSCs as measured by increased lipid vacuoles (oil red O stain). The relative mRNA expression levels of adipocyte-specific transcription factors (C/EBPa and Pparg2) and the marker gene (Adiponectin and aP2) were all enhanced by R-2HG. In addition, the mRNA expression of granulocyte colony-stimulation factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly diminished in R-2HG treated MSCs. Furthermore, we used Infinium 450 K arrays to investigate the methylation patterns. Statistical analysis of the methylation data identified 154 differentially methylated CpGs. Interestingly, these CpGs consistently showed pronounced methylation differences between healthy controls and R-2HG treated MSCs. Compared
with controls, we found hypermethylation in 117 genes and hypomethylation in 37 genes in R-2HG groups. Ingenuity pathway analysis implicated several signaling pathways involved in cell differentiation and stem cell maintenance including the WNT and TGF-beta pathways. Conclusion: There is evidence indicated that osteoblast lineage cells have been shown to support HSCs, however, adipocytes play an inhibitory role in HSC maintenance. We show here that the oncometabolite R-2HG inhibited osteoblastic differentiation, promoted the adipogenic differentiation and induced a pronounced DNA hypermethylation status of MSCs. Our results suggest that bone marrow microenvironment may be affected by the metabolite R-2HG, providing a novel insight into the proto-oncogenic role of IDH mutation. Disclosure of Interest: None declared. P061 Analysis of the immunoregulatory activity of Chemerin produced by Mesenchymal Stromal Cells P. Vinci1,*, A. Bastone2, S. Schiarea3, E. Dander1, A. Del Prete4, M. Salmona2, S. Sozzani4, A. Biondi5, G. D’Amico1 1 Centro di Ricerca "M.Tettamanti", Clinica Pediatrica, Universita` degli studi di Milano-Bicocca, Fondazione MBBM/Ospedale S. Gerardo, Monza, 2Dipartimento di Biochimica delle proteine, 3 Dipartimento di Ambiente e Salute, Istituto per le Ricerche Farmacologiche Mario Negri, Milano, 4Dipartimento di Patologia Generale e Immunologia, Universita` degli Studi di Brescia, Brescia, 5Clinica Pediatrica, Universita` degli studi di MilanoBicocca, Fondazione MBBM/Ospedale S. Gerardo, Monza, Italy Introduction: In the last years, several studies have shown that Mesenchymal Stromal Cells (MSC) can be widely used for treating inflammatory diseases. However, the mechanisms underlying their capacity to inhibit inflammation are not fully understood. Chemerin is an immunoregulatory protein with chemotactic activity, secreted as a precursor and converted into its active form through the proteolitic cleavage of the last six-seven amino acids at the C-terminal domain, by several serine and cysteine proteases of the inflammatory cascade. Materials (or patients) and methods: The production of Chemerin by MSC was evaluated by ELISA assays under basal conditions as well as after stimulation with IL6, IL1 and TNFa. In addition, Chemerin proteases expression was evaluated by RT-PCR under the same culture conditions. In order to understand if this protein could be involved in MSC immunoregulatory function, Chemerin produced by MSC (MSC-Chem) was purified from MSC supernatant and functional and biochemical analysis were performed. The chemotactic activity of MSC-Chem was evaluated in the presence or absence of different proteases by using a pre-B cell line expressing the human ChemR23 receptor (L1.2-ChemR23). The biochemical analysis was performed by LC/MS Mass Spectrometry. Results: MSC were able to produce Chemerin and its production was strongly increased after stimulation with inflammatory cytokines. RT-PCR analysis of Chemerin activating serine-cysteine proteases showed that MSC express low levels of Neutrophil Elastase compared to PBMC and its expression did not significantly increase after 24h, 48h or 72h of stimulation with inflammatory cytokines. MSC also expressed Cathepsin K, and its levels did not increase after stimulation with inflammatory cytokines. Functional analysis of MSC-Chem revealed that L1.2-ChemR23 cells were able to migrate in response to rh-Chemerin in a dose-depend manner until the concentration of 10 nM. However, at equivalent concentrations, rh-Chemerin was able to induce a stronger L1.2-ChemR23 migration compared to MSC-Chem, suggesting that in MSC supernatant only a fraction of the protein was in the active form. In accordance, the LC/MS Mass Spectrometry identified the active form of Chemerin (with the last peptide Chem144-Chem157) only in rh-Chemerin but not in MSC-Chem (Chem144-Chem163), confirming that MSC-Chem is mostly
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produced in its inactive form. Interestingly, the migration of L1.2-ChemR23 cells towards MSC-Chem pre-incubated with Neutrophil Elastase and Cathepsin L, resulted increased compared to MSC-Chem alone, indicating that MSC-Chem can be activated by proteases usually involved in inflammation. Conclusion: We demonstrated that MSC were able to produce Chemerin and directly cleave it in its active form, although only partially. We hypothesize that, when infused in vivo during inflammation, MSC produce Chemerin as a precursor, which is then converted in its active form by cysteine and serine proteases, highly expressed at peripheral inflamed tissues. Further in vivo studies are needed to clarify whether Chemerin activation can then induce ChemR23-expressing cells migration towards MSC, to better exert their antiinflammatory activity. Disclosure of Interest: None declared. P062 MSC Cavity Injection Boosts Th1 Subset and has Good Response in extensive cGVHD patients: A Prospective Multicentre Randomized Controlled trial W. Zheng1,*, Y. Luo1, S. Wang2, Q. Han3, M. Guo4, D. Zhou5, L. Yu6, H. Huang1 1 Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 2 Chinese PLA General Hospital, 3The Basic Medical Institute of Chinese Medical Sciences Academy, 4The Affiliated Hospital of Chinese PLA Academy of Military Medical sciences., 5Peking Union Medical College Hospital., 6Bone Marrow Transplantation Center, Chinese PLA General Hospital., Beijing, China Introduction: Since steroid-refractory chronic graft-versushost disease was still a great challenge, we conducted a prospective multicenter phase II study to confirm the efficacy of sequential marrow cavity injection of Mesenchymal stem cells (MSC) in extensive CGVHD patients. Materials (or patients) and methods: We recruited 40 patients from four transplantation centers from Jul. 2010 to Dec. 2014, who suffered extensive cGVHD in skin and/or liver, post allogeneic hematopoietic stem cell transplantation (HCT). 30 patients were in experimental group, who received four weekly sequential marrow cavity injection of MSC. 10 patients were in control group, who received traditional immunosuppressive therapy. MSC were cultured from MHC mismatched donors and offered by the Basic Medical Institute of Chinese Medical Sciences Academy. The count of MSC was 1-4*10E7 weekly, totally 4.8-16*10E7 every cycle (four weeks). Patients continued immunosuppressive therapy during and after MSC transfusion. We assessed the improvement of cGVHD sign/symptom, the relapse of hematological malignancies, the overall survival (OS), the progress free survival (PFS) in 1, 2, 3, 6, 12 months post MSC transfusion. At the same time, we detected T cell subsets of Th1/Th2/Th17, Tregs cells. Results: 1) The average follow-up time of control group was 23.6 months, whose overall response was 30%(3/10), including 1 patient (10%) excellent response. The mortality was 10%. 2) The average follow-up time of experimental group was 13.4 months, whose overall response was 90%(27/30), including 15 pts (50%) excellent response. The mortality was 6.7% (2/30), who died not of MSC transfusion, but of HCT complication. The response rates between experimental group and control group were significantly different. 3) Patients who respected to MSC transfusion showed improvement of skin thick, elasticity and movability. And some of them gain weight greatly. Some patients response to MSC transfusion quickly within 12 hours, the mean time of maximum response was six months. 4) Four patients received two cycles of MSC transfusion, one patient received three cycles of MSC transfusion, which didn’t increase the adverse effect, but positive correlated with the response. 5) Post MSC transfusion, the Th1 subset was increased significantly in response
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patients. The th1/th2 ratio was increased post MSC transfusion, and kept 2 to 6 months depends on response time. But for those three no response patients, (two were over-lap CGVHD), showed no difference between MSC transfusion. 6) Th17 subset was very low and no difference during MSC treatment. Conclusion: MSC transfusion by sequential marrow cavity injection was approved effective in treating extensive cGVHD, the response rate was 90%, increase the transfusion cycles could improved the effect. Th1 subset boost and reactivation post MSC transfusion may contribute to good response of cGVHD patients. Disclosure of Interest: None declared. P063 Human bone marrow adipocytes negatively regulate hematopoietic stem/progenitor cells through secreting adiponectin W. Lu1,*, W. Wang1, Y. Zhang1, S. Wang1, Y. Feng1, K. Liu1 1 Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Institute of Hematology, Peking University People’s Hospital, Beijing, China Introduction: Fatty bone marrow, in addition to hematopoietic hypocellularity, is a pathologic characteristic observed in Aplastic Anemia(AA), high-dose chemotherapy, poor graft function and other clinical hematological disorders. Recently, it is proved that bone marrow fat serves as a negative regulator in hematopoiesis. However, the exact role of adipocytes in the regulation of hematopoiesis remains a controversial subject and the mechanism by which adipocytes act on hematopoietic stem cell is still unclear. In our study, we set out to test the hypothesis that bone marrow adipocytes could affect hematopoietic stem/progenitor cells (HSPC) by adiponectin. Materials (or patients) and methods: A direct cell contact or transwell co-culture system of mobilized peripheral blood (mPB) CD34 þ with mesenchymal stem cells (MSC) or adipocytes was established. After 7 days of co-culturing, cell phenotype and cell cycle of HSPC was assessed by flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCT) analysis was performed to detected the mRNA levels of adiponectin, TNF-a, leptin and neuropilin-1. Meanwhile, the protein level of adiponectin was detected by Elisa. To further examine the role of adiponectin, an antiadiponectin antibody(15ug/ml) was added to the culture medium and further assessed the proliferation, differentiation of HSPC. Results: After co-culturing of mPB CD34 þ with MSC or adipocytes for 7 days, we first found that the fold expansion of total nucleated cells was significantly decreased after seeded on adipocytes compared to MSC [(17.76±2.88) vs. (26.15±2.09), Po0.05, n ¼ 4]. Moreover, the lower absolute levels and relative numbers of CD34 þ CD38 þ HPC was also decreased. Next, we tested the effect of adipocytes on HSPC in stromal transwell co-cultures. Consistently, we found that the proliferation and proportion of HPC was also decreased significantly in adipocyte group. Moreover, we found fewer cells in the G0 phase in CD34 þ HSPC co-cultured with adipocytes compared with control. To uncover the mechanism mediated in this process, we tested the expression of adipokine which was known to impact the expansion of HSPC by qRT-PCR. We found that the expression of adiponectin was increased by 60-fold in adipocytes compared with MSC (Po0.05). However, the expression of TNF-a, leptin, neuropilin-1 between adipocytes and MSC revealed no significant difference. Furthermore, the adiponectin protein level was also proved increased in the culture medium of adipocytes group. Studies by other groups proved that adiponectin is a negative regulator of myeloid progenitor cells and is critical for the maintenance of HSC. To further comfirmed the effect of adiponectin in the regulation of HSPC, CD34 þ HSPC was co-cultured with adipocytes in the absent or present of anti-adiponectin antibody (15ug/ml). Interestingly, adipocytes decreased the proliferation and
proportion of HSPC after co-culture for 7days, an effect that was partially reversed by addition of the antiadiponectin antibody. Conclusion: In conclusion, our study demonstrated that bone marrow adipocytes is more than just a tissue filler. It could negatively regulate the proliferation and differentiation of HSPC through secreting adiponectin. Furthermore, it also suggested that the hematopoietic inhibition imposed by fatty bone marrow may be improved by targeting adiponectin. Disclosure of Interest: None declared.
Stem cell donor I P064 Abstract Withdrawn
P065 The Impact of HLA Matching on the Clinical Outcome of Hematopoietic Stem Cell Transplantation from Volunteer Unrelated Donors According to the Uniform, Prospective Transplant Program of the Rome Transplant Network A. Picardi1,*, L. Cudillo1, I. Mangione1, G. De Angelis1, B. Mariotti1, F. Di Piazza1, M. Cantonetti2, E. Ceresoli1, C. Rapanotti3, P. De Fabritiis4, T. Dentamaro4, M. C. Tirindelli5, O. Annibali5, A. Mengarelli6, F. Marchesi6, A. Chierichini7, B. Anaclerico7, E. Montefusco8, A. Ferrari8, M. Andreani9, M. Testi9, R. Cerretti 1, W. Arcese 1 on behalf of on Behalf of Rome Transplant Network. 1 Hematology Division - Stem Cell Transplant Unit, 2Hematology Division, 3Laboratory Medicine, TOR VERGATA UNIVERSITY OF ROME, 4Hematology, S.Eugenio Hospital, 5Hematology, University Campus Bio Medico, 6Hematology, Regina Elena National Cancer Institute, 7Hematology, S.Giovanni Addolorata, 8Hematology, S.Andrea Hospital, Sapienza University, 9Laboratory of Immunogenetics and Transplant Biology, IME Foundation, Rome, Italy Introduction: The impact of HLA matching in unrelated Hematopoietic Stem Cell Transplant (HSCT) is still subject of debate. All the available results come from retrospective, registry based meta-analysis on large cohorts of patients (pts), but not homogeneous for policy of donor selection, conditioning regimen or GVHD prophylaxis. Herein, the clinical outcome of allogeneic HSCT from volunteer unrelated donor (VUD) has been analyzed according to a prospective and uniform transplant platform predefined in terms of donor selection, conditioning regimen, GVHD prophylaxis and time to transplant. Materials (or patients) and methods: In order to identify an alternative donor for patients lacking an HLA identical sibling, the Rome Transplant Network (RTN) has adopted a unique searching strategy following a hierarchical algorithm (1st VUD; 2nd Cord Blood; 3rd Haplo) on the basis of established selection criteria. According to this policy, VUD was identified for at least 8/10 high resolution A-B-C-DRB1-DQB1 matching within 3 months. The same conditioning regimen consisting of myeloablative (MAC) or reduced intensity (RIC) version of the Thiotepa, Busilvex and Fludarabine combination was used. GVHD prophylaxis is modulated according to the type of donor, but identical for each transplant category. From December 2007 to date, 87 consecutive pts, with a median age of 42 yrs (range, 18-66), affected by malignant haematological diseases underwent HSCT(MAC ¼ 64 and RIC ¼ 23pts) from VUD in early (n ¼ 49) or advanced (n ¼ 38) stage. GVHD prophylaxis consists of the CSA þ MTX association þ ATG at total dose of 20 mg/kg. The donor-recipient pairs were divided
according to the HLA matching with equivalence between antigenic and allelic level. Pts characteristics were not statistically different in the 3 groups with 0, 1 or 2 HLA disparities. Two analysis were performed: HLA matched (n ¼ 46) vs all mismatched (n ¼ 41) pairs and HLA matched vs 1 (n ¼ 27) vs 2 (n ¼ 14) mismatched pairs. Results: With a median follow up of 31 months (range, 3-74), the 5-yrs probability of overall (OS) and disease free survival (DFS) for all pts was 57±6% and 41±6%, respectively. By comparing matched vs mismatched HSCT, no significant difference in terms of 5-yrs cumulative incidence (CI) of TRM (28±7% vs 31±8%), CI of grade III-IV aGVHD (4±3% vs 5±3%), CI of extensive cGVHD (11±6% vs 5±4%), OS (56±8% vs 57±9%) and DFS (37±8% vs 45±9%) was observed. By comparing matched vs 1 vs 2 mismatched HSCT, the results were significant better in terms of CI of TRM (28±7%, 48±11% and 0%; P ¼ .02), OS (56±8%, 38±11% and 92±7%; P ¼ .03) and DFS (37±8%, 30±10% and 76±12; P ¼ .03) for pts receiving graft with 2 HLA disparities, for whom the relapse rate was lower without statistically significance. No significant difference was observed in terms of engraftment, aGVHD and cGVHD. Furthermore, the analysis showed that differences at DQB1, alone or combined, did not have any impact on HSCT outcome. Conclusion: Despite the small number of pts, these results confirm the effectiveness of the uniform program of the RTN policy for VUD transplants. The analysis on a larger number of pts will better clarify reasons of the particularly encouraging results observed in 2 HLA disparities subset. To date, the transplant platform for Z8/10 HLA antigen criteria of donor selection remains unchanged. Disclosure of Interest: None declared. P066 Improving Selection for Bone Marrow Donors with a Predictive Collection Score in HLA-Haploidentical Transplantation with Post-Infusion Cyclophosphamide A. Vai1,*, S. Bramanti1, R. Crocchiolo1, B. Sarina1, L. Morabito1, L. Giordano1, I. Timofeeva1, R. Capizzuto1, A. Santoro1, L. Castagna1 1 Humanitas Cancer Center, Rozzano (MI), Italy Introduction: Many transplantation outcomes have been demonstrated to be associated with the infused total nucleated cell (TNC) dose/kg of recipient body weight (BW). Donor bone marrow density (BMD) is directly related to the TNC dose/kg of recipient BW. Materials (or patients) and methods: We retrospectively analyzed the clinical data of 65 consecutive haploidentical (haplo) bone marrow (BM) donors at our center between 2009 and 2013. The target cell dose for haplo T-cell replete transplantation with post-infusion cyclophosphamide was 4x108 TNC/kg of recipient BW. Linear regression model was adopted and a recursive tree was estimated to build up a prognostic score.
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Table 1.
Donors’ characteristics
n ¼ 65 Median age Gender M/F Donor type Sibling Parent Child Cousin Median BMI Median WBC count (x103/mm3)
46 (18-71) 39/26
27 20 17 1 25.1 7.2
(42%) (31%) (26%) (2%) (17.6-37.9) (4.7-14.5)
Results: Donors’ characteristics are shown in table 1. Body mass index (BMI) and white blood cell (WBC) count were directly associated with BMD (Po0.001). 3 mayor prognostic categories were identified. As shown in the image, the probability to obtain a collection with a high BMD was firstly predicted by BMI (BMI Z30, BMD mean ¼ 25.8 TNC/mL x106). Secondly, it was possible to split donors with BMI o30 into two categories according to WBC count (WBC o8 x103/mm3: BMD mean ¼ 18.4 TNC/mL x106; WBC Z8 x103/mm3: BMD mean ¼ 23.1 TNC/mL x106). Moreover, BMD of the first collected bag directly correlated with overall BMD (R2 ¼ 0.69, Po0.001). Conclusion: This retrospective study on 65 haplo BM donors was aimed to identify donor-related variables associated with high BMD, in order to build up a predictive score for optimal collection. BMI Z30 and WBC count Z8 were the only variables independently related to donor BMD. Although in the clinical practice BMD is not a commonly used parameter, it is a valid surrogate of donor TNC content in the harvest. Given the impact of infused TNC dose on many transplantation outcomes, donor-related features affecting TNC quantity obtainable with the harvest could support the choice of the best-suitable donor, after selection on the basis of HLAmatching, gender, age, CMV serostatus and body weight ratio between recipient and potential donor. Disclosure of Interest: None declared.
Materials (or patients) and methods: We analysed 161 transplants, 121 for acute myeloid leukemia and 40 for acute lymphoblastic leukemia. Disease status at transplant was high-risk 1st to 3rd complete remission in 111 patients and chemo-resistant relapse in 50. Patients received TBI-based conditioning regimens and haploidentical, T-cell-depleted peripheral blood hematopoietic stem cells. No post transplant pharmacologic immune-suppressive GvHD prophylaxis was given. Results: In the absence of donor-versus-recipient NK cell alloreactivity, donor activating KIRs had no effects on outcomes. In the 69 transplants with donor-versus-recipient NK cell alloreactivity, transplantation from donors with KIR2DS1 and/or KIR3DS1 was associated with reduced risk of nonrelapse mortality, largely infection-related, (KIR2DS1 present versus absent: HR: 0.25, P ¼ .01; KIR3DS1 present versus absent: HR: 0.18, P ¼ .006) and better event-free survival (KIR2DS1 present versus absent: HR of death: 0.31, P ¼ .011; KIR3DS1 present versus absent: HR of death: 0.30, P ¼ .008) in multivariate analyses. After transplantation from donors with KIR2DS1 and/or KIR3DS1, the infection rate in the first six months after transplant was B50% lower (P ¼ .005). In vitro analyses showed that KIR2DS1 triggered NK cells to release IFN-g upon interaction with KIR ligand-mismatched, HLA-C2/ C2 DCs that were pulsed with LPS or Aspergillus Fumigatus. IFN-g release was inhibited when KIR2DS1-HLA-C2 interaction was blocked. Conclusion: 1) In vitro analyses suggest that activating KIR engagement by HLA ligands enhanced NK cell IFN-g production and this might improve immunity to infections. 2) Clinical data show that transplantation from NK alloreactive donors with the activating KIR2DS1 and/or KIR3DS1 genes reduces infection rates and infection-related mortality and improves survival. In T-cell depleted haploidentical transplantation specific donor factors were shown to impact on survival: KIR ligand mismatches and maternal donors. The present study identifies yet another: NK alloreactive donors carrying KIR2DS1 and/or KIR3DS1. As they constitute B40% of the NK alloreactive donor pool, searching for them may become an additional criterion in donor selection. Disclosure of Interest: None declared.
P067 Haploidentical hematopoietic transplantation from KIR ligand-mismatched donors with activating KIRs reduces non-relapse mortality A. Mancusi1,*, L. Ruggeri1, E. Urbani1, A. Pierini1, M. S. Massei1, A. Carotti1, A. Terenzi1, F. Falzetti1, A. Tosti1, F. Topini1, M. Stern2, F. Aversa3, M. F. Martelli1, A. Velardi1 1 University of Perugia, Perugia, Italy, 2University of Basel, Basel, Switzerland, 3University of Parma, Parma, Italy
P068 Passive transfer of male microchimeric cells from female donors to female HSCT patients: a case report A. van Halteren1,*, J. van der Griendt2, B. Kemps-Mols3, M. Eikmans3, E. Marijt2, T. Netelenbos3, M. Griffioen2, A. Lankester1, M. van Tol1 1 pediatrics, 2hematology, 3Immunohematology & Blood Transfusion, LEIDEN UNIVERSITY MEDICAL CENTER, Leiden, Netherlands
Introduction: In T cell-depleted peripheral blood stem cell haploidentical transplants, donor NK cells play a beneficial role in outcomes. NK cell function is regulated by inhibitory receptors (KIRs) for HLA class I allele groups (KIR ligands), that is, HLA-C group 1, HLA-C group 2 and HLA-Bw4 alleles. Because of KIR ligand mismatches in the GvH direction, donor NK cells are released from inhibition by recipient HLA and mediate donor-versus-recipient NK cell alloreactions which control leukemia relapse and improve survival. Homologues of the inhibitory KIRs, with shorter cytoplasmic tails, activating KIRs transduce signals which activate NK cells. Activating KIRs are heterogeneously expressed in the population. Approximately 25% of whites are homozygous for Group A haplotypes which predominantly contain inhibitory KIR genes. The others are either heterozygous or homozygous for Group B haplotypes that also carry combinations of activating KIR genes (KIR2DS1, 2, 3, 5 and 3DS1). Little is known about their ligands except that KIR2DS1 binds HLA-C group 2 molecules (HLA-C2). We investigated the role of donor activating KIRs in haploidentical T cell-depleted transplantation for acute leukemia.
Introduction: Male microchimerism is frequently acquired by female donors through feto-maternal or trans-maternal cell exchange. We earlier assessed a high prevalence of male CD34 þ stem cells and myeloid-committed cells in female peripheral blood stem cell (PBSC) grafts and in lymphocytes collected after PBSC donation for the purpose of donor lymphocyte infusion (DLI). The impact of passively transferred chimeric cells on HSCT outcome is unknown. Antibodies against Y-chromosome encoded proteins are often induced in male recipients of female grafts. Unexpectedly, we also measured a strong antibody response against a Y chromosome-encoded protein (DDX3Y) in a female AML patient who underwent myeloablative conditioning (TBI/cyclo) followed by infusion of T-cell depleted PBSC (20 mg Alemtuzumab injected into the collection bag) and two consecutive DLI from her HLA identical sister. We analysed whether the DDX3Y-specific antibody response in this female HSCT patient could be explained by the co-presence of passively acquired male microchimerism. Materials (or patients) and methods: The level of male DNA was determined by sensitive real-time PCR specific for a
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multicopy locus on the Y chromosome (DYS-1, detection limit: 1 male cell in 100.000 female cells) in longitudinally collected peripheral blood (PB) and bone marrow (BM) samples as well as in lymphoid and myeloid cells derived thereof. Standard clinical monitoring was used to assess post-HSCT hematopoietic recovery which was registered in the patient’s electronic files. Results: Male DNA was detected in two or more hematopoietic subsets derived respectively from donor PB and BM samples collected before the start of G-CSF treatment and from the DLI product. Multilineage male microchimerism was also detected in flow-separated blood cells collected from the patient at d þ 42 after PBSC infusion. From d þ 47 on, PB lymphocyte counts increased and the level of male microchimerism started to decline until male DNA became undetectable at d þ 196. The patient developed skin Graftversus-Host Disease (GVHD) around d þ 30 which required prolonged topical treatment and a short pulse of orally applied steroids to resolve around d þ 150. After DLI at d þ 272 and d þ 399 respectively, male cells re-appeared for a short while but without any clinical signs of GVHD. Again, male microchimerism became undetectable which was followed by the appearance of DDX3Y-specific IgG antibodies in the patient’s serum. The alloreactive nature of this the novo antibody response is supported by the observation that 1) DDX3Yspecific antibodies were not detected in patient or donor serum samples collected before HSCT and 2) DDX3Y-specific antibodies did not cross react with the homologue X chromosome-encoded DDX3X protein. Conclusion: We showed for the first time that male microchimerism and DDX3Y-specific antibodies can be detected during the course of a HLA identical female-tofemale HSCT procedure. Using PCR specific for unique HLA or other genetic markers which have been selected according to the genotyping results of the entire family, we are currently generating more evidence that the male microchimerism associated with anti-male alloreactivity in this female HSCT patient is passively acquired from her female sibling donor. Disclosure of Interest: None declared. P069 Second or further donations of hematopoietic stem cell for volunteer donors: the experience of the Italian Bone Marrow Donor Registry (IBMDR) A. Vassanelli1,*, N. Sacchi2, S. Pollichieni2 1 transfusion medicine, azienda ospedaliera univeristaria integrata verona, Verona, 2IBMDR, Ospedale Galliera, Genova, Italy Introduction: The Italian Bone Marrow Donor Registry (IBMDR), established in Italy in 1989, with the role of providing an unrelated volunteer to haematological patients waiting for a transplant and who do not have the ideal donor (an identical sibling), ensure that the TC activates a search exclusively for those patients where a HSCT represents a medically acceptable procedure, for pathologies that are defined, with annual revisions, by the EBMT. Haematopoietic Stem Cell donation can be carried out from both bone marrow (BM) and peripheral blood stem cell (PBSC) after administration of G-CSF, for a single recipient only. The TC may ask for the HSC collection only if the transplant is immediately performed, not for cryopreservation In some circumstances, an additional donation for the same patient may be required: a mononuclear cell collection from peripheral blood (for DLI), in the case of relapse, or HSC in the case of non-engraftment or of poor graft function. The volunteer, who has already donated peripheral HSC should, preferably, donate marrow HSC and, in any case, can only be submitted again for growth factor stimulation for HSC mobilization, if at least 12 months have passed since the first donation. A second HSC donation and a second or further lymphocyte donation requests are submitted to the SIMTI Committee
(Committee of Societa` Italiana Medicina Trasfusionale e Immunoematologia, a "second opinion" committee designated by the pertinent scientific society, with the aims to protect donor welfare and safety). A donor follow-up, even if no particular events have occurred, is performed at established times (up to ten years for bone marrow and PBSC donation and up to 6 months for lymphocyte donation). Materials (or patients) and methods: We evaluated all the requests of second or further HSC or lymphocyte donation, analyzing the experience of Italian Registry and the incidence of a second donation request Results: From 1989 to 2013, ‘‘first’’ HSC products donated by Italian donors were 2999 (2160 BM and 839 PBSC): 5% of these donors were asked for a ‘‘second’’ donation (55% DLI, 45% HSC). After PBSC donation the incidence of a ‘‘second’’ donation is 8,3% (87% for DLI, 13% for HSC: BM or second PBSC), after BM donation the incidence of a ‘‘second’’ donation is 2,7% (73% asked for PBSC donation, 22% for DLI and 5% for BM). On 20% of the lymphocyte donation, donor were asked to donate a further lymphocyte donation (31 donation for these donors); all of these requests were authorized by SIMTI Commettee. No donors were authorized for more than two HSC donation Conclusion: Analyzing Italian Bone Marrow Donor Registry experience, after HSC donation, the incidence of a ‘‘second’’ donation request is 5%, more after PBSC donation than after BM donation (8,3% vs 2,7%); when PBSC donation is a first donation, the request of a’’second’’ donation is more frequently for DLI (87% of requests), and when first donation is BM, donor is asked for a second donation less frequently (2,7%), but this request is usually for PBSC (73% of requests). Disclosure of Interest: None declared. P070 Haploidentical TCRab and CD19 Depleted Stem Cell Transplant for Primary Immunodeficiency – 6 cases B. Morillo-Gutierrez1,*, Z. Nademi1, J. Dunn2, D. Barge3, S. Hambleton4, M. Abinun5, T. Flood5, A. J. Cant5, A. Gennery4, M. Slatter5 1 Department of Paediatric Immunology, Great north Children´s Hospital, 2Hematopoietic Stem cell Laboratory, 3Regional Immunology Laboratory, 4Institute of Cellular Medicine. Newcastle University, 5Department of Paediatric Immunology. Great North Children´s Hospital, Newcastle Upon Tyne, United Kingdom Introduction: For many patients with Primary immunodeficiency (PID), Hematopoietic stem cell transplant (HSCT) is the only curative treatment available. If there is no HLA identical donor the use of HLA-haploidentical family donors is problematic due to the increased occurrence of graft versus host disease (GVHD) and graft rejection particularly for patients with forms of PID other than severe combined immune deficiency. Various T-cell depletion strategies have been used, but some can lead to slow immune reconstitution with the consequent risk of morbidity and mortality from infection. Depletion of T cell receptor alpha and beta (TCRab) and CD 19 þ cells, with selection of TCRgd T-cells and preservation of NK, myeloid and plasmacytoid dendritic cells which aid engraftment and also decrease the risk of infection, has been shown to be a promising new technique. Materials (or patients) and methods: Prospective collection of the data from the paediatric patients with different PIDs treated using this technique in our HSCT unit at Great North Children´s Hospital, Newcatle Upon Tyne, UK. Results: 6 patients were treated in our unit from September 2012 to July 2014. All had a successful haploidentical TCRab and CD19 depleted HSCT. All 6 had viral reactivation but are alive and well 4-24 months post transplant, with chimerism 490% and good immune reconstitution. See table for more details.
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Table 1. Diagnosis
Conditioning
1.WAS 12m Father 2.Artemis 18m 2nd HSCT Mother 3.DOCK 8 7y Father 4.CGD 3y Father
ATG Bu/flu/TT Campath T/Flu/TT
5.CID 12m Mother 6. CgC 6m Father
GVHD Neut GVHD prophylaxis
Viral
þ 15 None
CMV
CsA/MMF
Tacro/MMF þ 12 Skin I
Adeno
Adeno CMV
Campath T/Flu/TT
CsA/MMF
þ 12 None
Campath T/Flu/TT
CsA/MMF
þ 14 Skin I Adeno,CMV EBV,HHV6
T/Flu/TT
CsA/MMF
None
CsA/MMF
þ 11 Skin II NA None
Multivariate analysis of Survival
Follow up
N
RR (95% CI)
P-value
2 yr 100% 1 yr 100%
HLA Match 10/10 Match 1 mm 41 mm
878 239 77
1.00 1.22 (1.2-1.5) 1.46 (1.1-1.9)
0.033 0.009
1 yr 100%
Patient Donor CMV Match Mismatch
863 331
1.00 1.37 (1.2-1.6)
o0.0001
Patient age o40y 440y
572 622
1.00 1.26 (1.1-1.5)
0.006
1014 180
1.00 1.36 (1.1-1.7)
0.003
Donor Age o30y 430y
372 822
1.00 1.17 (0.99-1.4)
0.069
ERA 96-99 00-03 04-07 08-11
142 421 345 286
1.00 0.85 (0.7-1.1) 0.80 (0.6-1.1) 0.83 (0.6-1.1)
0.18 0.09 0.21
Disease Risk - EBMT Good Intermediate Poor
557 444 193
1.00 1.34 (1.1-1.6) 1.33 (1.1-1.7)
0.001 0.01
6m CD15 100%,T79% CMV 4m 100% Adeno 4m Rotavirus T 93% CD15 9%
Conclusion: We report here 6 patients who have had a successful haploidentical TCRab and CD19 depleted HSCT. Pending the results of larger series, this could be a promising technique in haploidentical HSCT with early and sustained engraftment, early immune recovery and low risk of GVHD for PID patients. References: Locatelli F, Bauquet A, Palumbo G, Moretta F, Bertaina A. Negative depletion of a/b T cells and of CD19 þ B lymphocytes: A novel frontier to optimize the effect of innate immunity in HLA-mismatched hematopoietic stem cell transplantation. Immunology letters 155 (2013) 21–23 Bertaina A, Merli P, Rutella S, Pagliara D, Bernardo ME, Masetti R, et al. HLA-haploidentical stem cell transplantation after removal of ab þ T and B cells in children with nonmalignant disorders. Blood. July 2014; 124(5): 822–826 Kharya G, Nademi Z, Leahy TR, Dunn J, Barge D, Schulz A, et al. Haploidentical T-cell alpha beta receptor and CD19-depleted stem cell transplant for Wiskott-Aldrich syndrome. J Allergy Clin Immunol. June 2014. Epub Disclosure of Interest: None declared. P071 HLA–DQB1 and –DPB1 mismatches are significantly associated with worse survival in unrelated donor haematopoietic stem cell transplantation B. E. Shaw1,2,*, N. Mayor1, R. Szydlo1, W. Bultitude1, K. Kirkland3, J. Perry3, A. Clark4, S. Mackinnon5, D. Marks6, A. Pagliuca7, M. Potter8, N. Russell9, K. Thomson10, A. Madrigal1, S. G. E. Marsh1 1 Anthony Nolan Research Institute, London, United Kingdom, 2 CIBMTR, Milwaukee, United States, 3BSBMT, London, 4The Beatson, West of Scotland Cancer Centre, Glasgow, 5University College London, Royal Free Campus, London, 6University Hospitals Bristol NHS Trust, Bristol, 7Kings College Hospital, 8 Royal Marsden Hospital, London, 9Centre for Clinical Haematology, Nottingham University Hospital, Nottingham, 10University College London Hospitals, London, United Kingdom Introduction: HLA matching is known to be important in unrelated donor (UD) stem cell transplantation to ensure the best patient outcomes. Historically five HLA loci were tested, HLA-A, -B, -C, -DRB1 and –DQB1. More recently, routine matching for –DQB1 has become less frequent and in some centres is not performed at all. Conversely, -DPB1 typing is much more frequently performed and matching at both an allele and T-cell epitope (TCE) level may be considered. Materials (or patients) and methods: We performed a study to analyse the impact of donor factors (HLA, CMV, age, gender and ABO matching) on survival of patients receiving an UD haematopoietic stem cell transplant. We included 1271 UK recipients transplanted between 1995-2011. All patients
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Previous autos 0 40
had received a transplant for a malignant disease (acute leukaemia/MDS, 63%). Disease type and risk were categorised by the EBMT risk score and 580, 457 and 199 were characterised as good, intermediate and poor risk respectively. The median follow up of 611 surviving patients was 5.5y (range 0.2-15.4). The median age of the cohort at transplant was 40.6y (range 0.9-71.9). 661 and 585 patients had myeloablative and reduced intensity conditioning respectively, with Alemtuzumab in 94%. PBSC were used in 54% of patients. The median donor age was 34.9 (range 19-60.4) with 80% being male and 20% female. The patient/donor CMV status was pos/pos (n ¼ 238), pos/neg (n ¼ 244), neg/neg (n ¼ 647), neg/pos (n ¼ 101). Results: Patients matched for 10/10 HLA alleles had significantly better overall survival than those matched at 9/10 or o9/10 (median survival in years: 2.7 vs 1.2 vs 0.9 respectively, P ¼ 0.001). This finding was retained in multivariate analysis (Table 1). Considering only the 9/10 matched patients, mismatching for HLA-B (P ¼ 0.01) and –DQB1 (P ¼ 0.03) resulted in a significantly worse survival, while mismatching for HLA-A (P ¼ 0.1), -C (P ¼ 0.2) or DRB1 (P ¼ 0.7) resulted in no difference. HLA-DPB1 TCE mismatching showed a significantly worse survival when compared to either DPB1 TCE or allele matched pairs (1.3 vs 2.6 yrs, P ¼ 0.01). Donor gender and ABO matching status did not impact on patient outcome in multivariate analysis. Conclusion: In conclusion, our results show that matching for HLA-DQB1 remains important in this cohort of patients, and that this should be considered pre-transplant. We also reconfirm the importance of matching for DPB1 if a choice of donors is available. Finally, donor CMV status and age should also be considered in the selection. Disclosure of Interest: None declared.
P072 Allogeneic versus Autologous Hematopoietic Stem Cell Transplantation for non promyelocytic Acute Myeloid Leukemia: 30 years experience in a single center C. Montes-Gaisan1,*, A. Bermu´dez1, G. Pe´rez1, A. Cuesta1, C. de Miguel1, C. Richard1, E. Conde1 1 Hematology, Hospital Universitario Marque´s de Valdecilla, Santander, Spain Introduction: The optimal post-remission treatment for Acute Myeloid Leukemia (AML) remains uncertain. Traditional comparisons of Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) versus Autologous Hematopoietic Stem Cell Transplantation (auto-HSCT) noted higher TRM(Treatment Related Mortality) but lower CIR (Cumulative Incidence of Relapse), with discordant results in Overall Survival (OS). Materials (or patients) and methods: Retrospective study of 275 patients diagnosed of AML (Acute Promyelocytic Leukemia excluded) who underwent Hematopoietic Stem Cell Transplantation (HSCT) in our center between 1982 and 2011. We compared OS and Disease Free Survival (DFS), CIR and TRM when receiving allo or auto-HSCT. Kaplan Meier curves and log-rank test were used for OS and DFS whereas cumulative incidences were used for CIR and TRM. Multivariate analysis with Cox Regression was used for controlling confounding factors. Patient and disease characteristics in allo and auto-HSCT were respectively: median age of 37,8(IQR 3052) and 44,5(IQR 36-56), secondary AML in 20,2% and 9,8%, failure after Induction course in 15,8% and 2,8%, complete remission pre-HCST in 87,1% and 97,3% and year of HSCT 42005 in 46,9% and 14,3% .There was no statistical difference in any other characteristic (cytogenetic risk and WBC count at diagnosis, included). Results: The OS in the earliest period (o1997) for allo and autoHSCT was respectively: 39,6% and 60,8% at 1 year and 28,3% and 45,1% at 5 years. And the OS in the latest one (Z1997) was respectively: 65,6% and 70,3% at 1 year and 46,8% and 47,7% at 5 years (image1). Any significant results in the multivariate analysis neither in the log-rank test regarding OS differences between allo and auto-HSCT were observed. Covariates such as elderly, elevated WBC count at diagnosis, high cytogenetic risk, early period of HSCT or secondary AML showed worse prognosis, but only MRD was a strongly outcome predictor. The DFS in the earliest period for allo and auto-HSCT was respectively: 50,0% and 65,2% at 1 year and 37,5% and 45,6% at 5 years. And the DFS in the latest one was respectively: 66,9% and 63,2% at 1 year and 51,7%and 46,5% at 5 years. 74% of the patients who underwent auto-HSCT have ECOG 0 the date of the last follow-up and 6 patients over 14 who relapsed after auto-HSCT were rescued with allo-HSCT. The CIR in allo and auto-HSCT was respectively: 18% and 32% at 1 year and 24% and 50% at 5 years (HR ¼ 0,36, P ¼ 0,01), with no statistical difference if the HSCT was perfomed before or after 1997(start to quantify the pre-HSCT minimal residual disease by flow
citometry). The cumulative incidence of TRM in the earliest period for allo and auto-HSCT was respectively: 30% and 7% at 1 year and 35% and 9% at 5 years. However, the cumulative incidence of TRM in the latest one was respectively: 16% and 2% at 1 year and 25% and 4% at 5 years (HR ¼ 2,87, P ¼ 0,01). Conclusion: In our series, we have found no statistical difference between allo and auto-HSCT in OS and DFS in any period. Despite the higher CIR, we have observed a higher survival in auto-HSCT in the earliest period which turns to get similar nowadays because of the decrease in TRM, more significative in allo-HSCT. References: European groups: HOVON, BGMT, MRC and EORTC. Disclosure of Interest: None declared. P073 JACIE accreditation significantly improves compliance with international recommendations for related donor care in EBMT transplant centres C. Anthias1,*, P. V. O’Donnell2, D. M. Kiefer3, M. A. Pulsipher4, S. C. Goldstein5, B. R. Logan6, D. L. Confer3, B. E. Shaw6 1 Anthony Nolan Research Institute, London, United Kingdom, 2 Fred Hutchinson Cancer Research Center, Seattle, 3(CIBMTR) Center for International Blood and Marrow Transplant Research, National Marrow Donor Program/ Be The Match, Minneapolis, 4 Huntsman Cancer Institute, University of Utah, Salt Lake City, 5 University of Michigan, Ann Arbor, 6(CIBMTR) Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, United States Introduction: Previous surveys in Europe and the US have highlighted discrepancies between the care of unrelated donors (UDs) and related donors (RDs). FACT-JACIE Standards have addressed some of these issues including requirements for independent donor assessment and guidelines for donor care. We hypothesised that changes in FACT-JACIE standards have significantly impacted related donor practices. The Donor Health and Safety Working Committee of CIBMTR undertook a survey of international transplant centres (EBMT and CIBMTR members) to address this issue. Materials (or patients) and methods: An internet-based questionnaire was administered via a secure hyperlink (surveymonkey.com) August-November 2014. Program directors of EBMT and US CIBMTR allogeneic transplant member centres (excluding UK) received an email invitation to participate with a request to forward the survey to whichever physician was responsible for the care of related donors at that centre. Non-responders received 3 further reminders. Centres performing solely paediatric allogeneic
Table 1.
Differences between accredited and non-accredited
centres
Written policy for RD care Recipient is informed of donor HLA results before donor Recipient’s physician is simultaneously responsible for donor Defined donor eligibility criteria exist Policy regarding number of RD apheresis procedures (per donation) Policy regarding volume of marrow to harvest Policy regarding subsequent donations Process for credentialing physicians performing marrow harvests Long term donor follow up
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JACIE
NonJACIE
P
98.4%
83%
0.004 0.038
15%
35%
0.014
93%
75%
0.029
73%
55%
0.057
64%
66%
0.984
45%
35%
0.286
76%
60%
0.014
34%
14%
0.05
transplants were excluded. Based on practice differences, CIBMTR and EBMT centre results were analysed and presented separately. Results: 125 EBMT centres returned evaluable responses. 51% responding centres were fully JACIE accredited. The key findings are shown in Table 1. We found that JACIE accredited centres significantly more frequently complied with international recommended care standards including the existence of a written policy for RD care, defined donor eligibility criteria, and a process for credentialing physicians performing marrow harvests. In other areas JACIE accreditation did not impact practice: In 71% centres BM harvest were performed by the same transplant physicians responsible for the recipient, 35% centres had no defined limit for marrow harvests, and 36% had no limit to the number of apheresis procedures performed. Conclusion: In conclusion, we found significant differences in related donor care between accredited and non-accredited centres. Although accredited centres performed better in some areas, even they failed to meet some standards (eg. in 15% centres care for a donor and their recipient still overlaps). In areas of care not addressed by JACIE standards (e.g. the existence of policies defining limits of donation), both accredited and non-accredited centres failed to meet accepted international UD practice. Through accreditation, centres have improved related donor practice, but there remain clear areas where practice could be improved. Disclosure of Interest: None declared.
not possible in all cases. In addition all centres now reported they had a written policy for the care of related donors. When evaluating areas addressed by other FACT standards, we found that all centres provided RDs with some information prior to HLA typing, however only 52.7% provided written information. 96% centres provided follow-up to 1 week postdonation, but only 13% followed-up RDs to 1 year, and no centre performed follow-up beyond one year. We also addressed other areas, not currently covered in FACT standards but where accepted international unrelated donor guidance provided a benchmark. 32% of transplant centres, did not assess donor health before HLA typing and, where assessed, this consisted of verbal open-ended questions in 65%. 24% centres did not verify that donors were willing to donate before proceeding with HLA typing, 43% centres either disclosed donor HLA results to the recipient first, or had no consistent practice regarding result disclosure. In 47% centres, marrow harvests were performed by the physicians responsible for the recipient. Conclusion: In conclusion, changes to FACT standards have improved practice since 2007. However, related donor followup falls below international recommendations published by the WBMT and this survey identified further important areas in which care received by RDs falls below that of UDs. Efforts are needed to improve these practices in related donor centres, which would be best effected by enhancing regulatory standards. Disclosure of Interest: None declared.
P074 Significant improvements in the practice patterns of related donor care in US transplant centres C. Anthias1,*, P. V. O’Donnell2, D. M. Kiefer3, M. A. Pulsipher4, S. C. Goldstein5, B. R. Logan6, D. L. Confer3, B. E. Shaw6 1 Anthony Nolan Research Institute, London, United Kingdom, 2 Fred Hutchinson Cancer Research Center, Seattle, 3(CIBMTR) Center for International Blood and Marrow Transplant Research, National Marrow Donor Program/ Be The Match, Minneapolis, 4 Huntsman Cancer Institute, University of Utah, Salt Lake City, 5 University of Michigan, Ann Arbor, 6(CIBMTR) Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, United States
P075 Activating KIR2DS1/2 positive receptors on donor NK cells are associated with an improved overall survival and reduced relapse incidence in C1-ligand negative patients with myeloid malignancies after unrelated haematopoietic stem cell transplantation C. Zollikofer1,2,*, D. Fu¨rst2,3, C. Tsamadou1,2, D. Niederwieser4, D. Bunjes5, G. Wulf6, M. Pfreundschuh7, E. Wagner8, G. Stuhler9, B. Glass10, S. Hubert1,2, J. Mytilineos1,2,11 1 Institute of Transfusion Medicine, University of Ulm, Germany , 2 Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service, Baden Wuerttenberg – Hessen, Ulm, Germany, 3Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, Ulm, 4Department of Hematology/Oncology, University of Leipzig, Germany, Leipzig, 5 Department of Hematology/Oncology, University Clinic Ulm, Germany, Ulm, 6Department of Hematology/Oncology, GeorgAugust-University Go¨ttingen, Germany, Go¨ttingen, 7Department Internal Medicine I, Universita¨tsklinikum des Saarlandes, Homburg, Germany, Homburg, 8Department of Medicine III, Johannes Gutenberg-University Mainz, Germany, Mainz, 9Centre for Bone Marrow and Blood Stem Cell Transplantation, Deutsche Klinik fu¨r Diagnostik, Wiesbaden, Germany , Wiesbaden, 10Department of Medicine II, University Hospital Wu¨rzburg, Germany, Wu¨rzburg, 11 DRST – German Registry for Stem Cell Transplantation, Ulm, Germany, Ulm, Germany
Introduction: A survey conducted in 2007 (O’Donnell et al. Blood 2010) investigated practice patterns in the care of related donors (RDs) in the US, and reported a potential conflict of interest in 470% centres (i.e. where a physician had simultaneous responsibility for the care of a RD and their recipient). In addition, 5% centres lacked written policies for RD care. The 5th Edition of the FACT-JACIE Standards addressed these issues. In order to assess the impact of the changes, we undertook a follow-up survey of US transplant centers. We took this opportunity to explore, in both the US and Europe, other areas where we hypothesised that RD care may differ from that of unrelated donors (UDS). Materials (or patients) and methods: An internet-based questionnaire was administered via a secure hyperlink (surveymonkey.com) August-November 2014. Program directors of EBMT and US CIBMTR allogeneic transplant member centres (excluding UK) received an email invitation to participate with a request to forward the survey to whichever physician was responsible for the care of RDs at that centre. Non-responders received 3 further reminders. Centres performing solely paediatric allogeneic transplants were excluded. Based on practice differences, CIBMTR and EBMT centre results were analysed and presented separately. Results: In the US CIBMTR cohort, 76 individual centers responded (response rate 40%), 72 are FACT accredited. We saw significant improvements since the earlier survey particularly with respect to potential conflict of interest in donor-recipient care. Over 93% of centres report that their standard was to specifically separate the care of donors and recipients. However 32% of these centres stated that this was
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Introduction: Natural Killer cells (NK) are lymphocytes that have the potential to recognize and lyse cells with aberrant HLA expression profiles which escaped T-cell mediated lysis. Communication between NK and leukemic cells is facilitated, among others, by killer cell immunoglobulin-like receptors (KIR) on the surface of NK cells and their respective HLA-class I ligands. KIRs influence NK-cell activity by mediating activating or inhibitory signalling, whereas activating signals and missing inhibition both lead to target cell lysis. Activating KIRs may have an immune-modulatory effect on the outcome of HSCT. In this study we show that patients with myeloid malignancies that do not express a C1 ligand (high risk group) suffer from impaired outcome after HSCT. Moreover, we demonstrate the beneficial influence of activating donor KIR2DS1/2 on this high risk group. Materials (or patients) and methods: Patients and donors were high resolution typed for HLA-A, -B, -C, -DRB1, and -DQB1. KIR-typing was performed through PCR-SSP. HLA-C alleles were
grouped in respect to amino acid 80 into C1 (asparagine) and C2 (lysine) ligands. Patient malignancies were AML, CML or MDS. Donors were at least 18 years old, and T-cell replete graft source was either peripheral blood stem cells (95.5 %) or bone marrow (4.5 %). Clinical data for this study where obtained through the German Registry for Haematopoietic Stem Cell Transplantation (DRST). Overall survival (OS), disease free survival (DFS), relapse incidence (RI), and treatment-related mortality (TRM) were assessed using Kaplan-Meier analysis, competing risks regression and extended Cox regression models. Survival endpoints were investigated with respect to HLA-matching grades by separate analyses of 9/10, and 10/10 matched transplant pairs (KIR2DS1 analysis), or by performing stratification for HLA-compatibility (KIR2DS2 analysis). Results: C1 negative patients (n ¼ 200) showed inferior OS (HR ¼ 1.41, CI ¼ 1.14-1.74, P ¼ 0.0012), DFS (HR ¼ 1.27, CI ¼ 1.05-1.53, P ¼ 0.015), RI (HR ¼ 1.33, CI ¼ 1.01-1.75, P ¼ 0.04) and TRM (HR ¼ 1.41, CI ¼ 1.01-1.96, P ¼ 0.04) when compared to C1 carriers (n ¼ 1246). Within this high risk patient group, however, RI was improved if a KIR2DS1 expressing donor was used (HR ¼ 0.26, CI ¼ 0.11-0.63, P ¼ 0.003, n ¼ 74) when compared to a control group of patients transplanted with a KIR2DS1 negative donor (n ¼ 126). Furthermore, in single HLA-mismatched transplants, OS was improved when transplanted with a KIR2DS2 positive donor (9/10: HR ¼ 0.29, CI ¼ 0.09-0.92, P ¼ 0.03, n ¼ 70; 10/10: HR ¼ 1.04, CI ¼ 0.51-2.12, P ¼ 0.91, n ¼ 33). Conclusion: Our findings can be explained by impaired donor derived NK-cell activity in C1-ligand negative patients. As the first KIRs to be expressed are KIR2DL2 and KIR2DL3 which recognize C1-ligands, the early onset of donor NK-cells in these patients is consequently non-reactive against C1-ligand negative recipient cells. Furthermore, C1-restricted NK-cells form bigger populations, react faster to interferon g secretion, and degranulate CD107a more potently. Our study shows, that activating signals derived from KIR2DS1/2 stimulation can overcome this impaired NK-cell response. We therefore suggest inclusion of KIR2DS1/2-genotyping in the unrelated donor search algorithm of C1 ligand negative patients with myeloid malignancies. Disclosure of Interest: None declared. P076 ab/CD19 negative immunoselection for haploidentical transplants in pediatric setting C. Del Fante1,*, M. Zecca2, G. Viarengo1, L. Rubert2, G. Giorgiani2, F. Bonetti2, C. Perotti1 1 Immunohaematology and Transfusion Service, Centre for transplant immunology , 2Oncoematologia Pediatrica, Fondazione IRCCS Policlinico S.Matteo, Pavia, Italy Introduction: Positive CD34 þ immunomagnetic cell selection (ICS) has been used since 90’s to prevent/avoid GVHD in haploidentical stem cell transplant (Haplo-HSCT) and overcome the HLA barrier. Recently, Miltenyi Biotec (Germany) released a new cell depletion system able to remove ab þ /CD19 þ lymphocytes from the graft while retaining TCRgd þ , NK þ and other cell subsets. This platform allows an optimal T cell depletion coupled with a B cell depletion that prevents the onset of post transplant EBV-associated lymphoproliferative disease, in order to maintain anti-tumor and anti-infectious properties. Materials (or patients) and methods: We retrospectively analyzed 18 negative ab þ /CD19 þ and 10 CD34 þ positive ICS (performed on leukapheretic fraction if CD34 þ cell dose/ kg in the graft was o10x106) performed from Oct 2013 to Dec 2014 from peripheral blood (PB) mobilized haploidentical donors (11 mothers, 6 fathers) for hematopoietic stem cell transplantation in children affected with different hematological diseases. ICS (Clinimacs Miltenyi Biotech) was performed according to the manufacturer’s instructions. PB CD34 þ cell number at apheresis collection, cell recovery after immunomagnetic cell selection, total amount of cells infused, engraftment and transplant related mortality were recorded.
Results: CD34 þ cells (mean 86,6/ul, range 35.5-107.2) were collected during 24 leukapheretic procedures from 17 mobilized haploidentical donors. No major side effects were recorded during leukapheretic mobilization and collection. Mean CD34 þ cell recovery from negative ICS was 82.8% (range 64.8-99), mean Log ab/T and CD19 þ /B depletion was 4.46 (range 1.18-7.4) and 5.15 (range 2.1-7.6), respectively. Overall, median graft cell content (x10^6/Kg recipient, considering also CD34 positive ICS when appropriate) was the following: CD34 þ cells 18.24 (min 10.85-max 31.09), ab cells 0.013 (min 0.0001-max 0.14), gd cells 16.21, B lympocytes 0.18 (min 0.0001-max 1.44), NK 36.9 (min 7.61-max 194.4), NK-T 5.58 (min 0.47-66.6). The pediatric HSCT cohort included 11 males and 6 females, median weight 27 kg (range: min 4.7max 80). Recipients’ underlying disease was ALL in 8 pts (47%), AML in 4 (23.5%), SCID in 3 (18%), FA and JMML in 1 pt (6%), respectively. No adverse events upon HSC infusion was recorded. No graft failure was observed. Median days to PMN and PLT engraftment was 14 (min 9-max 23) and 13 (min 9-max 41), respectively. So far, 1 patient developed aGVHD gradeII-IV, that resolved with steroid treatment, 1 patient died before day 100 due to multiorgan failure, and 2 patients experienced leukemia relapse. Conclusion: Our preliminary data show that ab þ / CD19 þ negative ICS is effective in depleting ab þ and CD19 þ lymphocytes and results in a sustained engraftment in pediatric haploidentical setting. A longer pts’ follow-up will be required for a meaningful evaluation of GVHD and infectious disease incidence, anti-tumor efficacy and immune reconstitution. Disclosure of Interest: None declared. P077 In vivo T cell depletion with antithymocyte globulin reduces the incidence of acute graft-versus-host-disease (GVHD) in matched unrelated donor stem cell transplantation without affecting chronic GVHD, relapse risk and survival D. KARAKASIS1,*, I. BATSIS2, F. PANITSAS1, E. XENOU1, Z. BOUSSIOU2, A. XIROKOSTA1, S. KARAKATSANIS1, P. KALOGIANNIDIS2, V. PARDALIS1, A. KAISARI1, D. MALLOURI2, E. GIANNAKI2, A. LOIDORIS1, M. BOUZANI1, C. SMIAS2, I. MARKOU1, J. APOSTOLIDIS1, S. GIGANTES1, I. SAKELLARI2, I. BALTADAKIS1, A. ANAGNOSTOPOULOS2, N. HARHALAKIS1 1 BMT Unit, Evangelismos Hospital, Athens, 2BMT Unit, Papanikolaou Hospital, Thessaloniki, Greece Introduction: There is conflicting data regarding the role of antithymocyte globulin (ATG) in the outcome of matched unrelated donor hematopoietic stem cell transplantation (MUD-HSCT). The aim of this study was to retrospectively analyze the impact of the inclusion of ATG in the conditioning regimen on the outcomes of MUD-HSCT in the era of donor selection by allele-level HLA typing. Materials (or patients) and methods: From 01/2001 until 12/ 2013, 256 adult patients (M/F: 149/107) underwent MUD-HSCT at two Greek transplant centers for myeloid (n ¼ 162) or lymphoid (n ¼ 94) malignancies. The median age of patients was 41 (range, 14-66) years. Donor/recipient HLA allele match was 8/8 (n ¼ 130) or 7/8 (n ¼ 126) at HLA-A, -B, -C, and -DRB1 loci. The conditioning regimen was mainly myeloablative (n ¼ 213), and peripheral blood was the source of the graft in 95% of cases. ATG (Thymoglobulin) was administered in 121/ 256 (47.3%) patients according to each center policy, at a total dose of 2.5-5mg/Kg BW. An ATG vs. no-ATG comparison was made in terms of engraftment, acute or chronic GvHD, relapse, non-relapse mortality (NRM), overall survival (OS) and diseasefree survival (DFS). Results: Patient and transplant characteristics were balanced between the two patient groups (ATG vs. no ATG), except for age (median, 39 vs. 43 years, respectively, P ¼ 0.008) and degree of HLA mismatch (58.7% vs. 40.7%, respectively, P ¼ 0.004). Engraftment occurred at a cumulative incidence
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(CI) of 96.4% without any difference between the two patient groups. ATG administration was associated with a statistically significant reduction in the incidence of grade II-IV acute GvHD (HR: 0.64, 95% CI: 0.44-0.92, P ¼ 0.017). In multivariate analysis, ATG administration was the only additional risk factor besides HLA match for grade II-IV acute GVHD. ATG administration had no impact on chronic GvHD incidence, relapse incidence, NRM, DFS and OS. HSCT-specific comorbidity-age index, degree of HLA match and EBMT score emerged as the major independent risk factors for NRM, OS and DFS in multivariate analysis. Conclusion: Our results do not suggest any considerable effect of in vivo T cell depletion with ATG on survival after MUD-HSCT. However, the observed significant reduction in the incidence of acute GvHD may argue in favor of the use of ATG, especially in the context of HLA mismatched MUD-HSCT. Disclosure of Interest: None declared. P078 Long-term Overall and Event-free Survival after RIC Allogeneic Transplantation in Acute Leukemias in a Single Centre: Impact of Age and Donor Type? S. Wittnebel1, S. Michiels1, S. Anoun1, M. C. Ngirabacu1, N. Meuleman1, G. Claes1, P. Huynh1, P. Crombez1, M. F. Jaivenois1, P. Lewalle1, D. Bron1,* 1 Hematology, INSTITUT JULES BORDET, ULB, Brussels, Belgium Introduction: Between Jan 2000 and Dec 2014, we transplanted 71 patients (Pts) using RIC- conditioning allogeneic transplant for acute leukemias (AML and ALL) and myelodysplastic syndromes (MDS) not eligible for myeloablative transplantation. Clinicians are usually scared to transplant older patients with MUD because of the higher risk of Treatment-related mortality (TRM). We thus reviewed retrospectively the impact of age and donor-choice (familial related donor (RD) versus matched unrelated donors (MUD) ) on the post-transplant outcome of the pts. Materials (or patients) and methods: We retrospectively studied in our cohort of 71 evaluable RIC transplant pts, the overall survival (OS) and event-free survival (EFS), according to ages (r 54yo vs Z55yo) and donor types (RD vs MUD). Conditioning consisted mainly of combinations of BusulfanFludarabin-ATG or Endoxan-Fludarabine-ATG. Immunosuppression was a combination of cyclosporine (CSA)/mycofenolate mofetil (MMF), Tacrolimus (TAC)/ MMF or TAC/ Sirolimus. Chimerism analyses were performed on d30, d90 and d180. Both study-groups were well balanced concerning transplant conditioning regimens, (ATG or not) RD vs MUD donors, type of leukemia and status of disease at transplant. Our local EBMT database was the source for this analysis. Results: 34 de novo AML (20 r54 yr and 14 Z55 yr), 30 secondary AML and MDS (15 r54 yr and 15 Z55 yr) and 7 ALL (5 r54 yr and 2 Z55 yr) were enrolled in our trial. 24 pts received HLA identical RD in the group r54yr and 14 pts in the group Z55 yr. The others pts received MUD transplant from HLA 9 or 10/10 donors (16 in the group r 54yr and 17 in the group Z 55yr). The median follow-up was 22 (3-93) months. Engraftment rate was 100% in the studied cohort. [S1] TRM before d100 was 18,3% (15% r54 yo and 22,6% Z55yyo). Grade II-IV Acute GVHD (aGVHD) at d90 were similar in both age populations (16,6%). Chronic GVHD rates are similar in both age groups (24%). [S2] There was a clear, even though not statistical, significant, difference in 5years OS when stratifying the two subgroups (r54 yr versus Z55 yr) dependent on donor-types (pts r 54yr : RD: 31,6%; and MUD: pts 38%; Z 55yr : RD: 27,8% and MUD 44,2%). This trend in a better OS in older MUD pts could be explained partially by a lower relapse-rate in the group Z 55 yr with an MUD as shown by EFS/5yrs (pts r 54 yr : RD: 32%; r 54yr MUD: 19%; pts Z 55yr RD: 21% and Z 55yr MUD 38,5 %) suggesting the benefit of a greater GVHD/GVL-effect in the MUD transplantation. Conclusion: Our observations confirm that:
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1) RIC transplantation is a curative approach in acute leukemia whatever the age when the pts are fit enough to tolerate the transplant. 2) Despite a higher TRM compared to younger patients, older patients seem to particularly benefit from MUD in terms of OS, probably due to a greater GVHD/GvL effect in the MUD setting leading to an increased EFS. Disclosure of Interest: None declared. P079 Impact of HLA matching on the risk of primary graft failure and acute GVHD in patients after allogeneic unrelated HSCT E. Kuzmich1,*, A. Alyanskiy1, A. Vitrischak1, E. Morozova1, S. Bondarenko1, L. Zubarovskaya1, B. Afanasyev1 1 First Pavlov State Medical University of St. Petersburg, Raisa Gorbacheva Memorial Institute for Pediatric Oncology, Hematology and Transplantation, St-Petersburg, Russian Federation Introduction: Primary graft failure and acute GVHD are the serious complications following unrelated HSCT. The aim of the study was to determine of the immunogenetic risk factors of these complications actual for our cohort patients. Materials (or patients) and methods: We retrospectively analyzed the results of 390 HSCT which were performed to the patients with various hematological diseases between January 2005 and December 2012. All donor/recipient pairs were typed for HLA-A, -B, -C, -DRB1, -DQB1 by PCR-SSP, PCR- SSOP, SBT methods on the high resolution level. Statistical analysis was done by SAS Enterprise Guide v. 5.1. Fischer’s Exact Test was used for comparison of the complications risk in HLAmatched and HLA-mismatched groups. Logistic regression model was used for the analysis of the association between HLA and non-HLA variables and risk of complications. Clinical variables included age of patient (continuous variable), type of disease, risk of leukemia relapse (standard, high, and diseases other than leukemia), donor/recipient gender-matching, donor/recipient CMV status, stem cell source, number of CD34 þ cells, conditioning regimen, aGVHD prevention regimen. Results: 290 patients received a 10/10 allele-matched transplant. 80 patients had a 9/10 allele- matched transplant (single mismatch for HLA-A: 27.5%, HLA-B: 23.7%, HLA-C: 32.5%, HLA-DRB1: 6.3%, HLA-DQB1: 10%). 20 patients received an equal and less than 8/10 allele-matched transplant. The single class I, class II allele mismatches didn’t impact on the frequency of primary graft failure. The multiple class I allele mismatches in the donor/recipient pairs were associated with higher risk of the primary non-engraftment (RR 3.42, CI 1.279.18). Other factors associated with primary graft failure were: using of bone marrow as the source of hematopoietic cells, positive CMV status of the donor in the case of the recipients with negative CMV status, type of disease. The risk of grades III-IV aGVHD was influenced by the number of mismatches, individual class I and class II loci disparities in the recipient. Recipients with single mismatches had an increased hazard of grades III-IV aGVHD compared with matched recipients (RR ¼ 1.98; CI 1.42-2.76; Po0.001). R ecipients with multiple class I, class II mismatches had the highest hazard (RR ¼ 2.71; CI 1.65-4.45; P ¼ 0.007). We analyzed the role of individual class I and class II loci. An increased risk of grades III-IV aGVHD was associated with the single HLADRB1 allele mismatches (RR ¼ 2.89; CI 1.57-5.29; P ¼ 0.03), single HLA-C allele mismatches (RR ¼ 2.36; CI 1.52-3.67; P ¼ 0.004, single HLA-B allele mismatches (RR ¼ 2.16; CI 1.30-3.61; P ¼ 0.02). Other factors associated with severe aGVHD were: using of peripheral blood stem cells as the source of hematopoietic cells, myeloablative conditioning regimens. Conclusion: The study results can be useful for the selection of the optimum donor for carrying out unrelated allogeneic HSCT. Disclosure of Interest: None declared.
P080 Hematopoietic stem cell donors – analysis of motivation and satisfaction throughout the donation process E. Snarski1,*, P. Kawka1, T. Torosian2, M. Achremczyk1, K. Skwierawska1, A. Waszczuk - Gajda1, E. Urbanowska1, W. Wiktor - J˛edrzejczak1 1 Hematology, Oncology and Internal Diseaes, Medical University of Warsaw, 2DKMS Poland, DKMS Poland, Warszawa, Poland Introduction: In recent years the number of Polish potential registered unrelated hematopoietic stem cell donors has increased to over 700 000. This caused substantial rise in the number of collections for recipients in Poland and worldwide. So far there is relatively little experience and knowledge about Polish donors: their motivation, the ways of recruitment, satisfaction during the donation process were never evaluated. Better understanding of these factors can contribute to improvement in methodology of recruitment, information and donation. The aim of our study was to assess these issues by approaching unrelated HSC donors at major centre in Poland. Materials (or patients) and methods: The unrelated HSC donors filled on line questionnaire at the two steps of donation process: first at the Final Clearance visit, and second immediately following the donation. The questionnaire was made available online for the ease of donors. Major questions included in the questionnaire: What are the main motivating factors to become a donor? How did donors register? What are the most important information sources? What are the biggest problems during donation? What are the biggest fears of donors? Results: At the time of abstract submission 93 questionnaires were submitted. The average age of responding donors was 32 years. Sixty six% of the donors registered declared that they wanted to help other people. The most important factors in recruitment were local action (21%), information from the Internet (23%), information from TV (19%) and prior recruitment of family member (15%). The major recruitment took place through the Internet (43%) and in local recruitment actions (39%). The biggest fears prior to the donation were: complications during donation (39%), pain (19%) and problems with work and family life that could be caused by donation (9%). Ninety eight% of donors felt adequately informed prior to donation and stated that the nurse (35%) at the apheresis centre and the qualifying doctor (33%) were the most important sources of information. The donation has been performed in 89% of donors from the peripheral blood and in 11% from the bone marrow. Forty% of donors have been also earlier blood donors. The level of pain during the PBSC donation was 1.2 in 10 point scale. Ninety four% felt that the information received prior to harvesting adequately described procedure. Ninety seven% were satisfied to give stem cells to the other person and 61% felt that the donation will bring a positive change to their lives. Eighty two% of donors expressed desire to meet recipient of their cells in the future, including 20% of donors who wished to meet the recipient immediately after the donation, 20% ready to wait at least for a year, and 23% ready to meet the recipient after 2 years. All declared consent to repeat collection procedure if it was needed. The major complaints concerning the procedure were: need for injections of G-CSF (33%), length of the procedure (30%) and pain (7%). Conclusion: Taken together these results create a positive view of donation process in Poland. The donors are generally well informed, the procedure does not cause significant problems and all the donors declared that they would donate one more time if needed. The major complaints are due to the technical parts of procedure – injections of G-CSF and length of apheresis. Disclosure of Interest: None declared.
P081 Prevalence of donor specific anti-HLA antibody in T cell-depleted haploidentical hematopoietic stem cell transplantation E. S. Choi1,*, H. J. Im1, J. K. Suh1, S. W. Lee1, K. N. Koh1, S. Jang2, H. B. Oh2, J. J. Seo1 1 Department of Pediatric Hematology and Oncology, 2Department of Laboratory Medicine, Asan Medical Center, SEOUL, Korea, Republic Of Introduction: Recently, the role of a donor specific anti-HLA antibody (DSA) in hematopoietic cell transplantation (HCT) has been emphasized as a consequence of the considerably increased cases of HLA-mismatched HCT including cord blood transplantation and haploidentical hematopoietic cell transplantation (HHCT) as well as unrelated HCT. The presence of DSA has been associated with an increased risk of graft failure (GF) after HCT. However, prevalence of DSA and clinically relevant level of DSA for GF in T cell-depleted HHCT remain unknown. Materials (or patients) and methods: We prospectively evaluated anti-HLA antibodies using the Luminex-based single antigen assay for 28 patients with malignant disease undergoing ex vivo T cell-depleted HHCT between Oct. 2011and Aug. 2014. Twenty-five patients had hematologic malignancy (7 ALL, 12 AML, 1 MLL, 1 MDS, 2 JMML, 2 NHL) and 3 had solid tumors. Two (1 ALL, 1 AML) of 28 enrolled patients received two rounds of HHCT for relapse after their first HHCT. Results: Of 30 transplants tested, anti-HLA antibody was detected in 17 (56.7%) patients. Of the positive cases, 4 (4/30, 13.3%) were positive against the donor HLA-antigen. Among the 4 cases with DSA, 2 had antibodies against HLA class I antigen, one against HLA class II, and one against both HLA class I and II. The peak levels of DSA were 1143, 1273, 4422 and 5564 of mean fluorescence intensity (MFI), respectively. None received any treatment in attempt to decrease DSA levels before transplants. Of a total of 30 HCTs, none experienced GF with achieving neutrophil engraftment at a median of 9 days (range, 9-13). In addition, the median times of neutrophil and platelet engraftment were similar regardless of the DSA status (10 versus 9 days for neutrophil, P40.05; 16 versus 18 days for platelet, P40.05). Conclusion: In conclusion, our study demonstrated that DSA up to MFI 5560 was not associated with increased risk of GF even without any interventions prior to T cell-depleted HHCT. However, further study including more cases is necessary to determine the optimal recipient’s MFI cut-off value for haploidentical family donor selection in T cell-depleted HHCT. Disclosure of Interest: None declared. P082 Impact of mismatches in the HLA system on the outcome after unrelated allogeneic stem cell transplantation E. Bojtarova1,*, B. Ziakova1, F. Al Sabty1, L. Sopko1, M. Kusikova1, D. Stemnicka1, M. Mistrik1, A. Batorova1 1 Department of Hematology and Transfusiology, LFUK, University Hospital Bratislava, Bratislava, Slovakia Introduction: Unrelated allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat patients (pts) with malignant and non-malignant hematological disorders, who miss their identical sibling. Better outcome of unrelated HSCT is achieved by knowledge of human leukocyte antigen (HLA) system and fast and precise tissue typisation techniques development. As the ‘10/10 matched’ (that is, high-resolution identity at loci HLA-A, -B, -C, -DRB1 and -DQB1) donor is not always available, alternative donor with single antigen/allele mismatch (9/10) is acceptable and HSCT might be succesfull as well, mainly in young pts. The aim of this analysis is to evaluate, whether the grade of HLA mismatch influenced the outcome of unrelated HSCT at our clinic. Materials (or patients) and methods: We retrospectively evaluated results of unrelated HSCT in 138 consecutive pts between February 1999 and May 2014 and compared grade of
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HLA match between donor and recipient. Results were evaluated to 31st August 2014. 88 pts received graft from 10/10 HLA identical unrelated donors, 50 pts from mismatched unrelated donors: 41pts with 9/10 (i.e. single antigen/allel mismatch) and 9 pts with 8/10 (i.e.two mismatches) HLA mismatch. Patient groups were comparative in age, primary diagnosis and peripheral blood use as the source of stem cells in majority of pts. The median age of pts at the time of HSCT was 37 (range, 18-68) years. AML/ALL was indication for HSCT in 84 pts, CML/MPN in 17 pts, MDS in 18 pts, SAA/PNH in 9 pts, MM/CLL/lymphoma in 10 pts. Median follow-up was 12, 11 or 8 months in ‘10/10 matched’, ‘9/10’ or ‘8/10 mismatched’ groups. Results: Median time to neutrophile engraftment (i.e. neutrophil count exceeding 0.5x10^9/l ) was 18 (range, 11-51) days in ‘10/10 match’, 16 (range, 13-32) days in ‘9/10’ and 20 (range, 14-21) days in ‘8/10 mismatched’groups. Graft failure occurred in 8 vs 0 vs 2 pts in ‘10/10’ vs ‘9/10’ vs ‘8/10’ HLA group. Acute graft versus host disease (GvHD) grades II to IV was observed in 32% of pts in ‘10/10’ vs 54% of pts in ‘9/10’ vs 75% of pts in ‘8/10’ group (P ¼ 0,01), chronic GvHD in 26% vs 41% vs 43% (P ¼ 0,2). Relaps incidence was observed in 24/83 (29%) evaluable pts in ‘10/10’, in 7/39 (18%) evaluable pts in ‘9/ 10’ and in 2/8 (25%) evaluable pts with ‘8/10’groups (P ¼ 0,41). 49/88 (56%) pts transplanted from ‘10/10 matched’ unrelated donors, 18/41 (44%) pts from donor with single mismatch and 4/9 (44%) pts from donor with two HLA mismatches are alive to 31.8.2014. Survival probability in 5 years (according to Kaplan-Meier) was 50%, 40% and 44% (P ¼ 0,254), with median survival of 24 vs 27 vs 19 months. Conclusion: Single or two antigen/allele mismatches between donor and recipient significantly increased the occurence of acute GvHD, non-significantly occurence of chronic GvHD, but didn’t negatively effect the egraftment, relaps incidence and overall survival in pts after unrelated HSCT. Disclosure of Interest: None declared. P083 Prospective study of T-cell -replete Haploidentical matched hematopoietic stem cell transplantation (HSCT) for patients with high risk hematological malignancies G. M. Al Dawsari1,*, S. MOHAMED1, F. ALMOHAREB1, N. CHAUDHRI1, F. ALSHARIF1, H. ALZAHRANI1, W. RASHEED1, A. HANBALI1, M. SHAHEEN1, F. ALFRAIH1, T. ELHASSAN1, M. Aljurf1 1 ONCOLOGY, KFSHRC, RIYADH, Saudi Arabia Introduction: HLA-matched sibling donors have been the standard for allogeneic HSCT, but about 35 %of our patients in Saudi Arabia have no an HLA- matched sibling donors. The chance of finding an HLA-matched unrelated donor from international registries is very low because of different ethnicity. Haploidentical HSCT with modern approach with post- transplant high dose cyclophosphamide (PT-CY) became an attractive approach with promising results, and make transplant available foe almost every patient. Here, we report the outcome results of our initial 11 patients. Materials (or patients) and methods: Analysis of patient’s data who underwent Haploidentical HSCT in prospective phase II study from January 2013 – September 2014. Eligibility include patients with high risk hematological malignancies in complete remission, less than or equal to 55 years of age without a suitable matched related donor , lack of fully matched unrelated donor (MUD) and Karnofsky performance of 70-100 %. Donor selection criteria included age of 18 years or more, a maximum 5 antigen mismatched is accepted and donor were excluded if the recipient’ serum contain anti- donor HLA antibodies. Myeloablative conditioning regimen consisted of total body irradiation (TBI) and fludarabine and thiotepa, busulfan and fludarabine (TBF). GVHD prophylaxis consist of PT-CY (50mg/kg) on day þ 3 and þ 5 and cyclosporine from day ( þ 4 to 180) and mycophenolate mofetil for 28 days. Results: A total of 11 patients received haploidentical HSCT and patients characteristics are shown in Table 1, with a
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median follow up of 6.5 months (range 1.75-16). The median time to both neutrophil and platelet engraftment (ANC40.5 109/L; platelet 420 109/L) were 19.5(15-24) and 30(17-150) respectively. The 100-day cumulative incidence (CIs) of acute GVHD grade II-VI was 59% and CIs of chronic GVHD was 55%.The CI of relapse was 20% with death in remission as competing event. The overall survival (OS) is 36%. Conclusion: Haploidentical HSCT represent a good alternative option for patients with high risk hematological malignancies who do not have suitable HLA- matched donors as it provide good result in term of engraftment of ANC and PLT and a good OS rate in patients with high risk diseases. GVHD remain a major obstacle in this type of transplant in our series when compared with published literature, for which we are currently investigating the addition of small dose ATG to our protocol. Disclosure of Interest: None declared. P084 Observational, prospective study on allogeneic family stem cell donors: results from a multi-center Italian study G. Andreola1,*, M. Martino2, T. Moscato2, M. Vacca3, F. M. Dallavalle4, S. Leoncino4, G. Mancini5, M. Montanari5, A. Babic1, M. Negri1, D. Laszlo1 1 Mobilization & Collection Stem Cell Unit, Division of Hematoncology, Istituto Europeo di Oncologia, Milano, 2Hematology and Stem Cell Transplant Unit, AO ‘‘Bianchi Melacrino Morelli", Reggio Calabria, 3Immunohematology and Trasfusion Unit, AO San Camillo Forlanini, Roma, 4Donation Center and Therapeutic Apheresis Unit, ASO ‘‘SS Antonio e Biagio e C. Arrigo’’, Alessandria, 5Hematology Clinic, Ospedali Riuniti Umberto I, Ancona, Italy Introduction: Allogeneic peripheral blood stem cell (PBSC) transplantation is a more convenient procedure than bone marrow (BM) for both donors and medical teams and shows similar outcomes allowing a faster hematologic recovery in recipients. However, adverse events (AE) and occasionally fatal complications within family donors have been reported. Complications are thought to be more frequent in family donors in respect to donors from international registries for whom suitability criteria and a 10-year(y) follow-up are standardized. Published retrospective studies usually lack long follow-up and prospective studies are urgently needed. We therefore designed a multicenter, observational, prospective study evaluating incidence and type of early and late complications in suitable family donors, in those judged as ‘‘not perfectly suitable’’ but undergoing PBSC collection anyway and in donors Z55 ys old; analysis of criteria excluding donors from PBSC and/or BM donation was also included. A complete blood cell count (CBC) after 7 days from
collection, a medical visit and a CBC after 30 days and a CBC yearly for the following 10 ys were required for each donor. Materials (or patients) and methods: From November 2011 to October 2014, 100 donors in 5 Italian centers were enrolled in the study. There were 51 males and 49 females; median age was 49 (18-70), 29 donors were Z55 ys old. Donors were parents in 8% of the cases, sibling in 80%, children in 12%; 71 donors were HLA-identical, 29 were HLA-haploidentical. A median of 4x106 CD34 þ /kg (range 4-6) of recipient body weight was requested. Results: According to IBMDR criteria, 71 (78%) donors resulted suitable for PBSC donation, 10 (10%) were not suitable for PBSC, 6(6%) were not suitable neither for PBSC nor for BM donation. Ten donors were evaluated as unsuitable for PBSC donation due to autoimmunity (40%), thrombofilic status (29%), not adequate peripheral access (10%), history of cardiac disease(30%); nevertheless 3 of them underwent mobilization anyway because no other donor was available or due to recipient’s related issues. Six donors were judged only partially suitable due to infections in 3, diabetes, splenomegaly and hypertension in the other 3. Lenograstim and filgrastim at 10 ug/kg were used in 96% and in 4% of donors respectively. PBSC collection was started after a median of 5 days (2-8) after GCSF administration. Mobilization-related AE were mostly bone pain (74%), headaches (33%), fatigue (30%), muscle pain (22%), splenomegaly not requiring GCSF interruption in 4%. Pre-leukapheresis white blood cell counts reached values 470.000/mm3 only in 3 donors. Median number of CD34 þ cells collected was 5.7 x106/kg (1.1-16) in a median number of 1 procedure (1-4); the target was reached in 84% of the cases and in 80% of donors Z55 y old. No donor required placement of a central venous catheter, red cell or platelet transfusion. Mild averse events during leukapheresis was reported in 11 donors (13.6%), moderate in 1 and severe in 1 donor Z55 ys. With a median follow-up of 18 months (1-36) no long-term adverse event were registered. Conclusion: PBSC mobilization and collections from family donors is a safe and well tolerated procedure and it allows an adequate collection of stem cells even in donors Z55 ys old as long as IBMDR criteria for donor selection are applied. Further data will be collected in order to reach a 10-y follow-up for each donor. Disclosure of Interest: None declared. P085 Hematopoietic Stem Cell Transplantation in Children Using Preimplantation Genetic Diagnosis for HLA-Matched Donor: a Turkish Multicenter Study ¨ ztu¨rk4, O. Gu¨rsel1, A. E. Ku¨rekc¸i1, A. Ku¨pesiz2, S. Anak3, G. O S. Aksoylar5, T. ˙Ileri6, B. Kus¸konmaz7, I. Eker1,*, M. Çetin7, G. T. Karasu8, Z. Kaya9, T. Fıs¸gın8, M. Ertem6, S. Kansoy5, M. A. Yes¸ilipek10 1 Pediatric Hematology, Gulhane Military Medical Faculty, Ankara, 2 Pediatric Hematology and Oncology, Akdeniz University, Antalya, 3Pediatric Hematology and Oncology, ˙Istanbul University, 4Pediatric Hematology and Oncology, Medikal Park Bahc¸elievler Hospital, ˙Istanbul, 5Pediatric Hematology and Oncology, Ege University, ˙Izmir, 6Pediatric Hematology and Oncology, Ankara University, 7Pediatric Hematology and Oncology, Hacettepe University, Ankara, 8Pediatric Hematology and Oncology, Bahc¸es¸ehir University Medical Faculty, ˙Istanbul, 9 Pediatric Hematology and Oncology, Gazi University, 10On Behalf of, Turkish Pediatric Bone Marrow Transplantation Study Group, Chair, Ankara, Turkey Introduction: Preimplantation genetic diagnosis (PGD) involves the diagnosis of a genetic disorder in embryo obtained through in vitro fertilization, selection of healthy embryos, and transferring them to the mother’s uterus. It has been used not only to avoid the risk of having an affected child, but also by using HLA matching together, it offers preselection of potential HLA-genoidentical healthy donor progeny for an affected sibling, who requires bone marrow
transplantation. Here, we share the hematopoietic stem cell transplantation (HSCT) results of 52 patients with benign or malign hematological diseases, metabolic diseases and immunodeficiencies, whose donors were their siblings borned with this technique in Turkey since 2008. To our knowledge, this is the largest case series in the literature performed to date about the results of the HSCT on the subject, which may thus provide valuable information for clinical outcomes of the transplantations with donors borned via this technique. Materials (or patients) and methods: Fifty two pediatric patients (23 female; 29 male) between 3-16 years old (median 8 years), underwent 53 HSCT at eleven different centers in Turkey, between February 2008 and January 2014. Their donors were their siblings borned via preimplantation HLA typing technique with or without mutation analysis. The most common indication for HSCT was thalassemia major (TM) (42 of all patients; 80%). Other indications for HSCT were acute lymphoblastic leukemia, acute myeloid leukemia, WiskottAldrich syndrome, juvenile myelomonocytic leukemia, Fanconi aplastic anemia, sickle cell disease and adrenoleukodystrophy. Stem cell source in all of the transplantations were bone marrow. In 37 of the transplantation procedures, umbilical cord bloods of the same donor were also used. Results: Except the two cases of TM with primary graft failure, 50 of 52 patients (96%) are primary disease free without any complication with a median follow-up period of 31 months (range 11–81 months). The median number of the IVF cycles, which was resulted with the birth of the donor was 2 (1-8) and the median time between the beginning of the first cycle and the transplantation was 3,7 (1-9) years. The median maternal age on the first IVF cycle was 30,3 (22-39) years. Median cost of the PGD/HLA typing and HSCT was 10.000 (1.600-480.000) and 35.000 (35.000-145.000) euros, respectively. The median total cost (HSCT plus PGD/HLA typing process) was 45.500 (38.000617.000) euros. Conclusion: Our experience showed successful HSCT results and reasonable cost rates for recepients using siblings who are the offsprings of pregnancies utilizing PGD and HLA matching technique. Transplant physicians should be aware of the feasibility and efficacy of the PGD/HLA typing method as well as the indications of its use. After establishing the indication for HSCT, a quick search for matched family donor (MFD) and successive matched unrelated donor (MUD) search in case no MFD available, should be performed. If there is no appropriate donor, either MFD or MUD, or HSCT from MUD carries significantly higher risks than the transplantation from MFD and if there is enough time in terms of primary disease, PGD/HLA typing technology should be considered and discussed with the family. However, it should be performed under strict supervision of national and local ethics committees. Disclosure of Interest: None declared. P086 Haploidentical stem cell transplantation with posttransplant Cyclophosphamide for patients with X-Linked Adrenoleukodystrophy and severe aplastic anemia: retrospective analysis I. Esteves1,*, J. Fernandes1, A. Ribeiro1, F. Santos1, A. Kondo1, C. Bonfim2, F. Kok3, J. Vargas1, L. Fernando Mantovani1, M. Rodrigues1, C. Bonet Bub1, F. Kerbauy1, N. Hamerschlak1 1 Hematology and Stem Cell Transplantation, Hospital Israelita Albert Einstein, Sao Paulo, 2Hematology and Stem Cell Transplantation, Hospital das Clinicas do Parana, Curitiba, 3 Neurology, Hospital das Clinicas de Sao Paulo, Sao Paulo, Brazil Introduction: X-linked adrenoleukodystrophy (X-ALD) and severe aplastic anemia (SAA) are non-malignant disorders. X-ADL is an inherited demyelinating disease caused by the deficiency of the ABCD1 gene and causes neurological disability during the first two decades of life. SAA is a rare acquired disease, commonly idiopathic and characterized by
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bone marrow failure. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only treatment that has shown to change the natural history of the X-ADL and is curative in patients with SAA. In the absence of a matched related or unrelated donor, haploidentical might be an option. Haploidentical HSCT using post-transplant cyclophosphamide (Cy) has been performed in malignant and non-malignant diseases Materials (or patients) and methods: We analyzed 8 patients with X-ALD and 7 patients with SAA treated with HSCT from haploidentical and post-transplant Cy from September 2011 to December 2014. Results: 8 patients with X-ALD (5-18 years) underwent haploidentical HSCT. Two patients received 2 allogeneic HSCT from different related donors and 1 patient received a second transplant after failure of a double cord blood transplant. Pretransplant MRI showed a median Loes score of X (range 2,518), all patients had neuropsychological evaluation with performance IQ above 90. Donors were the father (n ¼ 7), uncle (n ¼ 2) or brother (n ¼ 1). All patients received nonmyeloablative (NMA) regimen: fludarabine 150 mg/m2, Cy 29 mg/kg and total body irradiation (TBI) 2 Gy. Six patients also received rabbit antithymocyte globulin 4,5 mg/kg. Posttransplant immunosuppression consisted of Cy 50 mg/kg/d on days þ 3 and þ 4, tacrolimus and mycophenolate mofetil (MMF) starting on day þ 5. Seven patients engrafted and 1 patient had a primary graft failure and severe progression of disease. Two patients had secondary graft failure with progressive loss of donor chimerism (STR) and were successfully rescued with second haploidentical transplants using different related donors. Four patients had grade II-IV acute graft-versus-host disease and four patients had CMV reactivations (50%). One patient with grade III GVHD showed progression of neurologic symptoms of primary disease. Six patients are alive and engrafted from 3 to 11 months after transplant, with STR from 80 to 100% donor cells. The median of follow up of these patients was 424 days (102-789 days), the OS was 75% and the progression rate was 75%. We analyzed 7 patients with acquired SAA (7-39 years old) submitted to NMA with Flu/Cy/TBI associtated to MMF/tacrolimus/Cy as post transplant immunosuppression. The source was bone marrow for 6 patients and donors were mother (n ¼ 4) and siblings (n ¼ 3). All patients had neuthrophil engraftment and 1 had platelets graft failure and required thrombopoetin receptor agonist with response. Two (28.6%) patients had grade III-IV acute GVHD and chronic GVHD. There was no rejection and STR was 100% donor. The median of follow up was 355 days (111-651 days) and the OS was 71.4% and the DFS was 100%. Conclusion: Haploidentical HSCT with post-transplant Cy is an alternative for X-ALD and SAA lacking a suitable matched donor. Graft failure is still an obstacle that has to be prevented in NMA regimen. Patients with SAA have been submitted to IST before HSCT which has been a reason for low rate of graft failure when compared to X-ADL. Disclosure of Interest: None declared. P087 Haploidentical x Umbilical Cord blood Hematopoietic stem cell transplantation: a comparison between alternative donors I. Esteves1,2,*, F. Pires Santos Souza1, A. Alice Feitosa1, G. Fleury Perini1, R. Helman1, M. Afonso Torres1, A. Tiemi Kondo1, L. Nassif Kerbauy1, J. Folloni Fernandes1,3, F. Rodrigues Kerbauy1,2, N. Hamerschlak1 1 Hematology and Stem Cell Transplantation, Hospital Israelita Albert Einstein, 2Hematology and Stem Cell Transplantation, Escola Paulista de Medicina/ UNIFESP, 3Hematology and Stem Cell Transplantation, Instituto de tratamento do cancer Infantil/ ITACI, Sao Paulo, Brazil Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) from matched donors is used to treat malignant or
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non-malignant disorders. HSCT from Haploidentical donors and umbilical cord blood (UCB) can be an alternative in patients with no matched related donor (MRD) or matched unrelated donors (MUD). Materials (or patients) and methods: From April 2008 to July 2014, we analyzed patients that underwent haploidentical (n ¼ 42) and UCB HSCT (n ¼ 50). Results: Patients’ age ranged from 1-72 years old and most patients in the group UCB HSCT were younger than 18 years old. The median follow up time for UCB HSCT were 178,5 days (12-2183 days) and for haploidentical were 196 days (28-2333 days). In the haploidentical group, 23 patients had nonmalignant and 19 had malignants disorders and UCB group had 19 patients with non-malignant and 31 with malignant disorders. Patients in the UCB group received a nonmyeloablative conditioning regimen with fludarabine, cyclophosphamide (Cy) and TBI 200cGy in 24 patients (57%) and 24 patients (48%) were submitted to a myeloablative regimen (bussulfan and fludarabine or busulfan, fludarabine and thiotepa). The predominant source of cells in haploidentical group was bone marrow (n ¼ 39/ 92.8%) with a median total of infused nucleated cells x 108/Kg of 5.18 (4.367.75 x108/Kg). Patients submitted to UCB received a median total nucleated cell x 107/Kg of 9.5 (3.93 x 107- 4.12 x 107/Kg). Graft versus host disease (GVHD) prophylaxis consisted of tacrolimus/MMF plus post-transplant Cy in 36 (85.7%) patients submitted to haploidentical and calcineurin inhibitor plus MMF (68%) in UCB. Forty (80%) patients had neuthrophil engraftment in UCB group and 36 (85.7%) in haploidentical group. The median time of neuthrophil engraftment in UCB and haploidentical was 16 and 17 days respectively. 14.3% and 20% of patients that received haploidentical and UCB respectively had secondary graft rejection. Two (4.8%) patients developed hepatic synusoidal obstruction syndrome (SOS) in haploidentical group and 8 (16%) in UCB. Acute GVHD was observed in both groups: 28.6% in haploidentical and 46% in UCB. Chronic GVHD was similar in haploidentical and UCB groups (9.5% vs. 8%) respectively. The CMV reativation were similar in both groups (70%). The overall survival (OS) in haploidentical was 53.4% and 46.6% for UCB (P ¼ 0.055). Major cause of death in haploidentical and UCB group was infection in 63.6% vs. 73.9% respectively. The haploidentical and UCB groups relapse rate was 19% and 4% respectively. In multivariate analysis the graft failure and SOS had impact in OS in UCB HSCT (P ¼ 0.001) while graft failure had negative impact in OS in haploidentical group. The time of hospitalization at first year of HSCT was not different between groups. Conclusion: Graft failure was a serious complication in both groups, and conditioning regimen should be reviewed to improve the rates of engraftment. Haploidentical transplant with non-myeloablative regimens showed low rates of acute GVHD but it did not reduce graft failure. References: Chang YJ, Huang XJ. Haploidentical SCT: the mechanisms underlying the crossing of HLA barriers. Bone marrow transplantation 2014. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood 2013; 122(4): 491-8. Koh LP, Chao N. Haploidentical hematopoietic cell transplantation. Bone marrow transplantation 2008; 42 Suppl 1: S60-S63. Disclosure of Interest: None declared.
Graft-versus-host disease – clinical I P088 Platelet Gel for Treatment of Muco-Cutaneous Lesions Related to Graft-Versus-Host Disease: an Update from the Rome Transplant Network A. Picardi1,*, A. Di Veroli 1, A. Ferraro2, A. Lanti2, L. Cudillo1, R. Cerretti1, G. De Angelis1, B. Mariotti1, A. Biagi1, V. Molinari1, F. Trenta1, G. Adorno2, C. Bonchi2, W. Arcese 1 on behalf of Rome Transplant Network 1 Hematology Division - Stem Cell Transplant Unit, 2Transfusion Medicine , TOR VERGATA UNIVERSITY OF ROME, Rome, Italy Introduction: Platelet gel (PLT-gel) has been successfully used for the treatment of muco-cutaneous lesions associated with acute or chronic graft versus host disease (a/cGVHD) in patients undergoing an allogeneic transplant for haematological disorders (Platelet gel for treatment of mucocutaneous lesions related to graft-versus-host disease after allogeneic hematopoietic stem cell transplant. Picardi A, et al Transfusion. 2010 Feb; 50(2): 501–506). The rationale of its use in the transplant setting is based on its well known efficacy in facilitating tissue regeneration of diabetic/ surgical wounds through the releasing of growth factors such as bFGF and PDGF. Herein, we report the results in treating mucocutaneous GVHD associated ulcers with PLT-gel by extending our experience to 20 patients. Materials (or patients) and methods: Twenty patients with median age of 52 (range 38-75) and transplanted for hematological malignant disease developed acute (n ¼ 9) or chronic (n ¼ 11) GVHD complicated by muco-cutaneous lesions: skin grade I (n ¼ 4), skin grade II (n ¼ 5), oral mucosa (n ¼ 10), skin þ mouth (n ¼ 1). The patients were treated with local PLT-gel obtained from allogeneic hemocomponents processed with Vivostat System and applied over the lesions through spray diffusion. At time of PLT gel treatment, 17 out of 20 patients were on immunosuppressive therapy for their GVHD. The response rate was defined as ‘‘complete’’ in cases of total re-epithelization of ulcers and as ‘‘partial’’ when the appearance of granulation tissue on the target sites was not followed by the total re-epithelization, in presence of reduction size of 50% or more of the lesions. Results: Following the second PLT-gel application, the pain disappeared in all cases and the granulation tissue was observed in all patients with grade II lesions. One patient died too early of multiorgan failure and was not evaluable for the response. Fourteen out of 19 (74%) patients achieved a complete response after a median of 5 (range, 1-15) PLT-gel applications, the response was considered partial in 5 (26%) patients, who received a median of 5 (range, 3-9). Neither side effects nor local infections related to the PLT-gel therapy were documented. Conclusion: PLT-gel therapy by inducing pain discontinuation allows strong reduction of the analgesic drug administration and highly ameliorates the quality of patient life. Overall, this update confirms our previous data supporting safety and efficacy of PLTgel in the treatment of muco-cutaneous GVHD lesions. Disclosure of Interest: None declared. P089 Does defibrotide prophylaxis decrease the risk of acute graft versus host disease following allogeneic hematopoietic cell transplantation? A retrospective analysis A. I. E. Tekgu¨ndu¨z1,*, A. H. H. Kaya1, S. C. Bozdag2, S. Kocubaba1, O. Kayıkcı1, S. Namdaroglu1, B. Ugur1, F. Altuntas1 1 Hematology and bone marrow transplant unit, Ankara Oncology Hospital, 2Hematology and bone marrow transplant unit, Ankara University Hospital, Ankara, Turkey Introduction: Both acute (aGvHD) and chronic (cGvHD) graft versus host disease are important complications following allogeneic hematopoietic cell transplantation (allo-HCT)
decreasing survival and quality of life. Defibrotide is widely used agent for prophylaxis and treatment of hepatic venoocclusive disease (VOD). There are some preliminary evidence that defibrotidemay also have a role in decreasing risk of GvHD by preventing tissue damage. We aimed to investigate the role of defibrotide prophylaxis on development of aGvHD. Materials (or patients) and methods: All consecutive patients who received allo-HCT between January 2009 and January 2014 were included in the study. Medical records of patients were retrospectively analyzed.Patients were analyzed in two time frames according to availability of defibrotide. Patients who were transplanted in January 2009–October 2011 (conventional arm) received only low molecular weight heparin (LMWH), ursodeoxycholic acid (UDCA) and N-acetylcysteine (NAC) in terms of VOD prevention. All consecutive patients who undergone allo-HCT in November 2011–January 2014 period (defibrotide arm) received 10 mg/kg intravenous defibrotide prophylaxis for two weeks (from D þ 1 to D þ 14) in addition to conventional approach. Descriptive statistics are presented as median and range. A p value below 0.05 was considered to be statistically significant. Results: A total of 148 patients (conventional arm (n: 58); defibrotide arm (n:90) participated in the study. Median age of study cohort was 36 (17-58).The study groups were comparable in terms of gender, age, intensity of conditioning regimen, GvHD prophylaxis, use of TBI in conditioning and stem cell source. But significantly more patients in defibrotide arm (matched unrelated donor: 5, haploidentical donor: 11) received unrelated or mismatched donor HCT compared to conventional arm (matched related donor: 57; 1 Ag mismatched related donor: 1)(p:0.016). Acute GvHD was observed in 36 (40%) and 27 (47%) patients on defibrotide and conventional arms, respectively (p.0.431). Grade Z2 aGvHD were also similar in treatment arms (defibrotide arm: 15.5%; conventional arm:19%) (p:0.549). Conclusion: Our results indicate that defibrotide prophylaxis seem not to confer an advantage over conventional arm in decreasing aGvHD rate. Before drawing firm conclusions about the role of defibrotide on aGvHD development, some important facts should be bear in mind. First of all, the optimal dose, timing and duration of defibrotide in VOD prophylaxis in adult patients are undefined. The second point is that more patients on defibrotide arm received HCT from unrelated or mismatched related donors. Therefore it can be argued that higher dose and longer duration of defibrotide prophylaxis could reduce aGvHD rate in defibrotide arm if both groups were similar in term of HLA match. On the other hand, it can also be speculated that effect of defibrotide on GvHD may be seen later following HCT, by decreasing cGvHD rate. We hope that, properly designed prospective trials will define the role of defibrotide in GvHD prevention, if any. Disclosure of Interest: None declared. P090 Extracorporeal photopheresis in patients with acute and chronic GVHD modulates the cellular immune system A. Schmitt1,*, L. Wang1, A. Hu¨ckelhoven1, J. Hong1, A. D. Ho1, P. Dreger1, W. Kru¨ger2, S. Michael1 1 University Clinic Heidelberg, Heidelberg, 2University of Greifswald, Greifswald, Germany Introduction: Extracorporeal photopheresis (ECP) constitutes a promising therapeutic approach for both steroid-refractory acute and chronic graft-versus-host disease (GvHD). The mechanism of action in ECP might be elicited by modulation of lymphocyte subsets. We therefore analyzed the clinical and immunological effects during ECP therapy in 10 patients. Materials (or patients) and methods: Six patients with acute GvHD of the gut 1I-1IV received intensive ECP therapy over 12 weeks followed by ECP treatments at a lower frequency for up to 6 months. Despite therapy with steroids, calcineurin inhibitors, cyclosporine A and/or mycophenolate mofetil and pentostatin the patients suffered from extensive diarrhea due
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to histologically proven GvHD with negative microbiological results. Two patients with chronic GvHD of the skin and two patients with cGvHD of the lung with up to a triple immunosuppressive therapy received ECP treatment every 2 to 4 weeks. Immunological analyses of B-, T-, NK(T) cells and myeloid derived suppressor cells were performed. Results: Four of six patients with acute GvHD of the gut 1II-1IV showed a dramatic clinical improvement with reduction of stool volume thus allowing a tapering-out of steroids while on intensive ECP therapy. In parallel immunomonitoring demonstrated an increase of CD4 þ CD25hiFoxP3 þ regulatory T cells and a decrease of CD8 þ CD45RA þ CCR7- effector T cells. Patients with acute GvHD only 1I of the gut did not seem to profit from intensive ECP treatment. Patients with chronic GvHD kept clinically stable on ECP therapy with slow improvement over the time which might require even ECP treatment every four weeks over a period of several years. Conclusion: Intensive ECP treatment in acute GvHD might evoke a change in frequency of effector and regulatory T cells leading to an immune modulation with a subsequent chance to reduce immunosuppressive medication. Taken together, immunomonitoring of T cell subsets can help to elucidate the mechanism of action and might predict the feasibility to taper of immunosuppressive therapy. Disclosure of Interest: None declared. P091 Reduced-intensity conditioning (RIC) with high-dose rituximab and allogeneic stem cell transplantation for relapsed CD20 þ lymphomas: good disease control with low incidence of chronic GVHD A. Dodero1,*, B. Sarina2, G. Milone3, F. Patriarca4, A. Bosi5, A. Dominietto6, L. Farina1, R. Foa’7, E. Terruzzi8, F. Onida9, A. Rambaldi10, P. Corradini1 on behalf of on behalf of GITMO, Gruppo Italiano per il Trapianto di Midollo Osseo 1 Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano , 2Istituto Clinico Humanitas, Rozzano, 3Hematology, Ospedale Ferrarotto, Catania, 4Hematology, Azienda Ospedaliera Universitaria, Udine, 5Hematology, University of Firenze, Firenze, 6 Hematology, Ospedale San Martino, Genova, 7Hematology, Universita’ La Sapienza, Roma, 8Hematology, Ospedale San Gerardo, Monza, 9Hematology, Universita’ di Milano , Milano, 10Hematology, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy Introduction: The best conditioning regimen before allogeneic stem cell transplantation (alloSCT) for relapsed B-cell lymphomas remains to be clarified. The GITMO conducted a multicenter phase II trial (EUDRACT 2007- 003657- 87) to evaluate the effect of high dose rituximab before alloSCT. The primary objective of the study was the PFS at 2-years. Materials (or patients) and methods: 101 patients (pts) affected by relapsed/refractory B-cell lymphomas [n ¼ 57 indolent: 32 follicular lymphomas (FCL), 24 Chronic lymphocytic leukemia (CLL); n ¼ 44 aggressive lymphomas: 24 diffuse large B-cell lymphomas (DLBCL), 20 mantle cell lymphomas (MCL)] were enrolled. Pts received the preparative conditioning regimen consisting of thiotepa/cyclophosphamide/fludarabine and highdose rituximab (500 mg/m2 on day -6). GVHD prophylaxis consisted in cyclosporine and mini-methotrexate; ATG was added to those allografted from class I antigen mismatched sibling or unrelated donors. The proportions of transplantation from matched-related, mismatched-related, matched-unrelated, mismatched-unrelated were 53%, 1%, 34%, 12%, respectively. The majority of the pts were chemosensitive to the last salvage regimen (n ¼ 40 CR, n ¼ 45 PR, 84%). Results: With a median follow-up of 31 months (range,6-79.5 months), the estimated 2-years and 3-years OS and PFS were 74% and 62%, 65% and 48%, respectively. A similar 2-years OS was observed in pts who underwent sibling versus unrelated transplantation (71% versus 63%, P ¼ 0.6). In pts with a diagnosis of FCL, CLL, MCL and DLBCL the 3-years OS were 84%, 67%, 64%, 55%, respectively. The cumulative incidence (CI) of non-relapse mortality (NRM) at 2-years was 13% and
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20% in pts allografted from matched related versus unrelated donors (P ¼ 0.22), respectively. The CI of acute grade II-IV GVHD was not different between sibling and unrelated donors (27% versus 24%, P ¼ 0.91). There was a trend for a higher chronic GVHD CI in pts allografted from matched siblings as compared to unrelated donors (55% versus 29%, P ¼ 0.08). The overall CI incidence of extensive chronic GVHD and of systemic corticosteroid requiring chronic GVHD were 24% and 36% in recipients of sibling graft and 10% and 10% in recipients of unrelated grafts. The survival free of disease and chronic GVHD were 39% at 3 years with the majority of events occurring in the first 24 months post allografting. At 1 year post alloSCT, the administration of Rituximab did not impact T- but only B-cell immune reconstitution (median values CD4 þ , CD8 þ , CD19 þ were 499/ucl, 394/ucl, 88/ucl, respectively). Conclusion: RIC with rituximab is an interesting transplantation platform. In fact, we observed: i) long-term survival in heavily pretreated pts; ii) limited CI of extensive chronic GVHD and low use of systemic corticosteroid. This strategy should be compared with others transplantation platform used for relapsed CD20 þ lymphomas. Disclosure of Interest: None declared. P092 Oral chronic graft-versus-host disease outcomes and resource utilization: a subanalysis from the chronic GVHD Consortium A. Yuan1,2,*, X. Chai3, F. Martins4, C. Cutler5, S. Lee3, N. Treister1,2 1 Division of Oral Medicine and Dentistry, Brigham and Women’s Hospital, 2Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, 3Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, United States, 4Department of Oral Pathology, School of Dentistry, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 5Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, United States Introduction: Chronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. The objective of this study was to evaluate the predictive validity of oral cGVHD consensus assessments for clinical outcomes, including use of clinical services and prescription therapies across institutions with and without oral medicine (OM) centers. Materials (or patients) and methods: Patients enrolled in the cGVHD Consortium with oral cavity involvement were included, and additional mouth-specific cGVHD-associated outcome data was abstracted via retrospective chart review across five transplant centers. Utilizing longitudinal oral cGVHD ancillary therapy data, multivariable linear models were fit correlating mouth-related outcomes with ancillary care category. Results: A total of 79 patients with 656 documented oral visits were observed for a median duration of 7.1 months. Overall mouth scores were ‘‘mild’’ (54%), with ‘‘moderate’’ (15%) and ‘‘severe’’ (4%). The National Institutes of Health (NIH) oral cGVHD severity score corresponded the most with mouth involvement prompting treatment (o0.001). Ancillary therapies for oral cGVHD management were prescribed for 56% of patients for a median duration of 6.9 months. Other oral medicine agents (including sialogogues) were prescribed for 46% of patients, with the majority at OM centers. Compared to patients that did not receive oral ancillary therapy, patients treated with topical therapy were five times more likely to have NIH mouth involvement (Po0.001, OR 5.1, 95% CI: 2.212.0), and almost three times more likely to have mouth pain (P ¼ 0.01, OR 2.6, 95% CI: 1.1-5.8). The odds of receiving topical therapy from OM specialists were estimated to be 52% higher than from transplant physicians (P ¼ 0.03), and 39% higher than from midlevel providers (P ¼ 0.07). Conclusion: Utilization of clinical and prescription approaches for oral cGVHD therapy varies across institutions. Ancillary
therapies were prescribed for the majority of patients, who were more likely to have NIH mouth involvement and mouth pain. OM specialists were more likely to prescribe topical therapy than other providers. References: Rationale and design of the chronic GVHD cohort study: improving outcomes assessment in chronic GVHD. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 2011; 17: 1114–1120. Disclosure of Interest: None declared. P093 Impact of T-cell depletion techniques on post transplant graft versus host disease after allogeneic HSCT with myeloablative conditionning A. P. Grandjean1,*, F. Simonetta1, S. Masouridi-Levrat1, Y. Tirefort1, Y. Beauverd1, A. Simon1, M. Ansari2, C. Dantin1, E. Roosnek1, Y. Chalandon1 1 hematology, 2onco-hematology pediatrics, Geneva University Hospitals, Geneva, Switzerland Introduction: Graft versus host disease (GvHD) is known to be a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloHSCT). Different strategies are developed in attempt to reduce GvHD incidence. T-cell depletion using in vivo ATG or/and in vitro alemtuzumab represent potential strategies. In the present study we evaluated the incidence of GvHD using T-cell depletion in the bag with or without ATG administration as compared to non-Tcell depleted alloHSCT. Materials (or patients) and methods: We performed a retrospective study on 256 patients transplanted at our center from August 1997 to October 2013 with an alloHSCT from identical siblings or HLA 10/10 matched unrelated donors after myeloablative conditionning. Median patients age was 41 years (range 1-63), median EBMT score was 2 (range 0-6). One hundred nine patients received partial TCD (pTCD) grafts, consisting of in vitro alemtuzumab incubation before infusion followed on day þ 1 by an add-back of donor T CD3 þ cells. Thirty-four patients received in vivo ATG administration, consisting of either Thymoglobuline (Genzyme) or ATG-F (Fresenius). Sixty-nine patients had combined ATG and pTCD. Forty-four patients had no TCD. Log-rank Mantel-Cox test was used to compare the 1-year cumulative incidence of clinically significant, grade 2-4 acute GvHD (aGvHD) and 1- and 5-year cumulative incidence of chronic GvHD (cGvHD) as well as 5year transplant related mortality (TRM), relapse rate (RR) and overall survival (OS) in patients receiving partially T-cell depleted alloHSCT with or without ATG administration, with patients receiving non-T cell depleted alloHCST. Results: With a median follow up of 52 months (range 0.07220), grade 2-4 acute GvHD 1-year cumulative incidence was significantly decreased in patients receiving pTCD grafts combined with ATG (11%; Po0.0001) compared to patients receiving non-pTCD alloHSCT (58%). No effect of pTCD (37%, P ¼ 0.0537) grafts or ATG treatment alone (53%, P ¼ 0.9172) was observed on acute grade 2-4 GvHD incidence. Similarly, no significant effect on 1-year and 5-year cumulative incidence of cGvHD was observed for either ATG (15%, P ¼ 0.1132 and 15%, P ¼ 0.0902 repectively) or pTCD alone (27%, P ¼ 0.2631 and 28%, P ¼ 0.1942) compared to non-pTCD (40 % and 44%). Conversely, we observed a highly significant reduction in 1year and 5-year cumulative incidence cGvHD in patients receiving ATG combined with pTCD (11%, P ¼ 0.0010 and 11%, P ¼ 0.0004). 5-year TRM and RR were not significantly different between groups with TRM 8% (P ¼ 0.3569) for ATG and pTCD, 18% (P ¼ 0.6357) for pTCD, 28% (P ¼ 0.1388) for ATG alone compared to 12% for non TCD and RR respectively of 50% (P ¼ 0.7719), 45% (P ¼ 0.3511) and 28% (P ¼ 0.1388), compared to 51%. 5-year OS was found significantly worse with ATG alone (33%, P ¼ 0.0061) with no difference with ATG and pTCD (69%, P ¼ 0.4448) and pTCD alone (61%, P ¼ 0.7743) compared to non-TCD.
Conclusion: pTCD combined with ATG appears to decrease the incidence of acute and chronic GvHD in patients receiving allogeneic HCST with a myeloablative conditioning from sibling or HLA 10/10 matched unrelated donors without affecting 5-year TRM, RR or OS. These results extend our knowledge of effect of T-cell depletion strategies on transplant-related morbidity and mortality. Our findings need to be confirmed in larger prospective studies. Disclosure of Interest: None declared. P094 Cyclosporine Concentration and Incidence of Acute Graft versus Host Disease after Allogeneic Stem Cell Transplantation B. Josu1,*, L. Arrue2, A. Sainz2, I. Urreta3, N. Caminos2, E. Perez2, J. J. Ferreiro2, M. Artola2, I. Ceberio2 1 pharmacy, 2Hematology, 3Biostatistics and Epidemiology, Hospital Universitario Donostia, Donostia, Spain Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for hematological disorders. The combination of Cyclosporine-A (CsA) and metrotexate (Mtx) is the standard inmunosupresive prophylaxis in majority of transplant centers. We report our single-center retrospective analysis of 54 consecutive patients that underwent allo-HSCT with the aim to evaluate the impact of CsA concentration during the first month after allo-HSCT on the incidence of acute graft versus host-disease (GVHD). Materials (or patients) and methods: Between 2009-20013 fifty four patients ( M/F 28/26), median age 50 years (range: 21-72) were transplanted for Myeloid malignancies (78%), Lymphoid malignancies (18%) and Aplastic anemia (6%). at our center. Fourteen male patients underwent a transplant from a female donor. Conditioning regimen was myeloablative (M) in 26 patients and reduced intensity (RIC) in 28. All patients received CsA and Mtx as GVHD prophylaxis regimen. Donors were HLA matched (51) and mismatched (3) related. 85% of the patients received peripheral blood mobilized grafts. Patients who engrafted were evaluable for acute GVHD (aGVHD), and patients surviving at least 100 days were evaluable for chronic GVHD (cGVHD) based on NIH consensus criteria. The mean weekly CsA concentration was calculated using the different concentrations measured during a defined week. CsA doses were adjusted based on drug trough levels between 200-300 ng/mL and toxic side effects like nephrotoxicity. The probability of overall survival (OS) was calculated by the Kaplan-Meier method. Comparison of the groups with grade 0I acute GVHD (aGVHD) and grade II-IV acute GHVD was carried out using the Fisher test for categoric variables and the U-Man Whitney test for continuous data. Results: The median follow up was 1.74 years (range: 0.17-5.64). The median concentration of CsA in the blood at 1, 2, 3 and 4 weeks after allo-HSCT were 270 ng/mL (range: 144-593), 299 ng/mL (range: 165-508), 315 ng/mL(range: 117-814) and 268 ng/mL(range 62-650), respectively. The OS and relapse rates at 1 year after allo-HSCT were 81% and 22%, respectively. Grade II-IV aGVHD occurred in eleven patients (20%) at a median of 51 (range: 14-189) days after allo-HSCT. The incidence of severe grade 3-4 aGVHD and chronic GVHD were 6% and 39%, respectively. Univariate analysis of risk factors for grade II-IV aGVHD developments showed no statistical association between grade II-IV aGHVD and risk factors for aGVHD. Conclusion: It has been already reported that CsA concentrations should be monitored after allo-HSCT, because low CsA concentrations can increase risk of aGHVD. In our study we could not demonstrate a relationship between CsA trough blood concentrations and the risk of aGVHD. This might be because of the relatively limited size of our study
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population and the low incidence of overall and severe aGHVD in our cohort. Some authors have also failed to demonstrate a relationship between trough concentrations and aGVHD. It is not clear which CsA blood-level monitoring is the most appropriate. In the setting of solid organ transplant, some studies have shown that a peak-level (2 hours after and oral dose-C2) is the optimal method of monitoring CsA. Further prospective clinical trials are necessitated to evaluate the most appropriate way of CsA monitoring and its relationship with development of aGVHD. Disclosure of Interest: None declared. P095 Extracorporeal Photopheresis (ECP) As Second Line Treatment For Refractory Graft Versus Host Disease (GVHD): Preliminary Single Centre Experience M. Gonza´lez-Pardo1, B. Aguado1,*, C. Fernandez-Arandojo1, E. Acun˜a1, S. Guijarro1, A. Alegre1 1 Hematology, Hospital Universitario de La Princesa, MADRID, Spain Introduction: Steroid-refractory GVHD is a common cause of morbidity and mortality and remains a challenging therapeutic problem after allogeneic hematopoietic stem cell transplantation (HSCT). There is currently no standard of care for this complication. ECP is an immunomodulatory therapy which has been used in the treatment of acute and chronic GVHD. We present our experience of ECP as second line treatment option for GVHD post-HSCT. The aim of this analysis was to evaluate the clinical effect of ECP, its impact on intensity of immunosuppressive therapy, safety and tolerance of the procedure. Materials (or patients) and methods: Since May 2012 we have performed 197 ECP procedures with the Therakos Cellexs device that is an integrated ‘‘on line’’ system. 11 patients are evaluable with median age 55y (38-65) and weight 61kg (38-80) presented with acute (4 patients) or chronic (7 patients) therapy-dependent/refractory GVHD. Allogeneic HSCT was myeloablative in 9 cases and reduced intensity conditioning in two cases. Six patients were diagnosed of acute myeloid leukemia, two myelodisplastic syndromes, one acute lymphoblastic leukemia, one chronic lymphocytic leukemia and one non-Hodgkin’s lymphoma. The transplant was from an unrelated donor in 6 cases and 5 from a sibling donor, all of them HLA matched except for one with a missmatch in HLA A locus. All patients but one (with exclusive skin presentation) had multiorgan involvement, all suffered from skin changes in combination with liver, lung, oral or ocular disease. As first line treatment they all received esteroids plus cyclosporine or mycophenolate mofetil. Median ECP per patient was 11 (4-43). ECP procedures were performed for two consecutive days, in initial phase weekly (in those with aGVHD), or every two weeks (cGVHD) and then monthly according to clinical response. Median of ECP treatment duration was 3 months (1-30). Response was evaluated by clinical assessment and reduction in immunosuppression. Results: In the cGVHD group response was complete in 3/7 (43%), partial in 4/7 (57%), ECP led to Z 50% improvement in symptoms and signs of cGVHD and significant reduction or even withdrawal of esteroids was observed. The first response was seen in skin and mucosal symptoms. In the aGVHD group 2/4 patients (50%) had complete remission (one died of pneumonia) and 2/4 (50%) did not response. The tolerance to the procedure was excellent and the unique complications seen were related with two central venous catheter infections. Conclusion: In our preliminary experience, the ECP is associated with excellent tolerance, significant response rates and successful reduction of steroids in patients with cGVHD. More experience is required to assess response on aGVHD patients. References: Dignan FL et al. Diagnosis and management of chronic graft-versus-host disease. British Journal of Haematology, 2012; 158: 46–61.
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Merlin E et al. Extracorporeal photochemotherapy as second or first line therapy of acute GVHD?. Bone Marrow Transplantation 2010; 45: 963–965. Disclosure of Interest: None declared. P096 Graft-versus-host disease prophylaxis with high-dose post-transplantation cyclophosphomide in unrelated hematopoetic stem cell transplantation O. Pirogova1, I. Moiseev1, V. Vavilov1, O. Slesarchuk1, S. Bondarenko1, B. Afanasyev1,* 1 R.M. Gorbacheva Memorial Institute of Children Hematology, Oncology and Transplantation, First Saint-Petersburg State Medical University named I.P. Pavlov, Saint-Petersburg, Russia, Saint Petersburg, Russian Federation Introduction: Based on significant improvement in outcomes of haploidentical hematopoetic stem cell transplantation (HSCT) using post-transplantation cyclophosphamide (Cy), we conducted a pilot study to evaluate if this strategy could be translated to unrelated HSCT. Materials (or patients) and methods: The study group (Cy group) included 50 adult patients undergoing unrelated peripheral blood HSCT. Graft-versus-host disease (GVHD) prophylaxis for the Cy group consisted of Cy 50 mg/kg/day on day þ 3 and þ 4, tacrolimus (Tac) starting on day þ 5 and mycophenolate mofetil (MMF) from day þ 5 until day þ 35. Control group was a historical control of 176 patients with antithymocyte globulin (ATGAM) 60 mg/kg, Tac and MMF, or short-course methotrexate, as GVHD prophylaxis. Detailed patients’ characteristics and analysis of groups’ differences are presented in table 1. Results: The median follow-up was 155 days (range, 14 - 493) for the Cy group and 348 days (range, 5 - 1693) for the Control group. Engraftment was achieved in 92% vs 89.2% (P40.05) in the Cy and control groups respectively. In univariate and multivariate analyses Post-transplantation Cy was associated with significantly reduced incidence of grade II-IV (16% vs 46.6%, HR ¼ 0.242, 95% CI 0.120-0.516, P ¼ 0,0001) and grade III-IV (2% vs 23,3%, HR ¼ 0.053, 95% CI 0.007–0.387, P ¼ 0,004) acute GVHD. The cumulative incidence of chronic GVHD was not significantly different between groups (22% vs 39.3% P ¼ 0.27), but in the Cy group there was a significant reduction of severe chronic GVHD (0% vs 20.8%, P ¼ 0.005; the NIH global severity scoring of cGVHD). As a result there was significant reduction of 1-year nonrelapse mortality (10% vs 31.8%, HR ¼ 0.363, 95% CI 0.140–0.942, P ¼ 0.037), and improvement in 1-year overall survival (80% vs 53.4%, HR ¼ 0.448, 95% CI 0,227-0,886, P ¼ 0.021) and event-free
survival (74% vs 43.2%, HR ¼ 0.370, 95% CI 0.202–0.677, P ¼ 0.001). Moreover, the anti-leukemic effect of HSCT was not compromised and 1-year relapse rate was not different between groups (16% vs 24.4%, P ¼ 0.4). The median time to achieve an unsupported absolute neutrophil 4500 and platelet count 425 was not statistically different: 21 (range, 11-33) and 14 (range, 1039) days versus 21 (range, 8-70) and 14 (range, 8-87) days, respectively (P40.05). The toxicity of two GVHD prophylaxis regimens was comparable. There was a trend for a lower probability of fungal infections in the first 6 months posttransplantation in the Cy group (8% vs 18,2%,P ¼ 0,09), but the incidence of viral cystitis was significantly higher (22% vs 10.8%, HR ¼ 2.352, 95% CI 1.110-4.982, P ¼ 0.026). Conclusion: With relatively short follow-up post-transplantation Cy is a promising GVHD prophylaxis for unrelated HSCT. Larger studies with longer follow-up are required to confirm these preliminary results. Disclosure of Interest: None declared. P097 Urinary proteomics for aGvHD-prediction compared to ELISA for aGvHD-related proteins C. Human1,*, J. Metzger2, S. Ehrlich1, P. Schweier1, I. Tu¨ru¨chanow1, A. Ganser1, C. Falk3, E. M. Weissinger1 1 Hematology, Hemostasis, Oncology and Stem cell transplantation, Hannover Medical School, 2mosaiques GmbH, 3Integrated Research and Treatment Centre Transplantation – IFB-Tx, Hannover Medical School, Hannover, Germany Introduction: Graft-versus-host disease (GvHD) is a major complication after hematopoietic stem cell transplantation (HSCT). Prediction of aGvHD can be achieved by monitoring urine for excretion of peptides specific for aGvHD (aGvHDMS17) (1). In an effort to harmonize aGvHD prediction/ diagnosis, we analysed plasma and urine in parallel in more than 60 patients using an ELISA for published proteins(2) and Multiplex analysis of the angiogenesis panel (3). Materials (or patients) and methods: The test set consisted of 11 patients with and 12 without aGvHD, validation was performed on 31 patients. Plasma and urine were collected prospectively from 31 patients (1). While urine can be used as is and data can be normalzied using internal standard
peptides, plasma has to be serial diluted prior to analyses. Two peptides, CD99 and beta-2-microglobulin (b2m) can also be detected in plasma by ELISA. The ELISA-panel consisted of Elafin, IL-2 receptor alpha (IL-2sRa), REG-3a, ST2 and soluble TNF RI; IL-8 and hepatocyte growth factor (HGF) were analysed by multiplex and the outcome of all assays was compared. Results: While aGvHD_MS17 predicted pending aGvHD prior to the clinical diagnosis, most of the plasma biomarkers did not. In the test set IL-2R alpha, ST2 and sTNF RI, beta2-m and CD99 seemed predictive/diagnostic for aGvHD based on receiver operated characteristic (ROC) curves and areas under the curve (AUC) with more than 80% (Po0.01). CD99 (whole protein) was specific for chronic GvHD and could distinguish between patients with acute and chronic GvHD and those without with high sensitivity (70%) and specificity (93%) and AUC was 85% (P ¼ 0.0007). However, in the validation set most of the markers (especially Elafin, REG3-alpha) did not reach significance for prediction or even diagnosis of aGvHD at the time of biopsy proven aGvHD. Both markers are organ-specific – but limiting the analysis to patients with GvHD of the intestine (REG3-alpha) or skin (Elafin) did not increase sensitivity or specificity, albeit the number of patients analysed is still small. aGvHD_MS17 was positive for pending aGvHD, but none of the ELISA markers were reliable for diagnosis (cut off indicated by dotted line). Conclusion: Prediction of pending aGvHD using aGvHD_MS17 is possible with a sensitivity of 82% and specificity of 77% for aGvHD (1). In contrast, plasma biomarkers in our validation set lack sensitivity and specificity. Two biomarkers, CD99 and b2m could not be used to predict aGvHD-development when analysed in plasma. This indicates that the particular fragments excreted in urine are predictive, not changes in plasma protein levels. Taken together our results demonstrate that plasma monitoring of patients post-allogeneic HSCT seems less reliable and more time consuming and costly, as previously envisioned, than urinary proteomics for the prediction of aGvHD. References: 1. Weissinger EM, Metzger J, Dobbelstein C, et al. Leukemia 2014; 28(4): 842-852. 2. Paczesny S, Krijanovski OI, Braun TM, et al. Blood 2009; 8; 113(2): 273–278. 3. Luft T, Dietrich S, Falk C, et al. Blood 2011; 11;118(6): 1685– 1692.
[P097]
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Disclosure of Interest: C. Human: None declared, J. Metzger Employee of: mosaiques GmbH, S. Ehrlich: None declared, P. Schweier: None declared, I. Tu¨ru¨chanow: None declared, A. Ganser: None declared, C. Falk: None declared, E. Weissinger: None declared P098 The impact of donor specific anti-HLA antibodies on the outcome of allogeneic stem cell transplantation C. Kekik1,*, I. Yonal-Hindilerden2, F. Savran-Oguz1, S. Usta1, S. Temurhan1, D. Batu-Demir2, S. Kalayoglu-Besisik2, M. Aktan2, D. Sargin3 1 Istanbul University Istanbul Medical Faculty, Department of Medical Biology, 2Istanbul University Istanbul Medical Faculty, Department of Internal Medicine, Division of Hematology, 3 Medipol University, Department of Internal Medicine, Division of Hematology, Istanbul, Turkey Introduction: Graft failure and graft-versus-host disease (GVHD) are important life-threatening complications after allogeneic hematopoietic stem cell transplantation (AHSCT). In recent studies, GVHD and graft failure have been associated with the presence of donor specific anti-HLA antibodies in patients undergoing AHSCT from HLA-mismatched donors. We aimed to examine the impact of donor specific anti-HLA antibodies detected after AHSCT on the development of acute/chronic GVHD and graft failure. Materials (or patients) and methods: Anti-HLA antibodies were analysed by ELI˙SA/LUMINEX in a total of 74 patients (F/ M:30/44), who were transplanted at the Adult Hematopoietic Stem Cell Transplantation Unit of Istanbul Medical Faculty between 1996 and 2013. Results:
Anti-HLA class I (þ) Anti-HLA class II (þ)
aGVHD (þ) n:20
aGVHD (-) n:54
P value
cGVHD (þ) n:23
cGVHD (-) n:51
P value
4
7
40.05
1
10
40.05
3
4
40.05
1
6
40.05
12.1% of patients had an unrelated donor (10/10 matches, n ¼ 4; 9/10 matches, n ¼ 5) and the remaining patients had a matched sibling donor. Anti-HLA Class I antibodies were detected in 11 of 74 (14.8%) patients, 63% of whom were female. Anti-HLA Class II antibodies were detected in 7 of 74 (9.4%) patients, 71% of whom were female. 5 patients (6.7%) showed the copresence of anti-HLA Class I and II antibodies. The frequency of anti-HLA Class I antibodies was significantly higher than the copresence of anti-HLA Class I and II antibodies (14.8% and 6.7%, respectively; P ¼ 0.04). The frequency of antiHLA antibodies was significantly higher in female patients. Acute GVHD and chronic GVHD were observed in 27% (20/74) and 31% (23/74) of patients, respectively (Table 1). Ten of 74 patients (13.5%) died after AHSCT. Four of the patients carrying anti-HLA Class I antibodies and 2 of the patients showing the copresence of anti-HLA Class I and II antibodies died. Mortality rate was significantly higher in patients carrying anti-HLA Class I antibodies compared to those without antibodies (P ¼ 0.0361). There is no significant difference in mortality rate between patients with and without anti-HLA Class II antibodies (P40.05). Presence of anti-HLA antibodies did not contribute to development of acute/chronic GVHD and graft failure (P40.05). Conclusion: Our data demonstrate anti-HLA Class I antibodies have a significant impact on mortality. Previous studies have shown that presence of anti-HLA antibodies is associated with GVHD and graft failure in HLA-mismatched AHSCT. Our study aimed to evaluate the impact of anti-HLA antibodies on AHCST outcome regardless of HLA matching and consequently donor specific anti-HLA antibody status. In conclusion, our data point
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out that anti-HLA antibodies detected after AHSCT have no significantly effect on the development of GVHD and graft failure. Moreover, our results imply that anti-HLA Class I antibodies may have an influence on overall survival. We suggest that anti-HLA antibodies may play an important role on the outcome of HLA-matched AHSCT as demonstrated previously for HLA-mismatched AHSCT. Further studies including larger number of patients are warranted. References: 1- Taniguchi K, Yoshihara S, Maruya E, Ikegame K, Kaida K, Hayashi K, Kato R, Inoue T, Fujioka T, Tamaki H, Okada M, Onuma T, Fujii N, Kusunoki Y, Soma T, Saji H, Ogawa H. Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors. Bone Marrow Transplant. 2012 Oct; 47(10): 1338–1342. Disclosure of Interest: None declared.
P099 Extracorporeal photopheresis for cGVHD with lung involvement C. Del Fante1,*, P. Bernasconi2, L. Scudeller3, G. Viarengo1, A. A. Colombo2, D. Caldera2, F. Ripamonti2, C. Perotti1 1 Immunohaematology and Transfusion Service, Centre for transplant immunology, 2Division of Haematology, 3Biometry and Statistics, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Introduction: Lung GVHD (LG) is an important cause of morbidity and mortality despite immunosuppressive therapy (IST). Bronchiolitis obliterans syndrome (BOS-obstructive form) and restrictive lung disease (RLD-restrictive form) represent the two forms of lung dysfunction. Spirometry, and in particular Forced Expiratory Volume in 1 second (FEV1), is used to monitor lung function over time. We monitored FEV1 trend over time in 13 patients (pts) with different oncohematological disorders who underwent off-line extracorporeal photopheresis (ECP) for LG after allo-BMT. Materials (or patients) and methods: We retrospectively analyzed 13 pts (mean age 45, 46% females) who developed LG not responsive to standard therapy undergoing off-line ECP from 2012 to 2014. ECP was initially performed 2 times/week for 3 weeks, then 2 times every other week for 3 times, then maintained 2 times/month. Clinical conditions, spirometry and blood biochemistry were checked at least every 3 months, or more frequently whenever clinically indicated. Response was defined as stable/improved FEV1 from baseline. Results: Underlying disease was AML in 6 pts (46%), ALL in 2 (15%), AREB , HL, MF, NHL, THAL in 1 pts each. Cell source was bone marrow in 2 pts (15%) and peripheral blood in 11; donor was related in 7 (54%) and unrelated in 6. Conditioning was myeloablative in 10 and RIC in 3. cGvHD was classic in 7 pts (54%), overlap in 5 (38%) and late acute in 1. Median time from transplant to LG was 14 months. All pts had BOS; grade 3 developed in 8 pts (61%) and grade 2 in 5. Median time from LG to ECP was 3.4 months. Median FEV1 at ECP was 1.76 L (IQR 1.51-2.24). Cumulative follow-up under ECP was 130 pts-months. Median ECP number was 29 (min-max 956). ECP was well tolerated. Ten pts (77%) achieved a response, always preceded by a subjective improvement of dyspnoea. Median time to response was 4.6months (IQR 4.5-8.3). None died. Conclusion: Our preliminary results show that ECP in association to standard IST may be effective in slowing/ stopping lung function decline in pts with cGVHD-related BOS. References: Kooke KR. A window of opportunity for patients with late-onset pulmunary dysfunction after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2014;20:291-94. Disclosure of Interest: None declared.
P100 Parameters of Protein Metabolism and Thyroid Function As Predictors in a Scoring System for Acute and Chronic Graft-Versus-Host Disease C. Skert1,*, A. Turra1, M. Malagola1, S. Perucca1, V. Cancelli1, R. Daffini1, R. Ribolla1, C. Bergonzi1, C. Filı`1, C. Pagani1, A. Di Palma1, F. Cattina1, S. Bernardi1, D. Russo1 1 Chair of Hematology, Bone Marrow Transplant Unit, University of Brescia, AO Spedali Civili Brescia, Brescia, Italy Introduction: Several studies reported ‘‘classical’’ patient-, donor- and transplant characteristics, such as age, gender disparity, donor type, HLA-match, and source of stem cells as predictors for acute and chronic GVHD. However, no studies analysed these ‘‘classical’’ variables together with parameters of metabolic and endocrine functions, which may potentially influence the immune system. Patient-and transplant variables together with index of liver and thyroid function, and some parameters of protein and lipid metabolism were retrospectively evaluated at different time points after transplantation, in order to identify possible predictors of acute and chronic GVHD and to calculate a risk score. Materials (or patients) and methods: Clinical and transplant characteristics, number and type of infections before and after stem cell transplantation (SCT) were analysed in 161 patients. The following variables were also analysed pre-SCT, at day þ 7, þ 14, þ 21, þ 28, at þ 3 and þ 6 months: LDH, parameters of liver function; parameters of protein and lipid metabolism (serum albumin, urea, total protein, cholesterol, triglycerides); thyroid function tests; autoimmune parameters; body mass index. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, in order to solve the problem of the high number of variables in comparison with the relatively limited and heterogeneous pool of patients. Based on the regression coefficient of Cox analysis for each significant predictor, a scoring system for acute and chronic GVHD was calculated. Results: In multivariate analysis, diagnosis of Myelodisplastic Syndrome or Chronic Myeloid Leukemia (P ¼ 0.0004), conditioning regimen including Total Body Irradiation (P ¼ 0.0003), and pre transplantation urea 434 mg/dl with þ 21 day urea 454 mg/dl (P ¼ 0.0008) were evidenced as predictors for acute GVHD. Score values for each factor are 2, 1, and 1, respectively. The probability of acute GVHD ranged from 8% (score 0) to 98% (score 4). Female donor (P ¼ 0.0008), pre-SCT TSH values Z2 m/L with þ 28 day urea Z 39 mg/dl (P ¼ 0.02), þ 6 month total proteino5,5 g/dl with gamma-GT Z347 U/L (P ¼ 0.0001) resulted predictors for moderate/severe chronic GVHD, with score values of 1, 1, and 2 respectively. Risk of chronic GVHD at þ 6,5 month ranged from 3% (score 0) to 97% (score 4). Conclusion: Our study evidenced that factors other than ‘‘classical’’ ones may be associated to GVHD. Our scoring system includes routine-parameters, which are easily available in clinical practice. Urea levels depend on the balance between protein intake, endogenous catabolism and urinary excretion. The inflammatory microenvironment of GVHD promotes muscle catabolism, hence, urea levels do increase. Increased urea levels could be an indirect index of increased uremic toxins as well, which may stimulate the production of pro-inflammatory cytokines and the activation of leukocytes. On the other hand, increased urea levels and uremic toxins could derive from a dysregulated metabolism of the gut microbiome, which may influence immune system. Our findings, which require a prospective validation, suggest the usefulness of a deeper study of the complex network between metabolic/endocrine functions and immune system, in order to develop a holistic approach of the transplant management. Disclosure of Interest: None declared.
P101 Prophylaxis with Rifaximin preserves microbiome diversity in ASCT ¨ fner3, J. Hahn1, K. Peter1, D. Sporrer1,*, J. Ko¨stler2, A. Gessner2, P. O M. Kreutz1, W. Herr1, E. Holler1 1 Internal Medicine III, 2Microbiology, 3Functional Genomics, University Hospital Regensburg, Regensburg, Germany Introduction: Microbiome disruptions seem to be associated with intestinal acute graft versus host disease (aGvHD) and poor outcome after allogeneic stem cell transplantation (ASCT) [1]. In previous studies we found a major impact of broad spectrum antibiotics for prophylaxis and therapy of neutropenic infections on loss of intestinal bacterial diversity as measured by enterococcal overgrowth and low urinary indoxyl sulfate levels (UIS). Therefore, we switched antibiotic prophylaxis to Rifaximin, a non-absorbable gut decontamination, and now retrospectively analyzed its effect on intestinal diversity and clinical risk for neutropenic infections during ASCT. Materials (or patients) and methods: A total of 333 patients undergoing ASCT were included in the analysis. Two hundred patients received the conventional antibiotic prophylaxis with Ciprofloxacin and Metronidazole whereas 133 patients received Rifaximin at a dose of 200 mg twice daily. Prior to admission until four weeks after ASCT stool and urine specimens were collected weekly. Fecal enterococcal concentrations were measured using strain specific PCR and UIS levels were determined using chromatographytandem mass spectrometry. Enterococcal overgrowth was defined as conversion from negative to positive values or increase in copy numbers. Besides, we analyzed the incidence of infections in relation to the type of combined prophylaxis. Statistical analyses were performed using IBM SPSS Statistics 24. Results: In the Rifaximin group conversion to positivity for both germs, Enterococcus (E.) faecalis and E. faecium, was less frequent than in the standard group (P ¼ 0.05). Similarly, during ASCT, we observed higher UIS levels under the use of Rifaximin than under standard prophylaxis (Po0.001) (Table 1). The additional application of systemic antibiotics in neutropenia was similar in both groups with 93.2% of patients with Rifaximin and 91.0% of patients with standard prophylaxis. Even after application of systemic antibiotics, the protective effect of Rifaximin on bacterial diversity seemed to be maintained, as UIS levels were still higher than in patients receiving standard prophylaxis without systemic antibiosis. Regarding overt infectious complications we did not find higher infection rates, neither for gram-negative germs under Rifaximin. Simultaneously to the change of antibiotic decontamination a high dose supplementation with Vitamin (Vit) D had been initiated in our unit. As high Vit D levels seem not only to have tolerogenic immune effects but also to maintain bacterial diversity by induction of antimicrobial peptides, we also analysed the impact of VitD levels on bacterial diversity but did not observe an additional effect beyond Rifaximin. Conclusion: In conclusion, in a large cohort of patients undergoing ASCT we found a correlation between the use of Rifaximin and predictors of bacterial diversity such as lower intestinal enterococcal overgrowth and higher UIS levels.
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Additionally, Rifaximin seemed to exert a protective effect on intestinal microflora and to maintain a complex bacterial diversity despite the use of systemic antibiotics. Randomized studies are needed to define the definitive contribution of Rifaximin versus Vit D and to demonstrate the impact of this approach on overall outcome. References: 1. Taur, Y., et al., The effects of intestinal tract bacterial diversity on mortality following allogeneic hematopoietic stem cell transplantation. Blood, 2014; 124(7): 1174-1182. Disclosure of Interest: None declared. P102 Oral Candida species colonization in chronic Graft-versusHost Disease D. Dusek1,*, M. Mravak Stipetic2, M. Grce3, D. Pulanic4,5, L. Grkovic6, R. Serventi Seiwerth6, T. Klepac Pulanic7, R. Ceovic5,8, S. Bukovski9, M. Jelic9, M. Cindric3, L. Rajic5,10, E. Bilic5,10, Z. Peric5,6, N. Durakovic5,6, A. Vince1,5, R. Vrhovac5,6, S. Z. Pavletic11, D. Nemet5,6 1 University Hospital for Infectious Diseases, 2School of Dental Medicine University of Zagreb, 3Division of Molecular Medicine, Rudjer Boskovic Institute, 4Division of Hematology, Department of Internal Medicine, University Hospital Centre Zagreb, 5Medical School University of Zagreb, 6Division of Hematology, Department of Internal Medicine, University Hospital Center Zagreb, 7 Community Health Center Zagreb East, 8Department of Dermatovenerology, University Hospital Center Zagreb, 9Department of Microbiology, University Hospital for Infectious Diseases, 10 Department of Pediatrics, University Hospital Center Zagreb, Zagreb, Croatia, 11National Cancer Institute, National Institutes of Health, Bethesda, United States Introduction: Colonization with Candida sp has significant role in occurrence and pathogenesis of acute Graft-versusHost Disease (aGVHD) via induction of mucosal innate immunity (Th 17/IL 23 responses). However, data about prevalence and role of Candida sp in chronic Graft-versusHost Disease (cGVHD) are scarce. The aim of this study was to determine prevalence of oral colonization with different Candida sp in patients with cGVHD and possible association with cGVHD characteristics. Materials (or patients) and methods: This study enrolled cGVHD patients who are part of a larger multidisciplinary cGHVD project at the University Hospital Center Zagreb, Croatia. Patients were evaluated for clinical and cGVHD characteristics; severity of cGVHD and oral involvement were determined by using established NIH Consensus criteria. Swabs from oral cavity were collected from all patients. Specimens were analyzed by standard cultivation methods (Sabouraud and blood agar, identification by automated biochemical test). Molecular detection was performed by real-time in house PCR designed to distinguish 12 Candida species. Candida isolates were analyzed at proteomic level by Mass Spectrometry (MS/MS). Data were evaluated descriptively. Fisher’s exact test or Wilcoxon test were used to calculate statistical significance. Results: Twenty-one patient was enrolled, median age was 42 years 38% were male. In 15 patients (71%) underlying disease was AML, ALL or MDS, 3 patients had CML or MPN, 1 patient had severe aplastic anemia and 2 patients had lymphoma. Nine patients (43%) underwent myeloablative conditioning. Majority (71%) received PSBC as stem source. Sixteen patients (76%) had previous aGVHD. Ten patients (48%) had oral cGVHD; 6 patients had moderate or severe NIH oral cGVHD score. Nine patients (43%) had moderate and 12 (57%) had severe NIH global cGVHD score. Six patients (29%) were positive for C. albicans and 3 patients for C. krusei by standard cultivating methods. When using real- time PCR method, 13 patients (61%) were positive for C. albicans, 4 patients for C. krusei, 2 patients with C. dubliensis, 1 patient with C. tropicalis and 1 patient with C. glabrata. Coinfection with 2 or 3
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different Candida sp. was found in 6 patients. Ten samples were analyzed by proteomics with 5 samples confirmed as C. albicans. Although more patients were positive for C. albicans and C. non albicans sp by multiplex PCR than cultivation, this was not statistically significant (P ¼ 0.335 and 0.184, respectively). Nine patients with oral cGVHD (90%) had colonization with at least one Candida sp, while 7 patients without oral cGVHD (64%) had colonization with Candida sp (P ¼ 0.311). Ten patients with global severe cGVHD (83%) and 5 patients with moderate cGVHD (55.5%) had Candida sp colonization (P ¼ 0.331). Conclusion: Significant proportion of patients with cGVHD, especially patients with severe global cGVHD were colonized with Candida sp. Use of molecular methods is important in diagnosing C.non albicans (3 new species identified only by PCR). Majority (90%) of patients with oral cGVHD were colonized with Candida sp, however larger studies are needed to determine association between cGVHD global severity and organ involvement with Candida colonization. Considering significant proportion of cGVHD patients colonized with Candida sp, further studies investigating its possible role in pathogenesis of cGVHD, as well as possible prophylactic/ therapeutic are needed. Disclosure of Interest: None declared.
P103 The use of hyperbaric oxygen therapy in the treatment of hemorrhagic cystitis after allogeneic stem cells transplantation from unrelated donor D. Urbaniak-Kujda1,*, J. Dybko1, M. Biernat1,2, K. Kapelko-S"owik1, M. Laszkowska1, E. Stefanko1, M. Biedron´1, I. Deren´-Wagemann1, T. Wro´bel1, K. Kuliczkowski1 1 Department and Clinic of Haematology, Blood Neoplams and Bone Marrow Transplantation, Wroclaw Medical University, Wroc"aw, 2Deparment of Microbiology, Wroclaw Medical University, Wroclaw, Poland Introduction: Hemorrhagic cystitis (HC) is a diffuse inflammation of the bladder of an infectious or non-infectious etiology, causing bleeding of the bladder mucosa. HC may be caused by different factors including: medications: (busulfan, endoxan, idarubicin, carboplatin), radiation, viruses, and chemicals. Treatment guidelines of HC has not yet been established. Recent studies have demonstrated the efficiency of hyperbaric oxygen therapy (HOT) in patients suffered from HC after allogeneic HSCT (1,2). Materials (or patients) and methods: Patient characteristics is shown in Table 1. Material and methods: We retrospectively analyzed the effectiveness of HOT in 5 patients in the years from 2012 to 2014. Among patients there were 3 men, aged from 31 to 41 years and 2 women aged from 40 to 43 years. Patients characteristics is shown in Table 1. Results: Patient No
1
Age Sex Diagnosis Clinical (years) status
40
F
MDS
CR1
Condidtioning regimen
BuCyTym
2
41
M
AML
CR1
BuCyTym
3
39
M
ALL
CR1
TBI, CyTym
4
43
F
AML
CR1
BuCyTym
5
31
M
AML
CR1
BuCyTym
Source GvHD for prophylaxis alloHSCT PBSC, MUD PBSC, MUD PBSC, MUD PBSC, MUD PBSC, MUD
C, MTX, Gluc C, MTX, Gluc C, MTX, Gluc C, MTX, Gluc C, MTX, Gluc
Abbreviations: C ¼ cyclosporine, Tym ¼ tymoglobuline, Gluc ¼ glucocorticoids, MUD ¼ matched unlrelated donor, MTX ¼ metotrexate,
Results: The median time to onset of HC after allogeneic stem cell transplantation was 22 days (range, 11-55 days). In one patient, the symptoms did not appear until the day 55 after transplantation. Despite immunosuppressive therapy, all patients had macroscopic hematuria and GvHD. The BKV and ADV DNA were detected in urine and plasma samples in 2 patients, ADV DNA was detected in urine and plasma in 2 other patients, and in the one patient, BKV was detected in both samples, respectively. The patients were treated with HOT (2.5 atmospheres for 60 minutes, 5 days per week) after treatment failure of bladder drainage flow, hyaluronic acid administered intravesically, as well as antiviral treatment. All patients showed complete resolution of hematuria and eradicaton of the virus after a median of 13 sessions (range, 11-30) of HOT. Conclusion: HBO may be an alternative and promising therapy in the treatment of severe hemorrhagic cystitis. References: 1.Savva-Bordalo J., Pinho Vaz C., Sousa M., Branca R., Campilho F., Resende R., Baldaque I., Camacho O., Campos A. Clinical effectiveness of hyperbaric oxygen therapy for BKvirus-associated hemorrhagic cystitis after allogeneic bone marrow transplantation. Bone Marrow Transplnatation 2012; 47: 1095–1098. 2. Dellis A., Deliveliotis Ch., Kalentzos V., Vavasis P., Skolarikos A. Is there a role for hyperbaric oxygen as primary treatment for grade IV radiation-induced haemorrhagic cystitis? A prospective pilot-feasibility study and review of literatur˛e. Int Braz J Urol. 2014; 40: 296–305. Disclosure of Interest: None declared.
better OS in comparison to those with lack of response. Also, pts with aGvHD grade IV had significantly worse five-year outcome. Conclusion: aGvHD have significant impact on the morbidity after allo SCT (incidence is 58,5%). Predictors of incomplete response to treatment are advanced grade of aGvHD, PB as a source of SC, sex mismatch (female donor to male recipient), donor- recipient ABO incompatibility and CML at diagnosis. Pts with advanced grade of aGvHD have less probability of OS. Disclosure of Interest: None declared.
P104 Acute Graft- versus-Host Disease predictors and outcomeour experience D. Stamatovic1,*, M. Elez1, O. Tarabar1, S. Marjanovic1, B. Balint2, O. Tasic Radic3, Z. Tatomirovic3, A. Zivanovic Ivic1, L. Tukic1 1 Clinic of hematology, 2Institute for transfuziology, 3Institute for pathology, Military Medical Academy, Belgrade, Serbia
P105 Von Willebrand Factor and Factor VIII as potential biomarkers of chronic Graft-versus-Host Disease D. Pulanic1,*, L. Grkovic2, R. Serventi Seiwerth2, M. Mravak Stipetic3, E. Bilic4, R. Ceovic5, Z. Peric1, L. Rajic6, N. Durakovic1, N. Matic2, T. Klepac Pulanic7, I. Petricek8, T. Vukic9, E. Bilic6, D. Dusek10, E. Prenc11, I. O. Prah11, I. Bojanic12, M. Grce13, R. Zadro14, D. Batinic14, R. Vrhovac1, S. Z. Pavletic15, D. Nemet1 1 Division of Hematology, Department of Internal Medicine, University Hospital Center Zagreb and Medical School University of Zagreb, 2Division of Hematology, Department of Internal Medicine, University Hospital Center Zagreb, 3School of Dental Medicine University of Zagreb, 4Department of Neurology, 5 Department of Dermatology, 6Department of Pediatrics, University Hospital Center Zagreb and Medical School University of Zagreb, 7Community Health Center Zagreb East, 8Department of Ophtalmology, 9Department of Rheumatology and Rehabilitation, University Hospital Center Zagreb, 10University Hospital for Infectious Diseases, 11Medical School University of Zagreb, 12 Department of Transfusion Medicine, University Hospital Center Zagreb, 13Division of Molecular Medicine, Rudjer Boskovic Instute, 14 Department of Laboratory Medicine, University Hospital Center Zagreb and Medical School University of Zagreb, Zagreb, Croatia, 15 National Cancer Institute, National Institutes of Health, Bethesda, United States
Introduction: Acute Graft-versus-Host disease (aGvHD) is an immunologycally mediated inflammatory reaction which remains one of the major limiting factor in succesful allogeneic hematopoietic stem cell transplantation (HSCT). The disease should be defined based on a clinical and pathological features and not solely on the day of its appearence. Standard treatment for aGvHD consist of corticosteroids, although there is a lack of consensus over incidence and predictors of outcome. The goal of our retrospective study were to estimate incidence, type and severity of aGvHD, risk factors for treatment outcome and impact of aGvHD on the overall survival (OS). Materials (or patients) and methods: We have analyzed 222 patients (pts), female/ male ratio 90/132, average age 29 (range 9- 58) with different hematological malignancies (sAA 27, CML 33, AML 65, ALL 70, MDS/ MPN 14, HL/ NHL 6, MM 3, GrSa 3, Thalassemia major 1) in whom we have performed allogeneic HSCT from the identical sibling donors in the previous 15 years. Conditioning regimens were adjusted to the primary diseases and stem cell source was peripheral blood (PB) in 141 and bone marrow (BM) in 81 pts. GvHD prophylaxis was mostly combination of Cyclosporin A (CsA) and Metothrexate (Mtx). Results: aGvHD of any grade was documented in 130 (58,5%) pts, while grade II/IV had 83 (37,4%) pts with onset approximately on the day þ 41. (range 12- 179), while 10 pts (4,9%) had hyper acute form (before day þ 14.). Mostly involved organs were skin (50%), GIT (23,7%), liver (16,2%) or combinations. Predictors of incomplete response to steroid therapy were: advanced grade of aGvHD (II vs. III/IV; P ¼ 0.03), sex mismatch between donor and recipient (female to male vs. other; P ¼ 0.03), stem cell source (BM vs. PB; P ¼ 0.04) and diagnosis (CML vs. other; P ¼ 0.04). Pts with aGvHD that had favorable response to therapy had significantly
Introduction: Predictive, diagnostic, and risk stratification biomarkers of chronic Graft-versus-Host disease (cGVHD) are critically needed to improve treatment of this serious late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Since elevated von Willebrand’s factor (VWF) and factor VIII (FVIII) levels have been described as indicators of endothelial dysfunction and inflammation in different settings, the goal of this study was to assess possible role of VWF and FVIII as potential biomarkers of cGVHD. Materials (or patients) and methods: Multidisciplinary cGVHD clinical infrastructure was organized in 2013 at the Division of Hematology, University Hospital Center Zagreb, Croatia, using established cGVHD-related scales and measurements in collaboration with the National Cancer Institute, National Institutes of Health (NIH), USA. An extensive demography, history, physical and laboratory evaluations were obtained in this cross-sectional prospective study using standardized methods, including measurement of VWF:Ag, VWF:RCo, and FVIII. Descriptive statistic and non-parametric analyses were performed, results with p-valueso0.05 were considered as statistically significant, and variables with Spearman rank correlation coefficient 0.5o|r|o0.7 were considered as moderately correlated. Results: Twenty cGVHD patients were assessed (median age 38.5 years, 55% females). Median time from HSCT to study enrollment was 16.4 months. Median time from HSCT to cGVHD diagnosis was 9.5 months, and median time from cGVHD diagnosis to study enrollment was 5.5 months. Majority of patients (90%) were transplanted for hematologic malignancies. Myeloablative conditioning was used in 50%, 11 (55%) received peripheral blood stem cells, and 12 (60%) transplants were from matched related donors. Eight patients had de novo cGVHD, 5 quiescent, 7 progressive, and 16 (80%)
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classic cGVHD. Ten (50%) patients had moderate and 10 (50%) severe NIH global cGVHD score. Most involved organs were skin, lung, liver, and eyes. At the time of evaluation, clinician’s impression was in 50% that cGVHD was active. Elevated level of FVIII (median 191%, range 52-297%, reference range 50-149%) and VWF:Ag (median 208%, range 108-600%, reference range 50-160%) was found. Median level of VWF:RCo was 138% (range 75-547%, reference range 50-150%). Patients after unrelated HSCT had higher FVIII, VWF:Ag and VWF:RCo. Shorter time from cGVHD diagnosis to study enrollment was associated with higher VWF:Ag (P ¼ 0.015) and VWF:RCo (P ¼ 0.018). NIH cGVHD liver score (P ¼ 0.013), average NIH score (r ¼ 0.535), AST (r ¼ 0.668), ALT (r ¼ 0.603), beta-2 microglobulin (r ¼ 0.510), triglyceride (r ¼ 0.596) and cGVHD activity by therapeutic intent (P ¼ 0.019) were associated with higher FVIII. NIH cGVHD mouth score (P ¼ 0.016), clinician impression of cGVHD activity (P ¼ 0.023) and AST (r ¼ 0.563) were associated with higher VWF:Ag. Ferritin (r ¼ 0.506) was associated with higher VWF:RCo, while cGVHD activity by therapeutic intent, beta-2 microglobulin, D-dimers, and triglyceride were associated with both higher VWF:Ag and VWFR:Co. Other markers of inflammation (C-reactive protein, fibrinogen, erythrocyte sedimentation rate) did not correlate neither with VWF:Ag, VWF:RCo nor with FVIII. Conclusion: Results of this pilot study suggest that VWF and FVIII could represent interesting candidate biomarkers of cGVHD. However, these need to be further investigated in larger studies. Disclosure of Interest: None declared. P106 Protein expression in the setting of GvHD: CXCL9, ELAFIN AND REG3a E. Metafuni1,*, S. Giammarco1, D. De Ritis1, F. Sora`1, L. Laurenti1, S. Sica1, P. Chiusolo1 1 Hematology Department, Universita` Cattolica del Sacro Cuore, Rome, Italy Introduction: Graft-versus-host disease (GvHD) is the major life-threatening complication after allogeneic stem cell transplantation (SCT). There are no validated laboratory tests able to substitute the histopathology in GvHD diagnosis. In the last years, many authors focused their attention on serum protein expression in the setting of GvHD. Materials (or patients) and methods: We enrolled 36 patients (pts), 14F/22M, submitted to SCT in our centre from april 2010 to December 2013. Underlying diseases were: 5 ALL, 1 CLL, 18 AML, 1 CML, 4 NHL, 2 MPN and 5 MDS. Standard conditioning was performed in 12 pts and reduced intensity conditioning in 24 pts. Median number of CD34 þ in the graft was 5.3*106/Kg. Stem cell source were: 34 PB, 1 CB and 1 BM. Donor was sibling in 13 cases and MUD in the remaining 23 pts. GvHD prophylaxis: 28 CSA þ MTX, 7 CSA þ MMF, 1 Tacrolimus þ MTX. ATG was used in MUD
[P106]
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transplants. Statistical analysis was performed using IBM SPSS Statistics 22. Results: Acute GvHD (aGvHD) was detected in 24 pts at a median of 17 days after SCT: 9 grade I, 8 grade II, 5 grade III and 2 grade IV. Organ involvement: skin in 13 pts, skin and liver in 3 pts, skin and gut in 5 pts, skin, gut and liver in 3 pts. Chronic GvHD (cGvHD) was identified in 19 pts: 6 mild, 8 moderate and 5 severe grade. Organ involvement: skin and gut (1 pt), skin (7 pts), skin and liver (2 pts), skin, liver and gut (2 pts), liver (2 pts), gut and liver (1 pt), gut, skin and eyes (1 pt), 1 skin and mouth (1 pt), skin, liver, eyes and mouth (1 pt), skin and lungs (1 pt). CXCL9 and Elafin level were higher in pts who developed aGvHD than in the others: 462 pg/ml (range 2-1820) vs 215 pg/ ml (range 50-422) for CXCL9 (t-test, P ¼ 0.041) and 69000 pg/ ml (range 16000-165100) vs 41782 pg/ml (range 6125-92050) for Elafin (t-test, P ¼ 0.033). Figure 1. Among pts with aGvHD, CXCL9 and REG3a level was higher for skin involvement rather than hepatic or intestinal one: 499 pg/ml (range 2-1820) vs 51 pg/ml (range 6-96) for CXCL9 (P ¼ 0.002) and 1360 ng/ml (range 207-8443) vs 345 ng/ml (range 393-297) for REG3a (ttest, P ¼ 0.018). In the setting of cGvHD, REG3a level was higher for hepatic involvement rather than intestinal or cutaneous one, 791 ng/ml (range 2967-8443) vs 297 ng/ml (range 207-1241) (Mann Whitney, P ¼ 0.0157). Conclusion: CXCL9, Elafin and REG3a serum concentrations showed a correlation with the development of GvHD. Serum levels of these proteins were higher at the onset of aGvHD compared to pts who did not develop this complication. Moreover, we identified an organ-specificity in the setting of GvHD. In particular, CXCL9 and Elafin were overexpressed in pts with skin aGvHD, while REG3a was higher in pts with cutaneous aGvHD and hepatic cGvHD. In conclusion, our results suggest a potential role of these proteins as biomarker of GvHD, both for onset and organ involvement. However, there data need confirmation in a larger cohort of pts. Disclosure of Interest: None declared. P107 Gastro-intestinal microbiome diversity changes after allogeneic stem cell transplantation E. Metafuni1,*, F. Paroni Sterbini2, S. Giammarco1, S. Sica1, L. Masucci2, P. Chiusolo1 1 Hematology Department, 2Microbiology Department, Universita` Cattolica del Sacro Cuore, Rome, Italy Introduction: The immune response induced by gastrointestinal (GI) microbiome translocation through the intestinal barrier after stem cell transplantation (SCT) conditioning chemotherapy plays a key role in the pathophysiology of graft-versus-host disease (GvHD). Severe GI GvHD was closely related to an increased transplant related mortality (TRM). Recently, some data have been published regarding the marked reduction of microbiome diversity after SCT, and its
[P107]
relationship with post-SCT outcomes like as GvHD, infections and TRM. Materials (or patients) and methods: The aim of our study was to analyze the microbiome diversity before and after transplantation. We enrolled 8 patient (pts), 3F/5M, with AML submitted to allogeneic SCT (allo-SCT). A myeloablative conditioning was performed in 5 pts, while the other 3 received a reduced intensity regimen. Median dose of CD34 þ was 6.3*106/Kg, obtained in all but one (BM) cases from PB of sibling donor in 4 cases and MUD in the others. GvHD prophylaxis was performed with CSA þ MTX in 6 pts and CSA þ MMF in 2 pts. ATG were added for MUD transplantation. The control group included 3 pts with MM submitted to autologous SCT (auto-SCT). Timing for stool samples collection were: basal, day þ 15 and GvHD onset. Samples were stored at -801C until use. DNA was isolated with QIAamp DNA Stool Mini kit. The hypervariable V1-V3 region of the 16s RNA gene was amplified by PCR and sequenced on 454 GS Junior Titanium. Taxonomic composition was defined according to Greengenes database. IBM SPSS Statistics 22 was used for statistical analysis. Results: Five pts developed acute GvHD with the follow organ involvement: skin and gut (1), gut (1), skin (2) and liver (1). No difference was identified between auto-SCT and allo-SCT basal microbiome. At day þ 15 after auto-SCT, we detected a reduction of bacteroidetes and an increase of Proteobacteria (Wilcoxon matched-pairs signed rank test, P ¼ 0.0071). In alloSCT, at day þ 15 we identified an increase of Bacteroidetes and a reduction of Firmicutes (Paired t-test, P ¼ 0.0023). At the onset of GvHD, pts showed a reduction of Bacteroidetes and an increase of Firmicutes and Proteobacteria (t-test, P ¼ 0.0020). On the contrary, Bacteroidetes were more represented in pts with GI GvHD than in the others (t-test, P ¼ 0.0001). Figure 1. Conclusion: After auto-SCT there was an increase of Proteobacteria (Klebsiella, Proteus, Acinetobacter, Haemophilus, Pseudomonas, Enterobacteriaceae) and reduction of Bacteroidetes (Bacteroides, Saprospirae, Prevotella). After allo-SCT there was an increase of Bacteroidetes and Firmicutes (Bacilli, Lactobacilli, Clostridium, Enterococci, Streptococci). Moreover, pts who developed GvHD harboured more Firmicutes and Proteobacteria and less Bacteroidetes than pts without this complication. However, in pts with GI GvHD
Bacteroidetes were more represented than in pts with liver or skin involvement, although reduced if compared with the basal setting. Disclosure of Interest: None declared. P108 Prospective validation of a chronic GvHD-Specific Proteome Pattern (cGvHD-MS14) Post-Allogeneic Hematopoietic Stem Cell Transplantation E. M. Weissinger1,*, C. Human1, D. Wolf2, J. Metzger3, H. Greinix4, A. Dickinson5, I. Tu¨ru¨chanow1, P. Schweier6, H. Kreipe7, J. Raad3, A. Durban3, D. Ihlenburg-Schwarz1, Z. Kuzmina4, E. Holler2, J. Krauter8,9, C. Koenecke1, M. Stadler1, A. Ganser1 1 Hematology, Oncology, Hannover Medical School, Hannover, 2 University of Regensburg, Regensburg, 3mosaiques, Hannover, Germany, 4Medical University of Vienna, Wien, Austria, 5University of Newcastle, Newcastle, United Kingdom, 6Hematology, Oncology, Medical University of Vienna, 7Pathology, 8Hannover Medical School, Hannover, 9Hematology, Oncology, Klinikum Braunschweig, Braunschweig, Germany Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematologic malignancies and non-malignant hematopoietic disorders, but is associated with significant morbidity and mortality due to acute and chronic graft-versus-host disease (GvHD). Chronic GvHD (cGvHD) occurs with increasing frequency, hampering quality of life of patients post-allogeneic HSCT and leading to increased morbidity and mortality even years after transplant. Diagnosis of cGvHD is based on clinical features and histology. Here we report the generation of cGvHD-specific proteomic pattern (cGvHD-MS14) using capillary electrophoresis and mass spectrometry (CE-MS) in order to diagnose cGvHD, to differentiate acute from cGvHD, and to predict onset and severity of cGvHD prior to its clinical diagnosis as a noninvasive, unbiased laboratory test. Materials (or patients) and methods: cGvHDMS-14 was prospectively evaluated on 487 patients (1198 urine samples) after allogeneic HSCT at MHH and 3 collaborating transplant centres. The majority of the patients had acute leukemias prior to transplantation (n ¼ 304), were not in CR/CP (n ¼ 237) and were transplanted from matched (n ¼ 387) unrelated or
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[P108]
related donors (MUD n ¼ 317; MRD n ¼ 170). Reduced intensity conditioning regimens were used for 322 patients and the majority (n ¼ 363) received immunosuppressive antibodies as GVHD-prophylaxis prior to transplantation and a calcineurininhibitor based prophylaxis afterwards. Results: Prospective and blinded evaluation revealed the correct classification of patients developing cGvHD with a sensitivity of 78% and a specificity of 71% at time of diagnosis (Figure 1). Differentiation between late onset acute GvHD and cGvHD was achieved in 3 patients in this validation set. Acute GvHD prior to day 100 is not recognized by cGvHD-MS14, since aGvHD-specific peptides had been excluded during cGvHD-pattern generation. The pattern consists of 14 differentially excreted peptides, differentiating cGvHD from immune tolerant patients. Four of 14 peptides have been sequenced to date, 2 are fragments of collagen 1, 1 is from inter-alpha trypsin inhibitor heavy chain 4 and 1 is a fragment of the fibrinogen beta-chain. Conclusion: The proteomic pattern of urine enables diagnosis of cGvHD as well as differentiation of acute versus cGvHD. Further prospective evaluation of the cGvHD-specific pattern cGvHD-MS14 for organ specificity as well as severity prediction is currently ongoing. Taken together our results indicate that diagnosis of cGvHD is possible using CE/MS analysis of prospectively collected urine samples with high sensitivity and specificity. References: Weissinger EM, Metzger J, Dobbelstein C, et al Proteomic peptide profiling for preemptive diagnosis of acute graft-versus-host disease after allogeneic stem cell transplantation. Leukemia. 2014; 28(4): 842–852 Disclosure of Interest: E. Weissinger: None declared, C. Human: None declared, D. Wolf: None declared, J. Metzger Employee of: mosaiques GmbH, H. Greinix: None declared, A. Dickinson: None declared, I. Tu¨ru¨chanow: None declared, P. Schweier: None declared, H. Kreipe: None declared, J. Raad Employee of: mosaiques, A. Durban Employee of: mosaiques, D. Ihlenburg-Schwarz: None declared, Z. Kuzmina: None declared, E. Holler: None declared, J. Krauter: None declared, C. Koenecke: None declared, M. Stadler: None declared, A. Ganser: None declared
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P109 Prospective validation of a chronic GVHD-specific proteome pattern (cGVHD-MS14) post allogeneic hematopoietic stem cell transplantation E. Mischak-Weissinger1,*, C. Human1, D. Wolf2, H. Greinix3, J. Metzger4, A. M. Dickinson5, P. Schweier1, I. Tu¨ru¨chanow1, D. Ihlenburg-Schwarz1, H. Kreipe1, A. Krons4, H. Diedrich1, Z. Kuzmina3, E. Holler6, J. Krauter1, M. Stadler1, A. Ganser1 1 HANNOVER MEDICAL SCHOOL, Hannover, 2Innere Med. III, University of Regensburg, Regensburg, Germany, 3Medical University of Vienna, Wien, Austria, 4mosaiques diagnostics GmbH, Hannover, Germany, 5University of Newcastle, Newcastle, United Kingdom, 6University of Regensburg, Regensburg, Germany Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematologic malignancies and non-malignant hematopoietic disorders, but is associated with significant morbidity and mortality with focus on acute and chronic graft-versus-host disease (GvHD). Chronic GvHD (cGvHD) occurs with increasing frequency, hampering quality of life of patients post-allogeneic HSCT and leading to increased morbidity and mortality even years after transplant. Diagnosis of cGvHD is based on clinical features and histology. Here we report the generation of cGvHD-specific proteomic pattern (cGvHD-MS14) using capillary electrophoresis and mass spectrometry (CE-MS) in order to diagnose cGvHD, to differentiate acute from cGvHD, and to predict onset and severity of cGvHD prior to its clinical diagnosis as a non-invasive, unbiased laboratory test. Materials (or patients) and methods: This pattern was prospectively evaluated on 487 patients (1198 urine samples) after allogeneic HSCT at MHH and 3 collaborating transplant centres. The majority of the patients had acute leukemias prior to transplantation (n ¼ 304), were not in CR/CP (n ¼ 237) and were transplanted from matched (n ¼ 387) unrelated or related donors (MUD n ¼ 317; MRD n ¼ 170). Reduced intensity conditioning regimens were used for 322 patients and the majority (n ¼ 363) received immunosuppressive antibodies as GVHD-prophylaxis prior to transplantation and a calcineurininhibitor based prophylaxis afterwards. Results: Prospective and blinded evaluation revealed the correct classification of patients developing cGvHD with a sensitivity of 78% and a specificity of 71% at time of diagnosis (Figure 1). Differentiation between late onset acute GvHD and cGvHD was achieved in 3 patients in this validation set. Acute
GvHD prior to day 100 is not recognized by cGvHD-MS14, since aGvHD-specific peptides had been excluded during cGvHD-pattern generation. The pattern consists of 14 differentially excreted peptides, differentiating cGvHD from immunotolerant patients. Four of 14 peptides have been sequenced to date, 2 are fragments of collagen 1, 1 is from inter-alpha trypsin inhibitor heavy chain 4 and 1 is a fragment of the fibrinogen -chain. Conclusion: The proteomic pattern of urine enables diagnosis of cGvHD as well as differentiation of acute versus cGvHD. Further prospective evaluation of the cGvHD-specific pattern cGvHD-MS14 for organ specificity as well as severity prediction is currently ongoing. References: Weissinger E, Dobbelstein C, Metzger J, et al., Leukemia 2014 Disclosure of Interest: E. Mischak-Weissinger Conflict with: married with co-owner and founder of mosaiques, C. Human: None declared, D. Wolf: None declared, H. Greinix: None declared, J. Metzger: None declared, A. M. Dickinson: None declared, P. Schweier: None declared, I. Tu¨ru¨chanow: None declared, D. Ihlenburg-Schwarz: None declared, H. Kreipe: None declared, A. Krons Employee of: mosaiques GmbH, H. Diedrich: None declared, Z. Kuzmina: None declared, E. Holler: None declared, J. Krauter: None declared, M. Stadler: None declared, A. Ganser: None declared P110 Genital chronic Graft-versus-Host Disease in women - a population based prospective study E. Smith Knutsson1,*, Y. Bjo¨rk2, A.-K. Broman3, L. Helstro¨m4, M. Nicklasson5, K. Sundfeldt6, M. Brune7 1 Dept of Obstetrics and Gynecology, Nu Hospital Group, Trollha¨ttan, Sahlgrenska Academy, University of Gothenburg, Trollha¨ttan, 2Sahlgrenska University Hospital, Institution of Medicine, Sahlgrenska Academy, University of Gothenburg, Go¨teborg, 3NU-Hospital Organisation, Institution of Medicine, University of Gothenburg, Trollha¨ttan, 4dept of Clinical Science and Education , Karolinska Institutet, Stockholm, 5Sahlgrenska Akademin, Institution of Medicine, University of Gothenburg, 6 Dept of Obstetrics and Gynecology, Sahlgrenska University Hospital, Sahlgrenska Academy, University of Gothenburg, 7 Sahlgrenska University Hospital, University of Gothenburg, Go¨teborg, Sweden Introduction: Typically, chronic Graft-versus-Host Disease (cGvHD) causes inflammation and fibrosis in conjunctival and oral mucosa. In a recent cross-sectional population-based study we accounted for a high prevalence (52%) also of genital cGvHD with 21% cases having vaginal synechiae or stenosis. Concomitant symptoms included dryness, pain, and dyspareunia (Smith Knutsson et al 2014). We hypothesized that frequent gynecological examinations and early intervention with local treatment may improve signs and symptoms of genital cGvHD and reduce the risk of severe genital sequel in allografted women. Here we report preliminary results. Materials (or patients) and methods: In a prospective study in Region Va¨stra Go¨taland, Sweden we included 41 pts, median age 49 (17-66) yrs, i.e. 79% of all allografted women 2005/09 to 2010/02. Thirty pts were followed Z36 months, 11 pts were lost (7 relapsed, 4 other causes). At each visit, pts were asked for genital symptoms and had a structured examination with photo-documentation. In 29 pts 62 biopsies were obtained. Clinical scorings of genital cGvHD were made as per NIH criteria (Filipovich et al 2005), and vaginal strings were considered diagnostic signs. Extra-genital and genital cGvHD clinical scoring were combined for the assessment of global cGvHD. Local immunosuppressive ointments (clobetasol, tacrolimus) were prescribed at a structured scheme modified by each patient´s symptoms and signs. In case of vaginal cGvHD, a dilator was adviced to keep the vagina open. Estrogen was given locally to all pts and systemically in premature
menopause. Systemic corticoid steroids were prescribed by the hematologist. Results: The diagnosis of genital cGvHD was made at least once in 21 pts (51%), at a median 7 (1-30) mo after Tx; the cumulative incidence at 6, 12, 24 and 36 mo being 27%, 51%, 65% and 70%, respectively. Both vulvar and vaginal cGvHD developed in 11 pts, with start in the vulva in 8 pts, the vagina in 2, and at both locals in one pt. Ten pts had genital GvHD only in the vulva (n ¼ 5) or in the vagina (n ¼ 5). Two pts had only genital cGvHD; no other cGvHD. Partial stenosis developed in 3 pts in spite of ongoing systemic prednisone. Twenty pts did not develope genital cGvHD up to 36 mo (n ¼ 11), or up to lost follow-up (4-30 mo). Thirteen pts were treated locally. Median time to treatment was 10 mo. One pt developed a total vaginal stenosis. Conclusion: The aim of this study was to try and reduce signs, symptoms and sequel of genital cGvHD, a frequent complication to alloSCT. In our previous cross-sectional study with a median follow-up of 80 months, 9/42 pts had developed vaginal stenosis before 36 mo post Tx, whereas in this prospective, interventional study we observed on only one stenosis before 36 months. Notably, the high frequency of severe global cGvHD in the previous study was mainly due to severe genital cGvHD, thus illustrating that poor gynecological care may considerably contribute to impaired quality of life in women after alloSCT. From the results of our two studies with a high prevalence and high cumulative incidence of genital cGvHD, we conclude that early and regular gynecological control is important and may reduce the frequency of severe genital cGvHD. Disclosure of Interest: None declared. P111 Impaired NK cell subset reconstitution correlates with development of acute GVHD following allogeneic stem cell transplantation E. Ullrich1,2,3,*, S. Bakhtiar2, E. Salzmann-Manrique2, S. Gerstner1,3, M. Bremm1,2, A. Mackensen3, P. Bader1,2, P. Hoffmann4,5, E. Holler4,5, M. Edinger4,5, D. Wolff4,5 1 LOEWE Center for Cell and Gene Therapy, 2Department for Children and Adolescents Medicine, Division for Stem Cell Transplantation and Immunology, Goethe University Frankfurt, Frankfurt, 3Department of Internal Medicine 5, Hematology & Oncology, Friedrich-Alexander-University Erlangen, Erlangen, 4 Internal Medicine III, Hematology & Oncology, University Hospital Regensburg, 5Regensburg Center for Interventional Immunology, University of Regensburg, Regensburg, Germany Introduction: Allogeneic stem cell transplantation (alloSCT) often remains the only curative treatment for hematological disorders. However, its success is frequently limited by acute and chronic graft-versus-host disease (GVHD) causing significant morbidity and mortality. One of the major challenges of alloSCT is to reduce the incidence and severity of GVHD while boosting the graft-versus-leukemia effect (GVL). In the setting of alloSCT, reconstitution of natural killer (NK) cells is of notable interest due to their known capability to induce GVL without GVHD. This analysis is a single center prospective study performed at the University Hospital Regensburg investigating the possible correlation between the regeneration of NK cell subsets and the incidence of acute GVHD (aGVHD) during the first 200 days following alloSCT with focus on immature NK cells (CD14-CD3-CD56highCD16dim), mature cytotoxic NK cells (CD14-CD3-CD56dimCD16high), and the ratio between these two populations (CD56dim:CD56high). Materials (or patients) and methods: Data were collected from March 2009 to January 2012, and 345 samples of 111 patients were analyzed by FACS. In median 3 samples per patient were analyzed (1 - 9 samples). Median age of the cohort was 50 years (17 – 71 yrs.). Statistical analysis was performed using both B-spline linear and logistic regression.
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Results: While 43 (38.7%) patients did not develop any GVHD during the first 200 days, 68 (61.2%) patients developed aGVHD (grade 1 n ¼ 28, grade 2 n ¼ 23, grade 3 n ¼ 14, grade 4 n ¼ 3) in median at day 28 (9-182) after alloSCT. No differences in NK cell distribution were detectable before alloSCT. In the longitudinal analysis of immune reconstitution after alloSCT, there was a clear association between early expansion of the total NK cell population and lower incidence of aGVHD (P ¼ 0.024). In particular, the onset of aGVHD was accompanied by a lack of the CD56high NK cell subset and reconstitution of CD56high NK cells significantly correlated with a lower incidence of aGVHD (P ¼ 0.0003). Remarkably, we observed a significant inverse correlation between the severity of aGVHD and the frequency of CD56high NK cells during aGVHD. Conclusion: It is well known that NK cell reconstitution following alloSCT goes along with higher numbers of CD56high that further differentiate into cytotoxic CD56dimCD16high NK cells. In our study, aGVHD not only correlates with reduced numbers of total NK cells, but specifically with an impaired early expansion of CD56high NK cells. In sum, these data let suggest a negative impact of aGVHD on early NK cell immune reconstitution, maturation and NK subset distribution. In addition, monitoring of early NK cell reconstitution after SCT may help to identify patients at risk for the development of severe aGVHD. Disclosure of Interest: None declared.
Results: The drug was well-tolerated with no severe side effects: the most frequent side effect was mild hypotension and headache. In 15/19 cases we observed a significant clinical benefit after a median of 6 cycles (range 3-12). The Modified Rodnan Skin score, calculated before and after 1-year treatment, was 27 and 8, respectively. Iloprost courses were given in Outpatient Department for a median of 20 courses, the first 12 every month. All the 15 responsive pts received a maintenance therapy with 3-4 treatments a year. Further improvement was otherwise observed over the subsequent courses. At present, one patient not responsive to Iloprost has died because of sepsis. All the 15 responsive pts are alive in continuous complete remission, with satisfactory improvement of quality of life. Conclusion: In our study Iloprost seems to be one of the most effective drugs in reverting resistant/refractory sclerodermic cGVHD and in reducing the extension of the skin lesions. Improvements were observed in 15/19 pts (79%), after a median of 6 months of treatment. The Rodnan Skin Score is a practical and easy tool to assess skin involvement and to follow clinical outcome. It’s to be noted that the vast majority of the pts of this report received stem cells from an HLA identical sibling, even if in the same period the majority of transplants were done from unrelated donors. Disclosure of Interest: None declared.
P112 Synthetic PGI-2 (Iloprost) treatment in patients with resistant sclerodermic chronic GVHD (cGVHD) after Allogeneic Hematopoietic Stem Cell Transplantation F. Benedetti1,*, M. Sorio1, C. Tecchio1, A. Andreini1, A. Artuso1, D. de Sabata1, G. Turri1, I. Ferrarini1 1 Bone Marrow Transplant Unit, AZIENDA OSPEDALIERA UNIVERSITARIA INTEGRATA VERONA, Verona, Italy
P113 Improved outcome of patients developing Ocular chronic Graft Versus Host Disease and treated with Autologous Platelet-Rich Plasma (PRP) eye drops F. Monaco1,*, F. Zallio2, M. R. Astori3, L. Mazzucco4, G. Drago1, M. Rapetti5, G. Catania1, D. Dolcino3, R. Guaschino4, M. Ladetto1, M. Pini1 1 Hematology, 2AZIENDA OSPEDALIERA SS. ANTONIO E BIAGIO E CESARE ARRIGO, ALESSANDRIA, Alessandria, Italy, 3Opthalmology, 4Transfusion Center, 5Pharmacy Department, AZIENDA OSPEDALIERA SS. ANTONIO E BIAGIO E CESARE ARRIGO, ALESSANDRIA, Alessandria, Italy
Introduction: Scleroderma is a late manifestation of chronic GVHD, presenting as progressive tightness of the skin and hampering patients’ quality of life. Iloprost, a synthetic PGI-2, has been proving effective in the therapy of idiopathic systemic sclerosis, which has a similar pathogenesis to sclerodermic cGVHD. Materials (or patients) and methods: From 2001 to 2013 we observed 19 pts affected by diffuse or limited sclerodermic cGVHD. During the study period 353 allogeneic transplants were performed, 162 from HLA identical sibling and 191 from unrelated donor. The patients (9 AML, 3 MM, 4 ALL, 2 NHL and 1 MFI) were transplanted in 12 cases with classical conditioning regimen (FTBI 1200 cGy plus Cytoxan 120 mg/Kg or BusulfanCytoxan), in 7 cases with reduced intensity conditioning regimen. In 17/19 cases the donor was an HLA identical sibling. The source of stem cells was bone marrow in 3 cases and peripheral blood in 16 cases. Severe cGVHD with scleroderma was observed at a median of 21 months from HSCT (range 8-39), presenting as diffuse skin changes (tightness and thickening of the face, neck, hands, thorax, legs, feet and abdomen), cutaneous dyschromia, digital pitting scars and functional joint impairment. In 15 cases it was classified as de novo cGVHD. Most of the pts showed involvement of organs other than the skin: xerophthalmia in 13, xerostomia in 8 cases, liver in 5 pts, gastrointestinal tract in 4 and lung in 1 patient. In 4 pts these alterations were linked with itching and pain and in 4 cases diffuse scleroderma was associated with severe discomfort in tendons or muscles. In all cases cGVHD severely affected quality of life. The pts failed at least 2 immunosuppressive regimens including CSA, azathioprine, mycophenolate mofetil, steroids, repeated cycles of PUVA with little or no effect on the skin. Iloprost, 50 microgram a day over 8 hours IV continuous infusion for 5 days every month, was started after a median of 6 months from the appearance of scleroderma (range 6-13).
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Introduction: Despite significant advances, graft versus host disease (GVHD) remains the most important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Chronic GVHD is the most common long-term complication, but it is also considered the strongest predictor for improved outcome through a graft-versus Leukemia effect. Ocular involvement occurs in 40-60% of patients with cGHVD and is found to significantly impair the quality of life. Treatment recommendations focus on relieving ocular symptoms. On the basis of encouraging results reported previously with autologous serum eye drops in autoimmune diseases, we started this approach in collaboration with Ophthalmic Division and Transfusion Centre; furthermore we retrospectively investigated the rate of onset of ocular cGVHD in our population submitted to allogeneic transplant and the possible association with improved outcome. Materials (or patients) and methods: The study was conducted at the Hematologic Division of the Hospital of Alessandria, with the collaboration of the Ophtalmological Division and the Transfusion Centre. Since 2005 all patients submitted to alloSCT for hematologic malignancies and with a NIH eye involvement score 41 were treated with autologous Platelet Rich Plasma (PRP) eye drops; ophtalmologist evaluation (at day þ 30, þ 180 and þ 360 from the start of the therapy) included 3 different parameters: SUBJECTIVE SYMPTOMS, analyzed using the NIH Eye Score; OBJECTIVE TEST: Schirmer test, Tear Film Break Up Time and Best Visual Acuity; CORNEAL DAMAGE: anterior segment, fluorescein and lissamine score. PRP eye drops were prepared under laminar flow hood, in sterile single dose, stored at – 201C and instilled 6 times/day for one year.
Results: Between 2005 and 2014, 29 patients developed ocular GvHD. Pretransplantation diagnosis included: acute myeloid leukemia/myelodisplastic syndrome (50%), acute lymphoblastic leukemia (13%), lymphoproliferative malignancies (37%). 25 patients were classified as NIH score 2, and 4 patients as score 3. All but two patients (one interruption for local reaction and one for hematologic progression) completed the one-year schedule. Considering NIH criteria, 10 patients resolved their symptoms, with a NIH score of 0 (zero) at day þ 360. At 30 days 86% and 43% had an improvement of symptoms (S) and ocular signs (T), respectively and 0% had corneal damage; at 180 days, 71% and 57% improved their symptoms (S) and ocular signs (T), respectively and 14% had corneal damage; at 365 days, 86% and 57% kept on an amelioration of their symptoms (S) and ocular signs (T), respectively, while 7% had corneal damage. Compared with a control population submitted to alloSCT in the same period, we found that 5-year overall survival (OS) of the patients with ocular cGVHD was projected at 65% and is significantly better than OS of patients with GVHD other than ocular and without cGVHD (58% and 50%, respectively). Conclusion: Therapy with autologous PRP eye drops shows an improvement of symptoms and corneal damage already since 30 days, and its efficacy is maintained throughout the year of treatment. Therefore autologous PRP eye drops could represent a valid alternative option to conventional therapy in the management of ocular cGVHD. Moreover we found that developing ocular GVHD is a kind of protection for overall survival in patients submitted to alloSCT. Disclosure of Interest: None declared. P114 Neutrophil and monocyte CD64 expression provides limited diagnostic value for detection of Graft-versus-Host disease or viral infections in patients after allogeneic Hematopoietic Stem Cell Transplantation F. Simonetta1,*, A. Pradier1, S. Masouridi-Levrat1, C. Bosshard1, C. Dantin1, C. Hogan1, T. Matthes1, Y. Chalandon1, E. Roosnek1 1 Hematology, Geneva University Hospitals, Geneva, Switzerland Introduction: CD64 expression by neutrophils and monocytes has been proposed as a diagnostic marker of infection or inflammation. In the present study we evaluated the diagnostic power of CD64 levels for identification of viral infections or Graft versus Host Disease (GvHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Materials (or patients) and methods: We performed a prospective study on 295 blood samples obtained at different time points from 80 patients after allogeneic HSCT. We
measured CD64 levels on mature CD10-positive neutrophils (nCD64) and on monocytes (mCD64) as well as plasma levels of C-reactive protein (CRP) in HSCT recipients without complications or with viral infections or GvHD. Receiver operating characteristic curve analysis was performed to determine the discriminatory potential of nCD64, mCD64 and CRP for diagnosis of viral infections and GvHD in HSCT recipients. Results: nCD64 but not mCD64 was significantly up-regulated during early phases after HSCT even in the absence of infectious complications. Levels of nCD64 [MFI: 3.26 (2.07-5.37) vs 2.89 (1.89-4.48), P ¼ 0.0079] and mCD64 [MFI: 38.5 (29.751.5) vs 32.1 (20.2-52.0), P ¼ 0.0078] in patients with GvHD were significantly higher than in patients without but we did not detect significantly higher nCD64 and mCD64 levels in patients with viral infections. Hence, nCD64 and mCD64 expression had only a weak discriminatory power for detection of viral infections or GvHD. nCD64 and mCD64 had an area under the curve (AUC) value of 0.557 and 0.503, respectively, for viral infections and of 0.605 and 0.631 for GvHD. Importantly, nCD64 and mCD64 levels were not more discriminatory than CRP-levels (AUC of 0.570 for viral infections and of 0.629 for GvHD). Conclusion: nCD64 and mCD64 levels are increased during GvHD but have only a low diagnostic potential for the occurrence of GvHD or viral infections and their use for diagnostic purposes cannot be recommended. Disclosure of Interest: None declared. P115 Once weekly extracorporale photopheresis for treatment of steroid-refractory or relapsed chronic graft versus host disease F. Ayuk1,*, M. Christopeit1, M. Guellstorf1, H. Lellek1, T. Wolbers1, U.-M. Von Pein1, C. Wolschke1, N. Kro¨ger1 1 Clinic for Stem Cell Transplantation, UNIVERSITY MEDICAL CENTER HAMBURG-EPPENDORF, Hamburg, Germany Introduction: Extracorporale photopheresis is increasingly used to treat patients with chronic graft-versus-host disease (cGVHD), however published data on its efficacy is limited. While the optimal treatment schedule is unknown, most centres start with a twice-weekly treatment. Materials (or patients) and methods: We performed a retrospective analysis of 78 patients who received ECP at our centre for treatment of steroid-refractory or relapsed cGVHD. Primary end point was overall survival (OS) at 5 years, secondary end point was treatment response at 6 months after start of ECP treatment. Patients received ECP once weekly for at least 4 weeks. Thereafter, responding patients continued ECP every two weeks while non-responding patients continued on the once weekly schedule until 15 weeks. ECP was performed using UVAR-XTS or Cellex machines. Results: In all, 48 of 78 patients (62%) responded to treatment with 46 (59%) PR and 2 (3%) CR. 3 year and 5 year overall survival (OS) were 80% and 66% respectively. Responders had significantly better 5yr OS compared to non-responders (85% vs. 48%, P ¼ 0.01). Platelet counts at start of ECP treatment did not impact treatment response (P ¼ 0.76), however platelet countso100.000/ul vs. 4/ ¼ 100.000 was associated with poorer 5yr OS of 44% vs. 71%, P ¼ 0.01. In multivariate analysis for OS including platelet counts, steroid dose, patient age and cGVHD NIH grade (all at start of ECP), low platelet counts retained negative impact (RR: 0.34, 95% CI 0.11-1.00, P ¼ 0.05) while patient age below median (53yrs) was associated with better OS (RR: 2.8, 95% CI 0.96-8.25, P ¼ 0.06). Cumulative incidence of non-relapse mortality (NRM) at 2 years was higher for patients with low vs. high platelet counts (29% vs. 10%, P ¼ 0.03) and for older vs. younger patients (23% vs. 5%, P ¼ 0.003). Relapse incidence did not differ according to platelet counts (P ¼ 0.53) or patient age (P ¼ 0.1) We identified 9 patients who received ECP as the only immuno-modulatory treatment, 8 of whom attained a PR and none a CR. Seven out
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of 8 patients with eye involvement, 3 out of 5 with skin and none of 3 with joint involvement experienced PR of organspecific symptoms. Conclusion: In this relatively large study we found that the once-weekly schedule of ECP is effective in treatment of patients with relapsed or refractory cGVHD. It is noteworthy that ECP did not overcome the negative impact of low platelet counts on survival. Disclosure of Interest: None declared. P116 Unrelated cord blood (UCB) transplantation for AML: higher incidence of acute GVHD and lower survival in male patients transplanted with female unrelated cord blood. A report from Eurocord and the ALWP of the EBMT F. Baron1,*, M. Labopin2, A. Ruggeri3, M. Mohty4, G. Sanz5, N. Milpied6, A. Bacigalupo7, A. Rambaldi8, F. Bonifazi9, A. Bosi10, J. Sierra11, I. Yakoub-Agha12, J. M. Ribera Santasusana13, E. Gluckman14, A. Nagler15 on behalf of Acute Leukemia Working Party of the EBMT 1 University of Liege, Liege, Belgium, 2EBMT Paris Office, Hospital Saint Antoine, 3Eurocord, Hospital Saint Louis, AP-HP, and IUH University Paris VII, 4AP-HP, He´matologie Clinique et The´rapie Cellulaire, Hoˆpital Saint-Antoine, Paris, France, 5Servicio de Hematologia, Hospital Universitario La Fe, Valencia, Spain, 6 CHU Bordeaux - Hoˆpital Haut-leveque, Pessac, France, 7Department of Haematology II, Ospedale San Martino, Genova, 8 Hematology and Bone Marrow Transplant Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, 9Institute of Hematology & Medical Oncology, Bologna University, S.OrsolaMalpighi Hospital, Bologna, 10BMT Unit Department of Hematology, Ospedale di Careggi, Firenze, Italy, 11Hematology Department, Hospital Santa Creu i Sant Pau, Barcelona, Spain, 12UAM allo-CSH, Hopital HURIEZ, CHRU, Lille, France, 13Hospital Universitari Germans Trias i Pujol, Barcelona, Spain, 14Eurocord, Hospital Saint Louis, AP-HP, and IUH University Paris VII, France Monacord, Centre Scientifique de Monaco, Paris, France, 15 Division of Hematology and Bone Marrow Transplantation, The Chaim Sheba Medical Center, Tel-Hashomer, Israel Introduction: Despite major improvements in the field in the last decade nonrelapse mortality (NRM) has remained the main cause of failure of unrelated cord blood (UCB) transplantation for acute myeloid leukemia (AML). In the setting of allogeneic HLA-matched bone marrow or peripheral blood stem cell transplantation, transplanting male patients with grafts from female donor has been associated with a higher incidence of graft-versus-host disease (GVHD) and of nonrelapse mortality (NRM), and a lower overall survival (OS). This is due to recognition by female donor immune cells of minor histocompatibility antigens encoded by genes on the recipient Y chromosome that are polymorphic to their X-chromosome homologue. The aim of the current analysis was to compare transplantation outcomes in male patients given female UCB versus other gender combinations. Materials (or patients) and methods: Data from 552 consecutive patients with AML given a single UCB transplantation between 2000 and 2014 were included. Among them, 131 patients were male patients given female UCB, 119 patients were male patients receiving male UCB, and 302 were female patients. Male patients given female UCB were less often transplanted following a reduced-intensity conditioning (24% versus 35%, P ¼ 0.03) and received less total nucleated cells (2.47 vs 2.8 x106/Kg, P ¼ 0.001) than other patients. Results: Male patients given female UCB had a trend for a higher incidence of grade II-IV acute GVHD (33% versus 25%, P ¼ 0.08) but for a lower incidence of chronic GVHD (16% versus 25%, P ¼ 0.11), a trend for a higher incidence of NRM (41% vs 33%, P ¼ 0.06), and a lower leukemia-free survival (LFS, 30% vs 41%, P ¼ 0.01) and OS (33% versus 45%, P ¼ 0.008) than other patients. Restricting the analyses to male recipients, those given female UCB versus male UCB had a higher incidence of grade II-IV acute GVHD (33% versus 27%), a
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similar 2-year probability of NRM (40.8 vs 36.6%; P ¼ 0.41), and a suggestion for worse LFS (29.9 vs 40.7%, P ¼ 0.11) and OS (33 vs 42.4%; P ¼ 0.10). In multivariate analyses taking into consideration all patients for which data on HLA-matching and cell dose transplanted were fully available (n ¼ 363), male patients transplanted with a female UCB had a trend for a higher incidence of grade III-IV acute GVHD (HR ¼ 2.0, P ¼ 0.06), a trend for a higher NRM (HR ¼ 1.5, P ¼ 0.06) and a worse LFS (HR ¼ 1.4, P ¼ 0.04) and OS (HR ¼ 1.3, P ¼ 0.06). Conclusion: Our data suggest that male patients transplanted with female cord blood might have higher risk of acute GVHD and of NRM leading to worse LFS and OS. These results should be confirmed in other large cohorts of patients before used for determining the choice of a single UCB unit. Disclosure of Interest: None declared. P117 IL-10 producing B-10 cells can be identified as early as 30 days post an allogeneic stem cell transplant and are enriched in the transitional B-cell compartment G. Chakupurakal1,*, M. Garcia-Marquez1, A. Shimabukuro-Vornhagen1, S. Theurich1,2, C. Scheid1, M. Hallek1, U. Holtick1, M. von Bergwelt-Baildon1 1 Department of Medicine I, Department of Medicine I, University Hospitals Cologne, Kerpener str 62, Germany D 50937, 2MaxPlanck-Institute for Metabolism Research, University Hospials Cologne, Cologne, Germany Introduction: Allogeneic stem cell transplantation (alloSCT) is the curative therapeutic option for a variety of haematalogical malignancies. Graft versus Host disease (GvHD), with an incidence of around 40-60%, remains a major post transplant complication associated with severe morbidity and mortality. The role of B-cells and especially the Interleukin-10 (IL-10) producing regulatory B-cells (B10-cells) in the pathophysiology of GvHD is not clearly understood to date. We studied B cells in allogeneic transplant recipients in the early post transplant phase. Materials (or patients) and methods: We prospectively collected samples, after obtaining informed consent, on 89 patients, transplanted at the University of Cologne on days 30, 90 and 150 post transplant and 10 healthy donors. Patients were transplanted mostly for haematological malignancies (n ¼ 88). Male: Female ¼ 48:41. All received reduced intensity conditioning regimens (90%), 34/89 (38%) received antithymocyte globulin (ATG) in addition to a calcineurin inhibitor with mycophenolate mofetil or methotrexate as GvHD prophylaxis. 41 (46%) had no GvHD, and 9 (10%), 8 (9%), 17 (19%)and 14 (16%) had grade 1-4 GvHD as per the NIH classification respectively. Results: Day 30 post transplant the total B-cell percentages and numbers in transplant recipients were significantly reduced in comparison to the controls (P ¼ o0.0001).The majority of the B-cells had a naı¨ve phenotype, Transitional B-cells were significantly more in transplant recipients (P ¼ 0.02) No differences were observed in the memory B-cell or CD24high CD27high compartments in comparison to controls. B10-cells were studied in the cohort 30 days post transplant and could be demonstrated in transplant recipients (n ¼ 16, 8 with acute GvHD) irrespective of their GvHD status. For data analysis, 4 patients without GvHD had to be discarded due o0.1% B-cells. The percentages of B10-cells were significantly reduced in the transplant recipients on comparison with the control cohort (P ¼ o0.0002) (Figure 1a). The CD24high CD27high B10-cell compartment was significantly smaller in the patient cohort (P ¼ 0.054) (Figure 1b). CD24highCD38hightransitional B10-cell subset with a mean percentage of 9.82±9.157 accounted for the largest subset of the cells analysed. Data on chimerism analysis at the time of sampling showed that the majority of patients (n ¼ 15/16; 93%) had 495% donor chimerism. implying that the B10-cells are predominantly donor derived.
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Conclusion: This is the first longitudinal study of B-cells and B-cell subsets including CD24high CD38high transitional B-cells and CD24high CD27high B-cells commencing as early as 30 days post an alloSCT. The contribution of transitional B-cell phenotype to the total number of B-cells ,as early as 30 days post transplant, is a novel observation. This is the first report demonstrating B10-cells in stem cell transplant recipients in the early post transplant (30 days) period and are enriched within the transitional cell compartment.Unlike the observations in mice, the B10-cells are donor derived. The mean percentage value of B10-cells in our patient cohort 1.6± 1.1was similar to that seen in patients with chronic GvHD, by Khoder et al. The extremely low number of B10-cells in this early post transplant phase may explain the lack of significance between the patients with and without GvHD in our cohort. Disclosure of Interest: None declared. P118 Changes in T and B- cells in steroid refractory GvHD patients following treatment with a CD25-antibody G. Chakupurakal1,*, M. Garcia-Marquez1, A. Shimabukuro.vornhagen1, H. Schloesser1, U. Holtick1, C. Scheid1, M. von BergweltBaildon1 1 Department of Medicine I, Department of Medicine I, University Hospitals Cologne, Kerpener str 62, Germany D 50937, Cologne, Germany Introduction: Allogeneic stem cell transplantation is the therapeutic option for a variety of malignant and nonmalignant haematological diseases. Graft versus Host Disease (GvHD) is a common post transplant complication. In 40% of these patients, GvHD is steroid refractory and associated with a mortality of around 60%. Basiliximab is a chimeric murine – human antibody also selective for interleukin -2 receptor (IL2R) with a half life of 7 days. Phase 2 studies have demonstrated its superior efficacy in treating patients with steroid refractory GvHD(1). We administered Basiliximab in 14 patients with steroid refractory GvHD with a median age of 41 (range 20-69). M: F 7:7. We aimed to study the in vivo T- and B-cell changes following Basiliximab administration as this would be an ideal platform to monitor the alterations in the regulatory T and B-cell compartment. Materials (or patients) and methods: All patients but one 13/ 14 received PBSC from unrelated donors and 6/13 had mismatched unrelated donors. 7/14 achieved a complete response to treatment. PBMCs were obtained from all donors, after informed consent, prior to and after weekly administration of Basiliximab 40mg for 4 weeks. Control samples were obtained from patients with steroid responsive acute GvHD. Results: The total number of CD3 þ as well as CD4 þ and CD8 þ T-cells remained constant during treatment and no change was observed on comparison with the controls. The, naive (CD45RA þ , CCR7 þ ), central memory T-cell (CD45RA-,
CCR7 þ ), effector (CD45RA þ , CCR7-) and effector memory (CD45RA-, CCR7-) compartments remained constant, T-cell population decreased, though statistically not significant, during the treatment period. Gagliani et al 4 demonstrated that regulatory type 1 T-cells can be identified by the co-expression of CD49b and Lag3. The % CD49d þ , Lag3 þ T-cells was lower than that identified in the control population and decreased during the treatment period (statistically significant). No changes were seen in the % CD19 þ , CD20 þ B-cells , activated (CD20 þ , CD86 þ ) and anergic B-cell subsets (CD20 þ , CD21-) during the observation period. The % of, CD24high, CD27high regulatory B-cells were found to be twice that seen in the controls. With treatment a decrease was seen in this population.The CD27high, CD38high transitional B-cells were also found to be higher than that seen in the controls. No change was observed in this cohort with treatment. Conclusion: This is the first attempt to study the in-vivo changes induced by a CD25 antibody in patients with steroid refractory GvHD. We conclude that this antibody not only depletes the alloreactive CD25 þ T, B and NK cell population but also alters the regulatory T-cell and B-cell subsets in comparison to patients with steroid responsive GvHD. The role of regulatory B-cells in enabling this need to be studied further. References: 1. Massenkeil G, Rackwitz S, Genvresse I, Rosen O, Dorken B, Arnold R. Basiliximab is well tolerated and effective in the treatment of steroid-refractory acute graft-versus-host disease after allogeneic stem cell transplantation. Bone Marrow Transplant. 2002 Dec;30(12):899-903. 2. Gagliani N, Magnani CF, Huberr S, Gianolini ME, Pala M, Licona-Limon P, et al. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells Nat Med 2013 Jun 19(6): 739-746. Disclosure of Interest: None declared.
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P119 GVHD-Associated Early Impairment of Marrow Function Is Frequent after Allogeneic Transplantation and Is a Predictor of Treatment Related Mortality and Overall Survival G. Milone1,*, G. Avola1, M. G. Camuglia1, A. Di Marco1, S. Leotta1, A. Cupri1, A. Spadaro1, D. Berritta1, A. Romano1, M. Parisi1, G. Tripepi2 1 Hematology and BMT UNIT, Azienda Policlinico Vittorio Emanuele, Catania, 2Istituto di Fisiologia Clinica, CNR, Reggio Calabria, Italy Introduction: We have previously reported that a-GVHD is an important determinant of primary engraftment. Aim of our study was now to measure GVHD-associated marrow impairment and whether ‘‘a-GVHD-associated marrow impairment’’ is useful in predicting transplant outcome. Materials (or patients) and methods: In a prospective series of allogeneic transplants, we determined the frequency of CFU-GM and BFU-E in marrow at day þ 18. Findings were related to a-GVHD and to Treatment Related Mortality (2-yTRM), Relapse Rate (RR) and Overall Survival (OS). So far, 72 patients have been evaluated, the majority were affected by Acute Leukemia (73%), in various phase of disease (advanced 57%). Our series included matched family (62%) or unrelated donors (38%). In MUD transplants, HLA matching was: 8/8: 26% ; 7/8: 52%; other: 22%. Results: Patients who, within day þ 90, were diagnosed with a-GVHD (overall grade ¼ /4 21), had a lower growth of CFUGM (91/10e5 plated cells) in respect to patients without a-GVHD (185./10e5), P ¼ 0.01. Similarly, BFU-E growth was lower in marrow of patients with a-GVHD in respect to patients without a-GVHD, P ¼ 0.0007. In univariate analysis, in addition of a-GVHD, febrile neutropenia was the only other factor associated to a reduction of frequency of marrow CFU-GM in the bone marrow at day þ 18 (P ¼ 0.08) while all other factors tested resulted not important: ‘‘HSC source’’, ‘‘Donor Type’’, ‘‘Dose of CD34 þ cells infused’’, ‘‘Doses of MTX actually administered’’, ‘‘Phase of disease’’. To assess importance of clonogenic cell measurement at day þ 18, on transplant outcome, patients were divided in two groups based on CFUGM growth in respect to median (‘‘CFU-GM below median’’ and ‘‘CFU-GM over median’’). These two groups were comparable for patient’s age (P ¼ 0.6), percentage of patients transplanted in advanced phase (P ¼ 0.8) and percentage of patients who received MUD transplant (P ¼ 0.6). Patients having a frequency of marrow ‘‘CFU-GM over median’’ had a cumulative incidence of TRM significantly lower than those showing ‘‘CFU-GM below the median’’ (respectively, 4.5% vs 30%, Gray test P ¼ 0.01). Cumulative incidence of RR was not significantly different in the two groups ‘‘CFU-GM over median’’ and ‘‘CFU-GM below the median’’ (RR: 31% vs 34%, P ¼ 0.3). In our series, 2-y -TRM was higher also after MUD than after HLA-ID Family donor transplants (TRM: 30% vs 10%), this difference in univariate analysis was not significant (Gray test
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P ¼ 0.2). In univariate analysis, predictors of OS at 2 years were: i) phase of disease at transplant’’: early phase: 80%; advanced phase: 42%, (P ¼ 0.03); ii) donor type (HLA ID Family donor tx OS: 78%; MUD tx OS: 52%, P ¼ 0.10) and iii) Frequency of CFUGM ("CFU-GM greater than median" had an OS of 81%; "CFUGM below the median" had an OS of 40%, P ¼ 0.001). In multivariate Cox proportional hazard analysis, a frequency of marrow ‘‘CFU-GM below median’’ remained an independent factor important in worsening OS (HR:8.4; P ¼ 0.0006), together with MUD donor (HR 5.3; P ¼ 0.002). Phase of disease was also important and Early phase was associated with improved OS (HR: 0.27; P ¼ 0.04). Conclusion: a-GVHD impairs early marrow function and it is the principal determinant of frequency of clonogenic precursors cells found in marrow at day þ 18. Marrow-CFU-GM frequency at day þ 18 may be used an early and simple biomarker for prediction of TRM and OS. Disclosure of Interest: None declared. P120 The beneficial impact of incorporation of low-dose antithymocyte globulin (rabbit) 5mg/kg to standard graftversus-host disease prophylaxis in matched or mismatched unrelated allogeneic stem cell transplantation I. Batsis1,*, D. Mallouri1, Z. Bousiou1, V. Constantinou1, C. Apostolou1, K. Tsirou1, P. Kaloyannidis1, C. Smias1, E. Yannaki1, A. Fylaktou2, A. Anagnostopoulos1, I. Sakellari1 1 Haematology department - BMT unit, George Papanicolaou Hospital, 2Immunology department, Hippokration hospital, Thessaloniki, Greece Introduction: The use of in-vivo T-cell depletion in the setting of unrelated transplants is still controversial since randomised studies have shown a reduction in severe graft versus host disease (GVHD) occurrence but no difference in overall survival. Hence, there is evidence for reduction in late morbidity and mortality. Moreover, the dose of prophylactic anti-thymocyte globulin (ATG) used in various studies ranges and the optimal dose is not established. Materials (or patients) and methods: In a prospective, single centre study, ATG (rabbit) has been used as standardised part of the conditioning in a uniform dose of 5mg/kg (total dose) and was infused on days -2 and -1. We analysed the outcome of all consecutive patients (pts) who underwent matched or mismatched unrelated alloHCT for hematologic malignancies. In total 92 pts (36 female, 56 male) suffering from acute leukaemia (79), MDS/MPDs (6), multiple myeloma (1), lymphoma (4) and CML (1) underwent allogeneic haematopoetic cell transplantation (alloHCT) from matched (8/8, n ¼ 53) or allele or antigen mismatched (7/8, n ¼ 37 and 6/8, n ¼ 2) unrelated donors. The grafts were peripheral stem cells in 84 and bone marrow in 8 pts, with median dose of CD34 þ cells infused 6.4 (0.78-17.74) 106/kg. Disease phase was classified as early in 37, intermediate in 23 and advanced in 32 pts. Anti-
GVHD prophylaxis consisted of cyclosporine or tacrolimus plus methotrexate in myeloablative transplants (85) and mycophenolate mofetil in RIC (7). Results: With a median follow-up of 20 months (0.8-139), overall survival (OS) was 59.8%, relapse rate (RR) 25% and treatment related mortality (TRM) 22.8%. The incidence of acute GVHD was 47.8%, grade Z II 34.7% (matched 32%, mismatched 38.4%, P ¼ 0.5), grade III-IV 14% (matched 9.4%, mismatched 20.5%, P ¼ 0.27) and of chronic 65% (extensive 40.2%, matched 41.5%, mismatched 38.4%, P ¼ 0.2). The matched and mismatched pts were comparable in age, sex and disease phase (x2 test). In univariate analysis no significant difference was found in the two groups in terms of incidence and severity of acute and chronic GVHD. Overall survival was superior in matched alloHCT (69.8% vs 46.1%, P ¼ 0.02) and this was attributed to the higher rate of TRM in mismatched transplants (35.8% vs 13.2%, P ¼ 0.16) while RR was similar (24.5% and 25.6%, P ¼ 0.9). In multivariate analysis incorporating sex, disease phase, mismatched donor, intensity of conditioning regimen and grade Z II acute and chronic GVHD, significant unfavourable factors for OS were grade Z II acute GVHD (P ¼ 0.04), male sex (P ¼ 0.04) and advanced disease phase (P ¼ 0.01). For relapse the most powerful factor was disease phase (early vs any other, P ¼ 0.009) while chronic GVHD was also significant for lower relapse probability (P ¼ 0.01). In terms of TRM, none of the above included factors was statistically significant. Conclusion: Though it is well known that unrelated alloHCT is a higher risk procedure than sibling transplants, the addition of ATG at a relatively low dose of 5mg/kg can lead to a high OS rate and acceptable TRM even in the mismatched transplants. Disclosure of Interest: None declared. P121 Acquired hemophilia A in a patient with chronic GVHD after cord blood transplantation successfully treated with rituximab I. Donnini1,*, C. Nozzoli1, R. Angarano2, C. Bazzini3, F. Bacci3, S. Linari4, S. Guidi1, A. Bosi5 1 Bone Marrow Transplantation, AOU Careggi, Florence, 2Hematology Unit, National Cancer Centre ‘‘Giovanni Paolo II’’, Bari, 3 High Dependency Unit – Internal and Emergency Medicine Department, 4Center for Bleeding Disorders, Department of Hearth and Vessels, 5Hematology Unit, AOU Careggi, Florence, Italy Introduction: Acquired Hemophilia A (AHA) is a rare bleeding disorder related to the development of inhibitor autoantibodies against endogenous factor VIII1 and also described in recipients of allogeneic-Hematopoietic stem cell transplantation (alloHSCT) as expression of Graft versus Host Disease (GvHD)2. Materials (or patients) and methods: We report a case of AHA in a recipient of umbilical cord blood transplantation (UCBT) for acute myeloid leukemia (AML) M2 FAB, NPM1/FLT3 neg/neg. 40-year old male who underwent UCBT with a single cord blood unit 4/6 HLA compatible with total nucleated cells 3x107/Kg and CD34 þ 0.3x 105/Kg. Conditioning regimen consisted of Thiotepa 5mg/Kg on days -7, -6, Busulphan 3.2 mg/Kg on days -5, -4, -3 and Fludarabine 50 mg/m2 on days -5, -4, -3. Anti Thymocyte Globulin, Ciclosporine (CSA) and Mycophenolate Mofetile (MMF) were given for GvHD prophylaxis. Nine months after UCBT the patient experienced cutaneous lichenoid GvHD requiring CSA and steroid treatment. After interruption of steroid treatment, the patient developed a relapse of cutaneous GvHD grade III with eczema feature and started again treatment with steroid and Extracorporeal Photopheresis (ECP). Twenty two months after UCBT the patient experienced sequential spontaneous muscular hematomas in the right gluteus, calf and over the left arm;respiratory/hemodynamic parameters were normal.A thoracic and abdominal CT scan excluded the presence of
muscular and visceral hematomas. Laboratory exams were relevant for normocytic anemia (Hb 8.4 g/dL), prolonged activated partial thromboplastin time (aPTT) (60.5 s, normal range: 22.0-38.0 s), decreased FVIII activity (4%, normal value: 60-150%) and presence of FVIII inhibitors with a low titer of 1,6 Bethesda Units (BU)/mL (normal value o0,6 BU/mL). White blood total, platelet count, prothrombin time, fibrinogen and creatinine clearance were normal; dilute Russell’s viper venom time (dRVVT) for Lupus anticoagulant, antiphospholipid and antinuclear antibodies were negative. No evidence of AML relapse was documented and the chimerism was fully donor. The patient was treated with infusion of plasma derived FVIII (Emoclot D.I., Kedrion) 5.000 International Unit (IU) every 12 hours, associated to methylprednisolone 1 mg/Kg/day. After seven days of steroids treatment without an increase of FVIII activity, we started a second line therapy with Rituximab 375 mg/m2 weekly schedule for a total of 4 infusions. Results: After one week from the first dose there was a marked improvement of aPTT and FVIII activity (Figure 1), with a reduction in hematomas size and related pain. The last evaluation of aPTT, factor VIII and FVIII inhibitor values, performed 6 months after Rituximab was initiated, showed an excellent recovery (respectively 24.8, 168% of activity and 0.1 BU/mL); the patients didn’t experience cGVHD relapse or any clinical complication, included new bleedings. Conclusion: This case report confirms the role of Rituximab in the treatment of AHA as expression of cGVHD and to our knowledge, this is the first case of AHA in recipient UCBT. References: 1. Shetty S, Bhave M, Ghosh K. Aquired haemophilia A: Diagnosis, aetiology, clinical spectrum and treatment options. Autoimmunity reviews 10/2011; 311-316. 2. Wieliczka ML, Ganguly S et al. Rituximab as a treatment for factor VIII inhibitor in a patient with chronic GVHD.Bone Marrow Transplant. 2014 Apr;49(4):588. Disclosure of Interest: None declared. P122 Models for Effective Acute Graft Versus Host Disease Prophylaxis after Allogeneic Bone Marrow Transplantation with Mesenchymal Multipotent Stromal Cells I. Shipunova1,*, N. Petinati1, A. Bigildeev1, N. Sats1, N. Drize1, L. Kuzmina2, E. Parovichnikova2, V. Savchenko2 1 lab. physiology of hematopoiesis, 2Bone Marrow Transplant Department, National Research Center For Hematology, Moscow, Russian Federation Introduction: Graft-versus-host disease (GVHD) is often accompanied allogeneic bone marrow transplantation (alloBMT). As the conventional methods of GVHD prophylaxis are often inefficient new method involving the use of donors’
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multipotent mesenchymal stromal cells (MSC) was developed. In some cases prophylaxis of acute GVHD (aGVHD) failed. The reasons of the failure could be either the result of particular qualities of donor-recipient interaction, patient status or characteristics of MSC samples. The results of the aGVHD prophylaxis with donors’ MSC injections after allo-BMT in patients with hematological malignancies were analyzed. The growth and differentiation characteristics, relative expression levels of different genes were investigated in all MSC samples. Materials (or patients) and methods: The study included 88 patients received allo-BMT from related donors after informed consent. The patients were divided into 2 groups: the first received standard prophylaxis of aGVHD and the second were additionally infused with MSC from the bone marrow of corresponding hematopoietic stem cells donor. MSC were cultivated in aMEM with 4% donors’ platelet lysate. MSC were administered intravenously when the blood counts indicated recovery (peripheral blood leukocytes reached 1x109/l). MSCs and colony-forming unit-fibroblasts (CFU-Fs) from the bone marrow of those donors were analyzed. For this purpose MSC were cultivated in standard conditions (aMEM, 10% fetal calf serum) for 5 passages. Relative expression level (REL) of 30 genes involved in proliferation, differentiation and immunomodulation was estimated by RT-qPCR. Results: The infusion of MSC reduced the incidence of aGVHD 2 times and increased the 5 years overall survival of patients (P ¼ 0.047). Six of 44 MSC samples had been ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index), the MSC properties of these donors found no significant differences between the MSC, effective and ineffective for preventing aGVHD. The combination of predictors that characterize the most suitable for the prevention of aGVHD MSC samples was revealed by multiple logistic regression analysis. A few models calculating the probability of the success of MSC samples application was proposed: logit(P) ¼ a þ bx þ cy þ dz, where logit(P) ¼ ln[P/(1P)], P – probability of successful prophylaxis, x, y and z – REL of corresponding genes in tested MSC sample. These models define 55-71% of effective MSC samples as successful. Threshold for success was considered as 0.9. These models included REL of following genes: FGFR1, PDGFRb, PPARg, IGF1, IDO1, TGFb1 and CHF. Only REL of FGFR1 and PDGFRb were significantly increased in MSC successfully prevented the development of aGVHD comparing with ineffective cases, REL of other genes mattered only in the framework of the models. Conclusion: These data confirm the presence of hierarchy as well as heterogeneity in MSC population. The high variability of all analyzed characteristics among MSC from different donors was shown. The mathematical models revealed the combination of parameters enabling to distinguish effective and ineffective MSC samples. By proposed models ineffective MSC samples could be discharged and replaced. Such strategy hopefully will prevent the development of aGVHD in the maximum number of patients. Disclosure of Interest: None declared. P123 Association of tacrolimus concentrations with incidence of acute graft-versus-host disease and relapse after matched allogeneic hematopoetic stem cell transplantation in adult patients I. Moiseev1,*, N. Senina1, V. Vavilov1, O. Slesarchuk1, S. Bondarenko1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Hematology and Transplantation, First State Medical University named I.P. Pavlov, Saint-Petersburg, Russian Federation Introduction: A number of studies have highlighted the significance of early cyclosporine concentrations for outcomes of allogeneic hematopoetic stem cell transplantation
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(HSCT). Data on the significance of tacrolimus pharmacokinetics is limited, so we retrospectively assessed impact of tacrolimus concentrations on the incidence of acute graft-versus-host disease (aGVHD), 2-year relapse rate (RR), non-relapse mortality (NRM) and overall survival (OS) after HSCT. Materials (or patients) and methods: Concentrations were obtained from 153 consecutive patients who received 10/10 matched related or unrelated allogeneic HSCT between 2010 and 2013. GVHD prophylaxis consisted of tacrolimus, shortcourse methotrexate or mycophenolate mofetil (MMF). In unrelated HSCTs antithymocyte globulin (ATGAM) 60 mg/kg was added. Median follow-up was 342 days (range 28-1250 days). Detailed patient and transplantation characteristics are presented in Table 1. Tacrolimus concentration was measured 2-4 times weekly. Concentrations in the first 50 days after HSCT were analyzed. Results: In the ROC analysis geometric means of concentrations during the first 21 days (conc21Days) after HSCT had the highest predictive values for grade II-IV aGVHD (AUC 0.62) and relapse (AUC 0.58), compared to 30- and 50-day concentrations. The ROC-determined conc21Days cut-offs were o9.1 ng/ml for aGVHD and 413.1 ng/ml for relapse. In the univariate and multivariate analyses patients with conc21Days o9.1 ng/ml had increased incidence of grade II-IV aGVHD (51% vs 25%, HR 0.47, 95%CI 1.01-3.28, P ¼ 0.047) and NRM (31% vs 15%, HR 2.19, 95%CI 1.00-4.80, P ¼ 0.05). On the contrary, conc21Days 413.1 ng/ml was associated with increased RR (65% vs 35%, HR 2.45, 95%CI 1.19-5.06, P ¼ 0.015). In patients with conc21Days between 9.1 and 13.1 the above results translated into better OS compared to the rest of the group (69% vs 47%, HR 0.43, 95%CI 0.23-0.78, P ¼ 0.005). Conclusion: Conclusion: Based on this study, it is recommended to maintain tacrolimus concentrations within 9-13 ng/ml therapeutic range during first 21 days after HSCT. These results should be confirmed in prospective studies. Disclosure of Interest: None declared.
P124 Efficacy of Infliximab for the Treatment of Acute Gastrointestinal Graft-versus-Host Disease in Children after Allogeneic Hematopoietic Stem Cell Transplantation: A Single-Center Experience J. K. Suh1,*, S. W. Lee1, K.-N. Koh1, E. S. Choi1, H. J. Im1, J. J. Seo1 1 Pediatrics, Asan Medical Center Children’s Hospital, Seoul, Korea, Republic Of Introduction: Graft-versus-Host Disease (GVHD) is a major limitation of allogeneic hematopoietic stem cell transplantation (HSCT). Especially, the outcomes of steroid refractory acute GVHD is poor, irrespective of secondary therapy. Infliximab, a chimeric monoclonal antibody against tumor necrosis factor-a, has been reported to have activity against steroid refractory acute GVHD involving gastrointestinal tract (acute GI GVHD). Here we report a single-center experience with using infliximab as a treatment of steroid refractory acute GI GVHD. Materials (or patients) and methods: We retrospectively reviewed the medical records of 16 patients who had been treated with infliximab for stage II-IV acute GI GVHD after alloHSCT at Asan Medical Center from January 2005 to November 2014. The diagnosis of acute GI GVHD was made on clinical grounds with endoscopic biopsy. Severe acute GI GVHD patients, who did not respond to standard steroid therapy (methylprednisolone 2 mg/kg/day), were treated with infliximab (10 mg/kg, weekly). Results: Of total 16 patients who were treated with infliximab for acute GI GVHD, 6 patients (37%) had stage II and 1 (6%) had stage III and 9 (56%) had stage IV acute GI GVHD. Eleven patients (69%) diagnosed with malignancy (5 AML, 4 ALL, and 2 JMML) and 5 (31%) diagnosed with nonmalignancy (2 SAA, 1 MDS, 1 CGD, and 1 IPEX). Acute GI GVHD developed at median 22 days after transplantation (range: 7-54). Median time to start infliximab from onset of acute GI GVHD was 7 days (range 1-28). Median number of infliximab administration was 3 (range 2-10). All the 16 patients responded to infliximab; 13 (81%) achieved complete response (CR) and the remaining 3 (19%) achieved partial response (PR). None of the patients developed therapy-related adverse effects. Eventhough 6 bacterial infection, 3 respiratory viral infection and 8 CMV reactivation were documented after infliximab administration, all the cases were well controlled with standard anti-bacterial and anti-viral therapy. Of the 15 evaluable patients, 6 (38%) had chronic GVHD at a median of 16 months after transplantation (range: 4-52). Three patients died; 1 from chronic GVHD, 1 from acute GVHD and the remaining 1 from disease progression. All the dead patients had stage IV acute GI GVHD. At a median follow up of 24 months (range 4-52), 2 year-overall survival (OS) was 78.7%. In the analysis of responses to infliximab, time to start infliximab from the onset of acute GI GVHD (r10 days) and the stage of acute GI GVHD (r stage III) had positive effect on CR rate (Po0.001). Conclusion: Our study can suggest that the addition of infliximab to steroid refractory acute GI GVHD could be effective to achieve CR without life threatening adverse effect. A multi-center prospective trial is needed to confirm the efficacy of infliximab. Disclosure of Interest: None declared. P125 Impact of GvHD-prophylaxis regimen on incidence and severity of gastrointestinal toxicities and acute GvHD after allogeneic HSCT J. Niestadtko¨tter1,*, L. Mu¨ller1, C. Mu¨ller-Tidow1, T. Weber1 1 Klinik fu¨r Innere Medizin IV, Martin-Luther-Universita¨t Halle-Wittenberg, Halle, Germany Introduction: Besides methotrexate (MTX), two oral mycophenolate formulations, mycophenolate mofetil (MMF) and mycophenolate sodium (EC-MPS) are used for GvHD-
prophylaxis after allogeneic HSCT. Compared to MTX, MMFprophylaxis for GvHD is associated with a lower rate of mucositis but an equal incidence of acute GvHD. Analyses in solid organ transplant show that (1) the frequency of gastrointestinal toxicities (GI-tox) in EC-MPS is lower than in MMF and that (2) gastrointestinal EC-MPS resorption under proton pump inhibitor (PPI) co-medication is higher than of MMF. We hypothesize that this is also the case after allogeneic HSCT, and that GvHD-prophylaxis with EC-MPS will lead to a lower incidence of acute GvHD when compared to MTX and MMF. Materials (or patients) and methods: The rate and severity of GI-tox, acute GvHD, and the frequency of PPI-co-medication as well as dose reductions of methotrexate and mycophenolate formulations were retrospectively analyzed in a cohort of 102 patients after allogeneic HSCT. All patients received GvHDprophylaxis based on cyclosporine A plus MTX or plus one of the mentioned mycophenolate formulations. Patients treated with MMF or EC-MPS and occurrence of severe intestinal mucositis CTC 431 were bridged with MMF i.v. for this time interval. GI-tox CTC Z31 (nausea, emesis, mucositis, diarrhea, abdominal pain) was evaluated until 100 days after allogeneic HSCT or initiation of GvHD-treatment, and the overall incidence of acute GvHD was assessed. Results: In total, 32 patients (31%) received MTX, 40 (39%) MMF p.o. and 30 (29%) EC-MPS p.o. for GvHD-prophylaxis. In the MTX, MMF and EC-MPS-groups, the frequencies of GI-tox CTC Z31 were 31%, 18% and 17% for nausea, 9%, 13% and 7% for emesis, 56%, 10% and 23% for mucositis (MTX vs. mycophenolate formulations Po0.001), 47%, 43% and 33% for diarrhea and 3%, 3 and 17% for abdominal pain (MTX þ MMF vs. EC-MPS P ¼ 0.022). GvHD-prophylaxis was reduced due to GI-tox in 4 MTX and 1 MMF patient but none of the ECMPS treated patients. All patients received PPI-prophylaxis during the entire interval of GvHD-prophylaxis. The incidence of acute GvHD was 66% in the MTX group, 78% MMF group, and 47% in the EC-MPS group (MTX vs. MMF P ¼ 0.299, MTX vs. EC-MPS P ¼ 0.2, MMF vs. EC-MPS P ¼ 0.012, MTX þ MMF vs. ECMPS P ¼ 0.022). Conclusion: GvHD-prophylaxis with mycophenolate formulations was associated with a lower mucositis rate compared to MTX. When compared with MMF, GvHD-prophylaxis with ECMPS did not lead to lower GI-tox but was associated with a significant lower incidence of acute GvHD. This could be due to PPI co-medication lowering the intestinal uptake of MMF but not EC-MPS. Disclosure of Interest: None declared. P126 Incidence of severe chronic GVHD is reduced either by ATG or by statins K. Schmidt1,*, M.-A. Schwarzbich1, N. Lehners1, P. Dreger1, T. Luft1 1 University Hospital Heidelberg, Department of Haematology, Heidelberg, Germany Introduction: Severe chronic graft versus host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic stem cell transplantation (allo-SCT). The pathomechanism of cGVHD is currently not known, however, alloreactive T cells clearly play a role. Nevertheless, severe chronic GVHD poorly responds to immunosuppressive therapy similar to acute refractory GVHD. It has previously been shown that endothelial risk factors are associated with the risk of refractory acute GVHD. These factors include angiopoietin-2 (ANG2), serum nitrates and asymmetric dimethylarginine (ADMA), as well as single nucleotide polymorphisms (SNPs) in the thrombomodulin gene (THBD). In the presence of statins the incidence of refractory acute GVHD in patients with endothelial risk factors is reduced. In the present retrospective study we investigated whether the incidence of severe chronic GVHD (cGVHD) was also influenced by endothelial risk factors and statins.
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Materials (or patients) and methods: Patients were eligible if they were allo-grafted between June 2002 and December 2011 at our institution, and if their blood samples were available for nitrate, ANG2 and/or ADMA measurement at different landmarks (collected immediately before conditioning and on day þ 100 after allo-SCT). Cumulative incidence analysis of cause-specific hazards was performed. The occurrence of cGVHD was evaluated retrospectively by chart review applying clinical and histological criteria developed by the National Institute of Health’s consensus project. Results: Of a total of 329 eligible patients, 167 (51%) fulfilled the criteria for cGVHD, and 47 (14%) of them developed severe cGHVD. There was no correlation between cGVHD and antecedent acute GVHD (P ¼ 0.796). Moreover, there were no significant correlations between serum nitrates, ANG2, ADMA and thrombomodulin-(THBD)-SNPs and the risk of severe cGVHD. Mild and severe cGVHD were associated with a significantly prolonged overall survival (OS) compared to non-cGVHD patients (Po0.001) due to a reduced relapse rate (Po0.001). This effect was stable across all entities. Antithymocyte globulin (ATG), at our institution given in case of unrelated donor, reduced the risk of mild cGVHD (Po0,001), and of severe cGVHD (P ¼ 0,016) without increasing relapse rates. Statins, introduced as part of the supportive treatment for all patients who receive an allo-graft at our institution, increase the incidence of mild cGVHD in patients who had not received ATG (chi2 ¼ 0,039). Moreover, statins reduce the incidence of severe cGVHD in patients who had not received ATG (chi2 ¼ 0,011). Introduction of statins removed the protective effect of ATG on the incidence of severe cGVHD (P ¼ 0,558). Conclusion: Unlike refractory acute GVHD, endothelial risk factors were not associated with severe cGVHD. Our results suggest a different role of the endothelial component in the pathogenesis of acute vs chronic GVHD. Statins reduce the incidence of refractory acute GVHD which has been attributed to modulation of endothelial homeostasis. On the other hand, statins also have an impact on the occurrence of mild and severe cGVHD in the absence of ATG. Thus we hypothesize that the effect in cGVHD is executed by immunomodulation of the T-cell compartment. Within the limits of this retrospective study with consecutive patient cohorts, our data suggest that statins protect from severe chronic GVHD without reducing mild cGVHD. Disclosure of Interest: None declared. P127 Chronic Graft-versus-Host Disease Biomarker Discovery and Replication K. R. R. Schultz1,*, A. Kariminia2, S. Ivison3, S. G. Holton4, M.-J. Hebert5, M. E. Flowers6, P. Martin6, J. Rozmus3, P. Subrt7, B. Storer8, S. J. Lee8 1 Pediatric Hematology/Oncology/BMT, BC Children’s Hospital/ Univ. of BC, 2Pediatric hematology/Oncolcogy/BMT, BC Children’s/Child and Family Research Institute, 3Bc Children’s Hospital/CFRI, Vancouver, Canada, 4University of Minnesota, Blood and Marrow Transplant Program, Minneapolis, United States, 5Hopital Notre-Dae-Pavillion Mailloux, University of Montreal, Montreal, Canada, 6Clinical Research Division, Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, United States, 7BC Children’s Hospital and CFRI, Vancouver, Canada, 8Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, United States Introduction: Chronic graft-versus-host disease (cGVHD) remains one of the most significant long-term complications after allogeneic blood and marrow transplantation. Biomarkers that can act as diagnostic, prognostic and predictive markers for cGVHD are needed to improve the ability to treat cGVHD effectively. Many candidate cGVHD biomarkers have been identified with none validated for use in clinical practice. Larger replication studies are needed.
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Table 1. Univariate replication results for 13 candidate biomarkers Candidate biomarkers sBAFF CXCL10 ICAM-1 CXCL9 Anti-LG32 Gelsolin AGAy
N evaluable
Effect size1
Pvalue
173 173 173 162 173 173
1.2x 1.4x 1.2x 1.5x 0.7x 0.9x
0.11 0.06 0.06 0.03 0.05 0.17
AUC
0.60 0.59 0.61 0.60
1
Fold difference cGVHD vs. control, 2Anti-LG3 has been associated with renal transplant rejection.
Materials (or patients) and methods: We evaluated cGVHD biomarkers in a small adult discovery set (N ¼ 38, 21 cGVHD within one month of diagnosis and 17 controls) followed by a replication attempt in a larger cohort (N ¼ 173, 86 cGVHD and 87 controls). Samples were provided by the Chronic GVHD Consortium. Before proteomic analysis, the plasma samples were depleted for the 13 most common plasma proteins. Samples were tested at the University of Victoria Proteomics Facility (BC, Canada) with a MALDI-TOF-TOF methodology using iTRAQ labels as we have previously done. Multiple Reaction Monitoring-Mass Spectrometry (MRM) was used to measure 71 candidates identified by proteomic studies. Additional Luminex analysis was done for cytokines. Based on these results, as well as leading candidates in the published literature, 16 of the most promising markers were tested in an independent cohort of 86 cGVHD and 87 post BMT controls. Cases and controls were matched on time of sample collection (more or less than 9 months post BMT) and previous acute GVHD. Results: Proteomic analysis of the discovery cohort (N ¼ 38) with confirmation by MRM revealed two proteins of high interest as cGVHD diagnostic markers, aminopeptidase N (sCD13, P ¼ 0.004) and Peptidase Inhibitor 16 (PI-16, P ¼ 0.005) compared to controls. Luminex suggested that IL-2Ra (P ¼ 0.0007), IL-6 (P ¼ 0.05) and IL-8 (P ¼ 0.04) were all of interest. Enzymatic studies confirmed higher levels of aminopeptidase N in cGVHD. Of the 16 candidate biomarkers, assays for 3 biomarkers had technical difficulties and were deferred for later analysis (TNFa, IL-6, IL-8). Results for the other 13 candidates with p values under 0.02 are shown in the Table. Of the other 13 markers, aminopeptidase N, IL2Ra, PI-16, MCP1, Endothelin1, and Kallikerin had p values between 0.23 - 0.52. Results were similar when adjusted for time of sample collection, prior acute GVHD and conditioning with total body irradiation. Four biomarkers, CXCL10, ICAM-1, CXCL9, and antiLG3 (p 0.03 – 0.06) were marginally associated with cGVHD with ROC AUCs of 0.60, 0.59, 0.61 and 0.60, respectively and 0.63 for the four in combination. Conclusion: Despite strong associations observed in a discovery set using proteomic analysis with duplication by MRM as well as by a screening Luminex assay supplemented by enzymatic assays, attempted replication in a larger independent cohort did not replicate the original findings. Three candidate biomarkers could not be analyzed. This analysis confirms the need to replicate results of promising cGVHD biomarkers in larger independent cohorts. Disclosure of Interest: None declared.
P128 The activating NKG2C receptor is significantly reduced in NK cells after allogeneic stem cell transplantation in patients with severe graft-versus-host disease L. Kordelas1,*, P. A. Horn2, D. W. Beelen1, V. Rebmann2 1 Department of Bone Marrow Transplantation, 2Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany Introduction: Natural killer (NK) cells are important players of the innate immune system. The allo-reactivity of NK cells is regulated by a number of receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptor pair, which are both recognizing the non-classical human leukocyte antigen E (HLA-E). In allogeneic stem cell transplantation (alloSCT) donor-derived allo-reactive NK cells are important effectors mediating the killing of leukemia cells and patient’s dendritic cells thereby preventing leukemic relapses or graftversus-host responses. To analyze the contribution of these receptors to NK cell allo-reactivity, the NKG2A and NKG2C expression were monitored in 26 allo-SCT patients. Materials (or patients) and methods: The course of CD94/ NKG2C or CD94/NKG2A expression on NK cells were studied in 26 patients undergoing allo-SCT due to acute myeloid leukemia (AML, n ¼ 20), secondary AML (sAML, n ¼ 4), myelodysplastic syndrome (MDS, n ¼ 1) and T-Non-Hodgkin Lymphoma (T-NHL, n ¼ 1). EDTA samples were serially procured from the patients before (0) and 1, 2, 3, 4, 5, 6, 9 and 12 month(s) after allo-SCT. Receptor expression on NK cells were analyzed by flow cytometer using specific antibodies against human CD56, NKG2A, CD94 and NKG2C. The results obtained were correlated to occurrence of acute or chronic graft-versus-host disease (GvHD) and its severity after alloSCT. Results: The proportion of activating receptor pair CD94/ NKG2C on NK cells was significantly reduced in 10 patients
who had experienced severe acute GvHD (aGvHD) grade 2-4 in the first months after alloSCT (Table 1a, Po0.0001, two-way ANOVA) compared to 16 patients with no or only mild aGvHD. Moreover, the proportion of activating receptor pair CD94/ NKG2C on NK cells was lower in patients with extended chronic GvHD (cGvHD) compared to patients with no or only limited cGvHD (n ¼ 13, Figure 1b, Po0.0001, two-way ANOVA). At contrast there was no difference detectable between the proportion of the inhibitory NKG2A receptor on NK cells and the incidence of acute or chronic GvHD (Figure 1c and 1d). Conclusion: Thus, these results provide substantial evidence that the CD94/NKG2C receptor pair contributes to the alloreactivity of NK cells after allo-SCT and its influence on severe acute and chronic GvHD. Further investigation will have to clarify the role of HLA-E expression, which is the cognate ligand of CD94/NKG2C. References: This study was supported by a research grant by the Deutsche Jose´ Carreras Leuka¨mie-Stiftung e.V. Disclosure of Interest: None declared. P129 Association of NIH organ cGVHD scores with blood lymphocyte subsets – a pilot study L. Grkovic1,*, D. Pulanic1, Z. Peric1, D. Batinic1, R. Serventi Seiwerth1, M. Mravak Stipetic1, E. Bilic1, R. Ceovic1, L. Rajic1, N. Durakovic1, N. Matic1, T. Klepac Pulanic1, I. Petricek1, T. Vukic1, D. Ljubas1, E. Bilic1, D. Dusek 2, E. Prenc1, I. Prah1, I. Bojanic1, M. Grce3, R. Zadro1, R. Vrhovac1, S. Z. Pavletic4, D. Nemet1 1 UHC Zagreb, 2Infectious disease, University Hospital for Infectious Diseases, 3Ruder Boskovic Institute, Zagreb, Croatia, 4 National Cancer Institute, Bethesda, United States Introduction: Immune reconstitution after allogeneic stem cell transplantation is influenced by many factors including
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chronic graft versus host disease (cGVHD). Although cGVHD is a complex multifactorial disease and there is no satisfactory therapy, distinct clinical syndromes with specific organ manifestations may exist, indicating subpathologies, which ultimately may require differently tailored therapies. To address this question, we tested the associations of immunological monitoring parameters with individual organs involvement and other key cGVHD characteristics. Materials (or patients) and methods: After establishment of a multidisciplinary cGVHD team in 2013 at the UHC Zagreb, patients were enrolled into the prospective cross-sectional study that included subspecialists exam, extended clinical assessments, and laboratory data collection at the time of study entry. Five lymphocyte subsets, T-cell (CD3 þ , CD3 þ CD4 þ helper-Th, CD4 þ CD8 þ cytotoxic-Tc) B-cell (CD19 þ ) and natural killer (NK, CD16 þ CD56 þ ) cell were analyzed in peripheral blood by flow cytometry and correlated with demographic, transplant, cGVHD-related and laboratory data. Univariate analyses were undertaken to determine associations between lymphocytes subsets (high/low by median) and outcomes of interest. Results: Thirty adult and 3 pediatric patients were enrolled into the study. The median age was 40 years [9-72], 45% were female. Median time from transplant to study enrollment was 19 months [2-197]. The median time from cGVHD diagnosis to enrollment was 7 months [0.03-178] and the median time from transplant to cGVHD diagnosis was 10 months [1.8-29]. The majority of patients were transplanted for hematologic malignancies (85%). Eighteen patients (55%) underwent myeloablative conditioning and 45% received ATG, 70% had a related donor transplant and 64% received peripheral blood stem cells as graft source. Patients received a median of 2 [0–6] prior systemic therapies, 52% were on systemic immunosuppression, 55% had severe, 45% moderate NIH global score, 76% had previous acute GVHD and 85% were classified as classic. Patients who had previous acute GVHD had lower Th cells (P ¼ 0.02). Patients with progressive cGVHD onset had lower Th (P ¼ 0.009) and B cells (P ¼ 0.0007). Higher Th, B and NK cells were found in patients presenting with classic cGVHD compared to overlap. As expected, patients who were receiving moderate or high immunosuppression had lower B (P ¼ 0.005) and NK cells (P ¼ 0.03). Patients who had documented infections since cGVHD diagnosis had lower Th (P ¼ 0.01) cells. No significant associations were found between cGVHD severity (global NIH score) and activity, and subsets of lymphocytes. However, a statistically significant association was found between moderate or severe organ involvement (NIH score 2-3) and certain lymphocyte subsets in blood: higher NK (P ¼ 0.04) in lung, lower B (P ¼ 0.005) and NK (P ¼ 0.05) in liver, lower B (P ¼ 0.03) in mouth, and higher B cells in skin (P ¼ 0.02) and joint (0.004) cGVHD involvement. No difference was found in T cell subsets depending on different organ involvement. Conclusion: Several individual organs had distinct associations with lymphocyte subset numbers in patients with cGVHD. Immune status is clearly influenced by the intensity of immunosuppression but not by the global NIH severity or activity. These data provide rationale for further studying of chronic GVHD biology in conjunction with distinct clinical syndromes. Disclosure of Interest: None declared.
P130 Multipotent mesenchimal stromal cells as graft-versushost disease prophylaxis: a prospective randomised clinical trial L. Kuzmina1,*, E. Parovichnikova1, M. Drokov1, V. Vasilieva 1, T. Sorokina 1, N. Petinati 2, I. Shipounova 2, N. Drize 2, V. Savchenko1 1 Bone Marrow Transplant Department, 2lab. physiology of hematopoiesis, National Research Center For Hematology, Moscow, Russian Federation Introduction: Acute and chronic graft-versus-host disease (GvHD) develops in more than 50% patients after haemopoietic stem cell transplantation (HSCT) and remains one of the main causes of mortality. So new methods of GvHD prophylaxis are needed. The aim of the study was to investigate the efficacy and safety of multipotent mesenchymal stromal cells (MMSC) administration for graft-versus-host disease prophylaxis. Materials (or patients) and methods: Since February 2009 65 patients received stem cell transplantation from related donors were included in the randomized study (ClinicalTrials.gov:NCTO1941394) , with acute myeloid leukemia – 25, acute myelomonocytic leukemia – 7, chronic myeloid leukemia – 9, acute lymphocytic leukemia – 17, myelodysplastic syndrome – 5, chronic lymphocytic leukemia – 1, T-cell lymphoma – 1. Patients age was 18-63 years. Myeloablative conditioning was used in 43 patients, 22 patients received reduced intensity conditioning. Standard GvHD prophylaxis was cyclosporine A þ methotrexate, cyclosporine A þ methotrexate þ prednisolon, or cyclosporine A þ methotrexate þ mycophenolate mofetil. The observation time was 4-75 months. All patients were randomized in 2 groups: with standard GvHD prophylaxis (n ¼ 33) and those who received MMSC with standard GvHD prophylaxis (n ¼ 32). MMSC were administered in the moment of blood counts recovery (leukocytes more then 109 per liter). The dose was 0,9-1,39 106/kg. The day of administration was 17-54 after HSCT (median þ 24,5 day). MMSC were derived from the bone marrow of haematopoietic stem cells donors at the day of HSCT and cultured for 3-4 weeks in alpha-MEM with 4% human platelet lysate derived from peripheral blood of the same donors. Most of the patients had fever and chills during 24 hours after MMSC administration. There were no other complications. Acute GvHD was monitored during 100 days after HSCT. Results: In standard prophylaxis group acute GvHD II-IV developed in 21,9% patients, in MMSC group – in 9,4% patients (P ¼ 0,06). Chronic GvHD was diagnosed in 33,3% patients in the first group and in 25% patients in the second group. There was no difference in disease recurrence rate and graft failure rate. The overall mortality was 36,4% in standard prophylaxis group compared to 21,9% in MMSC group. Conclusion: The results of our study demonstrate the efficacy and safety of MMSC administration as graft-versus-host disease prophylaxis. Disclosure of Interest: None declared. P131 Tacrolimus (FK) compared to cyclosporine A (CyA) after haploidentical T cell replete stem cell transplantation (Haplo-SCT) with post-infusion cyclophosphamide (PT-Cy): a retrospective analysis L. Castagna1,*, S. Bramanti1, S. Furst2, B. Sarina1, R. Crocchiolo2, L. Giordano1, J. El -Cheikn2, A. Granata2, L. Morabito1, C. Faucher2, B. Mothy2, S. Harbi2, C. Chabannon2, C. Carlo Stella1, A. Santoro1, D. Blaise2 1 Bone Marrow Unit, Humanitas Cancer Center, Rozzano, Italy, 2 Bone Marrow Unit, Institue Paoli Calmette, Marseille, France Introduction: In this retrospective study, we analyzed 100 patients treated with Haplo-SCT with PT-Cy, and receiving FK or CyA.
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Table 2.
Main results in 2 groups FK þ BM vs CyA þ PBSC
CI 30 day ANC 40.5 x109/L CI 60 day PLT 420 x109/L G 3-4 aGVHD cGVHD OS@2 year EFS@2 year Relapse incidence NRM
FK þ BM N 42
CyA þ PBSC N 43
P
79% 76% 2% 13% 65% 22% 25% 17%
93% 85% 12% 10% 70% 29% 13% 15%
0.09 0.54 0.09 0.75 0.65 0.44 0.14 0.68
Materials (or patients) and methods: Conditioning regimen consisting of Fludarabine, Cyclophosphamide, and low dose TBI for all patients. GVHD prophylaxis consisted of Cy 50 mg/ kg on days þ 3 and þ 4, FK or CyA and MMF. FK (at a total dose of 1 mg) or CyA (3 mg/kg/day) were administered as a continuous infusion. MMF (45 mg/kg/day) was administered until day þ 35. FK, CyA and MMF were started on day þ 5. FK and CyA were tapered by day þ 100/ þ 180. Stem cell source was BM or peripheral blood stem cells (PBSC). Results: The cumulative incidence of engraftment was similar between FK and CyA groups: 30-days absolute neutrophil count 40.5x109/L was 79% vs 91% (p 0.3), and unsupported platelet count more than 20 000 at 60 days was 77% vs 84% (p 0.7), respectively. The incidence of grade 2-4 acute GVHD was 33% vs 48% (p 0.1) and the incidence of chronic GVHD was 13% vs 8% (p 0.9), respectively. The 2-year progression free survival (PFS) and overall survival (OS) were 53% vs 64% (p 0.2) and 65% vs 65%, respectively (p 0.7). The 2-year relapse incidence (RI) was 28% vs 12% (p 0.08) and the 2-year non relapse mortality was 16% vs 14% (p 0.7), respectively. In univariate analysis, only disease status (CR vs not CR) impacted the PFS, OS, and RI. Patients were regrouped in 3 cohorts (1 patient excluded because received FK þ PBSC): FK þ BM (n 42), CyA þ BM (n 14), and CyA þ PBSC (n 43). 1-year NRM was not different (17% vs 14% vs 15%, p 0.8) as well as the cumulative incidence of aGVHD and cGVHD. FK þ BM (as standard treatment) was compared to CyA þ PBSC and not significative differences were founded (Table 2). Conclusion: Although this is a retrospective study with potential selection bias, using CyA instead of standard FK did not seem to affect any major clinical outcome, with special interest for aGVHD and cGVHD. The NRM is acceptable in line with that published. Disease status before haplo SCT was the only prognostic factor for survival and relapse. Disclosure of Interest: None declared.
Infectious complications I P132 Immunogenicity of virosomal adjuvanted trivalent influenza vaccination in allogeneic stem cell transplant recipients A. Ambati1,2,*, S. Einarsdottir3, I. Magalhaes1,4, T. Poiret4, R. Bodenstein4, K. LeBlanc1,4, M. Brune3, M. Maeurer1,4, P. Ljungman2,5 1 Centre for allogeneic stem cell transplantation (CAST), Karolinska University Hospital, 2Dept of Medicine Huddinge, karolinska Institutet, Stockholm, Huddinge, 3Sahlgrenska University Hospital, Goteborg, 4Dept of Laboratory Medicine, karolinska Institutet, 5Department of Hematology, Karolinska University Hospital, Stockholm, Huddinge, Sweden Introduction: The immunogenicity after a single dose of adjuvanted trivalent virosomal vaccination Inflexal Vs was
evaluated in a study cohort of 21 hematopoietic stem cell transplant (HSCT) recipients vaccinated during the 2013-14 season. This immunogenicity was compared to a control cohort of 31 HSCT recipients who received a single dose of seasonal subunit vaccination over three seasons from 20102013 at two Swedish transplant centers. Materials (or patients) and methods: Whole blood IFNg (gamma-interferon) release assays were tested both prior to and 30 days after vaccination in response to influenza pandemic (pdm) (A/California/7/2009(H1N1), A/Victoria/210/ 2009(H3N2) and B/Brisbane/60/2008 monovalent bulks. Influenza Matrix 1 HLA-A*02- GILGFVTL and pdm H1 HLA-A*02 RLATGLRNV dextramers, to gauge for the absolute number of antigen-specific CD8 þ T-cells, and pdm 2009 hemagglutinin inhibition (HI) assays, to test for neutralizing serum antibodies, were used as immunological readouts. Results: The pdm HI titers were poor in both cohorts with only 13.3% (4/30) after the seasonal vaccination and 23% (5/21; P ¼ NS) after virosomal vaccination having protective titers (HI Z 40). The increase in IFNg production from pre to post vaccination, i.e the d response was calculated. The d response to pandemic H1N1 influenza (P ¼ 0.005) and influenza B antigens (P ¼ 0.01) were significantly elevated in blood from individuals who received the virosomal as compared to the seasonal vaccine. There was no difference in d IFNg production in response to H3N2 influenza antigens between the cohorts. The IFNg response to pdm H1N1 was stronger after vaccination with the virosomal (Po0.001) as compared the seasonal vaccine in patients vaccinated more than 6 month post HSCT. We detected a significant increase in the frequency of matrix 1 (GILGFVTL) dextramer specific CD8 T-cells after the virosomal vaccine (P ¼ 0.01). There were no differences in the hemagglutinin-specific CD8 T cells between the two cohorts. Conclusion: The virosomal vaccine was a stronger inducer of IFNg production after stimulation with pandemic H1N1 and influenza B antigens than the standard seasonal vaccine while there was no difference in the proportion of patients developing protective HI titers. Additional controlled studies are therefore indicated. Disclosure of Interest: None declared. P133 Sequence typing of human adenoviruses isolated from Polish allogeneic haematopoietic stem cell transplant recipients S. Rynans1, T. Dzieciatkowski1, M. Przybylski1, A. Tomaszewska2,*, G. W. Basak3, W. W. Jedrzejczak3, G. Mlynarczyk1 1 Chair and Department of Medical Microbiology, MEDICAL UNIVERSITY OF WARSAW, 2Department of Haematopoetic Stem Cell Transplantation, Institute of Haematology and Transfusion Medicine, 3Department of Haematology, Oncology and Internal Medicine, MEDICAL UNIVERSITY OF WARSAW, Warsaw, Poland Introduction: Human adenoviruses (HAdV) from species A, B and C are commonly recognized as pathogens causing severe morbidity and mortality in haematopoietic stem cell transplant (HSCT) recipients [1,2]. Materials (or patients) and methods: The purpose of present study was to determine adenoviral types that infect HSCT recipients in Poland, and try to specify whether these infections occurred due to primary infection or endogenous reactivation. Analysis of nucleotide sequences was used to type 40 clinical isolates of HAdV obtained from 40 alloHSCT recipients. Results: Using sequencing of hexon gene, we identified six different HAdV serotypes belonging to species B, C, and E. Possibility of HAdV cross-infection was on very low level, because patients infected with the same HAdV types, were not hospitalized at the same time. In almost all patients, anti-HAdV antibodies in IgG class were detected, which indicates a history of HAdV infection in the past. Clinical symptoms of
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respiratory tract infections occurred in 27.5% of individuals, and were mainly caused by HAdV-2 and HAdV-3. Graft-versushost disease (GvHD) was present in patients infected with species B and C, but grade II of GvHD was observed only in patients infected with HAdV-B Conclusion: The predominance of HAdV-C and common presence of anti-HAdV antibodies in IgG class may strongly suggest that most infections in present study were reactivations of HAdV persisting into patient’s mucosa-associated lymphoid tissues. References: 1. Mynarek M, Ganzenmueller T, Mueller-Heine A, Mielke C, Gonnermann A, Beier R, Sauer M, Eiz-Vesper B, Kohstall U, Sykora KW, Heim A, Maecker-Kolhoff B. Patient, virus, and treatment-related risk factors in pediatric adenovirus infection after stem cell transplantation: Results of a routine monitoring program. Biol Blood Marrow Transplant. 2014; 20(2): 250–256. 2. O¨hrmalm L, Wong M, Aust C, Ljungman P, Norbeck O, Broliden K, Tolfvenstam T. Viral findings in adult hematological patients with neutropenia. PLoS One 2012; 7(5): e36543. doi: 10.1371/journal.pone.0036543. Disclosure of Interest: None declared. P134 Human adenovirus infection in Polish patients during the pre-engraftment and the post-engraftment phase after allogeneic hematopoietic stem cell transplantation S. Rynans1, T. Dzieciatkowski1, M. Przybylski1, G. W. Basak2, A. Tomaszewska3,*, W. W. Jedrzejczak2, G. Mlynarczyk1 1 Chair and Department of Medical Microbiology, 2Department of Haematology, Oncology and Internal Medicine, MEDICAL UNIVERSITY OF WARSAW, 3Department of Haematopoetic Stem Cell Transplantation, Institute of Haematology and Transfusion Medicine, Warsaw, Poland Introduction: Human adenoviruses (HAdV) are one of causative agents of emerging infectious complications in immunocompromised patients [1]. The risk factors for HAdV infection include younger age, T-cell depletion and graftversus-host disease. HAdV infections occur mainly in postengraftment phase, however some studies show that HAdV DNA could be detected also in a few days after transplantation. Materials (or patients) and methods: A total number of 1128 serum samples coming from 100 adults, subjected to allogeneic HSCT was tested for presence of HAdV DNA, using real-time PCR assay. Monitoring of clinical status of all the patients and viral load in serum samples covered the period of 100 days after transplantation. The aim of this study was to identify differences between HAdV infections in the preengraftment and the post-engraftment phase after HSCT. Results: HAdV DNA was detected in 32 patients in the preengraftment phase and in 28 patients during post-engraftment period. Patients with HAdV infection in the preengraftment phase were more likely to have received myeloablative conditioning regimen and developed GvHD. Patients with HAdV infection in the post-engraftment phase showed slightly higher average viral load. Statistical significance was found for underlying disease, and younger age of recipients. Conclusion: There is a high frequency of detectable HAdV DNA in allogeneic HSCT recipients in Poland. Adenoviral infections could occur with the same frequency in the preengraftment and post-engraftment phase, therefore routine monitoring of its DNA for at least 100 days after alloHSCT is strongly recommended. References: 1. Matthes-Martin S, Feuchtinger T, Shaw PJ et al European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ECIL-4 (2011). Transplant Infect Dis 2012; 14: 555–563. ˛ 2. Rynans S, Dzieciatkowski T, Przybylski M et al (2014): Incidence of Adenoviral DNAemia in Polish Adults Undergoing
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Allogeneic Haematopoietic Stem Cell Transplantation. Arch Immunol Ther Exp (Warsz): DOI 10.1007/s00005-014-0320-z. Disclosure of Interest: None declared. P135 Amphotericin B lipid complex is efficient and safe when used empirically or preemptively for suspected fungal infections in patients with acute leukemia or undergoing hematopoietic stem cell transplantation : A single center experience R. Mughnieh1, T. Jisr2, A. Mugharbil1, A. Youssef1, R. Jalloul1, G. Nsouli1, A. Ibrahim1,* 1 Internal Medicine , 2Laboratory Medicine and Blood Bank, Makassed University hospital, Beirut, Lebanon Introduction: Patients (pts) with acute leukemia (AL) or undergoing hematopoetic stem cell transplantation (HSCT) carry a high risk for invasive fungal infections (IFI). Amphoterician B lipid complex (ABLC) is effective when used empirically or preemptively for IFI in such pts. Materials (or patients) and methods: In this retrospective study, we aim to evaluate the use of ABLC on an empiric or preemptive basis for the management of IFI in pts with AL or undergoing HSCT. We mainly focused on response rate and tolerability along with the adverse drug events associated with ABLC therapy; in addition to premedication combinations used in the prevention of drug delivery reactions (DDR). Results: In this retrospective study reviewing 3 years’ experience (between January 2011 and January 2014), a total of 78 pts received ABLC therapy for suspected IFI. Sixty-five percent were treated preemptively and 25% were treated empirically.The overall response rate was 56%.Nephrotoxicity occurred in 24%of pts and improved of serum creatinine was in 5.3%. Moderate hypokalemia was present in 60.8% of the pts and severe hypokalemia was present in 10% but was almost correctable. Eleven percent showed hepatotoxicity throughout ABLC therapy. Drug delivery reactions were observed in 27.8% of the pts. There was a tendency to decrease the incidence of infusion reactions upon using a combination of premedication drugs including paracetamol, steroids, pethidine and antihistamines. Overall mortality rate was c20%. Conclusion: This study suggests that ABLC used emptively or preemptively is an effective and safe treatment option of suspected IFI in pts with AL or undergoing HSCT. Disclosure of Interest: None declared. P136 Active surveillance and targeted antimicrobial therapy abate infectious mortality due to carbapenemaseproducing Klebsiella ssp in HSCT recipients: outline of a CPKs outbreak A. Forcina1,*, M. Noviello1, A. Bondanza1, A. Orsini2, C. Soliman1, P. Cichero3, R. Greco4, F. Lorentino1, E. Xue1, A. Biancardi5, P. Nizzero6, F. Giglio1, C. Messina1, M. G. Carrabba1, M. Bernardi1, J. Peccatori2, P. Scarpellini7, N. Mancini8, M. Clementi8, C. Corti2, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 2Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 3 Unit of Microbiology, 4Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, 5 IRCCS San Raffaele Scientific Institute, Milano, 6IRCCS San Raffaele Scientific Institute, 7Unit of Infectious Diseases, 8Unit of Microbiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Carbapenemase-producing Klebsiella ssp (CPKs) are considered emerging killers in immunocompromised pts, such as those undergoing hematopoietic stem cell transplantation (HSCT), accounting for high rates of morbidity and mortality. From literature data, CPKs-related mortality in HSCT is estimated to be in the 40-65% range. The management of
colonized or previously infected pts is therefore challenging in this setting, and is often considered a stringent criteria of noneligibility to the transplant itself. Materials (or patients) and methods: Following a CPKs outbreak in June 2012 at our Center, we surveyed the epidemiology of CPKs infections in a cohort of 430 HSCT pts from Jan 2011 to Oct 2014 (46 months). This included 129/430 (30%) autologous transplants and 301/430 (70%) allogeneic transplants (51% HLA-haploidentical, 29% matched unrelated, 19% HLA-identical and 1% cord-blood grafts). One-hundred seventy out of 301 allogeneic HSCT (56%) pts had active disease at the time of transplantation. After an initial period of observation (18 mo) in 180 pts, we modified our internal guidelines by implementing the following measures in 250 pts from Jul 2012 to Oct 2014 (28 mo): i) active surveillance for CPKs colonization through weekly rectal swabs; ii) reverse isolation of colonized pts; iii) early initiation of a combined empirical therapy with at least two antibiotics among: colistin, tigecyclin, gentamycin and meropenem in all CPKs-colonized febrile pts. Results: During the entire observation period, we recorded 17/430 (4%) CPKs infections (4 sepsis, 2 severe sepsis and 11 septic shocks). The infection was lethal in 9/17 (53%) cases. in case of septic shock day-30 mortality was 64%. All patients experiencing septic shock had grade IV neutropenia and 7/11 (64%) had advanced disease at the time of infection. The rate of CPKs infections was not significantly different between pts analyzed before (10/180; 6%) or after (7/250; 3%) implementing the specific measures (P ¼ ns). However while before June 2012, 8/10 pts had septic shock and 6/10 died (60%), after implementing the preventive measures from Jul 2012 onwards, 3/7 pts had septic shock, but only 1 pt died (14%), resulting in a significant reduction of CKPs-related mortality (P ¼ 0.01). Rectal swabs routinely performed from Jul 2012 onwards evidenced CPKs colonization in 30/250 (12%) pts. Positive rectal swab anticipated the onset of the infection in 4/ 17 pts (25%). Sixteen pts out of 430 (4%) had a positive rectal swab before HSCT. In this subgroup, only one pt developed a CPKs infection after the procedure, showing that having a positive CPKs rectal swab pre-transplant does not increase CPKs-related mortality (P ¼ 0.007). Conclusion: The implementation of an active surveillance through weekly rectal swabs, a prompt initiation of appropriate combined antimicrobial therapy and a timely reverse isolation in case of colonization, abates CKPs-related mortality and may improve the overall outcome of HSCT. CPKs colonization does not appear as a limiting factor in the application of HSCT. Disclosure of Interest: None declared. P137 Active surveillance of multidrug-resistant gram-negative bacterial infections with rectal swabs: analysis of 332 HSCT recipients A. Forcina1,*, A. Orsini2, A. Bondanza3, P. Cichero4, C. Ossi4, R. Greco5, F. Lorentino1, E. Xue1, M. G. Carrabba1, M. Bernardi1, J. Peccatori2, P. Scarpellini6, N. Mancini7, M. Clementi7, C. Corti2, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 2Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, 3Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milano, 4Unit of Microbiology, 5Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 6Unit of Infectious Diseases, 7Unit of Microbiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Infections due to multidrug-resistant (MDR) gram-negative bacteria have an increasing trend in hematopoietic stem cell transplantation (HSCT). Since the mortality of these infections in HSCT pts is very high, strategies of active surveillance capable of identifying MDR carrier patients are
warranted. The aim of this retrospective study was to determine the epidemiology of MDR gram-negative infections in a major HSCT clinic in Italy and the value of routine rectal swabs in active surveillance. Materials (or patients) and methods: From Jun 2012 – Oct 2014, we started a policy of active surveillance of MDR gramnegative bacterial infections through 2778 rectal swabs in 332 consecutive pts receiving allogeneic HSCT (190 pts; 57%) autologous HSCT (67 pts; 20%), or chemotherapy alone (75 pts; 23%). Underlying diseases were: 159 acute leukemias (48%), 73 Hodgkin and non-Hodgkin lymphomas (30%), 49 multiple myelomas (14%), 16 myelodysplastic syndromes (1%), 14 myeloproliferative neoplasms (1%), 7 others (o1%). In the case of allogeneic HSCT, 93 were HLA-haploidentical (49%), 57 matched unrelated (30%) and 38 HLA-identical (20%) and 2 cord blood grafts (1%). Pts were also carefully followed for infections, including 3245 blood cultures, and treated according to internal guidelines. Results: During the observation period, 404/3245 (12%) blood cultures were found positive, 65 (16%) for MDR gram-negative bacteria, 76 for gram-negative non-MDR (20%), 244 for grampositive (61%) and 15 for fungi (3%), for a total of 158/332 (46%) pts. MDR gram-negative bacterial infections developed in 36/332 positive pts (11%) pts (8 sepsis, 11 severe sepsis and 17 septic shocks), and were due to the following pathogens: 15 carbapenemase-producing Klebsiella. P. (42%), 7 P. Aeruginosa (20%), 6 ESBL E. Coli (17%), 5 Sten. Maltophilia (14%), 3 other (7%). Despite aggressive antibiotic treatment, 11/36 (31%) pts died. The risk of MDR gram-negative infections was higher in allogeneic compared with autologous HSCT pts (odd ratio: 2,4; confidence interval 0.8 – 7.1), as it was the mortality due to their complications (39% vs 0%). In the allogeneic HSCT group, this risk was not associated with the disease status at time of transplant (P ¼ 0.5) or with the occurrence of acute graft-versus-host disease (P ¼ 0.1). In a total of 114/332 (34%) pts, 296/2778 (11%) rectal swabs were positive for MDR gram-negative bacteria (11%). In retrospect, positive pts had undergone a higher number of swabs (average 10,4 ± 8,3 SD) compared with negative pts (average 7,3 ± 6,2 SD; P ¼ 0.0006), underlying the importance of multiple testing. Swabs were positive in 24/36 pts (67%) developing a MDR gram-negative bacterial infection due to the same pathogen, as documented by blood cultures, compared with 90/296 pts (30%) that did not develop the infection, showing a highly significant statistical association (P ¼ 0.0001). Moreover, positive rectal swabs anticipated sepsis in 11/36 pts (30%), allowing early initiation of antimicrobial targeted therapy. Conclusion: Active surveillance of MDR gram-negative bacterial infections through weekly rectal swabs in HSCT allows identifying pts at high risk for subsequent, potentially lethal, infectious complications. Positive rectal swab significantly associate with the development of MDR infection, allowing the timely application of prophylactic strategies in order to reduce infectious mortality. Disclosure of Interest: None declared. P138 Analysis of risk factors of viral reactivation after haploidentical and matched unrelated hematopoietic stem cell transplantations with TCR alpha/beta and СD19 depletion A. Laberko1,*, M. Maschan1, L. Shelikhova1, D. Balashov 1, J. Skvortsova1, E. Boyakova1, V. Kalinina2, D. Shasheleva1, M. Persiantseva1, G. Novichkova1, A. Maschan1 1 Hematopoietic stem cell transplantation, 2Molecular Biology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: Viral infection is one of the leading causes of morbidity and mortality in patients after T-depleted (both ex vivo and in vivo) hematopoietic stem cell transplantation. Data regarding the rates and risk factors for viral reactivation
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after novel type of selective T-depletion - TCR alpha/beta depletion are limited. Materials (or patients) and methods: In this retrospective study we analyzed the data of CMV and EBV reactivations in children undergoing HSCT with TCR alpha/beta and CD19 depletion in our center during 2012-2013. We performed 137 transplantations, 44 from haploidentical and 93 from MUD for 92 children with malignant disorders (36 ALL, 38 AML, 11 JMML, 4 lymphomas, 3 MDS) and 45 children with nonmalignant disorders (14 SAA, 8 congenital bone marrow failure, 22 PID, 1 B-thalassemia). The median age of patients was 6,8 years, m/f ratio - 93/44. One hundred eighteen of our patients were seropositive (all PIDs despite documentation were considered positive), 18 seronegative, serostatus of 1 patient and 2 donors before HSCT were unknown. Donor/ recipient status was: D-/R- in 11, D-/R þ in 51 , D þ /R þ in 65, D þ /R- in 7. Sixteen patients were EBV- and 107 EBV þ , EBV serostatuses of 14 patients are unknown. Conditioning regimen differed according to diagnosis. Patients with malignant disease and PID recieved treosulfan-based conditioning, patients with BMF - Cph/Fludarabine - based. All patients recieved ATG. The level of CMV and EBV DNA in blood was monitored by PCR during the first 100 days after transplantation weekly and then, depending on immune reconstitution, up to 1 year after HSCT. Preemptive therapy with gancyclovir was used according to standard guidelines. Results: The cumulative incidence of CMV reactivation was 0,53 (95% CI 0,44-0,63), of EBV viremia is 0,4 (95% CI 0,32-0,52). The median time to CMV reactivation is 5 weeks after HSCT, the median duration of reactivation is 3 weeks, the median number of reactivations is 1. There is also no discrepancy in CI of CMV reactivation depending on donor/recipient serostatus, age, sex, kind of donor and diagnosis. The only factor significantly influencing CI of CMV reactivation was acute GVHD with systemic steroid therapy – 0,71 (95% CI 0,58-0,87) for patients with GVHD and 0,42 (95% CI 0,33-0,54) without GVHD. The incidence of CMV disease is 0,05 (4 patients had CMV-pneumonia, 2 CMV-encephalitis and 2 CMV-choreoretinitis, 1 CMV-cystitis). CMV reactivation did not affect survival. The median time to EBV viremia is 13 weeks, the median duration of viremia is 2 weeks, the median number of reactivations is 1. There is also no significant difference in CI of EBV depending on age, sex, donor, diagnosis, GVHD manifestation and donor/recepient serostatus. There were no cases of PTLD, 5 patients had EBV disease (mononucleosis, high EBV DNA load) and received Rituximab treatment. EBV reactivation did not affect survival. Conclusion: Reactivation rate of CMV and EBV after TCR alpha/ beta depleted transplantation was not different from our previous experience in unmanipulated unrelated donor transplantation. Although CMV/EBV reactivations did not influence survival, these infections remain a significant unresolved medical problem in clinical transplantation. Disclosure of Interest: None declared. P139 BK Polyomavirus Nephropathy after allogeneic HSCT A. Schifferli1,*, T. Diesch-Furlanetto2, C. Rudin3, C. A. Levy4, H. H. Hirsch4 1 Paediatric Haematology /Oncology, 2Paediatric Haematology/ Oncology, University Chilren’s Hospital of Basel, 3Paediatric Nephrology, University Chirlen’s Hospital of Basel, 4Department of Biomedicine, University Hospital of Basel, Basel, Switzerland Introduction: BK polyomavirus (BKPyV) reactivation is associated with haemorrhagic cystitis in hematopoietic stem cell transplantation (HSCT) recipients. In kidney transplant recipients polyomavirus-associated nephropathy (PyVAN) is a major cause of renal allograft failure. In HSCT patients just a few cases of PyVAN have been reported so far. In a 2009 review (Lekakis LJ et al) only 6 cases were reported, all with severe renal failure. However in a more recent study of 68 patients by Oshrine et al (2013) BKPyV detection in urine and/or blood was
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not associated with changes in estimated glomerular filtration rate and the need for dialysis or mortality. Given these controversial results we retrospectively reviewed the charts and laboratory results of our own patients in order to analyse the association of BKPyV reactivation and other factors with deterioration of kidney function in the early phase after HSCT. Materials (or patients) and methods: Patients and methods: 21of 22 patients (mean age 14.3 years, range 2-22 years; 12 males) who underwent an allogeneic HSCT between January 2011 and September 2014 at the University Children’s Hospital of Basel (Switzerland) were included. BKPyV load dynamic in blood and urine was correlated with changes in serum creatinine, urine diagnostics, and with cyclosporine through levels for at least 30 days after HSCT. Results: Fifteen patients (71%) showed BKPyV reactivation, with peak urine loads after a mean of 28.6 days (range 12-94) posttransplant. Four patients (4/21) developed grad III-IV haemorrhagic cystitis (19%) after BK reactivation: one had dialysis-dependent kidney failure, two developed a moderate renal impairment and one didn´t show change in renal function, 3 (3/4) died following complications of HSCT. Of the remaining 17 patients, 11 patients had a BKPyV reactivation with no or microscopic haematuria (52%): 7 of them showed a reactivation in blood and urine (33%). In the remaining 4 patients BKPyV was only detected in urine (19%). Ten of these 11 patients with BKPyV reactivation had a relevant increase in serum creatinine (41.5 x baseline, or over the reference value) shortly after reactivation of BKPyV in urine (range days 5-38).The elevated creatinine level persisted for several weeks or months, and in only 2 patients the serum creatinine returned to baseline (minimal follow up of 3 months). Because of the moderate renal insufficiency no biopsy was performed. Two (2/11) patients had an elevated cyclosporine level (4325ug/l, target range:100-200 ug/l) for one respectively two days within the first 4 weeks after transplantation without an elevation of serum creatinine level. Six (29%) patients had no BKPyV reactivation, 2 of them had no impairment of renal function in the early post-transplant phase (4 weeks), 3 had transient elevation in serum creatinine probably associated with drug toxicities (calcineurin inhibitor, voriconazol), one patient had prolonged renal impairment over weeks but returned to baseline during follow up. Conclusion: Discussion: BKPyV reactivation is frequently associated with impaired renal function after allogeneic HSCT. Although drug toxicity is a frequent factor, BKPyV nephropathy is possibly underdiagnosed.To confirm our hypothesis a prospective study is needed. Disclosure of Interest: None declared. P140 Time course and sites of polyomavirus activation in patients following hematopoietic stem cell transplantation (HSCT) A. Chukhlovin1,*, L. Zubarovskaya1, S. Bondarenko1, Y. Eismont1, M. Vladovskaya1, A. Volkova1, B. Afanasyev1 1 R.Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantology, St.Petersburg State I.Pavlov Medical University, St.Petersburg, Russian Federation Introduction: Polyomaviruses comprise a group of latent pathogens that may be activated in immunosuppressed patients, e.g., following conditioning therapy after HSCT transplantation. Generally, BK virus (BKV) reactivation is associated with hemorrhagic cystitis, whereas JC virus (JCV) is thought to cause specific encephalopathies. Hence, the aim of our work was to assess time dependence of BK and JC reactivation post-HSCT. Materials (or patients) and methods: The study included 297 patients, mostly, with oncohematological disorders, 1 to 60 years old who underwent allogeneic HSCT from 2010 to 2013. The treatment was due to ALL (n ¼ 101), AML (n ¼ 105), MDS (n ¼ 31), CML (n ¼ 17), malignant lymphomas (n ¼ 23), aplastic
anemias (n ¼ 12), neuroblastoma (n ¼ 5), inherited metabolic diseases (n ¼ 4). Myeloablative conditioning regimens were applied in 35% of cases. HSCT was performed from related (16%), unrelated HLA-compatible (63%), or haploidentical donors (21%). Bone marrow or peripheral blood stem cells were used as a source of stem cells (resp., 47% and 53% of the cases). Due to sufficient risk of viral infection, the patients were given standard Acyclovir prophylaxis. To prevent and treat acute GvHD, cyclosporin A and methotrexate were routinely applied. DNA diagnostics of BKV and JCV was carried out in various clinical samples using genospecific PCR primers. Sensitivity of these qualitative assays was 41000 gene copies per mL, as compared to the reference data from quantitative PCR. Results: The overall PCR positivity for BKV in the patients at 0.5 to 6 months after HSCT was, respectively, 31%, 72%, 16%, and 21% in leukocytes, urine, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL). Appropriate prevalence rates for JC were 16%, 36%, 2%, and 19%. Strong correlations existed between BKV, JCV, and CMV positivity in the samples. Hence, BKV was most common in the urine sediments. When assessing time dynamics of BKV and JCV occurrence, higher detection rates were registered at 2 to 6 months post-HSCT, thus correlating with increased risk of hemorrhagic cystitis. It should be mentioned that JCV positivity was quite rare in the CSF samples. Meanwhile, JCV is common in BAL samples at later terms (4 to 6 months) following HSCT. Conclusion: Early polyomavirus reactivation post-HSCT is associated with immunocompromised state in the patients, thus causing a well-known BKV and JCV replication, both in urothelial cells and BAL. Later on, high incidence of JCV in bronchoalveolar cells may contribute to development of alveolitis and chronic GvHD, thus deserving further studies in this field. Disclosure of Interest: None declared. P141 Use of low dose Rituximab for treatment of EBV reactivation post allogeneic stem cell transplant: A single centre experience A. Publicover1,*, M. Prichard1, D. S. Richardson1, K. Hill1, C. Hurlock1, L. Jarvis1, H. Launders1, S. Main1, N. McKeag1, J. Newman1, K. H. Orchard1 1 Haematology, University Hospital Southampton, Southampton, United Kingdom Introduction: In patients who have undergone allogeneic stem cell transplantation (allo SCT), reactivation of Epstein-Barr virus (EBV) infected memory B cells, in the absence of EBVspecific T-cells can result in post-transplant lymphoproliferative disorder (PTLD). PTLD is fatal in over 50% of cases. Quantitative PCR can be used to monitor for EBV reactivation, allowing pre-emptive therapy to be given. The most common agent used is the anti-CD20 monoclonal antibody Rituximab, which targets B cells. A negative aspect of this treatment is profound and long-lasting B lymphocytopenia. Rituximab for EBV-reactivation has usually been given at a standard dose of 375mg/m2 for four once weekly doses but the minimum effective dose for control of EBV reactivation has not been determined. Here we compare effectiveness and post treatment immune reconstitution in patients treated with 375mg/m2 versus 100mg total dose. Materials (or patients) and methods: EBV reactivation is routinely monitored post allo SCT by whole blood PCR and treatment initiated if the number of EBV genome copies per ml of blood exceeds a threshold value. We wished to explore whether effective treatment could result from a lower dose of rituximab and consequently reduced treatment from 375mg/ m2 to a total dose of 100mg irrespective of patient size, repeated if necessary based on EBV PCR response. Between December 2009 and October 2014, 43 patients at our institution were treated for EBV reactivation, two of whom had biopsy proven PTLD. 26 received an initial Rituximab dose
of 375mg/m2 (Group A) and 17 an initial dose of 100mg (Group B). The mean age of the patients was 49 years in Group A and 54 years in Group B. The single largest indication for transplantation was AML/MDS in both groups (41% versus 53%). CD4, CD19 and total IgG pre, post, six months and one year post Rituximab were used as markers of immune reconstitution. Results: In Group A 50% and in Group B 35% of patients received more than one dose of Rituximab (mean 2.1 and 1.4 respectively). No patients in Group B have received more than 200mg in total. The mean total dose of Rituximab received was 1107mg in Group A and 135mg in Group B (P ¼ 9.6x10-7). Both patients with proven PTLD were in Group A and responded to Rituximab alone. Neither CD4, nor IgG levels in either group appear affected by Rituximab. Median CD19 levels pre, post, six and twelve months post Rituximab were 0.21x106, 0x106, 0x106 and 0.12x106 in Group A and 0.18x106, 0x106, 0.06x106 and 0.11x106 in Group B (no statistically significant differences between groups at any time point). Conclusion: We have demonstrated that a single dose of 100mg Rituximab reduces the EBV titre to below our treatment threshold for 460% of patients and a second dose of 100mg has proven sufficient for all patients. Immune reconstitution between the groups was not statistically different, but the numbers are small and this requires further follow up. Reducing the initial dose to 100mg would have significant implications for transplant centres both in terms of drug cost and infusion time. Having reduced to 100mg, it would be interesting to test further dose reduction, to establish the minimum effective dose. Our only patient with a CD19 level of 40.1x106 post first dose of Rituximab had a reduced dose due to an adverse reaction, but the EBV titre dropped to below our treatment threshold. Disclosure of Interest: None declared. P142 Molecular monitoring of Herpesviridae family members after allogeneic hematopoietic stem cell transplantation a single center experience A. M. Balan1,2, A. Oprisoni1,2,*, C. Jinca1,2, A. Isac2, A. Pascalau2, M. Serban2, S. Arghirescu1,2 1 Pediatrics, "Victor Babes" University of Medicine and Pharmacy, 2 BMT, Louis Turcanu Emergency Hospital for Children, Timisoara, Romania Introduction: Molecular monitoring and consecutive preemptive therapy after allogeneic hematopoietic stem cell transplantation (HSCT) managed to reduce the incidence and severity of viral diseases due to members of Herpesviridae family, but, unfortunately there are still unsolved issues in this field. Therefore we aimed to analyze the incidence of active Herpesviridae infections as well as the safety and efficacy of pre-emptive therapy. Materials (or patients) and methods: In a prospective observational study, we have performed weekly or once in two weeks quantitative RT-PCR for Herpesviridae family members (HSV1, HSV2, CMV, EBV, VZV, HHV-6 and HHV-7) in 53 allogeneic HSCT procedures. In the majority of HSCT procedures antiviral prophylaxis consisted of Acyclovir (n ¼ 50; 94.33%), whereas only 3 patients (5.66%), at high-risk for active CMV infection, received Ganciclovir (GCV) prophylactically. Thresholds for pre-emptive non-specific (reduction in immunosuppression) and/or specific (antiviral agents, monoclonal antibodies) measures were the following: 500 copies/mL for CMV, 103 copies/mL for EBV and HHV-6, and positivity of RTPCR (100 copies/mL) for the rest of the Herpesviridae viruses monitored. Results: At least one episode of active infection due to Herpesviridae viruses occurred in 34% of the HSCT procedures, more than one active infection being documented in 11.32% of the HSCT procedures with the following distribution: CMV þ EBV þ HHV-6 – 33.33%, CMV þ HHV-6 – 33.33%, CMV þ EBV – 16.66%, EBV þ HHV-6 – 16.66%. CMV was the most
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frequent pathogen detected, with an active CMV infection incidence rate of 26.41%. At the first positivity of CMV RT-PCR, the number of copies exceeded 500 copies/mL in 85.71% from the patients with active CMV infection. Results of pre-emptive therapy with GCV were the following: clearance of CMV viremia after an average time of 24.1 days, low recurrence rate of active CMV infection (16.66%) and a relatively high-rate of GCV-associated neutropenia (41.66%). Active HHV-6 infection was detected in 9.43% of the HSCT procedures, at a median time of 50.2±61.4 days and responded favorably to GCV therapy (given due to concomitant active CMV infection) with the exception of one patient who required administration of Foscarnet. In 13.2% of the HSCT procedures, EBV was detected through RT-PCR, all of the patients with active EBV infection having relatively low viral loads (190-2520 copies/mL) and responding favorably to reduction of immunosuppresion, without concomitant administration of Rituximab. Quantitative RT-PCR did not detect the other members of the Herpesviridae family – HSV1, HSV2, VZV and HHV-7 in blood samples analyzed. Conclusion: None of the patients included in our study developed viral diseases due to Herpesviridae family members and, moreover, overall survival was not influenced by the active Herpesviridae viruses infections. Although the results of our study are limited by the sample of patients analyzed, further evidence is brought to strengthen the safety and effectiveness of pre-emptive therapy after allogeneic HSCT, guided by molecular monitoring of Herpesviridae viruses. Although, generally initiated at low thresholds comparative to other published studies, pre-emptive therapy was not associated with significant adverse events, with the exception of GCV-associated neutropenia. Disclosure of Interest: None declared. P143 Hepatitis C virus infection triggers severe autoimmune hemolytic anemia in a transplanted toddler with HLH in XIAP deficiency: successful treatment with Sofosbuvir A. Jarisch1,*, J. Soerensen1, B. Kronenberger2, M. Meister1, E. Rettinger1, A. Willasch1, A. Berger3, T. Klingebiel1, P. Bader1 1 Pediatric Hematology and Oncology, University Children’s Hospital , 2Department of Medicine I, 3Institute of Medical Virology, University of Frankfurt, Frankfurt, Germany Introduction: Hemophagocytic Lymphohistiocytosis (HLH) is a multisystem inflammatory disorder due to cytokine overproduction from excessively activated lymphocytes and macrophages. X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and presents with highly variable clinical symptoms consistent with HLH. Materials (or patients) and methods: We report a Pakistani boy who developed postpartum symptoms of HLH (pancytopenia, hepatosplenomegaly, culture-negative fevers and pulmonary failure), and a XIAP deficiency was diagnosed. Despite treatment regarding HLH 2004 protocol his HLH remains refractory. At the age of 8 month the boy was successfully transplanted from a 10/10 matched unrelated donor using conditioning with fludarabine, melphalan, treosulfan and antithymocyte globulin. Viral screening prior to transplant was negative (especially HCV IgG and IgM). The posttransplant course was uncomplicated. Results: Six month post-transplant the patient developed a severe autoimmune hemolytic anemia (AIHA, minimum Hb 2.4 g/dl) refractory to immunoglobulins, corticosteroids and rituximab. IgG antibodies were present on red cells, also being positive for C3d, ADAMST 13 was normal. This constellation and a newly presenting thrombocytopenia were suggestive of atypical hemolytic uremic syndrome (aHUS). Treatment with eculizumab resulted in better tolerated red blood cell transfusions and an increase of Hb to 5 g/dl for few days, but did not control AIHA.
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Viral testing for Parvo B19, CMV, EBV, HHV-6, Adenovirus remains negative but was positive for HCV genotype 3a (1.040.000 iE/ml, with still negative serological findings in IgG and IgM). Due to previous allogeneic stem cell transplantation and severe autoimmune hemolytic anemia current standardof-care for hepatitis C with pegylated interferons and ribavirin was not feasible. Four weeks after presenting first symptoms of severe AIHA the patient still showed a severe AIHA, additionally an increase of bilirubin levels up to 12 mg/dl and rising HCV levels (26.900.000 iE/ml). At this time Sofosbuvir, a new nucleotide analog, HCV NS5B polymerase inhibitor, has become commercially available. Even though there was no previous experience with this compound in small children, we used Sofosbuvir in this critical situation. After one week of treatment with Sofosbuvir (100 mg/d) the Hb level was stabilized and the HCV level decreased to 40.000 iE/ml (267 IE/ml after 2 weeks of treatment). Ribavarin was added when Hb levels were stabilized 4 10 g/dl and the hepatitis C PCR became negative after 4 months after starting treatment. Sofosbuvir was well tolerated throughout treatment. Conclusion: Retrospective testing of cryopreserved blood samples from the patient and his mother confirmed intrauterine infection with HCV genotype 3a. This may have triggered HLH in this XIAP deficiency patient and may explain the missing response to HLH protocol. Disclosure of Interest: None declared. P144 Features of haemorrhagic cystitis in unmanipulated haploidentical transplant recipients A. Ruggeri1,*, G. Roth-Guepin1, G. Battipaglia1, A.-C. Mamez1, F. Malard1, A. Gomez1, E. Brissot1, R. Belhocine1, A. Vekhoff1, S. Lapusan1, F. Isnard1, O. Legrand1, J. Gozlan1, D. Boutolleau2, T. Ledraa1, M. Labopin1, M. T. Rubio1, M. Mohty1 1 Hoˆpital Saint Antoine, Hoˆpital Saint Antoine, 2Hoˆpitaux Universitaires Pitie´-Salpeˆtrie`re, Hoˆpitaux Universitaires Pitie´-Salpeˆtrie`re, Paris, France Introduction: Haemorrhagic cystitis (HC) is a common complication after allogeneic stem cell transplantation (alloSCT), associated with intensity of conditioning regimen, cyclophosphamide (Cy) metabolites, and BK virus infection. The incidence of HC after unmanipulated Haplo SCT ranges from 31 to 75%. Materials (or patients) and methods: Here, we analyzed 33 Haplo recipients using post Tx-Cy. Patients were transplanted for AML (n ¼ 18), ALL, (n ¼ 7), MDS (n ¼ 4), T cell lymphoma HTLV-I (n ¼ 1), Non Hodgkin (n ¼ 1), Hodgkin Lymphoma (n ¼ 1) and aplastic anemia (n ¼ 1). Results: Most of the patients (n ¼ 19, 53%) were transplanted in advanced disease status. Thirteen patients had a previous transplant (9 allo-SCT and 4 autologous). Median follow-up was 11 (range, 4-18) months. Eleven patients received a MAC with Thiotepa þ Busulfan þ Fludarabine, and patients with active disease received a sequential approach. All patients except 8 had ATG. GVHD prophylaxis consisted of CSA þ MMF and pTx-Cy (17 patients received a single dose of pTxCy on day þ 3 (BM recipients), and 16 had 2 days of pTx-Cy on day þ 3 and þ 5 (PBSC recipients)). All but 1 patient achieved neutrophil recovery at a median of 17 days. Cumulative incidence (CI) of platelet recovery at day 100 was 65%. Two patients had secondary graft failure (day þ 25 and day þ 43 after Haplo, respectively) and were rescued with a 2nd allo-SCT. Four patients had poor graft function and received a CD34 þ cell boost. CI of 100-day grade II-IV and grade III-IV acute GVHD was 44% and 32%, respectively. CI of HC (all grades) at day 180 was 62%. Twenty patients developed HC, in a median time after Haplo of 38 days. Eleven patients (55%) developed HC before platelet engraftment, (median 26 days).
HC was classified as grade 1 in 1 patient (5%), grade 2 in 6 patients (33%), grade 3 in 8 patients (38%) and grade 4 in 5 patients (24%). At the onset of HC BK virus was positive in blood and urine in 91% of patients. Treatment of HC consisted of hyperhydratation, intravenous Ig, antispasmodic and pain management for 54% of patients (n ¼ 11), 9 patients (46%) were treated with continuous bladder irrigation, and 4 received also iv cidofovir. Two patients required clots evacuation under cystoscopy. One patient was treated by intravescical instillation of potassium alum. All HC but 2 resolved. In the univariate analysis factors associated with HC were previous transplant (P ¼ 0.01) and occurrence of CMV reactivation before HC (P ¼ 0.05). Of 20 patients with HC, 15 had a CMV reactivation before HC. Grade II-IV acute GVHD was not associated with HC (P ¼ 0.62), of 20 patients with HC, 10 experienced acute GVHD grade II-IV and in 7 cases aGVHD occurred before HC. CI of day-180 viral infections was 73% (19 patients had CMV reactivation, 15, EBV reactivation). Two patients developed EBV PTLD (1 requiring chemotherapy). Five patients experienced symptomatic adenovirus infection and 1 a resistant HSV infection. HHV6 reactivation was documented in 16 patients, none requiring treatment. Two-year OS was 50%, 18 patients are alive (16 in remission). The occurrence of HC did not impact on OS (P ¼ 0.29). Nine patients relapsed (7 with AML, 1 MPD and 1 HD). Fifteen patients died, 11 of NRM ((GVHD n ¼ 7, infection n ¼ 4) and 4 of relapse. Conclusion: Incidence of HC after Haplo with pTx-Cy is high and is associated with morbidity, especially in the setting of high risk patients, who received a previous transplant, and with impaired immune reconstitution. Disclosure of Interest: None declared. P145 Subcutaneous administration of rituximab in patients with Epstein-Barr virus reactivation after allogeneic stem cell transplantation A. Klink1,*, V. Schmidt1, S. Dornaus1, N. Winkelmann1, H. G. Sayer2, M. von Lilienfeld-Toal1, A. Hochhaus1, I. Hilgendorf1 1 Department for Internal Medicine II (Hematology and Oncology), Jena University Hospital, Jena, 2Department for Internal Medicine IV (Hematology and Oncology), Helios Klinikum, Erfurt, Germany Introduction: Epstein-Barr virus (EBV) reactivation after allogeneic hematopoietic stem cell transplantation (alloHSCT) is most common between engraftment and the first 6 months after transplant and can develop into a life-threatening posttransplant Non-Hodgkin lymphoma. The CD-20 antibody Rituximab (R) as pre-emptive therapy is an important option alongside others such as tapering of immunosuppression and/ or use of adoptive EBV-specific cytotoxic T-cells. Subcutaneous (s.c.) R is approved for the treatment of follicular- and diffuse large B-Cell Lymphoma in Europe since March 2014. One advantage is the shorter time needed for patients with s.c. in the outpatient-setting. In a single center analysis, we report on 11 patients with EBV-reactivation after alloHSCT treated with R i.v. or s.c. Materials (or patients) and methods: Eleven patients (pts) [5 AML, 2 ALL, 2 MDS, 2 sAA] with a median age of 48 years [range, 24 – 59] underwent alloHSCT between 03/2013 and 08/ 2014 after myeloablative conditioning [6 pts] or reducedintensity conditioning [5 pts]. All ten pts with a graft from an unrelated donor (7 x 10/10; 2 x 9/10, 1 x 8/10 HLA-matched) received antithymocyte globulin [4 pts ATG -Freseniuss 10mg/kg x 4, 5 pts Thymoglobulin 2.5 mg/kg x 4, 1 patient Thymoglobulin 1.5 mg/kg x 4] as in vivo T-cell depletion during conditioning. One patient had a HLA-matched family donor and did not receive ATG. Five patients received bone marrow and 6 pts peripheral blood stem cells for transplantation. GVHD-prophylaxis consisted of cyclosprine þ methotrexate in 6 pts or cyclosporine þ mycophenolate in 5 pts EBV-serostatus was positive in all pts before transplantation, and one seronegative donor. Acute GVHD (grade I/II) was
observed in 4 pts. EBV reactivation occurred on median day 33 [range, 22-191]. At time of starting EBV pre-emptive therapy 10 out of 11 pts were still in need of immunosuppressive drugs. Results: Patients with an EBV reactivation of 4 10,000 copies/ ml in the peripheral blood were given pre-emptive R (375 mg/ m2) as i.v. application for the first dose. Thereafter, ten out of 11 patients dropped their EBV-copy load and four pts received a second and 2 pts a third application of R s.c. (1400 mg total dose) whereas seven pts received a second and one patient a third application of R i.v. at weekly intervals. The response to treatment was measured by quantitative determination of the polymerase chain reaction in the whole blood 7 days after each cycle pre-emptive R therapy and 30 days after the last application. If no EBV-Load (cut offo300 copies/ml) was demonstrated after the first dose, pts received only R application as consolidation. No moderate or severe side effects in the six s.c. applications were noted, in one patient a mild skin flair was observed. So far (12/2014) no second EBV reactivation was recorded in the s.c. group, whereas two reactivations occurred in the conventional i.v. group. Conclusion: Subcutaneous administration of R as pre-emptive therapy in EBV reactivation seems to be safe and feasible. The first results show no difference in efficacy between i.v. and s.c. regimen. The implementation of further studies regarding safety, virus reactivation, disease-free survival and overall survival in long-term follow-up is recommended. Disclosure of Interest: None declared. P146 Candida crusei Septisemia Presented with Skin Lesions After Bone Marrow Transplantation In a Patient with Aplastic Anemia N. Y. Ozbek1, A. D. Do¨nmez1,*, Z. Avcı1, S. Kirkiz1, P. Is¸ık1, N. Yaralı1, B. Tunc¸1 1 Pediatric Hematology/Oncology, Ankara Çocuk Sag˘lıg˘ı ve Hastalıkları Hematoloji Onkoloji EA Hastanesi, Ankara, Turkey Introduction: Systemic candidiasis is a serious problem in hospitalizad patients, especially treated with aggresive antimicrobials, chemotherapeutics, immunomodulating and transplantation therapies (1). Diagnosis of this fatal infection is often delayed or missed, leading to high mortality rates. Nonspesific clinical manifestations and absence of diagnostic tools are the most frequent reasons for this delay (2,3). Cutaneous presentation of sistemic candidiasis is rare, but appearence of lesions may help rapid diagnosis. Here we report an immuncompromised case of sistemic candidiasis with cutaneous manifestations. Materials (or patients) and methods: A 9 year old girl, with aplastic anemia, underwent second bone marrow transplantation from her 9/10 HLA-matched sister. Eight days after transplantation she developed fever and mucositis while using prophylactic flucanasol. Two days later, she also developed severe myalgia, arthralgia and cutaneous lesions. The lesions presented as erythematous macules, papules and nodules on the trunk, extremities and scalp (Picture 1). Skin biopsy showed minimal perivascular mononuclear inflammation. On fourth day of the fever, Candida crusei isolated from her periferic blood, Hickman catheter, as well as urine and skin biopsy cultures. Additional investigations did not show any other organ involvement including spleen and liver. The patient was given intravenous liposomal amphotericin B (12 days), caspofungin (18 days), and granulocyte transfusions. Her catheter was removed immediately. Results: After treatment, skin lesions resolved in one week. Control blood and urine cultures were negative. However, she could not achieve engraftment. She underwent a haploidentical transplantation. Conclusion: We would like to suggest that patients with peculiar dermatological findings should be carefully evaluated. Quick diagnosis by means of biopsy and culture may help
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clinicians to early diagnosis, especially in immunocompromised patients. References: 1. Grabowski R, Dugan E. Disseminated candidiasis in a patient with acute myelogenous leukemia. Cutis 2003; 71: 466–468 2. Pedraz J, Delgado-Jime´nez Y, Pe´rez-Gala S, et al. Cutaneous expression of systemic candidiasis. Clin Exp Dermatol. 2009; 34: 106–110. 3. Bae GY, Lee HW, Chang SE,Clinicopathologic review of 19 patients with systemic candidiasis with skin lesions. Int J Dermatol. 2005; 44: 550–555. Disclosure of Interest: None declared. P147 Is Systemic Primary Prophylaxis Against Yeasts Always Suggested after HLA-Identical Allogeneic Hematopoietic Stem Cell Transplantation? A Single Center Experience using Low-Dose Fluconazole as Gastrointestinal Tract Decontamination B. Sarina1,*, A. Vai1, S. Bramanti1, R. Crocchiolo1, L. Morabito1, A. Santoro1, L. Castagna1 1 Humanitas Cancer Center, Rozzano (MI), Italy Introduction: Invasive fungal infection (IFI) is a major cause of morbidity and mortality after HLA-identical allogeneic hematopoietic stem cell transplantation (HLA-id allo-HSCT). In standard risk patients (pts) anti-yeast active agents based primary antifungal prophylaxis is recommended. Materials (or patients) and methods: We analyzed 97 consecutive HLA-id related allo-HSCT recipients who performed transplantation between 1999 and 2013 and received low dose fluconazole (100 mg/day) as gastrointestinal tract (GIT) decontamination, starting from hospitalization until engraftment. The aim was to describe the incidence of fungal infections. Results: Main pts’ characteristics are shown in Table 1. During early phase (r40 days after allo-HSCT), 3 proven/probable (PP) IFIs were diagnosed and during late phase (41-100 days after allo-HSCT) 1 PP-IFI occurred. PP-IFI incidence rate was 3.0% and 1.6% during early and late phase, respectively. 2 patients had invasive lung aspergillosis. Possible IFIs were diagnosed in 8 patients within the first 100 days after allo-HSCT and they were treated with empiric antifungal therapy. After a median follow-up time of 86 months (range: 12-167), the overall survival was 43% and the non-relapse mortality was 16%. 100 days IFI-related mortality was 1.0%. Conclusion: In our series, the incidence of PP-IFI during early (3.0%) and late (1.6%) phase was lower than reported in randomized and prospective studies. These data suggest that systemic anti-yeasts prophylaxis could be avoided in selected HLA-id allo-HSCT recipients, also on the basis of the local
Table 1.
Patients’ characteristics n ¼ 97
Median age Gender M/F
46 (16-66) 59/38
Diagnosis Hodgkin’s lymphoma Non-Hodgkin’s lymphoma Acute leukemia Chronic leukemia Multiple myeloma Myelodisplastic syndrome Solid tumor
21 27 11 4 10 3 21
Conditioning regimen Myeloablative Nonmyeloablative Reduced-intensity conditioning
20 (21%) 6 (6%) 71 (73%)
epidemiology. However, it should be noted that most of pts received reduced-intensity conditioning regimens. Disclosure of Interest: None declared. P148 Impact of linezolid on hematopoietic engraftment in patients after umbilical cord blood stem cell transplantation T. Vilits1, B. Huber-Krassnitzer1,*, W. Zinke-Cerwenka1, P. Neumeister1, F. Quehenberger2, M. Hoenigl3, H. Sill1, A. Wo¨lfler1 1 Division of Hematology, 2Institute for Medical Informatics, Statistics, and Documentation, 3Section of Infectious Diseases and Tropical Medicine, Medical University of Graz, Graz, Austria Introduction: Linezolid is approved for the treatment of infections caused by otherwise resistant Gram-positive bacteria, but has been associated with hematologic toxicity in the general population. Thus, there are concerns about its potential myelotoxicity in the setting of allogeneic stem cell transplantation (SCT). Umbilical cord blood (UCB) SCT is a curative option in patients, who lack a suitable HLA-identical stem cell donor, but it is associated with delayed and sometimes incomplete hematologic recovery due to the low number of hematopoietic stem cells infused. Materials (or patients) and methods: We retrospectively analysed the data of 35 patients having undergone UCB-SCT and evaluated the impact of linezolid treatment on neutrophil and platelet engraftment as well as the occurrence of linezolid resistant bacteria. Results: 35 patients (12 female, 23 male) with underlying malignant hematologic diseases undergoing UCB-SCT were included in this study. 28 patients (80%) were treated with linezolid during the neutropenic phase and 7 patients (20%) received antibiotic regimens not including linezolid. The median number of days on linezolid therapy after UCB transplantation was 16 days (range 3-57 days). Clinical characteristics including the infused dose of CD34 þ cells were well balanced between both groups. Neutrophil and platelet engraftment were similar between both groups and did not differ in the time to engraftment as well as in the percentages of patients achieving predefined neutrophil and platelet counts. For example, in the linezolid group 21 out of 28 patients (75%) were able to engraft to absolute neutrophil counts of 1000/ml (ANC1000) after a median duration of 32 days (range 18- 67 days). In the alternative group 6 out of 7 patients (85.7%) achieved ANC1000 after a median of 35 days (range 25-52 days). Absolute platelet counts of 50.000/ml (PLT50) were reached in the linezolid group in 20 out of 28 patients (71.4%; median duration to engraftment 45 days (range 19- 105 days)), in the alternative group 5 out of 7 displayed PLT50 (71.4%; median duration 43 days (range 31121 days)). Linezolid-resistant enterococci were identified in routine cutaneous swabs of 3 out of 28 patients. Conclusion: Linezolid had no negative impact on neutrophil and platelet engraftment in patients having undergone UCBSCT. Thus, linezolid can be administered safely in such patients. However, the occurrence of linezolid resistant enterococci may be a matter of concern. Disclosure of Interest: None declared. P149 Evaluation of bacteremia characteristics in allogeneic hematopoietic stem cell transplantation without antibiotic prophylaxis B. Lopez Pereira1,*, M. Lopez Duarte1, L. Yan˜ez1, R. Lobeira1, A. Bermudez1, F. Antolin1, P. Rodriguez Cudin1, M. Colorado1, G. Martin Sanchez1, C. Richard1, E. Conde1 1 Hospital Universitario Marques de Valdecilla, Servicio Cantabro de Salud, santander, Spain
(22%) (28%) (11%) (4%) (10%) (3%) (22%)
Introduction: Bacterial bloodstream infections (BBSI) are a common complication in allogeneic hematopoietic stem cell
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transplantation (HSCT) with a high impact on terms of morbidity and mortality. Systematic use of antibiotic prophylaxis remains controversial. Materials (or patients) and methods: We retrospectively analyzed the cumulative incidence (CI) of bacteremia, incidence density of catheter related bacteremia , incidence of grampositive and gram-negative microorganisms (including multidrug-resistant gram-negative bacteria), risk factors associated with BBSI and mortality rate associated in a cohort of 82 patients underwent HSCT in our centre from January 2012 to June 2014 with a median follow-up of 12 months. All patients had a central venous catheter and any of them received antibiotic prophylaxis. Median age was 51 years and 51 patients were male. Main underlying disease was acute leukemia (35). The majority of patients (35%) had received 1 prior line of treatment and 52 patients (64%) were in CR. Sixty-one patients (74%) received a myeloablative-conditionig regimen. Stem cell source was bone marrow in 58 patients (71%). 41 patients (50%) had an unrelated donor and 26 (32%) had HLA mismatch. All patients except one (singenic HSCT) received a calcineurin inhihibitor þ / MTX or MMF. Median days until PMNl Z500/mm3 was 16 (9-33). Sixtyfour patients presented mucositis grade 3-4 and 32 patients needed parenteral nutrition. Forty-three patients developed acute II-IV GVHD (16 grade III-IV) and 43 chronic GVHD (extense 16). The median time of hospital stay during all the follow up were 41 (21-193) days. Results: We found 91 episodes of BBSI in 51 patients. Median bacteremia episodes per pacient was 2,4 (1-7). The CI of patients with bacteremia was 62,2% (if only catheter related bacteremia was considered the CI was 12,2%). With a median follow-up of 12 months, the incidence density of bacteremia was 9,85 per 1000 days of catheter and the incidence density of catheter related bacteremia was 6,6 per 1000 days of catheter . Gramnegative microorganism accounted for 50,5%. The main bacterial isolations were S. epidermidis (27), E. coli (15) and E. cloacae (8), P.aeruginosa (7), E.faecium (4). The incidence of extended –spectrum beta-lactamase-producing bacteria was 9% (1 case of E.Coli and 8 E.cloacae). We can’t consider mucositis, parenteral nutrition, conditionig regimen, acute and chronic GvHD predictive risk factors for bacteremia in our study. In our serie 29/82 (35%) patients died. Among 51 patients with BBSI, bacteremia was the main cause of death in 6 (11,7%). Mortality rate at 7 days after BBSI was 7%. Thirty-one (38%) patients in absence of prophylaxis don’t have any episode of bacteremia. Conclusion: The cumulative incidence of bacteremia is high (62,2%) in our serie with a increased incidence of gramnegative bacteria (50,5%). However, the incidence of multidrug resistant gram negative bacteria (9%) and the mortality rate at 7 days after BBSI (7%) were lower than other studies (1). The incidence density of catheter related bacteremia (6,6 per 1000 days of catheter) is similar to other series (2). References: 1. Bock AM. Bacteremia in Blood or Marrow Transplantation Patients. Biology and Bone of blood and marrow transplantation (2012). 2. Dettenkofer M. Surveillance of nosocomial infections in allogeneic and autologous bone marrow and peripheral blood stem-cell transplantation. Bone Marrow Transplantation (2003). Disclosure of Interest: None declared. P150 A Delphi method-based consensus for the management of IFI in HSCT recipients C. Vallejo1,*, I. Jarque2, J. Fortu´n3, J. Garcı´a4, J. C. Garcı´a5, J. Lo´pez3, M. Rovira6, M. Salavert2, L. Va´zquez7 on behalf of The Study Group of Delphi Method for IFI Management 1 Donostia Hospital, San Sebastian, 2La Fe Hospital, Valencia, 3 Ramo´n y Cajal Hospital, Madrid, Spain, 4La Paz Hospital, Madrid, Saint Kitts and Nevis, 5de Cruces Hospital, Vizcaya, 6H. Clinic i Provincial, Barcelona, 7Clı´nico de Salamanca Hospital, Salamanca, Spain Introduction: incidence and mortality of invasive fungal infection (IFI) in high risk hematologic patients (including
HSCT recipients) are still high. As a consequence, recommendations on strategies for prevention and treatment of IFI continue to be important. Objective: identify key antifungal strategies with high level of agreement among experts to improve the outcome and survival of patients at high risk of IFI. Materials (or patients) and methods: A national survey based on the Delphi technique in one phase was conducted in Spain in November 2014. The key questionnaire was based on a comprehensive list of several strategies on IFI management compiled from national and international guidelines. The study was conducted anonymously, using an electronic platform, by clinical hematologists with experience in IFI from different centers all over the country. High degree of consensus was defined as ‘‘always’’ or ‘‘frequent’’ answers. Results: 58 experts agreed to participate on the survey and of those, 38 (65.5%) completed the questionnaire. Routinary broad spectrum antifungal prophylaxis in patients with active GVHD achieved a consensus of 93% and in umbilical cord transplant of 91%. Liposomal amphotericin B was the treatment of choice for patients at risk of IFI when the pathogen was unknown (empirical) for 85% of the experts and also the drug of choice for the targeted treatment of mucormycosis in 96%. Patients on posaconazole prophylaxis, with fever and 2 consecutive positive AGA should receive liposomal amphotericin B for 95% of the experts. Antifungal combination therapy should be considered in specific clinical settings for 91% of the experts. Conclusion: Proper prevention and therapy of IFI in the HSCT recipients is critical for their outcome. The Delphi method is a robust tool to design suitable strategies for antifungal management in high risk patients. A high number of Spanish hematologists skilled on IFI participated in the project. Their recommendations may help to improve patients’ survival. Disclosure of Interest: C. Vallejo Funding from: This survey has been financed by Gilead, I. Jarque Funding from: This survey has been financed by Gilead, J. Fortu´n Funding from: This survey has been financed by Gilead, J. Garcı´a Funding from: This survey has been financed by Gilead, J. C. Garcı´a Funding from: This survey has been financed by Gilead, J. Lo´pez Funding from: This survey has been financed by Gilead, M. Rovira Funding from: This survey has been financed by Gilead, M. Salavert Funding from: This survey has been financed by Gilead, L. Va´zquez Funding from: This survey has been financed by Gilead. P151 E. coli bacteraemia is more common in autograft patients compared with allogeneic recipients- a single centre analysis C. Saha1,*, A. Kulasekararaj1, J. Wade2, N. Desai3, V. Potter1, A. Pagliuca1 1 Haematology, 2Microbiology, 3Infection Control, King’s College Hospital, London, United Kingdom Introduction: E. coli is one of the commonly isolated species in a clinical haematology unit. There is evidence of increased E. coli bacteraemia in patients receiving chemotherapy compared to that in transplant recipients. Materials (or patients) and methods: A retrospective audit was done on patients with E.coli bacteraemia, either acquired within 48 hours of admission to haematology wards or admitted within 4 days of discharge from hospital, in King’s College Hospital, London. The audit was initially done for the period of April 2013-March 2014 and then expanded retrospectively to August 2011. The data of eligible patients were collected from the electronic patient records. Results: Between April 2013 and March 2014, total 27 patients developed E. coli bacteraemia. 48% received chemotherapy, 44% autologous haematopoietic stem cell transplant (HSCT) and 1 patient had allogeneic transplant. Among all the autologous HSCT recipients during that time in this hospital,
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14% had hospital acquired E. coli bacteraemia. Six patients had carmustine (BCNU), etoposide, cytarabine, melphalan (BEAM) conditioning and 6 patients received high dose melphalan (HDM) conditioning. The onset of sepsis was between 7-9 days post-transplant. Neutropenia (o1x109/L) was found in 88% and lymphopenia (o0.5x109/L) in 85%. Other comorbidities included diarrhoea in 70% and oral mucositis in 30%. None of them received antibiotic prophylaxis for gut sterilisation. No death was reported during this period. During August 2011-March 2014, total 66 patients had hospital acquired E. coli bacteraemia. Of them 39% patients received chemotherapy, 52% had a transplant and the rest had nonmalignant haematological conditions. 73% in the transplant group received autologous HSCT and 27% received allogeneic transplant. Nine patients receiving BEAM conditioning had this complication compared to 16 patients receiving HDM conditioning. There was 1 death in the former group. Death was reported in 2 cases in the allogeneic group, and in 2 cases in the chemotherapy group. In 80% patients receiving autologous HSCT, sepsis onset was between 5-10 days. Risk factors included neutropenia (o1x109/L) in 83.3%, lymphopenia (o0.5x109/L) in 86.4%, diarrhoea with negative stool microscopy and culture in 58%, and oral mucositis in 24%. Seven patients received prophylactic antibiotics for gut sterilisation during the period of neutropenia as a part of allogeneic transplant protocol. E. coli bacteraemia among allogeneic transplant recipients was found to have reduced each year throughout the audit period (25% in 2011-2012, 17% in 2012-2013, 4% in 2013-2014) compared to a rising trend in the autologous transplant recipients (31% in 2011-2012, 35% in 2012-2013, 44% in 20132014). Conclusion: Incidence of E coli bacteraemia in patients receiving chemotherapy is similar to those with transplants, commoner among patients receiving autologous HSCT receiving conditioning regimens including melphalan, compared to those with allogeneic transplants. Diarrhoea was commonest associated symptoms suggesting melphalan induced mucositis of the gut and subsequent translocation of gut bacteria as the possible mechanism. Gut sterilisation with a short course of antibiotic (oral colistin or ciprofloxacin) may be tried, accepting the inherent risks associated with it. Disclosure of Interest: None declared. P152 Cost of CMV infection after allogeneic HSCT: How to anticipate on the cost-efficacy of new antivirals? C. Robin1,*, F. Hemery2, C. Dindorf3, J. Thillard3, L. Cabanne4, R. Redjoul4, F. Beckerich1, C. Rodriguez5, S. Maury1, I. Durand-Zaleski6, C. Cordonnier4,4 1 Hematology, Henri Mondor Hospital and UPEC, 2Medical Information, Henri Mondor Hospital, Cre´teil, 3URC-Eco , APHP ECEVE UMRS 1123 , Paris, 4Hematology, Henri Mondor University Hospital, 5Virology and INSERM U955 Team 18, Henri Mondor Hospital and UPEC, 6Public Health department Creteil and URCEco APHP ECEVE UMRS 1123 , APHP, Cre´teil, France Introduction: CMV infection and disease are main concerns after allogeneic HSCT. Pre-emptive therapy is the most common and easy strategy to reduce the risk of CMV disease. However, new antiviral drugs, CMV-vaccines and specific cellular therapy, may in a near future, encourage prophylactic strategies. In order to anticipate on the cost-efficacy of new anti-CMV compounds, we have estimated the cost of 1st year of transplant according to the pretransplant D/R CMV serologies and to onset of CMV infection. Materials (or patients) and methods: 208 consecutive allogeneic HSCT recipients transplanted in Creteil between 2008-2013 were included. The median follow-up was 18 months. The primary objective was to estimate the costs of the 12 months after HSCT and analyse CMV serology and infection (± disease) as predictor of costs. All patients except D-/Rwere weekly screened with pp65 Ag until Sept. 2009, or PCR
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from Oct. 2009, until d100 or end of immunosuppression. No patient received CMV prophylaxis. CMV-infected patients routinely receive pre-emptive ganciclovir or foscavir for X14 days. Medical data were collected from the original charts. Hospital resource utilization was retrieved via the linked hospital admissions and Diagnostic Related Groups from conditioning to 12 months after HSCT. Patients who needed a 2nd transplant (n ¼ 6) were censored at this date. Costs were estimated from the viewpoint of the payer using national 2013 tariffs. Analyses were performed for the entire population and according to CMV infection (X1 episode), vital status and pretransplant CMV serologies. Dichotomous variables were compared using the Chi-2 test, while continuous variables, described by mean and standard deviation, were assessed with a Student test, with a 0.05 significance level. Analyses were performed using Excel (2010, Microsoft) and SAS (9.3, SAS corp. NC) software. Results: Median age at transplant was 51 years, the sex ratio M/F 1.39. Patients were transplanted for acute leukaemia (69%), lymphoproliferative (22%) or myeloproliferative disorders (6%), or other (3%). The graft was from an HLA-id sibling (40%), unrelated donor (51%) or cord-blood unit (9%). The CMV D/R serology were / (28%), þ / (10%), / þ (28%) or þ / þ (34%). 71 (34%) patients developed X1 episode of CMV infection, at a mean of 85 days (median: 47 (7-802)). Only 1 case occurred after 12 months. 9 patients developed CMV disease. CMV-infection and death were significantly related (P ¼ 0.0389). The mean 1 y-cost of HSCT was 99 kilo-euros (KE) (range: 27-280; median: 92 [IC95%: 94105]). 89.5% of hospital resources were used during the first 6 mo of HSCT. The costs at 12 months did not correlate with D/R serology. When compared to non-infected patients, CMV-infected patients had longer duration of hospitalization (median: 115 [IC 95%: 102-127] vs 87 days [80-95], P ¼ 0.0002), and same duration of ICU stays (9 vs 11 days). Although the 1-year costs were not significantly different, the costs of the 6 first months were significantly higher in CMV infected-patients (97 vs 86 KE) (P ¼ 0.0291). Conclusion: CMV infection after allo-HSCT is significantly associated with a 1 y-longer hospitalization and with an increased cost of 11 KE at 6 months post-transplant. This difference between patients who do and those who do not develop CMV infection could be reduced by efficient prophylactic strategies. However, their choice should be guided by cost-effectiveness. Disclosure of Interest: None declared. P153 The clinical and financial impact of Respiratory Syncytial Virus infection post-Haematopoietic Stem Cell Transplantation C. M. Gorcea1,*, E. Tholouli1, A. Turner2, N. Flaum1, F. Dignan1 1 Department of Haematology, 2Department of Virology, Manchester Royal Infirmary, Central Manchester NHS Foundation Trust, Manchester, United Kingdom Introduction: Respiratory syncytial virus (RSV) is a common cause of respiratory viral infections and is associated with increased morbidity and mortality in patients undergoing haematopoietic stem cell transplantation (HSCT). Little is known about the economic burden associated with RSV infection. We sought to analyse the outcome and costs associated with RSV infection in a single UK centre. Materials (or patients) and methods: We undertook a retrospective case analysis of 48 consecutive adult patients diagnosed with RSV infection post HSCT between December 2010 and April 2014. Patient characteristics: male 28, median age 55 years (range 23 to 71), diagnosis: acute leukaemia 15, multiple myeloma 16, myelodysplasia 6, aplastic anaemia 5, chronic leukaemia 3, other 3. Conditioning regimen in the allogeneic HSCT group: 22 reduced intensity conditioning (RIC) regimen and 13 myeloablative conditioning. Stem cell source:
1 cord blood transplant, 14 sibling allograft, 20 volunteer unrelated donor. RSV infection was diagnosed by polymerase chain reaction (PCR); overall, 35/227 (17%) allogeneic and 13/180 (7%) autologous HSCT recipients were RSV positive. At diagnosis, 20 patients had an upper respiratory tract infection and 28 had a lower respiratory tract infection (LRTI). LRTI was the primary indication for treatment; patients at high risk of progression to LRTI also received therapy. In total, 42 patients received Ribavirin: orally 16; aerosolised 17; aerosolised and orally combined 5; aerosolised and IV combined 4. Six patients received no treatment. The median treatment duration was 7 days (range 5 to 47). Additional therapy: 13 immunoglobulin replacement, 33 concomitant antibiotic treatment and 11 also received antifungal treatment. Results: Twenty patients were admitted for RSV treatment: 6 required intensive care (ICU) admission, 4 requiring invasive ventilation. Fifteen patients were treated as out-patients. An additional 13 patients were diagnosed as in-patients and RSV was not felt to have extended their hospital stay. The median ward stay was 10 days (range 4 to 55) and median ICU stay 5 days (range 4 to 60). After a median follow-up period of 6.5 months (range 0 to 39): 33 patients are alive, 15 patients died. Causes of death: sepsis 3, relapsed disease 3, GVHD 2, gastrointestinal bleed 1, stroke 1. In 5 patients the cause of death was attributed to RSV infection, 4 having had previous ICU stay. No RSV-related deaths were recorded in the group treated with oral Ribavirin. Admission costs were calculated based on the 2014 hospital tariff. The treatment cost was calculated according to pharmacy prices and formulation for a 70 kg adult as follows: oral dose 1000 mg/day, intravenous formulation 33 mg/kg loading dose followed by 16 mg/kg and aerosolised preparation 6 gm/day. The total cost of ICU admission was d148,268 (h 187,146), ward admission cost of d122,100 (h154,116) and total treatment cost was d78,606 (h99,217). The overall cost for our cohort rose to the total sum of d 348,978 (h440,485). Conclusion: RSV in post HSCT patients remains a challenge due to the high frequency of infection and high rates and costs of readmission. Oral ribavirin may be an option for lower risk patients. Prompt initiation of treatment in high risk patients is essential due to increased morbidity and mortality associated with RSV- associated LRTI and may avoid unnecessary hospital admission. Disclosure of Interest: None declared. P154 Monitoring of blood BK virus is useful to predict BKrelated haemorrhagic cystitis in select high-risk postallogeneic transplant recipients C. Phipps1,*, A. Ghosh1, Y. C. Tan 1, A. Ho1, Y. T. Goh 1, S. Gopalakrishnan1, J. J. Lee1, Y. C. Linn1, W. Hwang1 1 Department of Haematology, Singapore General Hospital, Singapore, Singapore Introduction: Haemorrhagic cystitis (HC) in recipients of allogeneic stem cell transplantation (SCT) has been associated with latent BK-virus (BK) reactivation. Current treatment for BKHC is inadequate and relies on supportive care and measures to control bleeding. We initiated a plasma BK monitoring protocol in our allogeneic SCT patients on the basis that early detection or rising BK blood levels may predict for onset of BK-HC. Materials (or patients) and methods: Our primary objective was to determine whether blood BK monitoring would predict for the development of BK-HC. Secondary objectives were to define incidence of HC and BK reactivation, and identify patients at highest risk of BK-HC after allogeneic SCT. The monitoring protocol involved analysis of plasma before SCT and then weekly from day 14 to day 56 or until 2 consecutive negative results using quantitative polymerase chain reaction targeted at VP1 protein gene of BKV. These were paired with
urine microscopy samples. Only grade Z2 HC was considered positive. Results: The monitoring period involved 117 patients, 50 females and 67 males. Median age was 46 years (range 12 – 68) and median follow up in surviving patients, 17 months. Donor types were as follows: sibling, N ¼ 57; unrelated (URD), N ¼ 39; cord, N ¼ 17; haploidentical, N ¼ 4. Graft source was PBSC in 98, bone marrow in 2, cord blood in 17. Conditioning regimens consisted of conventional myeloablative (MAC), N ¼ 50; reduced-intensity (RIC), N ¼ 60; non-myeloablative, N ¼ 7. Antithymocyte globulin (ATG) was given to 42 patients. It was used routinely in all recipients of unrelated donor grafts, excluding cord units. Apart from ATG, post-transplant cyclophosphamide was given as T-cell depletion (TCD) in the haploidentical SCT recipients. BKV was detected in 39 patients (33%) at a similar rate in those receiving MAC and RIC regimens. HC was detected in 24 patients (21%) and was grade 2 in 17, grade 3 in 4, and grade 4 in 3 patients. Blood BK was positive in 15 of 24 patients with HC (P ¼ .009), giving an incidence of BK-HC of 13% . Ten of these were positive before the onset of HC but only 1 patient had rising BK titres coinciding with HC development. HC occurred at a median of 21 days (range 1-73). In non-BK related HC, median onset was 9 days and attributed to chemotherapy and thrombocytopaenia. BK-HC occurred at a median of 30 days (range 6 – 73). Univariate analysis identified ATG use, URD grafts, and TCD as significantly influencing the development of BK-HC. Only TCD was significantly related to BK-HC on multivariate analysis (P ¼ .009). It occurred in 22% of patients with TCD grafts. Conclusion: BK-HC causes significant morbidity and treatment of overt disease is difficult and usually lengthy. Our prospective monitoring of blood BK levels and development of HC revealed that of patients who developed BK-HC, 67% had detectable levels before the onset of overt HC. Furthermore, we identified TCD (in vivo in our series) as being significantly associated with BK-HC. Considering these results, monitoring of blood BK levels may be advisable in patients who have undergone T-cell depletion of their grafts with a view to reduce immunosuppression where possible and initiate treatment with the goal of halting progression to overt HC. Disclosure of Interest: None declared. P155 Multidrug-resistant bacterial pathogens in allogeneic hematopoietic cell transplantation – dismal outcome of multidrug-resistant Pseudomonas aeruginosa colonized patients D. Heidenreich1,*, F. Nolte1, S. Kreil1, M. Reinwald1, W.-K. Hofmann1, S. Klein1 1 III. Medizinische Klinik, UNIVERSITA¨TSMEDIZIN MANNHEIM, Mannheim, Germany Introduction: Multidrug-resistant bacterial pathogens (MRP) such as vancomycin-resistant enterococci (VRE) and extendedspectrum beta-lactamase producing bacteria (ESBL), particularly Pseudomonas aeruginosa are an emerging challenge in allogeneic hematopoietic cell transplantation (HCT). However, there are no data on the impact of MRP on the outcome after HCT in the literature. Thus, it was the aim of this study to analyze the issue of MRP in patients undergoing HCT. Materials (or patients) and methods: A total number of 72 consecutive patients who received the first HCT from 2010 to 2013 at our institution were retrospectively analyzed. The underlying diseases were AML (44), ALL (5), CML (4), MPN (2), lymphoma (5), MDS (9), and multiple myeloma (3). Conditioning was myeloablative in 23 and reduced intensity in 49 patients. Stem cell sources were peripheral blood (69) or bone marrow (3) from matched siblings (19), matched unrelated (45), mismatched (5) or haploidentical donors (3). As baseline investigation patients underwent a screening for MRP i.e. ESBL, VRE and MRSA. Swabs from nose, throat, axilla, urethra, anus,
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stool and urine were collected. Microbiological investigations such as cultures from blood, urine, swabs, stool or central venous catheters were performed whenever clinically needed. ESBL were categorized as multi-resistant gram-negative bacteria (4MRGN, resistant to cephalosporins, piperacillin, fluorochinolones and carbapenems) or as 3MRGN (resistant to 3 of these 4 drug groups). Results: 23/72 patients (32%) were colonized by MRP (MRP þ group) either at baseline (baseline MRP þ group, n ¼ 13, 18%) or at any other time point until day 100. 4 patients were positive for two MRP and 1 patient for 3 MRP. Detected MRP (n ¼ 29) were 3MRGN E. coli or Klebsiella pneumoniae (n ¼ 12), 4MRGN Pseudomonas aeruginosa (P. aeruginosa, n ¼ 4), 3MRGN P. aeruginosa (n ¼ 2), 4MRGN Stenotrophomonas maltophilia (n ¼ 2), 3MRGN Citrobacter freundii (n ¼ 1), VRE (n ¼ 7) and MRSA (n ¼ 1). Out of these 23 patients 8 developed an infection with MRP: Septicemia with 3MRGN E. coli or K. pneumoniae (n ¼ 4), septicemia with P. aeruginosa (4MRGN n ¼ 3) and one patient with VRE septicemia and 4MRGN P. aeruginosa pneumonia. Out of the 4 patients with 4MRGN P. aeruginosa infection 3 died (2 of these patients had been already colonized with 4MRGN P. aeruginosa at baseline). However, 2-year overall survival of MRP colonized versus noncolonized patients was essentially the same (63 vs. 67%, median follow-up 24 months). Day 100 NRM was higher in the baseline MRP þ group and in the entire MRP þ group in comparison with non-MRP colonized patients (23% and 17% vs. 10%, not statistically significant [ns]). Data for 2 year NRM were 33%, 22% and 17% (ns), respectively. The increased NRM of MRP þ patients was mainly due to the high NRM of patients infected by multidrug-resistant P. aeruginosa. Conclusion: Colonization or infection with 3MRGN gramnegative non-P. aeruginosa bacteria or by VRE has no negative impact on the outcome after HCT. Thus, HCT of patients colonized by MRP is feasible. However, patients colonized by multidrug-resistant P. aeruginosa seem to have a dismal outcome. HCT of these patients should be considered with care. We therefore suggest including screening for MRP in the pretransplant recipient work-up particularly identifying patients colonized by multi-resistant P. aeruginosa. Disclosure of Interest: None declared.
or VGV. 55 patients received treatment with CDF either because of failure to respond to GCV or VGV (n ¼ 43), CMV disease (n ¼ 7), or CMV infection occurring prior to neutrophil engraftment (n ¼ 5). The CMV specific CD8 þ T cell response was serially measured by HLA-CMV peptide tetramers in a sub-group of 20 patients. Results: 101/153 patients responded to GCV as first-line therapy. The likelihood of developing CMV disease was increased by use of T-cell depletion (P ¼ 0.033) and CMV / þ (D/R) serostatus (P ¼ 0.035). 44/55 patients treated with CDF achieved a durable response. 28 of 35 evaluable patients (80%) patients treated with CDF for GCV resistant CMV viremia achieved sustained (Z2 weeks) CMV PCR negativity. Responses were rapid with a median time to eradicate viremia of 15 days. CDF was generally well tolerated and only 2 patients experienced Grade 3-4 nephrotoxicity (NCI toxicity criteria 2.0). There was no difference in overall lymphocyte count, CD4, CD8 or gamma/delta T cells at any point post reactivation between patients that responded to GCV and those that did not. The number of CMV-specific CD8 þ T cells at D þ 50, D þ 100 and D þ 150 post transplant was lower in patients who required salvage therapy with CDF than in patients who responded to GCV. Importantly, no patient with undetectable CMV-specific CD8 þ T cells responded to GCV but 4/7 patients with absent CD8 þ T cells responded to CDF. Furthermore, low CMV-specific CD8 þ T cell numbers after the first episode of CMV reactivation are predictive of further CMV reactivations. The median CMV specific CD8 þ T cell count was 16 times higher in patients that experienced only one episode (n ¼ 13) of reactivation versus those that had multiple episodes (n ¼ 7) (P ¼ 0.007). Conclusion: CDF is a well-tolerated and effective salvage therapy for GCV resistant CMV viremia. In contrast to GCV it has the capacity to eradicate CMV infection in the absence of an antigen specific CD8 þ T cell response. CDF may also be used for secondary pre-emptive therapy in patients identified as at high rsk of recurrent CMV reactivation based on measurement of CMV-specific CD8 þ T cells at the end of the first episode of CMV reactivation. Disclosure of Interest: None declared.
P156 Cidofovir Treatment may benefit patients with low CMVspecific CD8 T cell levels after initial CMV reactivation post allogeneic stem cell transplant D. J. Lewis1,2,*, F. Farina1, P. Moss1,2, S. Nagra1, M. Raeiszadeh2,3, F. Chen1,2,3, R. Malladi1,2, C. Craddock1,2 1 Centre for Clinical Haematology, University Hospital Birmingham, 2School of Cancer Sciences, University of Birmingham, 3 R þ D Stem Cells and Immunotherapies, NHS Blood and Transplant, Birmingham, United Kingdom
P157 Peg-Filgrastim Versus Filgrastim In Prevention Of Febrile Neutropenia In Lymphoma Patients After Autologous Peripheral Blood Stem Cell Transplantation D. Radic-Kristo1,*, V. Zatezalo1, K. Misura Jakobac1, N. Gredelj Simec1, S. Jurenec2, N. Pavic´1, I. Mandac Rogulj1, M. Martinovic1, A. Planinc Peraica1, S. Ostojic Kolonic1 1 Department of hematology, 2Transfusion medicine, Clinical hospital ‘Merkur‘‘, Zagreb, Croatia
Introduction: Cytomegalovirus infection (CMV) remains a significant cause of morbidity and mortality following allogeneic stem cell transplantation (SCT). Up to 50% of CMV seropositive patients undergoing RIC have recurrent reactivation, associated with significant post-transplant complications. CMV-specific T cell measurements have been performed in a research setting using specific HLA-peptide tetramers, but are not yet routinely used in a clinical setting. Ganciclovir (GCV), and its orally available prodrug, valganciclovir (VGV) is established as standard of care in patients with CMV reactivation but the optimal treatment strategy for patients requiring second-line therapy such as with recurrent/ refractory viremia remains unknown. Cidofovir (CDF) demonstrates activity in CMV disease but its tolerability and activity in patients with GCV resistant CMV reactivation has not been studied. Materials (or patients) and methods: 397 patients at risk of CMV reactivation after allogeneic HSCT were sequentially monitored over a 7-year period. 156 patients required treatment of whom 152 received initial treatment with GCV
Introduction: Filgrastim and pegfilgrastim are widely used in prevention of febrile neutropenia after stem cell transplantation. The aim of this study is to compare the efficacy in prevention of febrile neutropenia using a single fixed dose of 6 mg of pegfilgrastim versus multiple daily administration of filgrastim 5 mcg/kg and to compare time to engraftment of leukocytes (defined as time to recovery of leukocytes to a value 41x109/L) after autoPBSCT. Materials (or patients) and methods: In this retrospective study we analyzed data of two groups of lymphoma patients who underwent autologous PBSCT. First group consisted of 47 patients (31 male, 16 female; median age 53, range 20-70), transplanted from January 2012 to June 2014, all of whom received pegfilgrastim single 6 mg dose on day 1 after autologous stem cell reinfusion. The second group is a historical cohort of 110 lymphoma patients (56 male, 54 female; median age 45, range 19-71) who had been transplanted between 2003 and August 2009 and received filgrastim 5 mcg/kg daily starting on day 1 until leukocyte
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and neutrophile engraftment was registered. Both groups had similar distribution of lymphoma subtypes; patients with Hodgkin’s disease represented largest subgroup followed by DLBCL, follicular lymphoma, and mantle cell lymphoma while other subtypes were less common. In both groups, all patients, received BEAM as a conditioning regimen before autologous PBSCT. In first group all patients received ciprofloxacin, acyclovir and fluconazole as antiinfective prophylaxis. In second group 54,5% of patients received ciprofloxacin and others received norfloxacin as antimicrobial prophylaxis. Most received fluconazole and ketoconazole (60% and 32% respectively) as antifungal prophylaxis while a few (8%) received other antifungal drugs. All patients received acyclovir. Results: In first group incidence of febrile neutropenia was 57,4%. Of those with febrile neutropenia, microorganism was isolated in 32,65% of cases. Median duration was 3 days (range 1-9), mean 3,18 days. Median time to engraftment of leukocytes was 9 days (4-19), mean 9,85 days. In historical cohort 64% of patients had FN, with median of 2 days ( 1-11), mean 3,36 days. Microorganism was isolated in 31,94% of cases. In this group median time to engraftment of leukocytes was 10 days (6-28), mean 10,28 days.There was no statistically significant difference between the two groups in incidence of FN or its duration, frequency of culture isolation nor in time to leukocyte engraftment. Conclusion: This study has shown no statistically significant differences in incidence nor duration of febrile neutropenia using pegfilgrastim vs filgrastim in lymphoma patients after autologous PBSCT. Disclosure of Interest: None declared. P158 The impact of carbapenem resistant klebsiella pneumonia related infections on the outcome of haematopoietic cell transplant recipients D. Mallouri1,*, E. Tsorlini2, V. Constantinou1, S. Papadimitriou1, M. Kaliou1, V. Kalaitzidou1, K. Panteliadou1, M. Iskas1, I. Batsis1, C. Smias1, D. Apostolou1, D. Sotiropoulos1, I. Sakellari1, A. Anagnostopoulos1 1 Haematology department - BMT unit, 2Microbiology Department, George Papanicolaou Hospital, Thessaloniki, Greece Introduction: Carbapenem resistant klebsiella pneumonia (KP) seems to be an emerging pathogen in haematologic malignancies. Little is known about its epidemiology and deleterious effect on haematopoietic cell transplant (HCT) recipients and potential excess of treatment related mortality. Materials (or patients) and methods: In this retrospective single centre analysis, we reviewed the medical data of all consecutive transplanted patients (pts) from 1/2009-10/2014. The total number of pts were 491; autologous (autoHCT) 266 and allogeneic (alloHCT) 225. Among them we identified 63 pts (21 females, 42 males) diagnosed with at least one KP isolate from the time of disease diagnosis to last follow up. The pts were suffering from acute leukemia (43), lymphoproliferative disease (14) multiple myeloma (3), MDS/MPDs (3) and the disease phase was early in 30, intermediate in 12, advanced in 21 pts, post median lines of treatment 3 (1-11). Twelve pts underwent autoHCT and 51 pts alloHCT (MUD 28, sibling 20, haploidentical 3). Results: In 17 pts (3.4%) a previous to HCT KP colonization was found from either perirectal area (5), urine (8), blood (3) or pancreatic fistula (1). Out of these 17 pts, 10 had infection diagnosed as KP associated that resolved before HCT. From the beginning of the conditioning and afterwards, KP associated infection was diagnosed in 40 pts (total, 8.1%), 4.1% of the autoHCT population (n ¼ 11) and 12.8% of the alloHCT population (n ¼ 29; 10 sibling, 16 MUD, 3 haploidentical). The main source of isolation was blood in 14, urine in 16, sputum in 8 and fecal in 2 pts. Twelve of these pts were previously colonized with KP. The pts developed sepsis (8),
bacteremia (6), pneumonia (10) and urine infection (16). The KP species were mainly refractory to many antibiotics and carbapenems and sensitive mostly to colistin (polymyxin E), gentamycine and tygecycline, while in 11 pts the KP species were refractory in all antibiotics tested. Eventually, 17 pts died from: infectious complications in association with graft versus host disease (GVHD) (4 pts), infectious complications in association with disease relapse (4 pts), refractory GVHD (2), relapsed/refractory disease (7 pts). The early deaths ( þ 100d) were only 2 and TRM was 6/40 pts. Conclusion: Through this time period is obvious that KP is an emerging pathogen and surveillance cultures leading to prophylactic decontamination treatment of infected patients is essential for this high risk population. The pre-transplantation colonization of pts does not seem to be an exclusion criterion and does not predict infection post-transplant. In both transplant modalities KP infection per se was not the sole cause of death since it was associated with heavily immunocompromised pts either due to refractory disease or severe GVHD. Disclosure of Interest: None declared. P159 Hemorragic cystitis after haploidentical stem cell transplantation: predisposing factors analysis D. Champ1,*, J. Gayoso1, M. Kwon1, P. Balsalobre1, D. Serrano1, A. C. Franco1, M. Bastos1, A. Pe´rez-Corral1, C. Pascual1, J. Anguita1, I. Bun˜o1, J. L. Dı´ez-Martı´n1 on behalf of Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n 1 Hematology, HGU Gregorio Maran˜o´n, Madrid, Spain Introduction: Hemorragic cystitis (HC) is a well known complication after hematopoietic stem cell transplantation related to the toxic effect of the conditioning regimen or viral reactivation/reinfection causing significant morbidity and even mortality to patients. HC has become an increasing problem after the widespread use of haploidentical transplantation (HAPLO) with post transplant cyclophosphamide (PT-Cy) as GvHD prophylaxis, but its real magnitude is not stablished. Our work studied the incidence and predisposing factors for HC in the context of HAPLO with PT-Cy in a single center. Materials (or patients) and methods: We retrospectively analyzed 60 consecutive adult patients receiving an HAPLO with PT-Cy in our center from Dec-2007 to July-2014, either after myeloablative (MAC) or reduced intensity conditioning (RIC). We evaluated patients and donors characteristics, graft source, conditioning regimen, GvHD prophylaxis, HC severity, onset and evolution, microbiologic isolations and HC treatment. Results: HC was diagnosed in 21/60 HAPLO recipients (35%) until day þ 100, median age was 34 years (range: 17-61) and 14/21 were males. HC severity was grade I (microscopic hematuria) in 5 (8%), grade II (macroscopic hematuria) in 6 (10%), grade III (hematuria with clots) in 5 (8%) and grade IV (clots retention, obstructive nephropathy) in 5 (8%). Median days of HC onset was 34 days post-HAPLO (interquartilic range 25-75% (IQR): 28-40) and median days of HC duration was 19 (IQR25-75%: 13-31), without clear relationship with severity. In 18/21 cases (86%) BK virus was isolated in urine samples and no microbiologic isolations were detected in 3/21 (14%). All patients had received fluid overload, diuretics and MESNA prior to PT-Cy and levofloxacin for HC prophylaxis, and received as HC treatment vigorous hydratation, diuretics, hemotherapy, analgesics and bladder irrigation in those with pain or obstruction due to clots. Hyaluronic acid washing was employed in 3, electrocoagulation in 3 and mesenchymal stem cells were used in 4 refractory HC with partial responses only. Antivirals and immunoglobulin replacement were not employed routinely. None of our patients died as direct consequence of HC. None significant relationship was found between patients and donor characteristics, underlying disease, graft source (BM vs PBSC), conditioning regimen
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(RIC vs MAC) or the presence of acute GvHD or steroids use. The only factor probably related to HC development was the presence of CMV reactivations, which was present in 17/21 patients with HC (81%) compared to 27/39 (69%) in patients without HC. The incidence of HC was higher during springsummer (14/21) compared to autumn-winter (7/21), and clustered distribution was observed. Conclusion: HC is a common complication in the setting of HAPLO with PT-Cy, mostly related to BK virus reactivations, that causes significant morbidity but not mortality in our series. Only CMV reactivation was found as a related factor for HC development, but it could also represent another manifestation of the immunosuppression status of the patients and not a real predisposing factor for HC. Epidemiological factors could be responsible for seasonal or clustered distribution. Disclosure of Interest: None declared. P160 Final results of the prospective study on the role of bed side ultrasound (BUS) in neutropenic enterocolites (NEC). Early US diagnosis reduces the mortality and changes the diagnostic criteria with a new discriminant statistical model E. Benedetti1,*, I. Bertaggia1, R. Morganti1, B. Bruno2, P. Lippolis3, F. Caracciolo4, E. Orciuolo1, G. Buda1, F. Cerri5, D. Caramella5, M. Petrini6 on behalf of NEC 1 Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Hematology Unit Ospedale S. Chiara Pisa, Pisa, 2U.O Ematologia Trapianti di midollo, University of Torino, Torino, 3UO Chirurgia D’Urgenza , Azienda OspedalieroUniversitaria Pisana, 4U.O Ematologia Trapianti di midollo, Hematology Unit Ospedale S. Chiara Pisa, 5Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, U.O. Radiologia Universitaria Pisa, 6Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Hematology Unit Ospedale S. Chiara Pisa, Pisa, Italy Introduction: Neutropenic enterocolitis (NEC) is a life threatening complication of leukemic and solid tumors patients (pts) treated with chemotherapy (CHT) with mortality rate up to 50%. NEC is characterized by abdominal pain (AP), fever (F) and diarrhoea (D). Ultrasound (US) was used to evaluate bowel-wall thickening (BWT), and 4 4 mm is considered diagnostic of NEC. Perforation occurs in 5%410% of cases. Early diagnosis is crucial to start conservative medical management (CMM) which appears the optimal strategy for most cases. We evaluated prospectively if Bed-side-US (BUS) can detect early signs of NEC and guide a prompt treatment (CMM or surgical) in order to reduce mortality Materials (or patients) and methods: In the last 7 years all pts admitted in Our Hematology/BMT Unit wards at University of Pisa (Italy), undergoing chemotherapy (CHT), autologous or allogeneic transplant (AutoTx,AlloTx) were enrolled. Abdominal US was performed, baseline before treatment, and as only one symptom (or a combination) appeared within 12h from onset: F and/or D and/or AP in CHT-related neutropenic pts. Results: 76 episodes out of 1680 neutropenic pts were identified (4.7%). Seven pts had 2 separate episodes of NEC. Disease diagnosis were HD (N ¼ 10), ALL (N ¼ 8), AML(N ¼ 21), MM (N ¼ 9) and NHL (N ¼ 28). Treatment received was intensive CHT (N ¼ 35), AutoTx (N ¼ 37) and AlloTx (N ¼ 4). At time of diagnosis (Dx) symptoms were: F þ AP þ D 48%, F þ D 4%, F þ AP 1%, AP þ D 34%,D 3%,AP 9%. F alone were never present at diagnosis of NEC. At Dx, F was absent in 35/76 NEC episodes (46%) and in 17/76 F never developed and 18/76 had delayed onset of F (from 10h to 72 h) from NEC Dx. There is a trend but not a statistical (St) significant difference in mortality among the 3 F groups (P ¼ 0.09). As control group (CG) we considered pts with CHT related mucositis and pts restaged with US during neutropenia in absence of symptoms. A total of N ¼ 509 pts were randomly chosen in the CG. None of them had BWT. Overall 11 pts died (14.5%) without a St difference
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between 1 or 2 episodes (P ¼ 0.309) of NEC. Treatment was CMM in 92% of pts, and was promptly started as BUS diagnosis was made. Mortality in pts treated with CMM was 11.5%. Six pts underwent surgery, guided by US features, during neutropenia, and 50% are alive. Median BWT was 8.6 mm in surviving pts (range 4.2-30mm) and 11mm in deceased (range 9.3-15mm). Authors have suggested BWT to be prognostic of outcome; in our study pts with 410mm had 60% survival. Median time to response from beginning of CMM was 24h and the first sign of was a decrease in AP, while median time to death was 26h (range 10.5-72h). The likelihood of NEC Dx in a discriminant St model (Bayes theoreme) for pts with BWT and AP ¼ 98.8%, AP þ D ¼ 99.9%, AP þ D þ F ¼ 100%, AP þ F ¼ 99.9%, D þ F ¼ 5%. Conclusion: BUS allowed to detect early signs of NEC and to start a prompt treatment, which was CMM in 92% with a 88.5% survival rate. With BUS pts do not live the isolation room. US guided surgical intervention with 50% survival rate. Images of BUS and CT were superimposable. Fever is not a condition sine qua non for NEC diagnosis. A prompt BUS in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical) reducing mortality. Disclosure of Interest: None declared. P161 Biological characterization of cytomegalovirus pp65 and IE1-specific CD4 þ and CD8 þ T cells from healthy subjects unfolds their heterogeneous profiles E. Albiero1,*, E. Amati2, E. Baumeister3, G. Astori2, H. Einsele3, G. U. Grigoleit3, F. Rodeghiero1 1 Hematology Project Foundation Research Laboratories, Department of Cell Therapy and Hematology, San Bortolo Hospital, 2 Advanced Cellular Therapy Laboratory, Department of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy, 3 Department of Internal Medicine II, Division of Hematology and Oncology, Julius Maximilian University Medical Center, Wu¨rzburg, Germany Introduction: Characterization of human cytomegalovirusspecific T cells (CMV-T) is of crucial importance for their potential use in adoptive immunotherapy (AI) after HSCT. In peripheral blood mononuclear cells (PBMCs) from CMVseropositive (CMV þ ) healthy subjects background frequencies of CMV-T are usually very low, hence the requirement for prolonged culture time and multiple stimulations to expand them. The evaluation of the end-culture specificity and composition have been sometimes neglected or difficult to assess in these settings. We explored the identity and the functionality of CMV-T enriched in short-term cultures after ex vivo stimulations with viral antigens (Ags). Materials (or patients) and methods: We induced mixed lymphocyte peptide cultures (MLPCs) to enrich cognate pp65and IE1-specific T cells from PBMCs obtained from CMV þ healthy donors (n ¼ 9) before selection by cytokine capture system or CD154 microbeads. CMV-T were characterized by flow cytometry analysis of T-cell subpopulations, intracellular cytokine staining combined with CD107a degranulation assay, CFSE-proliferation assay and cytokine production. Results: The median frequencies of pp65-specific CD3 þ CD8 þ cells (CTLs) was 0.17% (o0.01%–1.56%) and 0.66% (0.05%–3.61%) of the IE1-counterpart. Median frequency of pp65-specific CD3 þ CD4 þ (Th) cells was 0.34% (o0.012.89%) and 0.17% (o0.01-4.79%) of IE1-specific Th cells. CD107a degranulation by CTLs in PBMCs upon stimulation with pp65 and IE1 was 0.46% (o0.01-6.51%) for the former and 0.13% (o0.01-7.99%) for the latter. There was no correlation between the frequency of CMV-T in PBMCs and their cytotoxic potential. Ag-specific Th cells were negative for CD107a. Cell viability after MLPC_pp65 was 35.4% (6-84.3%) while 21.1% (2.3-81.7%) after MLPC_IE1 in spite of absolute CMV-T number at day 0. The rate of increase of pp65-specific T
cells was 1- to 33-fold while IE1-specific T cells ranged from 1to 961-fold under the same experimental conditions. The final cell culture product was mostly composed by Th cells. Good enrichment of CMV-T upon MLPC was feasible if a frequency of CD3 þ CMV-TZ10% was reached. The isolated CMV-T showed high Ag-specific proliferation (495%). Cytokine repertoire after MLPCs was IFN-g þ TNF-a þ polarized with high levels of IL4 and IL10, too. Expanded CMV-T had significantly lower level of late effector (Temra) CD8 þ cells compared with PBMCs before CMV stimulation (Po.001). Temra CD4 þ cells were less represented upon MLPC_IE1 (Po.05). CMV-T expansion was accompanied by a significant increase of naı¨ve/memory stem (Tn/scm; Po.001) CTLs while the Th counterpart significantly increased upon stimulation with IE1 (Po.005). Conclusion: We improved basis knowledge on CMV-T biology and functionality. Outcome was variable and showed donor and epitope dependency. We used pp65 and IE1 libraries as stimulating Ags to be independent from HLA-type and to be able to investigate T cell responses directed not only against single peptide epitopes. Differences in HLA and epitope dominance and variability in the relative number of CD3 þ effector cells and IFN-g/CD154 expression among healthy donors could reflect the observed individual CMV-specific cellular immunity. This heterogeneity raises points to be considered when approaching AI and should be taken into account in the attempt to define private predictors of putative individual responses. Disclosure of Interest: None declared. P162 Allogeneic Haematopoietic Stem Cell Transplantation in Hepatitis B Virus previously exposed recipients or donors: a safe procedure in antiviral era E. Ravano1,*, B. Forno1, E. Zucchetti1, P. Marenco1, G. Grillo1, I. Lotesoriere1, R. Cairoli1 1 BMT Unit, Niguarda Hospital, Milan, Italy Introduction: A previous exposition to hepatitis B Virus (HBV) is still frequent in donors and patients (pts) who could take advantage from a haematopoietic stem cell transplant (HSC Tx). Viral infection, reactivation or seroreversion can lead to chronic or fulminant hepatitis. Availability of antiviral drugs has certainly reduced risks and has changed standard prophylaxis procedures. Materials (or patients) and methods: To evaluate incidence and outcome of HBV þ Txs in our Centre, all the 161 consecutive pts who underwent an allogeneic HSC Tx between January 2009 and August 2014, were analysed. Those who were positive for HBsAb only were excludes as vaccinated. 29/161Txs (18%) were performed in HBV exposed: 6 in recipients HbsAg þ /HbcAb þ (3,7%), 20 in HbsAg-/ HbcAb þ (12,4%), and 3 in HBV-naı¨ve recipients who received HSC from a donor HBV þ (1 HbsAg þ /HbcAb þ , 2 HbsAg-/ HbcAb þ ). 1/29 died d þ 13 for septic shock, 28 are evaluable surviving over day þ 100 . Anti HBV prophylaxis was started in 27/28 pts before Tx and was stopped not earlier than 6 months after withdrawal of immunosuppression (presently reached only in 3 pts); 23 Txs were performed under Lamivudine, 3 received Entecavir and one Tenofovir. According to our standard follow-up procedure pts were regularly tested for liver function, serology and, when applicable, HBV genome and mutants search. Results: With a median follow-up of 767 days (range: 1811827), 17 pts (62%) are alive. 5 died for relapse, 7 for NRM (none of them for a cause HBV related). The only hepatic complication observed is in a HbsAg-/HbcAb þ pt who experienced a serious acute hepatitis (seroreversion) 6 months after stopping Lamivudine that occurred 1,5 year after Immunosoppression withdrawal; hepatitis resolved after Entecavir therapy. 14 pts are still in prophylaxis: none of them experienced virus reactivation, neither viral hepatitis. To be noted that 1 out of the 3 pts who lost their HbsAb after Tx, was
vaccinated day þ 1035 and reached a good HbsAb titre; so he could stop Lamivudine without any reactivation. Conclusion: With our policy for anti HBV prophylaxis, HBV positivity does not seem an exclusion cause for HSC Tx any longer, at least with our conditioning and GVHD prophylaxis (only 2 Txs were T-depleted with Thymoglobuline, and none received Rituximab). Further studies are needed to define best timing for stopping prophylaxis and a possible role of HBV vaccination after HSC Tx. Disclosure of Interest: None declared. P163 Laboratory surveillance of Herpes and polyoma viruses: results and clinical consequences E. Jaskula1,2,*, J. Lange2, A. Tarnowska2, M. Sedzimirska2, M. Mordak-Domagala2, D. Duda2, K. Suchnicki2, A. Lange1,2 1 L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland, 2Lower Silesian Center for Cellular Transplantation with National Bone Marrow Donor Registry, Wroclaw, Poland Introduction: Viral complications constitute one of the main threats post-HSCT, especially in patients who received transplants from unrelated donors. Therefore, all patients transplanted in our institution were followed for viral reactivation events including HHV6, CMV, EBV, polyoma BK, and JC. Materials (or patients) and methods: This study included 156 unrelated donor HSCTs. 90 and 66 patients received myeloablative or reduced intensity conditioning. All patients except 3 received anti-lymphocyte antibodies (ATG [142 patients] and Campath [21]). The latter patients received ganciclovir as a part of microbial prophylaxis (6 mg/kg b.w. for 5 days). Quantitative detection of Herpes virus DNA copies (real-time PCR), was performed routinely in one-week intervals shortly post-transplant and then in one-month intervals unless required earlier. Results: CMV reactivation events were less frequent shortly posttransplant than at later times ( 14% vs. 27%, P ¼ 0.003, before and after 30 days post-HSCT).Patients positive for CMV DNA copies (detected between 30-100 days post-HSCT) had higher levels of CD8 þ CD57 þ lymphocytes in the blood than counterparts lacking CMV reactivation at any time post-transplant (18.92 þ / 2.14% vs. 8.36 þ / 1.50%, P ¼ 0.0075; 186x106 þ / 41.86x106 vs. 84.15 þ / 32.53x106 cells/L, Po0.001). CMV reactivation was associated with the presence of aGvHD grade II-IV (22/52 vs. 22/89, P ¼ 0.031). In variance to CMV HHV6 reactivation events were rather more frequent early post-HSCT than at later times (15% vs. 6%, P ¼ 0.096). Skin rash was frequent in HHV6-positive patients but encephalitis was the main concern. Out of 12 encephalitis cases, 6 were HHV6-positive as compared to 22 positive cases out of 133 patients lacking encephalitis (P ¼ 0.015). EBV was seen at a similar frequency early and later post-HSCT and resulted in a higher percentage of CD4 þ CD57 þ cells in the blood (3.00 þ / 0.58% vs. 1.42 þ / 0.27%, P ¼ 0.033; 56.045x106 þ / 28.95x10^6 cells/L vs. 8.89x106 þ / 2.39x106 cells/L, P ¼ 0.005). EBV reactivation was seen more frequently in patients with aGvHD (15/52 vs. 13/75, P ¼ 0.044) and in those with encephalitis (6/12 vs. 21/130, P ¼ 0.004) or lymphoproliferative syndrome (7/16 vs. 19/130, P ¼ 0.004). Reactivation of polyoma viruses differed in terms of BK (earlier, median 41 days post-HSCT) and JC (later, median 80 days postHSCT). Conclusion: CMV reactivation seen rather later post-transplant and a lack of apparent clinical symptoms may be related to the effect of prophylactic and pre-emptive ganciclovir treatment. The prevalence of CD8 þ CD57 þ lymphocytes in the blood of CMV-positive cases documented the impact of the infection on the immune system. An increase in the proportions and numbers of CD4 þ CD57 þ lymphocytes is a rather novel observation and demonstrates the profound effect of the EBV virus on the lymph nodes.
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Reactivation of either HHV6 or EBV constituted the main risk factor of encephalitis. EBV is associated with lymphoproliferative syndrome in spite of the fact that EBV DNA positivity is usually pre-emptively treated with anti-CD20 antibodies. Overall survival is only marginally affected by reactivation of Herpes viruses, likely due to the pre-emptive treatment followed according to routine laboratory blood work, which includes viral surveillance. Supported by INNOMED/I/1/NCBR/2014 CellsTherapy NCBiR grant. Disclosure of Interest: None declared. P164 Incidence and outcome of infections after unrelated cord blood transplantation in patients with hematological malignancies: a long-term single center experience E. Scalzulli1,*, W. Barberi1, C. Girmenia1, G. Gentile1, G. F. Torelli1, D. Ballaro`1, A. M. Testi1, M. L. Moleti1, L. Lombardi1, M. Campanelli1, R. Foa`1, A. P. Iori1 1 Hematology, Policlinico Umberto I, Sapienza University Rome, Rome, Italy Introduction: Unrelated cord blood (UCB) has become an alternative stem cell source for patients with hematological malignancies requiring allogeneic transplantation and lacking a HLA-matched donor. Infections after an UCB transplantation (UCBT) represent a leading cause of morbidity and mortality. The aim of this study was to evaluate the incidence, risk factors and outcome of infections occurring after an UCBT. Materials (or patients) and methods: We performed a retrospective study in 75 patients who received a UCBT at our center between July 1995 and July 2013: there were 51 children (median age 9 years, range 1-17) and 24 adults (median age 28 years, range 18-60). Patients were affected by high risk hematological malignancies: 22 had acute myeloid leukemia, 47 acute lymphoblastic leukemia and 6 chronic myeloid leukemia. All patients received a myeloablative conditioning regimen, in vivo T-cell depletion with antilymphocyte serum, and cyclosporine plus corticosteroid as GVHD prophylaxis. Fluconazole, acyclovir and ciprofloxacin were administered as prophylaxis for fungal, viral and bacterial infections, respectively. All patients underwent CMV antigenemia or DNAemia monitoring, while EBV DNAemia monitoring was performed since 2002 for the last 40 patients. Results: The incidences of bacteremia, proven/probable invasive fungal diseases (IFD), CMV infection, EBV infection and herpes zoster disease were 88%, 21%, 61%, 20% and 17% of transplants, respectively. Out of 80 bacterial isolates, 44%
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were Gram negative and 56% Gram positive. IFDs were caused by filamentous fungi in 62% of cases. Gastrointestinal CMV disease was documented in 4% of patients with virus infection. Out of 8 patients with EBV infection, 3 developed a posttransplant lymphoproliferative disorder (PTLD) (fatal in 2 cases). The distribution of the infections in the early (days 140 from transplant), late (days 41- 100), very late (days 101 365) phases and after day 365 are detailed in Figure 1. Bacterial, fungal and CMV infections were documented mainly during the early engraftment phase, while EBV and herpes zoster infections were generally documented after day 100. The duration of pre-engraftment neutropenia did not correlate significantly with an increased infectious risk. Grade II-IV acute GVHD was significantly predictive of the development of an IFD (HR 0.28; IC: 0.09 – 0.87; P ¼ 0.029). At a median follow-up of 10 years, 30 of 75 (40%) patients are still alive. Infections represented the primary cause of death in 24% of patients. The attributable mortality rate for bacterial and fungal infections accounted for 4.5% and 31%, respectively. Conclusion: Our study confirms the high rate of infectious complications occurring after an UCBT. In agreement with other experiences, the early engraftment phase represented the period at higher risk of bacterial, fungal and CMV infections. After this time period, a moderate risk of developing IFDs persists up to 6 months, especially in patients with GVHD. Infections by viruses other than CMV have been more frequently documented after the very late phase posttransplant. These epidemiological findings should be considered for the definition of tailored anti-fungal prophylaxis and virological pre-emptive strategies. Disclosure of Interest: None declared. P165 Micafungin antifungal primary prophylaxis in 40 patients with Acute Myeloid Leukemia candidate to allogeneic tranplantation F. Lorentino1,*, R. Greco1, E. Xue1, A. Forcina1, F. Giglio1, M. Morelli1, C. Messina1, M. G. Carrabba1, M. Bernardi1, J. Peccatori1, S. Paolo1, C. Corti1, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milano, Italy Introduction: Primary prevention of invasive fungal infections (IFI) is a clinically relevant goal in patients with Acute Myeloid Leukemia candidate to allogeneic transplantation. In such population, the greatest proportion of IFI occurs during aplasia following induction chemotherapy (Pagano et al., 2010). Micafungin (MICA) may be an effective antifungal agent since
it has activity against all major Candida spp. and it demonstrates in vitro and in vivo activity against Aspergillus spp. Aim: to investigate the incidence of IFI occurring in pts with newly diagnosed AML undergoing induction chemotherapy (IC) receiving MICA as antifungal primary prophylaxis (APP). Materials (or patients) and methods: Since July 2011 to October 2014, we prospectively treated 40 consecutive pts with newly diagnosed AML: 20/40 pts were treated with ‘‘3 þ 7’’ IC, 12/40 pts with ‘‘FLAI’’ IC, 8/40 pts with ‘‘ICE’’ IC. MICA was started 1 day following the end of IC at the daily dose of 50 mg i.v. MICA was continued until neutrophil engraftment (PMN40.5x109/L for 3 consecutive days). Galactomannan was measured once a week using ELISA test and results were considered positive if Z0.5 in serum. IFI was defined as possible, proven or probable according to 2008 EORTC/MSG definitions. Results: None of our patients discontinued the treatment for drug-related adverse events. 30/40 pts concluded MICA treatment at neutrophil engraftment without signs of IFI. 10/ 40 pts (25%) switched to an alternative antifungal therapy as empirical treatment. Of these, possible (n ¼ 4), probable (n ¼ 3) and proven (n ¼ 1) IFI were diagnosed. Probable IFIs were pulmonary infections, 1 candidiasis and 2 invasive aspergillosis were diagnosed and treated with liposomal B amphotericine or voriconazole. The only proven IFI was a disseminated fusariosis treated with a combination of liposomal B amphotericine and voriconazole. All pts were neutropenic at the onset of IFI with a median duration of neutropenia of 31 days (14–73). All of them resolved the infection at neutrophil recovery after IC. Conclusion: In this 3-year study in 40 patients breakthrough proven or probable IFI occurred in 10%. This result is comparable with randomized clinical trials and several observational studies focused on posaconazole prophylaxis (graded A-I by the European Conference on Infections in Leukemia - ECIL- for antifungal primary prophylaxis during induction chemotherapy) which found breakthrough IFI ranging from 0 to 17%. Micafungin primary antifungal prophylaxis is safe and effective during induction chemotherapy in patients with AML candidate to allogeneic transplantation. Disclosure of Interest: None declared. P166 Incidence and course of clostridium difficile infections after autologous and allogeneic blood stem cell transplantation N. Nagorny1, C. MacKenzie2, E. Rachlis1, M. Kondakci1, A. Dienst1, D. Lopez y Niedenhoff1, T. Schroeder1, R. Fenk1, K. Pfeffer2, R. Haas1, G. Kobbe1,* 1 Department of Hematology, Oncology and Clinical Immunology, 2Department of Medical Microbiology and Hygiene, University Hospital Duesseldorf, Duesseldorf, Germany Introduction: Immunosuppressed individuals are at risk of opportunistic and nosocomial infections. Cancer patients who receive autologous or allogeneic blood stem cell transplantation frequently suffer from infections that may increase morbidity and mortality. The role of intestinal infection with Clostridium difficile (CDI) in this high-risk group has so far not been properly defined. Materials (or patients) and methods: We retrospectively identified all patients who had received a blood stem cell transplant and developed symptomatic CDI (diarrhea and positive test for C.-diff. toxin) after transplantation from a database of the Department of Medical Microbiology and Hygiene. Clinical data was retrospectively collected from the patients’ charts to describe the clinical cause of infection and identify prognostic factors. Results: Between 1/2006 and 10/2014 a total number of 4797 stool specimens were analyzed for C.-diff. toxin from 751 patients with diarrhea who had previously received an
autologous or allogeneic blood stem cell transplant. Sixty-five individuals (MM 25, AML 23, Lymphoma 9, MDS/MPS 7 and 1 germ cell tumor) tested positive, reflecting an infection rate of 8.7% among all transplant recipients with diarrhea during that time period (4,01 % for all autologous and 8,16 % for all allogeneic transplants). Five patients (8%) had a history of CDI before Tx. The detection rate was 6% in 2006, 8.3% in 2007, 10% in 2008, 6.7% in 2009, 5% in 2010, 4.2% in 2011, 4.1% in 2012 and 6.43 % in 2013. Median time from transplant to a positive test was 112 days (median, range 1-5501) in autologous and 155 (range 3-2831, P ¼ ns) in allogeneic recipients. When considering only the 1st year after transplant, infection occurred earlier after autoTx (median day þ 37, range 1-331) than after alloTx (median day þ 85, range 3-364, P ¼ 0.014) and in MM patients (median day þ 19, range 1-331) than in Lymphoma patients (median day þ 93, range 59-159, P ¼ 0.03) which may be the result of metronidazole prophylaxis in allografted patients until day þ 20 after transplant and different previous antibiotic exposure. Of 29 autoTx patients with CDI 21(72%) relapsed with their primary malignancy (11 before, 9 after CDI). Of 36 alloTx patients 18(50%) relapsed (9 before, 9 after CDI). As therapy for CDI 40 patients (61.5 %) received metronidazole, 12 (18.5%) oral vancomycin, 2 (3.1%) fidaxomycin, 7 (10.8%) unknown and 3 (4.6%) no therapy. No patient died of CDI. Sixteen patients (24.6%) had at least one CDI recurrence (12 after metronidazole, 1 after vancomycin, 1 after Fidoxomycin and 2 after unknown therapy). At day þ 30 after the 1st positive CDI test 93.1% and 94.5% of patients were alive after autoTx and alloTx, respectively. While overall mortality in CDI patients was similar after autoTX (55%, median FU 202 days, 4-2361) and alloTx (53%, median FU 365 days, 13223), causes of death differed (TRM 7% and DRM 48% after autoTX vs TRM 17%, DRM 36% after alloTx). Conclusion: Between 2006 and 2013 the rate of CDI among patients with diarrhea after blood stem cell transplantation varied from 4.1 to 10% and was related to diagnosis and type of transplant/prophylaxis. CDI recurrence occured frequently, especially after therapy with metronidazole. While CDI itself was not fatal, patients who developed CDI after blood stem cell transplantation resembled a high risk population with high treatment and disease related mortality. Disclosure of Interest: None declared. P167 False Positive Clostridium Difficile Toxin Testing From Stool Sample by Immunochromatographic Method in a Pediatric Patient With Hemorrhagic Colitis Clinical Findings After Bone Marrow Transplantation I. Eker1,*, O. Gu¨rsel1, O. Babacan1, M. Topdemir2, A. E. Ku¨rekc¸i1 1 Pediatric Hematology, 2Pediatrics, Gulhane Military Medical Faculty, Ankara, Turkey Introduction: Clostridium difficile infection (CDI) is one of the prominent causes of colitis associated with antibiotics in children. Patients, who are required the useage of long term and broad spectrum antibiotics, such as hematopoietic stem cell transplantation (HSCT) conditions, are particularly at risk. Here a pediatric HSCT recipient, who had hemorrhagic colitis clinical findings and false-positive Clostridium difficile toxin test in stool sample by immunochromatographic method is presented and diagnostic approach of pediatric HSCT patients in the suspicion of CDI is discussed. Materials (or patients) and methods: A 6 year old boy had undergone HSCT with the diagnosis of relapsed ALL in our HSCT Center. Full engraftment and donor type chimerism was achieved. But recent after the engraftments, grade IV graftversus-host disease and thrombotic microangiopathy developed. Clinical response was achieved with plasma exchange and immunosuppressive therapy. But during this period, the usage of broad spectrum antibiotics for a very long time was needed. He developed clinical findings presenting like haemorrhagic colitis with fever, abdominal pain, leukocytosis and bloody stools. CDI was thought and C.difficile toxin B was
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detected positive in stool sample with immunochromatographic method. Oral vancomycin therapy was started, but there weren’t any clinical improvement. Results: Although C.difficile toxin B was detected positive again with the same method in a different stool sample, the stool culture was negative. He had undergone endoscopy and colonoscopy, when he was clinically stable. Pathological and microbiological evaluation of biopsy material was normal. Because there were no evidence of CDI, vancomycin therapy was discontinued. The localization of the gastrointestinal bleeding (GIB) couldn’t be identified with nuclear medicine scan and transcatheter arteriography also, but there were seen angiodysplastic lakes in branchs of superior mesenteric artery. With the suspicion of these lakes may be the origin of GIB, superselective embolization was performed and GIB episodes were resolved completely without any complication. Occult GIB was also became negative after ten days of the embolization and he was discharged on the 130th day of HSCT. Conclusion: The gold standard for the diagnosis of CDI is the determination of toxins in cell culture. Tests made with the aim of examining the CDI quickly, detects Clostridium difficile toxin (CDT) or the enzyme glutamate dehydrogenase (GDH) at fecal specimens. Although the sensitivity of these tests is high, specificity is low. Rapid CDT test by immunochromatographic method is based on the principle of binding of the agent toxin to the latex particles coated with monoclonal antibodies. In the literature it is reported that, the positive predictive value of both CDT and GDH positivity in stool sample is 100% and the negative predictive value of both CDT and GDH negativity in stool sample is 95%. Therefore in pediatric HSCT patients, having Clostridium difficile colitis signs and symptoms, the usage of this two step algorithmic approach is recommended. Using of CDT and GDH detection tests together in stool samples for this two step algorithmic diagnostic approach, until the result of the gold standard tests, will reduce the false positivity cases as in our case. Disclosure of Interest: None declared. P168 Successful treatment of sino-orbital mucormycosis after allogeneic transplantation: the role of urgent surgical approach I. Batsis1,*, G. Venetis2, S. Papadimitriou1, Z. Bousiou1, N. Pasteli3, V. Constantinou1, D. Mallouri1,1, I. Sakellari1, A. Anagnostopoulos1 1 Haematology department - BMT unit, 2maxillofacial surgery, 3 Pathology, George Papanicolaou Hospital, Thessaloniki, Greece Introduction: Mucormycosis after allogeneic transplantation is a fulminant, life-threatening infection that has a high mortality rate without prompt combined therapy. Unrelated hematopoietic stem cell transplantation (HSCT), which requires long term immunosuppression for treatment of GVHD, predisposes to fatal infections. Eradication is challenging and needs combination of systemic antifungal treatment and surgical intervention. Materials (or patients) and methods: We present the clinical outcome of a 27 year old male who underwent HSCT from an unrelated donor for chronic myelogenous leukemia in second chronic phase in 29/1/2013 after TBI-Cy plus ATG 5mg/kg. He was treated with high doses of corticosteroids for acute GVHD (grade III), received two preemptive courses of Rituximab for EBV reactivation and 2 further lines of immunosuppression for chronic extensive GVHD. Six months post HSCT he was successfully treated for three months with voriconazole for possible aspergillus lung infection. Two months later he presented with unilateral blurred vision and he was started on Amphotericin B (5mg/kg) with the suspicion of mucormycosis. Results: Imaging study with MRI of the facial skull revealed involvement of all sinuses. After 5 days of Amphotericin B with a stable clinical status, he appeared with rapid progression including mass swelling of the right side of the face, proptosis
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of the orbit and visual loss. MRI scan revealed that the infection was expanded to the orbit and adjacent tissues. An urgent consultation by neurosurgeon and maxillofacial surgeon posed the risk for imminence brain expansion. The patient was then immediately admitted to surgery and sustained exenteration of the right orbit and extensive surgical debridement of necrotic tissues. Debridement was repeated twice and histology confirmed the diagnosis of mucormycosis. He continued receiving Amphotericin B three months after the surgery and at the same time corticosteroid dose was tapered as possible. Six months post-surgery and after resolution of the infection, he received reconstructive surgery with no further complications. Conclusion: We present a case of sinusoidal mucormycosis with a rapid spread to surrounding tissues and all the steps guided by ECIL recommendations that led to a successful management. Eradication of the infection includes surgical debridement, administration of systemic antifungals for sufficient time and an effort to reverse the poor immunological status in the deeply immunocompromised patient. Disclosure of Interest: None declared. P169 Use of micafungin for antifungal prophylaxis in patients undergoing allogenic hematopoietic stem cell transplantation (allo-HSCT) in Spain (GETH-MIC) C. Lo´pez-Sa´nchez1, I. Ruiz-Camps1,*, V. Go´mez2, J. Lo´pezJime´nez3, D. Serrano4, V. Rubio5, C. Solano6, D. Valca´rcel1, L. Va´zquez7 on behalf of Grupo Espan˜ol de Trasplante Hematopoye´tico (GETH) 1 Hospital Universitario Vall d’Hebron, Barcelona, 2Hospital Universitario de La Princesa, 3Hospital Universitario Ramo´n y Cajal, 4Hospital Universitario Gregorio Maran˜o´n, Madrid, 5Hospital Jerez de la Frontera, Ca´diz, 6Hospital Clı´nico Universersitario de Valencia, Valencia, 7Hospital Universitario de Salamanca, Salamanca, Spain Introduction: Echinocandins are highly effective antifungal agents against Candida and Aspergillus spp. Micafungin provides compared to other echinocandins better activity against some Candida spp. (specially C. glabrata) and also Aspergillus spp. All guidelines focused on antifungal prophylaxis recommend its use during neutropenic phase in hematopoietic stem cell transplant (HSCT) recipients. Moreover, its low profile of side effects and drug interactions makes micafungin a good alternative in those patients who need concomitant treatments, present hepatic insufficiency and also in those that do not tolerate oral drug administration. The aim of this study was to describe the experience with micafungin as primary prophylaxis during the neutropenic phase in patients undergoing allo-HSCT in a cohort of Transplant Spanish centres, and to evaluate it’s efficacy and tolerability in this population. Materials (or patients) and methods: retrospective multicentre observational study including all consecutive adult patients admitted for allo-HSCT to haematological units belonging to participating centres of the Grupo Espan˜ol de Trasplante Hematopoye´tico (GETH), from January 2010 to December 2013, which received micafungin as prophylactic treatment during the neutropenic phase. Patients that received less than 5 days of micafungin were excluded from the analysis. Failure of prophylaxis was considered in those patients who developed breakthrough invasive fungal infection (IFI) during prophylaxis with micafungin or early after. Results: Data from 240 patients from 13 HSCT units belonging to the GETH group were collected. Eighty-one patients were excuded from the analysis for different reasons. Finally, 159 patients were included for the analysis. Ninety-four (59%) were men. Mean age was 48 (± 13) years. Underlying disease was leukaemia in 82 (51.6%) patients, lymphoma in 34 (21.4%), myelodysplastic syndromes in 26 (16.4%), multiple myeloma in 8 (5%) and other diseases in 9 (5.7%) patients. One hundred and fifty-one (94.9%) patients had been submitted to
peripheral blood SCT, 6 (3.8%) to bone marrow SCT and 2 (1.3%) to umbilical cord SCT. Mean of says of prophylaxis with micafungin was 19 (± 9) days. Most patients (95.6%) received doses of 50mg per day, while the remaining ones received 100mg per day. During follow-up 7 (4.4%) patients developed breakthrough IFI, 1 proven IFI (Fusarium spp. in skin biopsy) and 6 probable IFI (positive galactomannan with suggestive radiological images), and one patient presented serious drug interactions that obliged micafungin cessation. The median of days of treatment with micafungin in these patients was 14 (10-25) days at a dose of 50mg per day. Four of these patients died: two with multiorganic failure, 1 patient because of venoocclusive disease and 1 because staphylococcal septic shock. Six other patients died because of different complications related to the underlying disease and it’s treatment. Finally, prophylaxis with micafungin was considered effective in 151 (94.9%) of the patients. Conclusion: According to our experience, micafungin is an appropriate alternative for antifungal prophylaxis in patients undergoing an allo-HSCT, because it’s efficacy, it’s low profile of drug interaction and side-effects. This study has been partially funded by Astellas. Disclosure of Interest: C. Lo´pez-Sa´nchez Funding from: Astellas, I. Ruiz-Camps Funding from: Astellas, V. Go´mez Funding from: Astellas, J. Lo´pez-Jime´nez Funding from: Astellas, D. Serrano Funding from: Astellas, V. Rubio Funding from: Astellas, C. Solano Funding from: Astellas, D. Valca´rcel Funding from: Astellas, L. Va´zquez Funding from: Astellas. P170 Fluoroquinolone Prophylaxis Reduces the Incidence of Severe Viral Hemorrhagic Cystitis in Cord Blood plus Third-Party Donor Allogeneic Transplant Recipients I. Sa´nchez-Ortega1,*, G. Bautista2, M. Kwon3, A. Va´zquez2, R. Fores2, A. De Laiglesia2, J. Gayoso3, P. Balsalobre3, D. Serrano3, M. Arnan1, B. Patin˜o1, A. Sureda1, J. L. Dı´ez-Martı´n3, R. Cabrera2, R. F. Duarte1 1 Catalan Institute of Oncology, Hospital Duran i Reynals, Barcelona, 2Hospital Universitario Puerta de Hierro, 3Hospital Universitario Gregorio Maran˜o´n, Madrid, Spain Introduction: Virus-associated hemorrhagic cystitis (HC) is a potential serious complication of allogeneic hematopoietic cell transplants (HCT), most commonly after myeloablative conditioning regimens and in cord blood HCT recipients. Prophylaxis with fluoroquinolones has been shown to reduce BK viral load and the incidence of severe (grades III-IV) HC in HLA matched sibling and unrelated volunteer donor allogeneic HCT. Here, we present our experience on the effectiveness and safety of fluoroquinolone prophylaxis of severe HC in recipients of cord blood supported with thirdparty donor hematopoietic cells (CB-TPD) HCT. Materials (or patients) and methods: 148 consecutive CBTPD HCT recipients from 03/1999 to 07/2014 in three Spanish centers were included in this study: 79 AML/MDS, 48 ALL, 11 CLPD, 8 CMPD and 2 others, 89 men (60%), median age 37 [16 - 64]. Two consecutive cohorts are included, with 28 patients receiving 60 days of sustained post-transplant fluoroquinolone prophylaxis (16 cases with 500 mg/12h oral ciprofloxacin and 12 cases with 500 mg/day levofloxacin) regardless of the initiation of IV broad-spectrum antibiotics, and 120 controls receiving no anti-HC prophylaxis. There were no differences between cohorts in patient demographics and transplant characteristics. Severe HC episodes included grades III (macroscopic hematuria with blood clots) and IV (massive macroscopic hematuria requiring instrumentation of the urinary tract or causing urinary retention) HC [Leung et al, BBMT 2005]. Results: Seventeen patients in the non-prophylaxis group developed severe HC (8 grade III and 9 grade IV) a median of 30 days after HCT, compared with no cases in the fluoroquinolone prophylaxis group (14.2% vs 0, P ¼ 0.043). Severe HC occurred after neutrophil engraftment in 88% of the
cases (15/17). Fifteen cases were related to BK virus, one case to adenovirus and one additional case had negative viruria. Severe HC episodes increased the hospital stay during which patients required supportive therapy (hyperhydratation, forced diuresis), blood and platelets transfusions and up to four lines of treatment including intravesical (n ¼ 3) and IV cidofovir (n ¼ 7), intravesical hialuronic acid (n ¼ 11), mesenchymal stem cells (n ¼ 2), cystoscopic cauterization (n ¼ 2), arterial embolization (n ¼ 1), and immunusoppression switch to sirolimus (n ¼ 2). Eleven patients achieved complete resolution of HC and survived the episode, while 6 died in refractoriness or partial response (significant improvement of symptoms of HC but persistence of gross hematuria). Severe HC-related mortality was 35.3% (6/17), and contributed a 5% to the non-relapse mortality of the cohort without prophylaxis (6/120). In addition to the absence of severe HC, all 28 cases in the fluoroquinolone cohort completed prophylaxis as planned and no fluoroquinolone-related adverse events were reported. Conclusion: Our results suggest that fluoroquinolone prophylaxis may reduce the incidence of severe HC in high-risk cord blood transplant recipients, the morbidity and resource use associated with it and improve patients’ outcome. They also confirm that the maintenance of fluoroquinolone prophylaxis during 60 days after transplantation regardless of IV broadspectrum antibiotics is safe and well tolerated. Disclosure of Interest: None declared. P171 A study of adherence to a vaccination schedule following adult allogenic haematopoietic stem cell transplants in UK transplant centre J. Meiring1,*, T. De Silva1, J. Snowden2 1 Department of Infection and Tropical Medicine, 2Dept of Haematology, Sheffield Teaching Hospitals, Sheffield, United Kingdom Introduction: In the past fifteen years significant advances have been made in the area of preventing infection in haemopoeitic stem cell transplant (HSCT) recipients. Despite this, infection is reported as the primary cause of death in 8% of autologous HSCT patients and 17%420% of allogenic HSCT patients. After HSCT, patients remain immunocompromised and are therefore susceptible to a range of infections. Patients lose vacine-induced immunity they may once have had through childhood vaccination programmes. Therefore in order to restore immunocompetence; exposure to these antigens via a standardised vaccination programme is essential. We assessed adherence to a local vaccination policy following allogeneic HSCT administered via primary care physicians. Materials (or patients) and methods: Between 2008 to 2010 we identified 65 patients that received a HSCT. 30 individuals survived for a period where they should have completed the full vaccination schedule. Primary care physicians were contacted and a full vaccination history attained. Where information was missing we consulted the centralised haematology late effects clinic letters. We also used these to document why vaccines were delayed or not administered. Results: Sufficient data was obtained for 25 patients. The lack of data for 5 patients was due to changes in GP practices with data not following patients and some practices being unwilling to release data to us. Of the 25 patients we did study, only six (24%) patients fully completed their vaccine schedule. Ten had documented reasons for failure, although graft versus host disease (GVHD), disease relapse and donor lymphocyte infusion were the main documented reasons. This left 9 patients who did not complete the schedule, with no documented reason. Looking sequentially through the data, those vaccines given at 12 months like the first DTP, pneumococcal polysaccharide and haemophilus vaccines had adherence of around 70%.
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However, vaccines given at 2 years were considerably lower with only 28% adherence, evident in the third DTP, the second pneumococcal and MMR vaccine. Seasonal influenza vaccine uptake was better with 72% receiving post-HSCT. Conclusion: There were limitations to this study, namely difficulty in accesing accurate data from 30 different sources. This limitation comes directly from the problem with the delivery of de-centralised care. Despite this the data collected showed that the implementation of the vaccine schedule is not effective. The numbers of patients recieving protective levels of vaccine were very low, , partly due to physician perception of GVHD being a contraindication to all vaccines. In light of this a centralised vaccine service that could run alongside a late effects clinic has been proposed with the input of infectious disease specialists. Also, updates to the current schedule have been advised bringing them in line with current EBMT and IDSA guidance. Specifically, using the pneumococal conjugate vaccine, starting schedules at 3 months and for the administration of non-live vaccines dismissing GVHD as a contra-indication to vaccine administration. We hope that these significant developments will improve uptake and ultimately the long term health of transplant recipients. Disclosure of Interest: None declared. P172 Viral Infection and Reactivation in Alemtuzumab Reduced Intensity Stem Cell Transplantation in lymphoid malignancies: a study from the Irish National Adult Stem Cell Transplant (NASCT) Unit J. M. Nolan1,2,*, C. Delaney1, G. Lee1, E. Higgins1, M. Nı´ Chonghaile1, E. Conneally1,2, C. L. Bacon1,2, P. V. Browne1,2, B. Crowley3, E. Vandenberghe1,2 1 Haematology, St James’ Hospital, Dublin 8, 2Haematology, University of Dublin, Trinity College, Dublin 2, 3Microbiology, St James’ Hospital, Dublin 8, Ireland Introduction: Alemtuzumab-based reduced intensity stem cell transplantation (A-RIC-SCT) was adopted by the Irish NASCT unit in 2003 for Chronic lymphocytic leukaemia (CLL), Non-Hodgkins lymphoma(NHL) and Hodgkins Lymphoma (HL). Viral infection-associated morbidity is a concern with this conditioning and we reviewed the risk factors, incidence and morbidity of CMV, EBV and respiratory viral (RV) infections following A-RIC-SCT. Materials (or patients) and methods: Lymphoid patients conditioned with A-RIC-SCT from 2006-2013 considered "at viral risk" were identified and the incidence, risk factors for infections and infection outcomes were reviewed. "At risk" patients included: donor/recipient CMV and/or EBV exposure and were assessed twice weekly by PCR from engraftment to 100 days or cessation of steroid immunosuppression. RVs including influenza, parainfluenza, adenovirus and RSV were detected by PCR on appropriate samples in patients with respiratory symptoms. Risk factors assessed were age, diagnosis, donor type, alemtuzumab dose, disease status at SCT and steroid use for graft versus host disease (GvH) using SPSS (Statistical Package for Social Sciences, version 22) software. Results: Fifty-eight patients (37M:21F) underwent transplantation for CLL (n ¼ 16), NHL (n ¼ 30) and HL (n ¼ 12) at a median of 47.5 years (range 18-60). Donors were HLA-matched siblings (41) 69% and unrelated volunteers (17)31% of whom 2 were mismatched. Alemtuzumab doses (in mg) were: 30(n ¼ 13,22%), 50 (n ¼ 10,17%) and 100(n ¼ 35,60%). Disease status at SCT was: CR(n ¼ 17) 29%, PR(n ¼ 35) 61% and stable disease (n ¼ 6) 10%. Steroid-treated GvH occurred in (24) 41% of patients. 21 patients (36%) were at CMV-risk, of whom 14 (67%) reactivated, prolonging SCT hospital stay in 8 patients for a median of 14 (range 3-21) days. CMV disease affected 2 patients (colitis, pneumonitis) who survived. 56 (91%) patients
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were EBV þ ve and 16(30%) reactivated, 3 required anti-CD20 therapy and one death occurred. RV detected were: adenovirus (13) 22%, RSV/Parainfluenza (15) 26% resulting in 2 death. None of the risk factors assessed were statistically significant. TRM deaths occurred in 10 (17%) patients as follows: bacterial/fungal infection (4), parainfluenza (1), RSV (1), PTLD (1), GvH (2), liver failure (1) with 1 infective death 4100 days. 12 (20%) patients died of disease, giving an overall survival of 62.1% (36 patients) with a median follow-up of 45 (range 11-109) months. Conclusion: CMV reactivation occurred in 2/3 of "at risk" patients causing significantly increased hospital stays, but no deaths. EBV reactivation occurred in 1/3 of "at risk" patients but resulted in 1 death. RV present a challenge for all SCT patients and resulted in 2 deaths in this series. A-RIC-SCT was not associated with an excess viral mortality rate in this series, but strategies to decrease risk include pre-emptive EBV viral reactivation treatment, reduction of alemtuzumab dose (not statistically significant in this small series) and earlier diagnosis and treatment of RV. Disclosure of Interest: None declared. P173 Incidence and outcome of infections in pediatric stem cell transplant centers: comparison with oncohematology patients in nationwide study J. Styczynski1,*, K. Czyzewski1, M. Wysocki1, O. GryniewiczKwiatkowska2, A. Kolodziejczyk-Gietka3, B. Dembowska2, D. Perek2, M. Salamonowicz3, L. Hutnik3, M. Matysiak3, A. Zaucha-Prazmo4, J. Kowalczyk4, L. Che"mecka-Wiktorczyk5, W. Balwierz5, K. Siewiera6, J. Fraczkiewicz6, K. Kalwak6, A. Chybicka6, R. Tomaszewska7, T. Szczepanski7, O. Zajac-Spychala8, J. Wachowiak8, N. Irga-Jaworska9, E. Drozynska9, M. Plonowski10, M. Krawczuk-Rybak10, T. Ociepa11, T. Urasinski11, F. Pierlejewski12, W. Mlynarski12, Z. Gamrot13, M. Woszczyk13, J. Gozdzik5, Z. Malas14, W. Badowska14, A. Urbanek-Dadela15, G. Karolczyk15, W. Stolpa16, G. Sobol16, L. Gil8 on behalf of Polish Society of Pediatric Hematology and Oncology 1 Department of Pediatric Hematology and Oncology, Collegium Medicum, University Hospital 1, Nicolaus Copernicus University, Bydgoszcz, 2Children’s Memorial Health Institute, 3Medical University, Warsaw, 4Medical University, Lublin, 5Jagiellonian University, Krakow, 6Medical University, Wroclaw, 7Medical University, Zabrze, 8Medical University, Poznan, 9Medical University, Gdansk, 10Medical University, Bialystok, 11Medical University, Szczecin, 12Medical University, Lodz, 13Pediatric Center, Chorzow, 14Children Hospital, Olsztyn, 15Children Hospital, Kielce, 16 Medical University, Katowice, Poland Introduction: Infections are the main cause of morbidity and mortality in children undergoing hematopoietic stem cell transplantation (HSCT) or treated for cancer in pediatric hematology and oncology (PHO) centers. The aim of this nationwide study was the analysis of incidence and outcome of bacterial, fungal and viral infections in pediatric HSCT centers, in comparison to PHO centers over a period of 24 months (iPhot-13 project). Materials (or patients) and methods: During analyzed period a total number of 308 HSCTs (232 allo, 76 auto; including 67 ALL, 47 AML, 28 NHL/HD, 60 solid tumors, 39 PID, 38 BMF, 21 other) were performed, and 1768 children were newlydiagnosed for malignancy (430 ALL, 81 AML, 110 HD, 108 NHL, 1012 solid tumors, 27 other) in participating centers. Following episodes were reported: microbiologically documented bacterial infections; proven/probable/possible IFI; latent and sporadic viral infections. Results: The overall incidence of bacterial, fungal and viral infections in HSCT patients was 33.9%, 22.8%, and 38.3%, respectively. The hazard risk of any infection was higher in comparison to PHO patients (HR ¼ 2.7, 95%CI-2.3-3.2, Po0.0001), especially during the first 30 days of therapy (after transplant or cancer diagnosis, respectively). The incidence
was higher in HSCT patients for bacterial (33.9% vs 26.3%, HR ¼ 1.4, P ¼ 0.0046), all fungal (22.8% vs 7.9%, HR ¼ 3.5, Po0.0001), proven/probable IFI (12.3% vs 2.9%, HR ¼ 4.6, Po0.0001), and viral infections (38.3% vs 3.5%, HR ¼ 15.7, Po0.0001). The incidence of IFI in ALL and AML both in HSCT and PHO setting was very high (ALL: 31.3% vs 13.0%, AML: 44.7% vs 43.2%). The incidence was higher in allo- than autoHSCT for bacterial (HR ¼ 1.4, P ¼ 0.2), fungal (HR ¼ 3.2, P ¼ 0.0028), and viral (HR ¼ 15.7, Po0.0001) infections. The rate of resistant bacteria was higher in HSCT patients both for G-negative (72.5% vs 59.2%), and G-positive (41.4% vs 20.5%) strains. The most often detected G-negative bacteria included: E.coli (25.9%), Enterobacter cloacae (22.9%), and Klebsiella pneumonia (16.8%); while Staphylococcus epidermidis (16.5%), Enterococcus faecium (29.6%), and Clostridium difficille (38.6%) among G-positive bacteria. Cumulative incidence of viral infections in allo-HSCT was: 28.0% for CMV, 18.5% for BKV, 15.5% for EBV, 9.5% for adenovirus, 2.6% for VZV, 0.9% for influenza, 0.9% for HHV6, and 0.3% for HBV. Survival rate from infection was lower than in PHO patients in bacterial (96.0% vs 98.2%), fungal (75.5% vs 94.6%), and each viral infection (except influenza). Conclusion: The incidence of infections in allo-HSCT patients is high, while in auto-HSCT is comparable to PHO patients. The rate of multi-drug resistant strains in HSCT patients is very high. The outcome of infections in allo-HSCT patients is still unsatisfactory. Intensified supportive anti-infectious therapy is one of the keys to success in transplant setting. Disclosure of Interest: None declared.
Early complications/late effects & quality of life I P174 Impact of non-transferrin-bound iron (NTBI) in comparison to serum ferritin as prognostic factor for outcome after allogeneic stem cell transplantation (SCT) A. Fritsch1,*, C. Langebrake1,2, C. Wolschke2, J. Kersten3, H. Rohde4, P. Nielsen5, N. Kro¨ger2 1 University Medical Centre Hamburg-Eppendorf, Pharmacy, 2 University Medical Centre Hamburg-Eppendorf, Stem Cell Transplantation, 3University Medical Centre Hamburg-Eppendorf, Department of Medical Biometry and Epidemiology, 4University Medical Centre Hamburg-Eppendorf, Institute of Medical Microbiology, 5University Medical Centre Hamburg-Eppendorf, Department of Biochemistry and Molecular Cell Biology, Hamburg, Germany Introduction: Iron overload (IO) prior to allogeneic SCT has shown to influence negatively outcome after allogeneic SCT, mainly because of a high non-relapse mortality. IO is usually measured by serum ferritin (SF), which however is not specific for IO. We hypothesized that increased risk of infections and tissue damage is due to reactive oxygen species and NTBI is a better marker to predict outcome rather than SF. Therefore, we performed a prospective observational trial evaluating the influence of NBTI in comparison to SF on the outcome of SCT patients, with regard to overall survival (OS), bacteraemia and invasive fungal infections (IFI). Materials (or patients) and methods: 95 consecutive patients who received allogeneic SCT for various different diseases at University Medical Centre Hamburg-Eppendorf between 09/2010 and 09/2011 were analysed. The median follow-up was 19.6 month. NTBI levels, transferrin saturation and SF were determined daily during the conditioning regimen and also on day þ 7 and þ 14 post-SCT. For further analyses, the area under the curve (AUC) was calculated up to the day of transplantation (NTBI-AUC d0) and up to day þ 14 (NTBI-AUC d14). After mobilising free iron with help of
nitrilotriacetic acid and centrifugal filtration, NTBI was measured by graphite furnace atomic absorption spectrometry. Results: Regarding OS the results indicate a nearly 2 fold increased risk of death (HR ¼ 1.729) for patients with a high NTBIAUC d0 compared to patients with a lower iron load, without reaching statistical significance (P ¼ 0.095). Even in multivariate analysis comprising common prognostic factors, patients with a high NTBI-AUC d0 had a nearly 2 fold risk of dying (HR ¼ 1.887), while failing again statistical significance (P ¼ 0.074). Regarding the rate of infection, per one mmol*d/l NTBI-AUC d0 there was a 4% higher risk of bacteraemia (HR ¼ 1.042, P ¼ 0.009) and a 7% higher risk of IFI (HR ¼ 1.070, Po0.001). The NTBI-AUC d14 appears to have none prognostic value. Furthermore, baseline SF had no significant influence on the OS (1000 mg/l cut-off: HR ¼ 1.554, P ¼ 0.283; 2500mg/l cut-off: HR ¼ 1.486, P ¼ 0.389) or bacteraemia (per 1000mg/l: HR ¼ 0.943, P ¼ 0.630), but on IFI (per 1000mg/l: HR ¼ 1.255, Po0.001). It was shown, that only 25% of the patients presented with positive NTBI values prior to their conditioning regimen. However, the reduced intensity regimen containing a preceded chemotherapy had the greatest iron releasing effect. Conclusion: This study provides further hints for the adverse effect of NTBI on OS and the rate of infections of SCT patients. It was shown that NTBI occurs during conditioning regimen even in patients without elevated SF or NTBI at baseline. Furthermore we could not determine a negative prognostic impact of baseline SF on OS or bacteraemia, bur on IFI. This results supports the hypothesis that SF is not an appropriate parameter for estimating iron release in SCT patients. The influence of SF shown in several previous studies is probably rather attributable to other effects than to iron toxicity. Additional studies with a more homogenous group of patients and a larger number of patients are necessary to evaluate the influence of NTBI on the outcome of SCT patients. It remains unclear whether iron is directly affecting the outcome or just reflecting the cytotoxicity as a surrogate parameter. Disclosure of Interest: None declared. P175 Graft-versus-host disease prophylaxis by early commencement of immunesuppression using methotrexate decreases pre-engraftment syndrome and accelerates engraftment after cord blood transplantation A. Iguchi1,*, M. Sugiyama1, Y. Terashita1, J. Ohshima1, T. Sato1, Y. Cho1, T. Ariga1 1 Pediatrics, HOKKAIDO UNIVERSITY, Sapporo, Japan Introduction: Graft failure is one of most serious problems in cord blood transplantation (CBT). The incidence of preengraftment syndrome (PES) after CBT is also high. PES is associated with transplantation-related complications such as acute graft-versus-host disease (GVHD) and respiratory failure. It has been suggested that the incidence of PES after CBT can be decreased by early commencement of immunosuppression after transplantation, but a randomized study has not been carried out. In our institute, GVHD prophylaxis after CBT was changed after January 2011. Between December 1997 and December 2010, 49 patients received prophylaxis against GVHD with cyclosporine (CsA) or tacrolimus (Tac) in combination with methylprednisolone from day þ 7 (mPSL group). Between January 2011 and November 2014, 18 patients received CsA or Tac with methotrexate on day þ 1 and þ 3 (MTX group). Thus, we analyzed engraftment and incidence of PES in CBT recipients in the Department of Pediatrics Hokkaido University Hospital. Materials (or patients) and methods: We reviewed medical records of 67 consecutive child CBT recipients between December 1997 and November 2014. Indications for SCT were hematological malignancies in 41 patients, solid tumor in 12, congenital immunodeficiency in 11, bone marrow failure in 2, and congenital metabolic diseases in one patient. A preparative conditioning regimen consisting of myeloaberative
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conditioning (MAC) was provided to 54 patients, reduced intensity conditioning (RIC) was used for 13 patients. Fortynine patients received prophylaxis against GVHD in mPSL group, and 18 patients in MTX group. PES was defined as noninfectious fever and skin rashes with or without evidence of fluid retention and/or respiratory distress occurring before neutrophil engraftment: an absolute neutrophil count of 40.5x10^9/l. Graft failure was defined as no donor hematopoiesis beyond day þ 28. Results: Engraftment was detected in 56 patients (83.6%). Although there was no significant difference between engraftment rate patients who received MAC and those who received RIC (45/54 (83.3%) and 11/13 (84.6%), respectively), engraftment rate in MTX group was significantly higher than that in mPSL group (18/18 (100%) and 38/49 (77.6%), respectively, P ¼ 0.029). Only four of the 11 patients with graft failure could be rescued by second transplantation. PES developed in 34 patients. The incidence of PES in patients receiving MAC was significantly higher than in the patients receiving RIC (P ¼ 0.043). Moreover, the incidence of PES in mPSL group was significantly higher than that in MTX group (P ¼ 0.029). Additional treatment of corticosteroids were required in 7 patients, but no patient died of PES. Conclusion: Although this study was a small scale analysis, the results showed that increase of engraftment rate and decrease in the incidence of early immune reactions such as PES could be achieved by early commencement of immunesuppression using methotrexate. Disclosure of Interest: None declared. P176 Elevated ferritin level at day þ 100 after allogeneic stem cell transplantation has a higher impact on overall survival and transplant related mortality than the pre-transplant ferritin level A. Casado1,*, M. Sanchez Escamilla1, L. Yan˜ez1, A. Insunza1, A. Bermu´dez1, E. Conde1 1 Hematology Department, Hospital Universitario Marques de Valdecilla, Santander, Spain Introduction: It is well known that iron overload is an important adverse prognostic factor for patients who undergo allogeneic stem cell transplantation (allo-SCT). Therefore, iron chelation before the procedure has been recommended in
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several guidelines. The aim of this study is to analyze the impact of a high ferritin level (Z1000 ng/ml) pretransplantation (Fe-PRE) and at day þ 100 (Fe-100) on overall survival (OS) and procedure related mortality (TRM) at one and two years after transplantation. Materials (or patients) and methods: We have evaluated retrospectively 518 patients who underwent an allo-SCT in our centre between January 2000 and December 2013. Ferritin levels are available pre-transplantation in 312 patients and at day þ 100 in 148 patients. No patient received iron chelation pre-transplantation or before day þ 100. Statistical analysis was performed with the SPSS v 15.0 software. Results: At day þ 100 post-transplantation, the ferritin level and the number of patients with a ferritin level Z1000 ng/ml were higher than before transplantation (2301 ng/ml vs 1437 ng/ml, Po0.001, and 75% vs 54%, Po0.001, respectively). Patients’ characteristics are shown in table 1. Compared with patients with a low ferritin level at day þ 100, patients with a ferritin level Z1000 ng/ml had delayed platelet engraftment (450 x 109/L 26 days vs 17 days, Po0.001), received more red cell transfusions (4 vs 2, Po0.001) and developed more extensive chronic GvHD (37.9% vs 10%, P ¼ 0.024). There were no significant differences in status at transplant, donor type, conditioning intensity, CD34 cell dose, neutrophil recovery or grade III-IV acute GvHD. With a median follow-up of 32 months, Fe-100 Z1000 ng/ml had a more negative impact on OS (54% vs 76%, P ¼ 0.009) than FePRE Z1000 ng/ml (62% vs 55%, P ¼ 0.593). The negative impact of Fe-100 Z1000 ng/ml on TRM at 1 year (17.6% vs 8.1%, P ¼ 0.129) and 2 years (20.8% vs 8.1%, P ¼ 0.068) was also higher than that of Fe-PRE Z1000 ng/ml (15.6% vs 14.4%, P ¼ 0.846, and 18.7% vs 16.1%, P ¼ 0.721, respectively). Conclusion: In our experience, a high ferritin level at day þ 100 showed a more negative impact on OS and on TRM at 1 and 2 years post-transplantation than the pre-transplant ferritin level. These results need to be confirmed in larger studies, and the possible benefit of early post-transplant iron chelation should be evaluated. Disclosure of Interest: None declared.
P177 Chemotherapy-induced nausea and vomiting in adult patients after hematopoietic stem cell transplantation: a survey from the Quality of Life Working Party of the Rome Transplant Network (RTN) A. Tendas1,*, O. Annibali2, F. Sollazzo3, A. D’Apolito1, S. Cacciaraichi4, C. Viggiani5, E. Conte3, V. Molinari6, A. Di Veroli6, A. Chierichini7, M. R. Mauroni6, C. Papa6, D. Saltarelli7, I. Carli7, F. Marchesi5, T. Dentamaro1, P. de Fabritiis1, W. Arcese6 on behalf of Quality of Life Working Party of the Rome Transplant Network 1 Hematology, S. Eugenio Hospital, 2Universita` Campus Bio-medico, Hematology, 3Hematology, Ospedale S. Andrea, 4Hematology, Universita` Campus Bio-medico, 5Hematology, Regina Elena National Cancer Institute, 6Hematology, Tor Vergata University, 7 Hematology, Ospedale S. Giovanni Addolorata, Rome, Italy Introduction: Chemotherapy-induced nausea and vomiting (CINV) has a major impact on quality of life (QoL) of patients undergoing hematopoietic stem cell transplantation (HSCT). Objective: to compare strategies for CINV prevention, monitoring, and treatment adopted in the 6 centers of the RTN. Materials (or patients) and methods: An assessment form had been filled out by each center including the following items: incidence, monitoring, prevention (with the detailed prophylaxis schedule for three broadly used conditioning regimens: MEL200, BEAM and FEAM) and treatment of CINV. Cost-effectiveness ratio of each prophylaxis scheme was represented by measuring the probability of inefficacy and the risk of side effects by comparing each individual prescription as recommended by MASCC/ESMO guidelines. Missing/underdosed prescriptions were counted and scored with a negative score, according to the predictable effectiveness reduction (-1 mild, -2 moderate, -3 strong); scores sum was designated as effectiveness reduction score (ERs). Not required prescriptions were counted and scored, according to different probability and severity of side effects (1 mild, 2 moderate, 3 severe); scores sum was designated as side effects score (SEs). Schedule cost was calculated and compared with the cost of a fully guidelines-compliant one. Relation between CINV incidence estimation and ERs was analyzed. Results: Forms were delivered and filled out on October 2013. Median (range) incidence of CINV was 57% (20-90). All centers declared CINV data recording on medical record (combined with electronic database recording in 1 center); 3/6 centers reported using corticosteroids; 6/6 using a new (1/6) or old (5/ 6) generation 5HTRA; no center commonly used aprepitant. The details of prophylaxis schedules for MEL200, BEAM and FEAM allowed the analysis of costs, effectiveness reduction and risk of side effects. The euro median cost was 74.46 h (28.46 - 128.86) for MEL200, 193.04 h (179.20 - 203.02) for BEAM and 238.84 h (192.62 - 354.64) for FEAM; the cost of a schedule designed on the guidelines amounting to 108.05 h, 68.32 h and 102.16 h for MEL200, BEAM and FEAM, respectively. The median missing/underdosed prescriptions was 6 (3-6), 5 (3-5) and 3 (0-8) for MEL200, BEAM and FEAM, respectively; the median ERs was -17.5 (-9 - -18), -10 (-6 - -14) and -6 (0 - -18) for MEL200, BEAM and FEAM, respectively. Median not-required prescription was 1.5 (1-6), 7 (7-8) and 7 (1-12), for MEL200, BEAM and FEAM, respectively; the median SEs was 3.5 (1-11), 21 (16-22) and 7 (3-26), for MEL200, BEAM and FEAM, respectively. Linear regression analysis showed a strong correlation between CINV incidence estimation and ERs (r40.8); Centers with higher median ERs (more negative) reported a higher estimation of CINV incidence (mean ¼ 83%) than Centers with lower median ERs (mean ¼ 30%). CINV treatment showed a great heterogeneity between the Centers: 5HTRA, metoclopramide and corticosteroids being used in 4 (66%), 6 (100%) and 3 (50%) Centers, respectively. Conclusion: The level of adherence to guidelines showed a wide margin of improvement; strict adherence could provide
direct cost saving as well as prophylaxis efficacy improvement, rescue therapy, indirect costs and, above all, symptom burden reduction. Disclosure of Interest: None declared. P178 Effect of pretransplant body mass index on the clinical outcome after allogeneic stem cell transplantation in patients with hematologic malignancies : a single center experience A. Gokmen1,*, E. Soydan1, M. Kurt Yuksel2, M. Kurdal1, D. Ozbek1, O. Arslan2, M. Ozcan2, O. Ilhan2 1 Department of Hematology, Medicana International Ankara Hospital, 2Department of Hematology, Ankara University, Faculty of Medicine, Ankara, Turkey Introduction: Obesity and overweigt is common in adut patients with hematologic malignancies undergoing hematopoetic stem cell transplantation (HCT). Some studies demonstrate that pretransplant body mass index may affect posttransplant outcome. The aim of the study was to assess the effect of pretransplant body mass index (BMI) on posttransplant outcome in adut patients undergoing HCT for hematologic malignancies. Materials (or patients) and methods: 82 patients ages 23 to 71 with hematologic malignancies who underwent allogeneic hematopoietic stem cell transplantation from matched related donor in 2013 and 2014 were analyzed retrospectively. Diagnosis included acute lymphoblastic leukemia, acut myeloblastic leukemia with first or second remission and active disease, myelodysplastic syndrome and chronic lymphocytic leukemia. Patients were devided into categories based on body mass index (BMI). BMI categories were defined as underweight, BMI o18.5 kg/ m2 ; normal, 18.5 r BMI o25 kg/ m2 ; overweight, 25 r BMIo30 kg/ m2, obese, BMI Z 30 kg/m2. Results: There were 33 patients in normal group ( group 1), 34 patients in overweight group ( group 2), 15 patients in obese group ( group 3). There was no patient who was underweight. Median age and sex were similar in all groups. There were no statistically significant difference among groups in time to neutrophil and platelet engraftment.The median time to neutrophil engraftment was 14 days in group 1, 16 days in group 2, 13 days in group 3 (p: 0.26). The median time to platelet engraftment was 14 days in group 1, 12 days in group 2, 12 days in group 3 (p:0.16). The rate of grade 2-4 acute GVHD was %31 in group 1, %34 in group 2,% 29 in group 3. (p:0.17). Treatment related mortality (TRM) were similar in all groups. TRM was %18 in group 1, %20 in group 2,% 20 in group 3. (p: 0.25). The causes of the death (TRM) were infection (n: 7), GVHD (n: 5), venocclusive disease (1), and pulmonory complication (3). Conclusion: We demonstrated that pretransplant BMI did not affect the major posttransplant clinical outcome (time to engraftment, GVHD, TRM). Prospective studies to fully evaluate the impact of BMI on transplant outcome is warranted. Disclosure of Interest: None declared. P179 Risk factors for early hepatic cytolysis during treatment with anti-thymocyte globulin (ATG) B. Aldweik1,*, R. Redjoul1, S. Bastuji-Garin2, F. Beckerich1, C. Robin1, C. Pautas1, A. Toma1, L. Cabanne1, S. Maury1, C. Cordonnier1 1 Hematology, 2Public Health Department, Henri Mondor University Hospital, Cre´teil, France Introduction: ATG is widely used for treatment of aplastic anemia, and in allogeneic HSCT for conditioning regimens or 2nd line treatment of acute GVHD. Early hepatic toxicity of ATG is poorly reported. Due to several consecutive cases, we aimed
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to assess the risk factors for developing early, acute hepatic cytolysis (HC) under ATG therapy. Materials (or patients) and methods: All consecutive hematology patients receiving ATG (2005-2012), whatever the indication, were retrospectively identified from the local pharmacy files. HC was defined according to the CTCAE v3 as moderate (ALT or AST42.5 and o5 UNL; grade 1-2) or severe (ALT or AST 45UNL; grade 3-4). Data collected from the medical charts included patient and disease characteristics, status, pre-ATG hepatic and renal function, lymphocyte counts, virologic tests, dose and type of ATG, concomitant drugs within the 8 days before and during ATG, immediate tolerance of ATG. Additionally, in case of HSCT, conditioning, donor, graft, donor and recipient CMV serology, and GVHD were collected. ATG was administered according to the official French recommendations, under hyperhydratation, IV dexchlorpheniramine, and methylprednisolone (1mg/kg/d from day-1 until the last day of ATG). Characteristics of the patients developing HC were compared to those of patients who did not, using X2 or exact Fisher test. All significance tests were 2-tailed. Po.05 was considered to be statistically significant. Po0.15 was considered as indicative of a trend. Results: 84 patients received ATG for treatment of aplastic anemia (n ¼ 17, 20%), HSCT conditioning (n ¼ 55, 65.5%) or treatment of aGVHD (n ¼ 12; 14.5%). HC was observed in 24 (28.5%; grade1-2: 9; grade 3-4: 15) patients during or within 10 days following ATG. Maximal ALT/AST levels reached 328xUNL. ATG was stopped during treatment in 3 patients, delayed without changing the total dose in 2, and pursued without change in the other cases. None patient developed hepatic failure. HC evolved favorably in 79% within 10 days and did not impact 3-month survival. However, all the 6 HCpatients who died had grade 3-4 HC. Two patients with HC developed veno-occlusive disease. Both died, one from leukemia relapse, the other one from multivisceral failure. Age, underlying disease, status, indication and type of ATG, pre ATG liver and renal function, immediate side effects, virologic markers, TBI, and concomitant administration of azoles or other hepatotoxic drugs were not associated with the risk of HC. There was a trend for an association between HC and recipient CMV-seropositivity, and liver GVHD. Two factors were significantly associated with a decreased risk of HC: lymphopeniao500/mm3 (6/53 (11.3%) vs 18/31 (58%), Po .001), and conditioning with amsacrine þ cytarabine (0/11 vs 24/73 (33%), P ¼ 0.029), administered in the setting of FLAMSA regimen. Conclusion: In our experience, the incidence of early ATGcytolysis was high (28.5%) and severe in 18%. We were unable to identify any significant risk factor for HC due to ATG. However, the 2 factors associated with a decreased risk (lymphopenia, and amsacrine þ cytarabine in conditioning) suggest that the more immunodepressed patients could be protected of the risk, and therefore that ATG cytolysis could be more an immuno-allergic phenomenon, rather than a direct toxicity. Although this side effect did not impact on survival, we plan to reinforce steroid administration during ATG to decrease this risk. Disclosure of Interest: None declared. P180 Autologuous peripheral blood stem cell transplantation associated with low transplant related mortality rates: Results of a single centre analysis of 856 cases B. Kragl1,*, A. Kraus1, D. Gla¨ser1, C. Groe-Thie1, C. Wittke1, S. Freitag1, M. Leitha¨user1, C. Kahl1, K. Borchert1, M. Freund1, C. Junghan1 1 Department of internal medicin 3, Hematology, Oncology, Palliative Care, University of Rostock, Rostock, Germany Introduction: Autologous peripheral blood stem cell transplantation (PBSCT) is a treatment option for various hematologic diseases such as multiple myeloma and lymphoma. As several new drugs for the treatment of these diseases are
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developed, it is of major importance to investigate continuously morbidities and mortalities associated with autologous PBSCT. Here we report on 856 autologous PBSCTs performed at a single centre academic institution with regards to the early mortality rates. Materials (or patients) and methods: Consecutive autologous PBSCTs performed at a single centre (University of Rostock) over a time period of 20 years were analysed in regards to their 30 day and 60 day mortality rates. Patient characteristics analysed were sex, age, date of transplant, CD34 þ cell number, type of conditioning regimen and underlying disease. Major endpoints were mortality rates at day 30 and day 60 after auto PBSCT. Results: 856 autologous PBSCTs were performed during a time span from 11/1994 to 12/2014. Median age of PBSCT recipients was 53 years (range 16 years to 74 years). Most patients were male (67.1%). Most common underlying diseases were plasma cell diseases (PCDs; n ¼ 419; 49.0% of all), lymphomas (n ¼ 288, 33.6% of all), leukemias (n ¼ 13, 1.5% of all) and others including solid tumours (n ¼ 136; 15.9 of all). Most common conditioning regimen were melphalan based (52.4%), followed by BCNU-based regimen (7.2%) and others including treosulfan based ones (40.4%). 30 day mortality for all entities was low (1.52%). A total of 13 patients died (1 PCD, 9 lymphomas, 3 other). 60 day mortality for all entities was 2.80%. 24 patients died: 4 with PCD, 15 with lymphoma, 5 other. 30 and 60 day mortality rates for the individual underlying diseases were as follows: PCD: 0.12% (1 pt.) and 0.48% (4 pts), Lymphoma: 1.05% (9 pts) and 1.75% (15 pts), Other including solid tumours: 0.35% (3 pts) and 0.58% (5 pts). Most common causes of death within the first 30 days were infections (9/13 pts; 69.23%). Conclusion: Autologous PBSCT is a common procedure in the treatment of PCDs and lymphomas. Treatment associated early mortality rates are low, especially in PCDs. Therefore we conclude that auto PBSCT can be safely administered in PCDs aiming at deeper remission rates. Second, our single center data indicates that mortality rates might be slightly compared to PCDs if PBSCTs are performed in lymphomas which might be due to the fact of the more advanced underlying diseases. References: Disclosure of Interest: None declared. P181 Survival outcome and long-term follow-up of Stem Cell Transplantation (SCT) in Acute Myeloid Leukemia (AML): a single-center experience C. Montes-Gaisan1,*, A. Bermu´dez1, G. Pe´rez1, A. Cuesta1, P. Ibarrondo1, C. Richard1, E. Conde1 1 Hematology, Hospital Universitario Marque´s de Valdecilla, Santander, Spain Introduction: Post-remission treatment for AML is very aggressive and, in many cases, a SCT is needed. Comparisons between Allo-SCT and Auto-SCT have always shown more Transplant Related Mortality (TRM) but less Cumulative Incidence of Relapse (CIR) in the first group. Our study describes, not only the long-term survival outcomes, but also the quality of life in long survivors. Materials (or patients) and methods: Retrospective study of 274 patients diagnosed with non-promyelocytic AML who underwent SCT between 1982 and 2011 in our center. Characteristics in the 162 Allo-SCT and the 112 Auto-SCT groups of patients were respectively: median age of 38 and 45 years old, secondary AML in 20% and 10%, refractory to Induction AML in 16% and 3%, pre-SCT status different from Complete Remission (CR) in 13% and 3% and year of SCT before 2005 in 53% and 86%. No significant differences between both groups were found in other risk factors as hyperleucocytosis at diagnosis or adverse cytogenetics.
Results: With a median follow-up of 55 months [2-316], Overall Survival (OS) until 1997 in Allo-SCT and Auto-SCT were respectively, 40% and 61% at 1 year and 28% and 45% at 5 years, but from 1997, 66% and 70% at 1 year and 47% and 48% at 5 years. TRM until 1997 in Allo-SCT and Auto-SCT were respectively, 30% and 7% at 1 year and 35% and 9% at 5 years, but from 1997, 16% and 2% at 1 year and 25% and 4% at 5 years. Allo-SCT: From the 162 patients, 72(44%) are alive by this moment, 43(60%) with ECOG 0, 21(29%) with ECOG 1 and the other 8(11%) with ECOG 2, basically because of graft versus host disease (GVHD present in 39 patients, 21 steroiddependent and 3 refractory to any treatment). All of them have been in CR during the last 2 years of follow-up. In contrast, 90(56%) patients have died: 52(58%) because of SCT complications (20 infections, 16 GVHD, 8 toxicity and 8 mixed causes), 33(37%) because of disease and 5(5%) because of other causes. With a median follow-up of 43 months [2-316], there have been 4 secondary neoplasm, all of them solid ones, which appeared with a median of 242 months [179-311] from SCT. None of them had previously received radiotherapy. Auto-SCT: From the 112 patients, 43(38%) are alive by this moment, 32(74%) with ECOG 0 and the other 11(26%) with ECOG 1. All of them have been in CR during the last 2 years of follow-up. In contrast, 69(62%) patients have died: 45(65%) because of disease, 14(20%) because of SCT complications and 10(15%) because of other causes. With a median follow-up of 93 months [5-230], there have been 6 secondary neoplasm, 5 of them hematologic ones, which appeared with a median of 90 months [76-115] from SCT. None of them had received radiotherapy, but previously treated hematopoietic stem cells. Conclusion: OS is lower in Allo-SCT during the first years [1982-1996], although it has a tendency towards OS in AutoSCT during the last ones [1997-2011] because of the decrease in TRM, which is more significative in Allo-SCT. Both procedures are efficient to treat AML (near 50% of the patients in both groups are alive at 5 years from SCT in recent years). The decrease in TRM until 2% in Auto-SCT makes it a good choice for older patients without risk factors. The development of secondary hematologic neoplasms is a not inconsiderable fact in Auto-SCT, with an incidence of 10% and a late mortality of 100%. Disclosure of Interest: None declared. P182 Higher Prevalence of HLA-Alloimmunization in Aplastic Anemia and MDS/MPN patients C. Tsamadou1,*, D. Fu¨rst1, C. Zollikofer1, H. Schrezenmeier1, J. Mytilineos1 1 German Red Cross Blood Transfusion Service, Baden-Wuerttemberg – Hessen, Ulm, Germany & Institute of Transfusion Medicine, University of Ulm, Germany, Ulm, Germany Introduction: According to current clinical data, the prevalence of preformed HLA Donor Specific Antibodies (DSA) may lead to graft rejection after Hematopoetic Stem Cell Transplantation (HSCT). The aim of the present study is to investigate the prevalence of HLA antibodies (abs) among different hematological disease groups in terms of identifying patients at higher risk for HLA-alloimmunization. Materials (or patients) and methods: 1405 patients scheduled for a haematopoetic stem cell transplantation (934 males& 471 females) with a median age of 58y were screened for HLA abs class I (CLI) and II (CLII) with ELISA and were stratified according to diagnose in 6 major disease groups (61 AA: Aplastic Anemia, 415 NHL: Non-Hodgkin Lymphoma, 33 HL: Hodgkin Lymphoma, 228 MM: Multiple Myeloma, 222 CL: Chronic Leukemia and 446 MDS/MPN: Myelodysplastic Syndrome/Myeloproliferative Neoplasms). Acute leukemia patients were not included in the study considering that patients with longer course of disease are more likely to be HLA-alloimmunized due to higher frequency of transfusions. Statistical significance was assessed via chi-square test and/or
Fisher’s exact test. The threshold for statistical significance was set to a p value of o0.05. R program v 3.1.0 was used for all statistical computations. Results: We found a statistically significant higher prevalence of HLA CLI and CLII abs in the AA (CLI:21.3%, Po0.001, CLII:16.4%, P ¼ 0.003) as well as in the MDS/MPN (CLI:15.7%, Po0.001, CLII:16.4%, Po0.001) group of patients in relation to patients in the other four groups which were subsequently classified as control group (CLI:7%, CLII:5.6%). It was also observed that female patients presented a higher rate of HLA alloimmunization (Po0.001). Subsequent analysis however, revealed no preferential distribution of patients according to sex in the various disease groups (P ¼ 0.21), and therefore female sex was excluded as possible confounding factor for our initial findings. Conclusion: Our data indicate that AA and MDS/MPN patients are more prone to develop HLA abs compared to those in other hematological disease groups. A plausible explanation could be the higher rate of blood and /or platelet transfusions among these patients. Past pregnancies can also explain why female patients had a significantly higher frequency of positive HLA-antibody results. Conclusively, based on our findings and on the existing data regarding the negative effect of patient’s HLA DSA on HSC engraftment, it would be reasonable to advise an initial screening for HLA abs in patients at higher risk for HLA-alloimmunization such as Aplastic Anemia or MDS/ MPN patients upon initiation of unrelated donor search. In cases when a fully matched donor is not an option, further identification of HLA abs and consideration of arising HLA antibody specificities upon donor selection would be strongly recommended. Disclosure of Interest: None declared. P183 Biosimilar G-CSF vs classical G-CSF in post allotransplant recovery. A case control study C. Targhetta1,*, D. Baronciani1, C. Depau1, F. Pilo1, M. Capasso2, E. Angelucci1 1 - Ospedale Oncologico di Riferimento Regionale ‘‘A. Businco’’, Cagliari, Italy., U.O. Ematologia-CTMO, Cagliari, 2Dept. of Molecular Medicine and Medical Biotechnologies CEINGE Biotecnologie Avanzate , University of Naples Federico II, Napoli, Italy Introduction: The use of biosimilarG-CSF was licensed by the European Medicines Agency (EMA) for all the registered indications of the originator G-CSF on the basis of extrapolation of efficacy and safety data without a specific clinical trial. Moreover the setting of allogeneic transplantation is unexplored. Because we believe that clinical data is the only cornerstone for clinical practice since February 2013 we started a case-control clinical study in patients undergoing allotransplant to verify biosimilars safety and clinical appropriateness in the post-transplantation hematopoietic recovery. Materials (or patients) and methods: We analyzed two cohorts comprising a total number of 86 patients suffering from various hematological diseases treated in our HSCT Centre. The first cohort included 43 consecutive patients undergoing allogeneic HCST between February 2013 and October 2014 who were treated with biosimilars of G-CSF (Tevagrastims or Ratiograstims or Zarzios). The second cohort (control group) included 43 consecutive patients who underwent allogeneic HSCT between January 2011 and December 2012 and received originator G-CSF (Granulokines). The injection of G-CSF began at day 7 after HLA identical sibling or MUD and at day 5 after haploidentical stem cell infusion. To compare the efficacy of the two G-CSF formulas we evaluated the day of engraftment and the incidence of graft failure in the two cohorts. Neutrophil engraftment was defined as a sustained (for more than 3 consecutive days) absolute neutrophil count of 500/ml without the use of growth factors. Primary graft failure was defined as failure to maintain absolute neutrophil count Z500/ml for 3 consecutive days following transplantation. Categorical variables have been
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evaluated by X2 test, continuous variables have been evaluated by Mann-Whitney test. Data are expressed as median with a range. Results: Study and control groups were matched for sex, type of disease, Karnofsky score, HCT-Comorbidity Index, status of disease at transplant, conditioning regimen, type of donor, HLA-matching, stem cell source, number of stem cells injected (P not significant). There were 41 and 40 evaluable patients in the study and control group respectively. White blood cell engraftment took place in median time of 13 (10-28) days in the study group and in 14 (10-22) days in the control group (P ¼ 0.205). Primary graft failure developed in 1 out of 41 and in 1 out of 40 evaluable patients in the two groups, respectively (P ¼ 0.4). To further enhance clinical relevance we evaluated on intention to treat basis the incidence of early infections that developed in 13/43 (30%) patients in the first cohort, in 18/43 (41%) in the second cohort (P ¼ 0.37). Conclusion: This study shows that biosimilars of G-CSF are equivalent to classical products in terms of safety and efficacy when used for white blood cell recovery after allogeneic hemopoietic stem cell transplantation. Disclosure of Interest: None declared. P184 Implementing International HCT Late Effects Screening Guidelines in Clinical Practice: Outcomes D. Greenfield1,*, V. Shanklin2, J. Snowden3 1 Cancer Services, Sheffield Teaching Hospitals NHS FT, 2Medical School, University of Sheffield, 3Haematology, Sheffield Teaching Hospitals NHS FT, Sheffield, United Kingdom Introduction: HCT survivors are at risk for developing late complications secondary to pre-, peri-, and post-transplantation exposures and risk factors. Updated recommendations for late effects screening for survivors of autologous and allogeneic HCT were published in 20121. The aim of this study was to assess the clinical efficacy of a screening clinic to identify late complications of HCT. Materials (or patients) and methods: Our centre implemented the internationally agreed guidelines in 2013 using a locally developed Standardised Operating Policy (SOP) through a nurse-led late effects screening clinic. 59 consecutive patients who attended the screening clinic for the first time during 1st Sept 2013 to 31st Aug 2014 were included in the audit. Data was extracted from the clinic letter annotated from the consultation and from results of the requested investigations as per the SOP. Presence or absence of each variable and investigation was coded and current results and actions were recorded in a spreadsheet. Audit approval confirmation was given by the Clinical Effectiveness Unit of our hospital trust. Results: 30 female and 29 male HCT survivors with a mean age of 49 years and a mean 6 years post-transplant attended the nurse-led late effects clinic as new patients in the time period. 49/65 were allogeneic HCTs. 54 had one transplant procedure, 4 had 2 and one had 3 procedures.13 patients received TBI radiotherapy. 43/59 primary indications for HCT were lymphoid diseases, 12/59 were myeloid diseases with 3/59 were solid tumour and 1/59 had an auto-immune disease. 134 late complications were identified in 59 patients with 114 requiring further action or referral, mean complication per patient ¼ 2. The most frequent late complications were pain 18/59 (31%), sleep disturbance 11/59(19%), fatigue 14/59(24%) and sexual function concerns 14/59(24%). Conclusion: To our knowledge this is the first time new international late effect screening guidelines have been audited. This new nurse-led screening clinics demonstrates clinical efficacy in identifying late complications using holistic screening. This will inevitably result in earlier detection of problems and may prevent and/or reduce morbidity therefore playing a central role in optimising quality of life for HCT survivors. Future research should look at complications and their link to certain treatments and whether these
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complications can be reduced using different transplantation exposures. References: 1. Majhail NS, Rizzo JD, Lee SJ, Aljurf M, Atsuta Y, Bonfim C, et al. Recommended screening and preventive practices for long-term survivors after haematopoietic cell transplantation. Biol Blood Marrow Transplant 2012 Mar 3; 18(3): 348–371. Disclosure of Interest: None declared. P185 Ruxolitinib for symptomatic patients with relapse of myelofibrosis after allogeneic hematopoietic transplantation D. Janson1,*, T. Stu¨big1, H. Alchalby1, F. Ayuk1, C. Wolschke1, M. Christopeit1, N. Kro¨ger1 1 University Medical Center, Hamburg, Germany, Department of Stem Cell Transplantation, Hamburg, Germany Introduction: The janus-activated kinase (JAK) 1/2 -inhibitor ruxolitinib is approved for the treatment of symptomatic myelofibrosis. Ruxolitinib can reduce constitutional symptoms and spleen size resulting in an improvement of the performance status and also in a potential prolonged survival. No data on ruxolitinib for relapsed patients after allogeneic hematopoietic transplantation have been reported so far. Materials (or patients) and methods: Five patients of our department with relapse or progressive disease of primary (n ¼ 3) or post ET/PV- (n ¼ 2) myelofibrosis were treated with ruxolitinib after allogeneic transplantation. The median age of the patients was 63 years (r: 50-72). Stem cell source had been PBSC (MRD n ¼ 2, MUD n ¼ 3) in all patients. The median time from allogeneic hematopoietic transplantation until the start of ruxolitinib was 8.8 months (r: 3.5-85.7). The median treatment duration was 268 days (r: 12-312). Ruxolitinib doses differed from 5mg to 20mg bid according to platelet counts. Two patients received additional DLI. One patient started ruxolitinib after non-responding to DLI and another patient received DLI due to decreasing chimerism during ruxolitinib treatment. All patients had mixed chimerism. Before starting ruxolitinib three patients had palpable splenomegaly and all had constitutional symptoms. Three patients had cGvHD (skin, liver or oral mucosa) at the time of treatment-start. Results: Constitutional symptoms improved in all patients (100%). The palpable spleen size was reduced in all three patients with splenomegaly (450% reduction n ¼ 2, 45% reduction n ¼ 1). In transfusion-dependent patients (n ¼ 3) a reduction of the transfusion frequency could be seen. No reduction of the JAK2-alllele-burden was seen in those patients with a JAK2V617F-mutation (n ¼ 2). During ruxolitinib treatment a further decline of donor chimerism was seen in all patients. In three out of four patients with an available bone marrow histology before and within the last days of ruxolitinib treatment the grade of bone marrow fibrosis aggravated. Two patients transformed to AML despite of response to spleen size and constitutional symptoms during ruxolitinib. Two patients with cGVHD had an improvement and even complete resolution of their GvHD. In one patient cutaneous GvHD (grade II) occurred during ruxolitinib treatment but immunosuppressive treatment had been stopped simultaneously. Within the time of ruxolitinib treatment no severe infectious complications were seen. Conclusion: Ruxolitinib is well tolerated, ameliorates MFspecific symptoms and reduces the spleen size in patients with relapse after allogeneic transplantation. Ruxolitinib does neither reduce the JAK2-allele burden nor does it lead to an increase of donor chimerism. Bone marrow fibrosis seems to aggravate whilst treatment with ruxolitinib. Transformation to AML was seen in two patients despite of clinical response to ruxolitinib. It is of note that in two patients an improvement of the cGvHD was seen. Disclosure of Interest: None declared.
P186 Central Venous Catheter Related Complications in a Pediatrıc Stem Cell Transplantation Unit F. Azik1, D. Gurlek Gokcebay1,*, P. Isik1, Z. Avci1, N. Ozbek1, B. Tavil1, D. Uc¸kan1, B. Tunc1 1 Pediatric Hematology, Ankara Children’s Hematology and Oncology Hospital, Ankara, Turkey
P187 CD4 þ cell count on day þ 30 predict overall survival and transplant related mortality in acute leukemia patients after alloPBSCT D. Pastore1,*, M. Delia1, A. Mestice1, P. Carluccio1, T. Perrone1, F. Gaudio1, A. Ricco1, P. Curci1, C. Pasciolla1, G. Specchia1 1 Hematology-Policlinico-Bari Italy, BARI, Italy
Introduction: Children undergoing hematopoietic stem cell transplantation (HSCT) require long term central venous catheters (CVC) for administrating of medications, blood products and blood sampling. The aim of the study is to assess infectious and mechanical complications of CVCs during HSCT. Materials (or patients) and methods: We retrospectively evaluated 95 consecutive patients between April 2010 and January 2013 who underwent HSCT at our Bone Marrow Transplantation Unit. Data included demographic information, CVC type and location of insertion, duration of insertion, occurrence of complications, antimicrobial therapy and causes of removal collected from medical records. Results: A total of 95 patients (42% girls, 58% boys) underwent 100 HSCT with median age of 8.9 years (range; 0.5-16.8). Ninety-two of the transplants were allogeneic, and 8 of them were autologous. Forty-five patients had malignant, 40 had non-malignant, and 10 had immunodeficiency disorders. Ninety-nine Hickman double lumen catheters were inserted in 91 (95.8%) of the patients for a total 6369 catheter days, with a median catheter life of 56 days (range; 23-201). Hickman catheters could not be inserted in 4 (4.2%) of the patients because of repeated CVC insertions or radiation therapy scars; they had nontunneled jugular or femoral catheters during HSCT. Forty CVC-related infections were recorded with a rate of 6.28/1000 catheter days. Coagulase negative staphylococci were the predominant microorganisms in 42.5% (17/40). Twenty-nine of the CVC’s (27%) were removed during the study period as 22 of them became infected, 5 had mechanical complications (3 ruptures, 1 malposition, 1 leakage), and 1 was revised because of blocking. One of the CVC which was blocked opened using tissue-plasminogen activator. Overall mechanical complication rate was 1.09/1000 catheter days. Enoxaparin at a dose of 0.8 mg/kg/day by subcutaneous injection was administrated as prophylaxis of veno-occlusive disease for median 34 days (total 3016 days, range; 11-54), and overall 5.2% (5 out of 95) of the patients developed CVC-associated thrombosis. Type of the CVC did not influence the frequency of mechanical or infectious complications. Children o8 years of age were more likely to develop infectious and mechanical complications leading to CVC removal (P ¼ 0.01). Conclusion: The rate of CVC-related infections were varying from 0.87 to 8.24, and mechanical complications were varying from 0.45 to 4.5 per 1000 catheter days in children with hemato-oncological diseases; which were in accordance with our study. Prior studies have suggested that thrombosis occurs in 4.4%49% of pediatric HSCT recipients. Although enoxaparin was used for prevention of VOD in this study, the thrombosis rate did not differ from another reports. In younger children with smaller size CVCs more frequently developed a complication leading to removal which shown in previous studies, it is important that the proper care of the CVC by nurses and parents. References: 1. Fratino G, Molinari AC, Parodi S, et al. Central venous catheter-related complications in children with oncological/hematological diseases: an observational study of 418 devices. Ann Oncol. 2005; 4: 648–654 2. Lagro SW, Verdonck LF, Borel Rinkes IH, Dekker AW. No effect of nadroparin prophylaxis in the prevention of central venous catheter (CVC)-associated thrombosis in bone marrow transplant recipients. Bone Marrow Transplant 2000; 26: 1103– 1106. Disclosure of Interest: None declared.
Introduction: Allogeneic peripheral stem cell transplantation (alloPBSCT) from a related or unrelated donor is a wellestablished strategy for patients with acute leukaemia. However, this procedure is associated with increased morbidity and mortality because of graft versus host disease (GvHD), immune reconstitution impairment and a high risk of infections. In our study we evaluated the lymphocyte subset recovery after alloPBSCT and its impact on transplant-related mortality (TRM) and overall survival (OS) in acute leukemia patients. Materials (or patients) and methods: We evaluated the immune reconstitution of CD3 þ /CD4 þ , CD3 þ /CD8 þ and NK cells performed at 30, 100, 180 and 360 days after alloPBSCT in 115 patients with acute leukaemia. Patients were transplanted with unmanipulated PBSC from an HLA matched related donor (MRD) (n ¼ 80) or an HLA (8/8) matched unrelated donor(MUD) (n ¼ 35). Median age was 38 years (range 18-61); diagnoses were acute myeloid leukaemia (n ¼ 90) and acute lymphoblastic leukaemia (n ¼ 25); 80% of patients underwent myeloablative conditioning (busulphan, cyclophosphamide in MRD and busulphan, cyclophosphamide and ATG in MUD) and 20% underwent reduced intensity conditioning (busulphan, fludarabine, ATG) Results: The median counts of CD3 þ /CD4 þ were 98, 160, 200, 262 ml at 30, 100,180 and 360 days, respectively. The median counts of CD3 þ /CD8 þ were 180, 350, 500 and 670 ml at 30, 100, 180 and 360 days, respectively. The median counts of NK cells were 110, 260, 270 and 260 mL at 30, 100, 180 and 360 days, respectively. Considering the entire patient population the median cell count rose above 200 ml after day 180 for CD3 þ /CD4 þ and after day 45 for CD8 þ cells; the median NK cells count rose above 100 ml after day 30. The median CD3 þ /CD4 þ cell count on day þ 30 for the entire patient population was 98 ml( range 20-190) and TRM at 2 years was significantly higher in patients not achieving this CD4 cell count (38% vs 15%, Po0.001); patients with a low CD3 þ / CD4 þ count (o 98 ml) on day þ 30 had a higher risk of dying of infections (30% vs 11%, P40.003). Median OS in patients not achieving CD4 þ of 98 ml at 30 days was 40 months while median OS in patients with more than 98 CD4 þ at 30 days was not reached. Univariate analysis at 100 days showed a significant association between CD4 cell recovery and transplant from a related donor (P ¼ 0.003), myeloablative conditioning (P ¼ 0.04) and absence of aGvHD (grade II-IV). In multivariate analysis, transplant from a related donor (P ¼ 0.05) and absence of aGvHD( grade II-IV) (P ¼ 0.002) were significantly associated with a better CD4 cell recovery. CD3 þ / CD8 þ and NK cells recovery did not correlate with a different TRM risk or OS. Conclusion: Our results support the relationship between immune reconstitution of CD4 þ , OS and TRM. The CD4 þ cell count on day þ 30 is able to predict OS and TRM after myeloablative alloPBSCT in acute leukemia patients. Disclosure of Interest: None declared.
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P188 Restrospective comparative analysis of clinical outcomes between elderly and young patients in allogeneic hematopoietic stem cell transplantation E. Chapchap1,*, L. N. Kerbauy1, F. Santos1, M. Rodrigues1, I. Esteves1, J. Sobrinho1, F. Kerbauy1, A. Ribeiro1, N. Hamerschlak1 1 Haematology and Oncology, Albert Einstein Hospital, Sao Paulo, Brazil Introduction: The number of allogeneic hematopoietic stem cell transplantations (Allo-SCT) has been rising for older patients in the past decade. This is mostly due to advances in treatment and supportive care, as the use of reduced intensity and non myloablative conditioning regimens together with post-transplant immunomodulatory strategies. In this retrospective study, we analyzed the outcomes of two groups: elderly group (EG) versus young group (YG) that underwent Allo-SCT, from January 2007 to June 2014, at Israelita Albert Einstein Hospital in Brazil, in the light of prognostic factors and toxicity. Materials (or patients) and methods: The two groups, YG (r55 years old) and EG (455y), were analyzed with the following endpoints: overall survival (OS); therapy-related mortality (TRM) in the first 100 days and after 1 year post transplant; admission to intensive care unit (ICU), need for renal replacement therapy (RRT) and total days as inpatient in the first year. STATA software (Kaplan-Meier curve and Cox regression) were used for statistical analysis. We also analyzed potential risk factors for mortality in the elderly group. Results: A total of 111 patients were analyzed, 75 in the young group (YG) and 36 in the elderly (EG). MDS and AML were the most common indications in both groups: 47.2% (YG) and 40.7% (EG). Half of patients in the EG underwent Allo-SCT with MRD and 60% in the YG. Haploidentical donors were used in the young group: 18 transplants (24%) and in 5 (13.9%) in the elderly. In both groups, the most frequent conditioning regimen was IV Busulfan and Fludarabine±ATG with adjusted busulfan dose per pharmacokinetics/AUC analysis. The OS rates in the YG and EG were 84% versus 75% (P ¼ 0.01) at D þ 100 and 71% vs 53% (P ¼ 0.01) at one year. TRM were higher in the EG at D þ 100: 17% vs 14% (P ¼ 0.04) and 29 % vs 17% (P ¼ 0.04) at one year, respectively. Admission to ICU was more common in the elderly 44% vs 23% (P ¼ 0.019). The requirement of RRT was higher in elderly: 15% vs 25% (P ¼ 0.18). The median days as inpatient in the first year for elderly versus young was 73 vs 60 (P ¼ 0.67). The cumulative GVHD incidence was similar in both groups: 52% vs 50% (P ¼ 0.88), while grade III-IV GVHD were 14% vs 11%. The relapse rate in the first year was: 23% vs 20% (P ¼ 0. 67). None of these variables: age older than 65y; conditioning regimen; graft source and the primary disease (AML/MDS versus others), had impact in OS or TRM in the elderly group. Conclusion: In our population, despite the mortality was higher in elderly group, this was not found to be related to relapse or GHVD. Probably, the co-morbidities and loss of functionality in the elderly group are important factors that impacted in the OS, since the TRM increase over time from 100 days to one year. Comorbidity index and functional assessment should be used in the elderly patients in order to define the best transplant candidate and treatment strategy choice before and after transplant. References: 1. Alatrash G et. al (2011) Myeloablativereducedtoxicity iv busulfan and allogeneic hematopoietic Fludarabinestem cell transplant for acute myeloid leukemia with patientes or myelodysplastic syndrome in the sixth through eighth decades of life. Biol Blood MarrowTransplant. 10,1490-1496. 2. Appelbaum F. (2011) Preparative regimens and ageism. Biol Blood Marrow Transplant 17(10), 1419–1420 Disclosure of Interest: None declared.
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P189 Veno occlusive disease of the liver after hematopoietic stem cell transplantation (HSCT) in pediatric patients: Padova experience E. Calore1,*, S. Rossin1, M. Pillon1, M. Tumino1, C. Mainardi1, T. Toffolutti2, S. Varotto1, R. Destro1, M. V. Gazzola1, G. Basso1, C. Messina1 1 Department of Pediatrics, Clinic of Pediatric Hematology Oncology, 2Institute of Radiology, Unit of Pediatric Radiology, University of Padova, Italy Introduction: Hepatic veno-occlusive disease (VOD) is one of the most severe complication due to HSCT. The incidence of VOD has been described between 10-60% in children, depending to many factors. Materials (or patients) and methods: We performed a prospective study analyzing 117 pediatric patients that underwent HSCT from 2011 to 2014 in the Bone Marrow Unit of Pediatric Onco-Hematology in Padova. Thirty nine patients underwent autologous HSCT, 88 allogeneic HSCT. Seven patients received more than one HSCT. The conditioning regimen was TBI based in 38 patients, busulfan based in 32. Ninety two patients were Caucasian, 6 Black, 5 Asiatic e 14 south American. Patients with one or more risk factors for VOD (58) were enrolled to receive intravenous Defibrotide (DF) prophylaxis, while the other patients (58) were enrolled to receive Heparin; 1 patient didn’t receive any prophylaxis. Results: VOD has been diagnosed in 4/117 patients (2 received autologous HSCT, 2 allogeneic HSCT) with an incidence of 3.4% using Seattle criteria, while using Baltimora criteria in 2/117 patients (1.7%). Inversion of portal flow has been found in all 4 patient at a median time of 5.5 days (range 0-7) after clinical diagnosis of VOD. Two children have been affected by severe VOD while the others 2 by a moderate VOD. All patients have been treated with rt-PA and Heparin after the occurrence of inversion of portal flow and it has been reverted in all patients with resolution of VOD in a median time of 15 days (range 9-22). No one had bleeding due to the therapy with rt-PA and no one died for VOD. Taking into account our statistical analysis, gender, primary disease, conditioning regimens, type of transplant (auto or allo), stem cell source, pre-existing hepatic disease, abdominal irradiation in the year before HSCT and ferritin level 42000 ng/ml are not risk factors for VOD. Non caucasian ethnicity is the risk factor for VOD (p-value ¼ 0,03). The 4 patients with VOD were in prophylaxis with DF. Comparing the two groups of patients in prophylaxis with Heparin and DF we saw that patients in prophylaxis with DF had lower incidence of hepatotoxicity (lower levels of AST/ ALT) than patients in prophylaxis with Eparina; we didn’t observe any difference about incidence and severity of acute Graft versus Host Disease (aGHVD) and Transplant related mortality (TRM). Conclusion: Our study shows that the incidence of VOD is lower than that reported in literature. Moreover the incidence of VOD is different depending on diagnostic criteria used (Seattle or Baltimore). All 4 patients developed inversion of portal flow and doppler US may be helpful in identification of the patients with VOD. All 4 patients with VOD were in prophylaxis with DF but they also had risk factors for VOD. From our analysis, non Caucasian ethnicity is the only risk factor for VOD. This data hasn’t been reported in literature and it requires a larger series to be confirmed. Prophylaxis type doesn’t affect on incidence and severity of aGVHD in patient with allogeneic HSCT. Disclosure of Interest: None declared.
P190 Peripherally Inserted Central Catheter (PICC) Power Groshong 5 Fr device for management of peripheral blood stem cell collection and transplantation in haematological patients: a single centre study from the Rome Transplant Network E. Conte1,*, A. Ferrari1, M. P. Bianchi1, M. Piedimonte1, C. Guglielmi1, V. Naso1, R. Palmieri1, D. Grigore1, F. Sollazzo1, M. Shafii Bafti2, D. Meo2, A. Tafuri1, W. Arcese3 on behalf of Rome Transplant Network 1 Haematology, Sant’Andrea Hospital, "Sapienza" University of Rome, 2Immunohematology and Transfusion Medicine, "Sapienza" University of Rome, 3Stem Cell Transplant Unit, Haematology, Tor Vergata University of Rome, Rome, Italy Introduction: PICC is inserted bedside, via peripheral vein such as cephalic, basilic or brachial veins, avoiding general anesthesia, sedation and surgical procedures. However, only few data are reported on PICC management in peripheral blood stem cell (PBSC) collection and transplantation. Herein, we are reporting our experience in using PICCs during these procedures. Materials (or patients) and methods: From October 2012 to November 2014 a single lumen silicone 5 FR Power Groshongs PICC (Bard Access, Usa), a new device recently available, was inserted in 48 haematological patients (28 F and 20 M; median age 54, range 24-68), affected by Acute Myeloid Leukemia (n ¼ 9), Non Hodgkin Lymphoma (n ¼ 18), Multiple Myeloma (n ¼ 20), Hodgkin Disease (n ¼ 1). Power Groshongs PICC was used to collect PBSC in 27 patients, 18 of whom later underwent an autologous stem cell transplantation (AuSCT). In the remaining 17 and 4 patients, PICC was used only in AuSCT or an allogeneic stem cell transplantation (AlloSCT), respectively. In all patients PICC was used for PBSC reinfusion. All PICCs were positioned and managed by a team of specifically trained physicians and nurses, at our Haematology Unit. Results: The 48 PICCs remained in place for an overall period of 7,099 days (median time 137 days, range 21-391). The infective complications observed were represented by 1 Central Line Associated Bloodstream Infection (CLABSI) by Staphylococcus Epidermidis and 9 Bloodstream Infections (BSI): 3 by Staphylococcus Epidermidis, 1 by Staphylococcus Haemolyticus and 5 by Escherichia Coli. The incidence of CLABSI and BSI were 2% (0.14 per 1000 PICCs days) and 18% (1.26 per 1000 PICCs days), respectively. PICCs were removed in 38/48 patients: 35 for completion of therapy (72%, 4.9 per 1000 PICCs days), 1 for BSI (2%, 0.14 per 1000 PICCs days), 1 for breakage (2%, 0.14 per 1000 PICCs days), 1 lumen occlusion (2%, 0.14 per 1000 PICCs days). The remaining 10 PICCs are still in place. Conclusion: In summary, Power Groshongs PICC 5 Fr seems a feasible, safe and effective alternative to tunnelled central venous catheters (CVCs) for PBSC harvesting (using this device to return blood components) and reinfusion. PICC resulted useful for the complete management of transplanted patients (administration of high dose chemotherapy, antibiotic agents, blood cell product transfusion and parenteral nutrition). The results obtained by the use of Power Groshongs PICC should be therefore compared with those of tunnelled CVCs, on a large number of patients and in a prospective randomized trial. The above proposed study may evaluate, in addition, quality of life and will be conducted by the same team composed by specifically trained physician and nurses. Disclosure of Interest: None declared.
P191 Clinical Impact And Prognosis About Multiple Transfusions Prior To Allogeneic Transplantation Of Haematopoietic Progenitors E. Alonso1,*, T. Botin2, J. R. Grifols1, M. Morgades2, C. Ferra`2, I. Rodrı´guez3, B. Xicoy2, J. M. Ribera2 1 Banc de Sang i Teixits, Hospital Germans Trias i Pujol, 2 Hematologic Clinic-Institut Catala` d’Oncologia, 3Red Cell Pathology Unit-Hematologic Clinic-Institut Catala` d’Oncologia, Hospital Germans Trias i Pujol-Institut de Recerca contra la Leucemia Josep Carreras, Badalona, Spain Introduction: Hyperferritinemia in patients who undergo allogeneic progenitor cell transplantation (alloHSCT) has been related to lower overall survival (OS) and higher non-relapsed mortality (NRM), probably due to iron overload toxicity in hepatic and cardiac cells, and to an increased risk of fungal and bacterial infections. The aim of this study was to analyse if the number of red blood cells (RBC) units received prior to alloHSCT could impact on the probability to develop infections (bacterial or fungal), hepatic sinusoidal obstruction syndrome (SOS), graft-versus-host disease (GVHD) and OS. Materials (or patients) and methods: Retrospective analysis of 88 patients submitted to alloHSCT from January 2008 to December 2013. We compared the patients who had been transfused more than 20 RBC units before alloHSCT (n ¼ 46) with the patients who had received less than 20 RBC units (n ¼ 42). We collected demographic and clinical data, transfusional history, value of serum ferritin and hepatic function previous and after alloHSCT (at 3, 6 and 12 months later), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) pre-alloHSCT, chelating treatment pre- and postalloHSCT, history of bacterial sepsis (two blood culture positives), invasive fungal infections (IFI) and the incidence of SOS and III-IV mucositis grade, GVHD type (acute and chronic), OS, as well as, NRM and relapse incidence (RI). Results: There were no statistically significant differences between two groups as to age, sex, diagnosis, ABO and HLA compatibility, progenitors source, donor type, pre-alloHSCT risk, type of conditioning, CD34 cells infused, Left Ventricular Ejection Fraction (LVEF) pre-alloHSCT, median of neutrophils recovery days neither chelation treatment pre-alloHSCT. There were statistically significant differences in ferritinemia prealloHSCT (higher in the most transfused group [3639 ng/mL vs. 1094 ng/mL, P ¼ 0.001]). A greater tendency to present SOS was observed in the group of patients who received more than 20 RBC units (9/46 vs. 3/42, P ¼ 0.090); without significant differences in III-IV mucositis grade, GVHD type and infections incidence between the two groups (but higher incidence of infections by multiresistant pathogens was observed in the more transfused group, without significant differences). The OS at 5 years was lower in patients who had received more than 20 RBC units (27% [95% CI, 14% - 40%] vs. 43% [25% 61%] P ¼ 0.04), without significant differences in RI (26% [12% -40%] vs. 17% [6%; 28%], P ¼ 0.427) or NRM (48% [30% -66%] vs. 47% [31% -63%], P ¼ 0.578). Conclusion: In this study, the patients who received more than 20 RBC units before alloHSCT, presented higher serum ferritin values, major SOS incidence and worst OS than the patients who received less than 20 RBC units before HSCT. So, it is important to insist on an adequate chelation treatment before submit a patient to alloHSCT, whenever possible. References: Sivgin et al. Ann Hematol 2013; 92: 577–586. Sivgin et al. Transfusion and Apheresis Science 2013 48: 103–108. Sakamoto et al. Int J Hematol 2013; 97: 109–116. Disclosure of Interest: None declared.
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P192 Analysis of early life-threatening complications in children and adolescents undergoing allogeneic hematopoietic stem cell transplantation Z. Szmit1, E. Gorczynska1,* on behalf of Kalwak K., Owoc-Lampach J , Mielcarek M., Ussowicz M., Kulej D., Musial J, Chybicka A. 1 Medical University, Wroclaw, Poland, Department of Pediatric Hematology, Oncology and Bone Marrow Transplantation, Wroclaw, Poland Introduction: Allogeneic hematopoietic stem cell transplant (allo-SCT) is a treatment method of cancer, bone marrow failure resistant to standard therapy, and immune or metabolic disorders. This procedure has high risk of life-threatening complications. The aim of the study was analysis of severe complications and treatment related mortality (TRM) in early period post allo-SCT. Materials (or patients) and methods: Medical records of 526 patients aged from 1month to 21 ys (median 9.4ys), who underwent first procedure of allo-SCT in Department of Pediatric STC, Hematology and Oncology in Wroc"aw were analyzed. Since 2007 range of diagnostic methods (regular determination of EBV, ADV, BKV by quantitative method of PCR, and fungal antigens) and infection prophylaxis (antifungal drugs including Aspergillus spp. and Ciprofloxacin for BKV-induced cystitis) were expanded, therefore the patients cohort was divided into two groups. Group I:231 patients transplanted between Jan.2002-Dec.2006 (59 matched sibling donor MSD ,135 matched donor MD, 37 mismatched donor MMD). Group II: 294 patients (74 MSD, 188 MD, 32 MMD) transplanted in the period Jan. 2007-Mar.2014. Frequency of early severe complications ( until day 180 ) such as: organ damage, infections, acute graft versus host disease (GvHD) and TRM were evaluated depending on type of stem cell donor (MSD vs alternative donor, including MD and MMD). Results: Early life-threatening complications in group I and group II were as follow: Fever of unknown origin (FUO) 64.9% vs 63% (P ¼ 0.59), severe toxic mucositis 47.2% vs 47% (P ¼ 0.95), bacterial infection 32.5% vs 21.8% (P ¼ 0.013), fungal infections 21% vs 14.5% (P ¼ 0.06), BKV induced haemorhagic cystitis (HC) 25.5% vs 15.3% (P ¼ 0.0046), CMV infection 28% vs 36.1% (P ¼ 0.0428), VOD 9.5% vs 7.1% (P ¼ 0.3234), neurological disorders 17.8% vs 15.3% (P ¼ 0.45), severe aGvHD 22% vs 24.1% (P ¼ 0.334), respiratory failure 23.8% vs 12.7% (P ¼ 0.0016), EBV-PTLD 6.1% vs 6.5% (P ¼ 0.88). TRM 19.9% vs 17.7% (P ¼ 0.67). After MSD transplantation major TRM causes in in group I and II were: sepsis 37.5% vs 18.2%, aGvHD 12.5% vs 18.2%, viral infections 12.5% vs 9% neurological disorders 12.5% vs 9%, VOD 0% vs 27.3%, MOF 0% vs 18.2% HC 12.5% vs 0% (P ¼ 0.2). Post transplantation from alternative donor, major causes of TRM in group I and II were as follow: aGvHD 7.8% vs 39% (P ¼ 0.0012), sepsis 26.4% vs 21.9%, MOF 13.5% vs 19.5%, viral infections 18.4% vs 7.3% (P ¼ 0.05), VOD 5.2% vs 2.4%, rejection 7.8% vs 0% (P ¼ 0.06), fungal infections 0% vs 4.9%. Conclusion: 1. Extended prophylaxis significantly decreased frequency of HC and systemic fungal infections. 2. Regular EBV diagnostics had no influence on occuring of EBV-PTLD. 3. Despite decrease in frequency of life-threatening complications no TRM decrease was noticed. Only the causes of death structure has changed. 4. As aGvHD is actually major cause of TRM in early period after allo-SCT it would be expedient to extend early diagnostic including monitoring of plasma GvHD biomarkers such as ST2. Disclosure of Interest: None declared.
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P193 Sistemic Inflamatory Response Syndrome (SIRS) associated to stem cell infusion in haploidentical transplantation recipients F. Lopez1,*, S. Alonso2, M. Manzanares3, O. Lopez-Godino1, O. Ferre´1, L. Garcia2, E. Perez-Lopez2, A. Redondo-Guijo2, F. Sanchez-Guijo2, L. Vazquez2, M. C. del Can˜izo2, D. Caballero2, L. Lopez-Corral2 1 HOSPITAL UNIVERSITARIO DE SALAMANCA, salamanca, Spain, 2 hematologia, HOSPITAL UNIVERSITARIO DE SALAMANCA, Salamanca, 3Hospital of Jerez, Jerez de la Frontera, Spain Introduction: Haploidentical stem cell transplantation has been used as an option for patients needing rapid allogeneic stem cell transplantation (SCT) in the absence of a sibling donor. Some specific side effects have been reported, some of them being considered a probable consequence of a cytokine release associated with activation of donr T cells after graft infusion Materials (or patients) and methods: This is a retrospective descriptive study in which we included all consecutive patients receiving haploidentical stem cell transplantation in our Hospital Results: 26 patients were included in this study. Median age was 58 years (17-68).Gender M/F ratio was 14/12. Underlying diseases were lymphoblastic (n ¼ 2, 8%) and myeloblastic leukemia/myelodysplasia (n ¼ 14, 54%), Hodgkin (n ¼ 7, 27%) and non-Hodgkin (n ¼ 1, 4%) lymphomas, myelofibrosis (N ¼ 2, 8%). Disease status at transplant was:complete response in 12 patients (46.2%), minor residual disease in 2 patients (7.6%), partial response in another patient (3.8%), aplasia without blasts in 3 patients (11.5%), active disease in 3 patients (11.5%), untreated relapse in 3 (11.5%). Some patients had received a prior transplant: ASCT 30%, and allogeneic 20%. 1 patient had graft failure after a previous cord transplant. Conditioning regimen consisted on busulfan (BUS), fludarabine (FLU) and cyclophosphamide (CTX) in 17 patients, and in BUS, FLU & thiothepa in 6. Three (11.5%) received total body irradiation. Graft versus host disease prophylaxis (GVHD) was tacrolimus (TCR) plus mycophenolate (MMF) starting on day þ 5, & CTX at days þ 3 & þ 4 in 23 patients (88.5%), those receiving non-myeloablative conditioning and days þ 3 & þ 5 in the remaining 3 (11.5%) who received myeloablative conditioning. MMF was dropped out at day þ 35 while TCR was maintained until day þ 80/100 if disease was in complete remission. 25 patients (96%) had fever around SC infusion day. All received antibiotics when fever was onset. In 7, fever started before infusion with an infectious origin; 18 (78%) developed fever during the first 24 hours after SC infusion. Only in one there was microbiological documentation and fever persisted until engraftment. From the remaining 17, in 14 (82%) fever resolved at day þ 4 or þ 5, while in the other 3, fever spontaneously disappeared within the first 24 hours. In all of these 17 patients, median PCR value at infusion day was 1 mg/dL (0-2) (upper limit 0,5), and it rose up to 7.5 mg/dL (2-19) at day þ 2 and went down to 4.0 mg/dL (0-9.1) at days þ 4 or þ 5. Conclusion: An important part of our haplo patients developed fever closed to stem cell infusion. Although all of them received antibiotics, the low PCR at fever onset suggest a noninfectious origin. Cytokine storm around stem cell infusion has been already reported previously, however our patients did not have other associated symptoms suggesting that several degrees of this cytokine release is possible in the haploidentical transplant setting. Biological studies are required for understanding these phenomena. References: 1. Luznik L, O’Donnell P, Ephraim JF: Post-transplantation cyclophosphamide for tolerance induction in HLA-haploidentical BMT. Semin Oncol. 2012; 39(6): 10
2. Colvin GA, Berz D, Ramanathan M, Winer ES, Fast L, Elfenbein GJ, Quesenberry PJ. Nonengraftment haploidentical cellular immunotherapy for refractory malignancies: tumor responses without chimerism. Biol Blood Marrow Transplant 2009; 15(4): 421–431. Disclosure of Interest: None declared. P194 Superior efficacy of piperacillin-tazobactam vs. ceftriaxone for the prophylaxis of neutropenic fever in lymphoma patients managed at-home after autologous stem cell transplantation conditioned with BEAM F. Fernandez Aviles1,*, M. Rovira1, C. Martı´nez1, M. Sua´rez-Lledo´1, N. Martı´nez1, A. Gaya1, C. Gallego1, A. Hernando1, S. Segura1, J. Gu¨ell1, A´. Urbano-Ispizua1 1 Haematology, HOSPITAL CLI´NIC I PROVINCIAL, Barcelona, Spain Introduction: Bacterial infections remain a major cause of morbidity in patients undergoing autologous stem cell transplantation (ASCT). In this study we analyze the efficacy of primary prophylaxis with either ceftriaxone (Ct) or piperacillin-tazobactam (P-T), in two consecutive cohorts of patients managed at-home receiving BEAM as preparative regimen. Materials (or patients) and methods: Between March 2007 and September 2014, 75 consecutive patients received an ASCT for Hodgkin and Non-Hodgkin lymphomas and all were managed at-home since day þ 1. All patients received the same conditioning regimen with BEAM (mg/m2): BCNU 300, etoposide 800, cytarabine 800 and melphalan 140. The first group of patients received prophylaxis with Ct 1 g/day i.v. (n ¼ 28; group A) and the remaining patients with P-T 4.5 g/8h i.v. (n ¼ 47; group B). Both started these antibiotics on day þ 1 and were maintained until febrile neutropenia or granulocyte count over 1x109/l. First-line therapy of neutropenic fever was P-T 4.5 g/6h i.v. in group A, and meropenem 1 g/8h i.v. in group B. The nurse visited the patient at-home once or twice daily, while the physician in the hospital visited the patient only in case of clinical complications. Indications for readmission to the hospital were: willingness of the patient or caregiver; uncontrolled nausea, vomiting or diarrhoea; mucositis requiring total parenteral nutrition or i.v. morphics; fever with focal infection or signs of severe sepsis. Results: There were no differences between the two groups with respect to the age of the patients, sex, performance status, diagnosis, stage of disease, and source of stem cells. The only difference was the median quantity of CD34 þ cell dose (x106/kg) infused: 3.8 (1.5-21) in group A and 5.9 (1.617.6) in group B; P ¼ 0.0001. After the transplant, there were no differences in the duration of neutropenia r0.5 and o0.1x109/l between groups. Whereas the incidence and intensity of oral and gastrointestinal toxicity (NCI-CTC-score) were similar in both groups, neutropenic fever occurred in 82% of patients in group A and 53% in B (P ¼ 0.02). Patients of group A required two daily visits by nurses at-home more frequently (64% vs. 30%; P ¼ 0.007) but not more physician visits in the hospital (14% vs. 16%). Bacterial infection was documented in 2 febrile episodes in group A and 5 in group B. Re-admission was required for persistent fever in 6 (21%) patients in group A, and 1 (2%) in group B (P ¼ 0.009). Conclusion: This study suggests that the use of prophylaxis with piperacillin-tazobactam reduces significantly the incidence of febrile neutropenia and the need for hospital readmission in patients receiving BEAM, optimizing both domiciliary and hospital resources. Disclosure of Interest: None declared.
P195 Mega-dose double conditioning chemotherapy of thiotepa and melphalan is feasible about renal function for the treatment of pediatric solid tumor F. Yamazaki1,*, C. Kiyotani1, Y. Shioda1, T. Osumi1, K. Terashima1, D. Tomizawa1, K. Matsumoto1 1 Children’s Cancer Center, National Center of Child Health and Development, Tokyo, Japan Introduction: Thiotepa (TT) and melphalan (LPAM) are alkylating anti-cancer drugs which are often used as conditioning regimen of high dose chemotherapy (HDC) for treating pediatric solid tumors. Hara et al. reported feasibility and efficacy of double conditioning regimen including TT and LPAM with one-week interval. Due to excessive dosage of LPAM, patients undergoing this therapy are at risk of renal insufficiency. Here, we retrospectively analyzed our institutional experience of the double-conditioning regimen for patients with solid tumors, focusing on renal toxicity and its efficacy to control tumors. Materials (or patients) and methods: Thirty-eight patients with solid tumors (neuroblastoma 18; brain tumor 13; other 7) were treated by the double-conditioning regimen with TT and LPAM followed by peripheral blood stem cells or bone marrow rescue between March 2003 and January 2010. The double conditioning regimen consisted of two cycles of TT and LPAM with a 1-week interval. TT (patients aged 42 years; 200 mg/ m2/day, o2 years; 8 mg/kg/day) and LPAM (patients aged 42 years; 70 mg/m2/day, o2 years; 1.5 mg/kg/day) were administered on days -12, -11, -5, and -4. When creatinine clearance (Ccr) waso100 ml/min/1.73m2 in patients aged 42 years, dosage was adjusted according to the following formula: given dose (mg/m2) ¼ (Ccr/100) 200 mg/m2/day (TT) or 70 mg/m2/day (LPAM). We analyzed renal function before and after HDC of the patients who had sufficient medical records and who did not received additional treatment for one year after HDC. Results: Four out of 27 patients experienced 1.5 times more elevation of serum creatinine level at one year post-HDC compared with one at pre-HDC, but only one patient had creatinine level higher than institutional normal range at one year post-HDC. The ratio of serum creatinine at one year after HDC to pre-HDC was 0.75-3.20 (median 1.19). This ratio was significantly higher in neuroblastoma patients (1.55; 95% CI, 1.18 to 1.92) than in other solid tumors (1.13; 95% CI, 1.05 to 1.21) (P ¼ 0.034). Ccr before HDC was associated with the elevation of serum creatinine level after HDC (P ¼ 0.041). We analyzed progression free survival (PFS) in 18 patients with neuroblastoma. Median follow-up time of the 9 patients who remain alive without progression is 5.3-12.0 years (median 7.8 years). PFS and overall survival (OS) at 5 years from diagnosis were 56% (95% CI, 33% to 79%) and 72% (95% CI, 52% to 93%), respectively. One patient died from transplant-related toxicity. Relapse occurred in 8 (44%) of 18 patients. Conclusion: HDC using the double conditioning regimen comprised of two cycles of TT and LPAM with a one-week interval is acceptable in renal toxicity and effective for treating high-risk neuroblastoma. Renal insufficiency after the double conditioning regimen could be predicted by the Ccr before HDC even in patients who received renal adjusted dose of TT and LPAM. For the patients with potential risk of renal insufficiency, such as patients with low Ccr, this mega-dose HDC should be used with meticulous care. References: Hara J et al. Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors. Bone Marrow Transplant. 1998; 22: 7-12 Disclosure of Interest: None declared.
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P196 Safety and feasibility of romiplostim treatment for patients with persistent thrombocytopenia after allogeneic stem cell transplantation G. Battipaglia1,2,*, A. Ruggeri1, A.-C. Mamez1, L. Drevon1, F. Malard1, A. Gomez1, E. Brissot1, R. Belhocine1, A. Vekhoff1, S. Lapusan1, F. Isnard1, O. Legrand1, T. Ledraa1, M. Labopin1, M.-T. Rubio1, M. Mohty1 1 Hoˆpital Saint-Antoine, Hoˆpital Saint-Antoine, Paris, France, 2 Federico II University of Naples, Federico II University of Naples, Naples, Italy Introduction: Prolonged thrombocytopenia after allogeneic stem-cell transplantation (SCT) is a common complication, with a negative impact on survival. It can occur as secondary failure platelet recovery (SFPR) defined as platelet (PLT) counts o20,000/mL or requiring transfusion support after achieving sustained counts Z50,000/mL without transfusions for 7 consecutive days after SCT. Furthermore, thrombocytopenia could lead to potentially lethal bleeding, thus needing the use of iterative PLT transfusions. There is a lack of effective and reliable methods to promote PLT recovery and to prevent hemorrhagic complications and PLT transfusion needs in SCT recipients. The use of the weekly subcutaneous recombinant thrombopoietin agonist romiplostim (Rm) could be an attractive option, but many concerns still exist about its safety and feasibility after SCT. Materials (or patients) and methods: We report the use of Rm in 7 thrombocytopenic patients (pts) who experienced thrombocytopenia after SCT. All pts were transplanted for high risk hematological malignancies (4 acute myeloid leukemia, 1 myelodysplastic syndrome, 1 non Hodgkin lymphoma, 1 T-cell leukemia/lymphoma) in complete remission. Results: Two pts had received a previous allo-SCT, and one an auto-SCT. Median follow up after SCT is 249 days (range 115-520). Median age at SCT was 58 (range 16-67) years. All pts received peripheral blood stem cell as stem cell source, 5 from an haploidentical donor, 1 a sibling donor and 1 a matched unrelated donor. All pts achieved neutrophil engraftment with full donor chimerism. Five pts had PLT engraftment in a median of 11 days after SCT (range 11-41),while 2 were on therapeutic PLT transfusions for concomitant hemorrhagic cystitis (HC). In the engrafted pts SFPR was observed at a median of 140 days after SCT (range 121-313) in 3 patients, while 2 pts developed HC with transfusions when PLTr50000/mL. Thrombocytopenia was of central origin in 4 pts, peripheral in 2 pts, mixed in 1 patient. Reason for starting Rm was therapeutic transfusion dependence in 4 pts with HC and SFPR in 3 pts. Median time between SFPR or therapeutic transfusion dependence and start of Rm was 23 and 37 days, respectively. Starting dose was 3 mg/kg with weekly escalation of 1 mg/kg till a maximal dose of 10 mg/kg (reached in 3 pts). Median number of injections was 7 (range 5-29). All SFPR pts achieved PLT 450000/mL without transfusion after a median of 2 injections (range 1-5), while HC and transfusion needs weren’t significantly improved by Rm. Three pts had concomitant aGVHD, 2 presented thrombotic thrombocytopenic purpura signs, 6 experienced CMV reactivation, 3 infectious complications, and 3 received concomitant myelotoxic therapies. Three non-responders pts received a CD34 þ boost due to overall poor graft function. PLT recovery was achieved in all but one, who needed to restart Rm. Conclusion: Overall Rm was well tolerated and no patient needed to discontinue drug treatment because of adverse events, making this agent a feasible therapeutic option after allo-SCT. As to its efficacy, we can speculate in SFPR that Rm improved PLT counts but in hemorrhagic patients it didn’t either reduce PLT transfusion needs or hemorrhagic syndrome. However, these preliminary results need to be confirmed in larger studies with longer follow-up to improve assessment of efficacy and safety profiles in this setting. Disclosure of Interest: None declared.
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P197 Erythrocytosis following allogeneic hematopoietic stem cell transplantation in six cases G. Pekcan1,*, E. Atilla1, P. Ataca1, P. Topcuoglu1, A. Uslu2, S. Civriz Bozdag1, M. Kurt Yuksel1, O. Arslan1, M. Ozcan1, O. Ilhan1, H. Akan1, M. Beksac1, G. Gurman1, S. K. Toprak1 1 ANKARA UNIVERSITY DEPARTMENT OF HEMATOLOGY, 2ANKARA UNIVERSITY DEPARTMENT OF INTERNAL MEDICINE, ANKARA, Turkey Introduction: Post transplant erythrocytosis (PTE) is not an expected complication after allogeneic hematopoietic stem cell transplantation (HSCT). Only Mahmood et al reported PTE in 3 aplastic anemia cases in literature (1). We present PTE in 3 acute myeloblastic leukemia (AML), 2 paroxysmal nocturnal hemoglobinuria (PNH) and 1 myelodysplastic syndrome (MDS) from 945 allogeneic HSCT in our center. Materials (or patients) and methods: Patient 1: AML, 38 years old, male, second complete remission, HLA fullmatch related donor, Cyclosporin (CsA) þ methotrexate (MTX) for graft versus host disease (GVHD) prophylaxis, myeloablative conditioning regimen. Grade 2 acute skin GVHD, liver chronic GVHD. In remission, no complications. Patient 2: PNH/bone marrow failure, 29 years old, male, HLA fullmatch related donor, CsA þ MTX for GVHD prophylaxis, reduced intensity conditioning regimen. Grade 1 acute skin GVHD. In remission, no complications. Patient 3: AML, 49 years old, male, second complete remission, HLA fullmatch related donor, CsA þ MTX for GVHD prophylaxis, myeloablative conditioning regimen. Grade 1 chronic skin, oral mucosa and liver GVHD. In remission, followed by skin, liver, eye and lung (bronchiolitis obliterans (BO)) GVHD. Patient 4: MDS, 17 years old, male, 1 antigen mismatch unrelated donor, CsA þ metilprednisolon for GVHD prophylaxis, myeloablative conditioning regimen. Grade 3 acute skin, liver and gastrointestinal GVHD. In remission, no complications. Patient 5: PNH/bone marrow failure, 26 years old, male, HLA fullmatch related donor, CsA þ mycofenolatemofetil (MMF) for GVHD prophylaxis, reduced intensity conditioning regimen. In remission, no complications. Patient 6: AML, 33 years old, male, second complete remission, HLA fullmatch related donor, CsA þ MTX for GVHD prophylaxis, myeloablative conditioning regimen. Grade 2 acute liver GVHD. In remission, no complications. Results: During follow-up, patients’ hemoglobin (gr/dL) and hematocrit (%) values are as listed: 17,9 and 52 in 1st patient at 5th year; 17,6 and 51 in 2nd patient at 3rd month; 17,5 and 54,5 in 3rd patient at 4th year ; 17,7 and 51,7 in 4th patient at 4th year, 17,5 and 55 in 5th patient at 3rd year; 17,6 and 51 in 6th patient at 6th year. None of them had either leukocytosis or thrombocytosis. All of the patients had hyperviscosity symptoms with no organomegaly. All the patient had erythropoietin levels in the upper limit of normal. No JAK2V617F mutation positivity was detected. None of the patients had pulmonary symptoms or oxygen requirement. Conclusion: Liver GVHD, BO and cyclosporine could lead to secondary erythrocytosis however there is no evidence for causality. All of our patients were male which is a risk factor for erythrocytosis similar to hypertension and smoking.Remarkably, all of the patient except one had erythrocytosis three years after allogeneic HSCT. Further surveillance of the cases will improve our knowledge over erythrocytosis. References: 1. Bone Marrow Transplantation 2011; 46: 1163– 1165. Disclosure of Interest: None declared. P198 Improving the re-vaccination programme for patients post allograft H. Leonard1,*, C. Anthias1 1 THE ROYAL MARSDEN , Sutton, United Kingdom Introduction: International consensus guidelines recommend commencing re-vaccinations at 3-6 months post-transplant,
even in patients on immune suppression. We carried out an audit to determine compliance with international revaccination guidelines at the Royal Marsden Hospital. Materials (or patients) and methods: Electronic patient records of 100 consecutive patients who received allogeneic transplants between Nov 2012 and March 2014 were retrospectively examined. Information collected included whether revaccinations had been commenced at 21st October 2014, number of months post transplant that revaccinations were commenced, and whether revaccinations schedules had been sent to general practitioners. Patient clinical status (remission, relapse and presence or absence of GVHD) was also recorded. Results: 22 patients had died or relapsed within 6 months of allograft and were therefore excluded from further analyses. Of the remaining 78 patients, 47 (60%) had commenced the revaccination programme. 7 (14%) of these were international patients who had received vaccinations in their home country. Of the 47 that had received vaccinations, practice was inconsistent with respect to timing of when the vaccinations commenced. The reasons for failure to initiate revaccinations in the remaining 31 patients (40%) were examined. These patients tended to be more complex, with recurrent hospital admissions. There was an inconsistent approach by the medical team to commencing vaccinations for patients with chronic GVH on immunosuppression, with some team members recommending delaying the start of the vaccination programme. Documentation of both discussion of the vaccination programme with patients and GPs and of when/if vaccinations had commenced was also variable and difficult to identify from patient records. Conclusion: This audit raises several issues with our approach to revaccination. There is currently no formal method of ensuring and documenting that vaccinations for post transplant patients are carried out. We have therefore proposed the following changes: Prior to transplantation, all patients will have a clinic appointment set up for 6 months post transplant with the post transplant CNS. At this appointment the rationale for revaccinations will be discussed with patients, a standard letter with the revaccination schedule will be posted to the GP with a request to arrange vaccinations. This will be then clearly documented. The post transplant CNS will follow up all patients by telephone 1 month after this 6-month appointment to ensure vaccinations have commenced and to answer any queries. For the international patients a reminder will be sent by e-mail to the referring consultant with a vaccination schedule. Lastly, to address the varied practice by the medical team in recommending timing of revaccination, the vaccination SOP will be reviewed; contraindications to vaccination agreed with the transplant lead and subsequent training of the medical team will be arranged, ensuring consistency in practice. We will re audit in 6 months to evaluate the effectiveness of these changes. Disclosure of Interest: None declared. P199 Successful treatment with dexamethasone palmitate for patients with post-transplant cytokine storm syndrome H. Sakaguchi1,*, K. Matsumoto2, K. Narita1, M. Hamada1, S. Kataoka1, N. Yoshida1, M. Ito3, K. Kato1 1 Department of Hematology and Oncology, Children’s Medical Center, Japanese Red Cross Nagoya First Hospital, Nagoya, 2 Children’s Cancer Center, National Center for Child Health and Development, Tokyo, 3Department of Pathology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan Introduction: Post-transplant cytokine storm syndrome (PTCSS) is an early complication after hematopoietic stem cell transplantation (HSCT) caused by a massive release of proinflammatory cytokines, macrophage colony-stimulating
factor, erythropoietin, products of degranulation and oxidative metabolism, in which activated macrophage play a key role, and consists of hyper acute graft versus host disease (GVHD), pre-engraftment immune reaction, engraftment syndrome, idiopathic pneumonia syndrome, and hemophagocytosis. Standard therapy for PTCSS is not established yet. Liposomeincorporated dexamethasone, dexamethasone palmitate (DP), is known to suppress activated macrophages by readily taken up via phagocytosis and strongly retained in the cytoplasm. In this retrospective study, we aimed to identify the incidence of PTCSS, evaluate the efficacy, feasibility, and clinical impact of DP treatment for PTCSS in HSCT. Materials (or patients) and methods: We reviewed retrospectively 139 consecutive HSCT (autograft, n ¼ 17; allograft, n ¼ 122) for children (median age at HSCT 8.6 years; range, 0.5–18.2) with hematological malignancies (n ¼ 93), solid tumors (n ¼ 15), and non-malignant disorders (n ¼ 31), performed in our institution between April 2006 and September 2014. Allografts were donated from family members (n ¼ 35), unrelated volunteer donors (n ¼ 39), or cord blood (n ¼ 48). Among patients with malignant disease, 44 patients received HSCT at high risked stage; third remission or non-remission. The diagnosis of PTCSS consists of non-infectious cause of fever more than 3days with skin rash, pulmonary infiltration, or bone marrow aspiration findings; proliferation of activated macrophage or phagocytosis at peri-engraftment period. DP treatment (2.5 mg/m2/day i.v. for 3 consecutive days) indicated for all patients diagnosed as PTCSS, and on demand DP treatment for refractory cases. Results: Forty-five patients (33%) were diagnosed as PTCSS and treated with DP for 3 days at median day 14 (range, 7–22) after HSCT. PTCSS was resolved in 39 of 45 cases; 6 patients needed additional methylprednisolone treatment. Of 45 patients with PTCSS, graft failure was concomitantly observed in 3 patients donated from sibling (n ¼ 1) and unrelated cord blood (n ¼ 2). The interval from PTCSS to engraftment: median, 5 days (range, 0–27 days). The incidence of acute GVHD for patients with PTCSS was as comparable as that of patients without PTCSS (12% versus 12%, P ¼ 0.99), however, the incidence of chronic GVHD of the former was higher than that of the latter (18% versus 8% in 5 years, P ¼ 0.11). The incidence of transplant related mortality for patients with PTCSS was as comparable as that of patients without PTCSS, on the other hand, relapse rate in malignant disease of the former was lower than that of the latter (32% versus 46% in 5 years, P ¼ 0.06). Finally, the overall survival of patients with PTCSS was superior to that of patients without PTCSS (76% versus 55% in 5 years, P ¼ 0.10), even in high risked patients’ cohort (57% versus 36% in 5 years, P ¼ 0.10). Conclusion: Our results suggested that DP could control the PTCSS, at the same time, could save the immune reaction which was important for graft versus leukemia effect; DP could control acute allo-reaction effectively, but not for later immune reaction. Prospective multi-institutional study is warranted to confirm our findings. Disclosure of Interest: None declared. P200 Evaluation of The Long-Term Effects of Hematopoietic Stem Cell Transplantation on Oral and Dental Health of Children After Transplantation for Benign and Malign Hematologic Diseases ¨ . M. Akgu¨n1, C. Yıldırım1, I. Eker2,*, O. Gu¨rsel2, O. Babacan2, O F. Bas¸ak1, A. E. Ku¨rekc¸i2 1 Pediatric Dentistry, 2Pediatric Hematology, Gulhane Military Medical Faculty, Ankara, Turkey Introduction: Because of the severe immunosuppression in chilren after hematopoietic stem cell transplantation (HSCT), any problem in the oral cavity may become life-threatening and may increase the complications, treatment costs and duration of hospitalization. In this study, pre-transplant and post-transplant dental health evaluation results of pediatric
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patients with benign and malign hematological diseases, who underwent HSCT at Gulhane Military Medical Faculty, Pediatric Hematopoietic Stem Cell Transplantation Center are presented. Materials (or patients) and methods: DMFT index (decayed, missing, filled teeth index), plaque index (PI), gingival index (GI) and bleeding on probing (BOP) scores, which were obtained from oral and dental health assessment of 37 HSCT patients were analyzed retrospectively. In our center, HSCT patients are evaluated for oral and dental health at pretransplant period and on post-transplant 100th day and 6th, 12th and 24 th months. Pedodontist, who made the evaluation of the patient at pretransplantation period, continued the all other evaluations on the time points above. Assessment was made with the reflector light by using mirror and periodontal probe. Results: The median age of the patients was 10 (1-18) years, 10 were female and 27 were male. Oral and dental health assessments of all patients were completed on the pretransplant period and on post-transplant 100 th day. But on post-transplant 6th month 94,6%, on 12 th month 81% and on 24 month only 33% of the patients’ oral and dental health assessments could be completed. The remaining of the asessments could not be done because of the incompliance of the other patients. The patients’ pre-transplant and posttransplant 100 th day and 6 th month oral and dental health evaluation results of DMFT index GI, PI and BOP were very similar. But patients’ decay teeth number, formation of plaques, gingival inflammation and bleeding scores were increasing, especially starting at 12th month evaluation and become more prominent at 24th. In other words, patients’ adherence to oral and dental health care were decreasing and proportional with these, oral and dental problems were increasing especially starting at 12th month after HSCT and become more prominent at 24th. Conclusion: Our findings showed an increase in oral, periodontal and gingival problems after HSCT, especially in the longterm period, which was consistent with other literatures on the issue. This is because of the decreasing importance, given to oral health at post-HSCT period. It is very important to take measures to ensure regular assesments of patients’ pedodontic monitoring and treatment of the problems in post transplant long term period, like as in the pre-transplant and post-transplant early periods. Preparation of guidelines and patient education programs about oral and dental health in HSCT, together by pedodontists and pediatricians; and regularly pedodontic evaluations on the early and late postHSCT periods are of extremely important for the success of transplantations. Disclosure of Interest: None declared.
[P200]
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P201 Comprehensive Pulmonary Rehabilitation in patients with pulmonary chronic graft versus host disease I. C. Song1,*, S. J. Jee2, S. W. Baek1, Y. S. Choi1, H. W. Ryu1, J. Y. Moon1, H. J. Lee1, H. J. Yun1, S. Kim1, K. H. Cho2, D. Y. Jo1 1 Internal Medicine, 2Rehabilitation Medicine, Chungnam National University Hospital, Daejeon, Korea, Republic Of Introduction: Pulmonary chronic graft versus host disease (cGVHD) is a serious late complication of allogeneic hematopoietic stem cell transplantation (HSCT), which hampers quality of life and increases mortality of survivors. Medical treatments have limited efficacy at improving pulmonary function. We investigated the therapeutic effects of comprehensive pulmonary rehabilitation (CPR) in patients with deteriorated lung function due to pulmonary cGVHD. Materials (or patients) and methods: We retrospectively analyzed the medical records of patients who had pulmonary cGVHD and participated in the CPR program, which consisted of aerobic exercise, inspiratory muscle strengthening, accessory respiratory muscle strengthening and mechanically assisted coughing. The subjects were planned to undergo maximal cardiopulmonary exercise test and a body composition test with bioelectrical impedance analysis at baseline and 3 months. Exercise testing parameters, skeletal muscle mass of lower extremities and body mass index (BMI) were analyzed. Results: Nine patients were assessed (two males, seven females; median age 40 (range 24–64) years). Donor lymphocyte infusion was performed in two patients. All patients had skin, mucosal and visceral manifestations, in addition to pulmonary cGVHD. When starting CPR, the median FEV1 was 25% (range 18% – 51%). Eight patients received concomitant corticosteroid and three received imatinib. At a median followup of 11 (range 2–16) months, the patients had participated in the CPR program for a median of 40.5 (range 10–148) days. Eight patients (88.9%) had completed more than 20 sessions of the CPR program. The rehabilitation programs were readily tolerable and had no adverse effects. The median change in the maximum oxygen consumption (VO2max) was 28.5% (range –31.8% to 60.1%) and the muscle mass of the lower extremities changed by 3.0% (range –0.2% to 24.1%). All four patients who had an increased muscle mass in the lower extremities had an increase in VO2max. There were no marked changes in BMI. Conclusion: Our comprehensive pulmonary rehabilitation program for patients with pulmonary cGVHD with limited lung function was feasible and improved the exercise capacity in a population of patients. Disclosure of Interest: None declared.
P202 Prospective, randomized trial of physical function in patients before and after haematopoietic stem cell transplantation H. Schumacher1, S. Stru¨we1, N. Greger1, P. Blaschke1, S. Freitag1, C. Junghanss1, I. Hilgendorf1,2,* 1 University Hospital Rostock, Rostock, 2Jena University Hospital, Jena, Germany Introduction: Physical and sports therapy during stationary treatment and aftercare operations, in patients receiving hematopoietic stem cell transplantation (HSCT), is becoming more and more important. Positive effects are known for strain dependent physical practice after HSCT. Nevertheless, there is lack of capacities and problem orientated therapies. Materials (or patients) and methods: Methods: Here we report on a prospective, randomized study comparing a multimedia sensor-based practice with a classical physiotherapeutic treatment in 30 HSCT recipients. Both groups performed the exercises under the supervision of a physical therapist. Physical function was evaluated at the date of hospital admission (T1), 14 days after HSCT (T2) and 30 days after HSCT (T3). The conditional status was assessed by Treadmill and a two minute walk test. Furthermore, a grip strength test was performed with a hydraulic hand dynamo meter and balance was evaluated using the Berg Balance Scale. Results: Patients after HSCT for hematologic malignancies were randomized into the control group receiving physiotherapeutic treatment (n ¼ 17) or the experimental group with exercising on the Nintendo Wii (n ¼ 13). The median age of patients in the control group was 60 (46–69) years and 55 (22– 69) years in the experimental group. Treadmill intervention showed a median performance of 100 W (25–125) (T1), 75 W (25–100) (T2) and 25 W (25 - 75) (T3) in the control group and 100 W (75 - 125), 50 W (50 - 125), 50 W (50 - 125) in the Nintendo Wii-group, respectively. In the two minute walk test the median distances in the control group were 166 m (105 211) (T1), 154 m (73 - 208) (T2), 147 m (105 - 204) (T3), while the experimental group walked an average distance of 166 m (134 - 210), 143 m (60 - 196) and 139 m (124 - 156). The grip strength test resulted in mean values of 35 kg (13 - 51) (T1), 34 kg (11 - 53) (T2), 27 kg (11 - 50) (T3) in the control group and 37 kg (19 - 61), 34 kg (13 - 60), 32 kg (21 - 53) in the experimental group, respectively. The control group scored a mean value of 55 (52 - 56) (T1), 53 (34 - 56) (T2), 53 (52 - 56) (T3) in Berg Balance Scale (max. 56) compared with 56 (54 56), 54 (47 - 56), 56 (55 - 56) in the experimental arm. Conclusion: Discussion: The functional status of patients decreased after HSCT in both groups and impairment remained at least until one month (T3) after HSCT. However, compared to classical physiotherapeutic treatment the group with sensor-based practice showed a slightly lower endurance, while none differences were observed in balance testing. Exergaming is feasible as well as enjoyable in the stationary setting of HSCT and may be used in addition to physiotherapeutic treatment. Disclosure of Interest: None declared. P203 Reduced Incidence of CMV Reactivation with Prophylactic Use of Ganciclovir in Allogeneic MUD Transplants Using Alemtuzumab as Part of Conditioning J. Vogelaar1,*, V. Rodriguez1, A. Nageswara Rao 1, J. Gourde1, S. Khan1 1 Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, United States Introduction: Cytomegalovirus (CMV) disease is a cause for morbidity and mortality in the allogeneic transplant setting. Ganciclovir is an effective treatment for CMV infection. However, given concern for myelosuppression and other side effects, there continues to be a debate between prophylactic
versus preemptive therapy in allogeneic transplant patients. At our institution, alemtuzumab (Campath) is part of the conditioning regimen for matched unrelated donor (MUD) transplants. Due to concern of increased risk of early and late CMV disease secondary to alemtuzumab-induced T-cell depletion, it is our standard practice to use ganciclovir prophylactically in these patients. In contrast, in patients undergoing matched related donor (MRD) transplantation, we use preemptive ganciclovir therapy upon CMV reactivation. Materials (or patients) and methods: We reviewed data in our allogeneic transplant patients (MUD transplants: n ¼ 42; MRD transplants: n ¼ 37) from 2003-2014. All patients with matched unrelated donors who were CMV seropositive, or CMV seronegative (-ve) with a seropositive ( þ ve) donor, were started on ganciclovir once their absolute neutrophil count (ANC) was greater than 1000 and continued until day þ 100. Patients with matched related donors and similar CMV status received preemptive ganciclovir upon CMV reactivation. Weekly CMV PCR was obtained for close followup. Results: Of the 42 patients with unrelated donors who received Campath, 66.6 % were at risk for reactivation. They included recipients CMV þ ve (n ¼ 14), recipients CMV-ve and donors CMV þ ve (n ¼ 6), recipients and donors CMV þ ve (n ¼ 8). Only four (9.5%) had CMV reactivation (days þ 9 through þ 60). Only one patient was on ganciclovir prophylaxis at the time of reactivation (day þ 60, started ganciclovir day þ 24). One patient was on ganciclovir prophylaxis for only two days prior to reactivation. The two remaining patients were not on ganciclovir prophylaxis due to an ANC of less than 1000. Among patients receiving prophylaxis, only one patient had discontinuation of ganciclovir due to myelosuppression. However, this patient’s cause of myelosuppression remains disputed as the patient had also been on cidofovir due to urine and plasma BK virus activity. This patient was started on ganciclovir on day þ 22 and both cidofovir and ganciclovir were discontinued on day þ 40 due to continued myelosuppression. Of the 37 patients who underwent MRD transplants and were followed closely with CMV PCR, two had CMV reactivation (5.6%), at day þ 26 and day þ 31. Both patients were started on ganciclovir. Unfortunately, one patient died seven days later due to veno-occlusive disease, multi-organ failure, and CMV viremia. The other patient completed a 14 day course of twice daily ganciclovir with resolution of CMV infection. Conclusion: Based on our review, ganciclovir prophylaxis reduced the incidence of CMV reactivation despite using Campath in MUD transplants. No patient had active CMV disease and there were no deaths due to CMV infection. The incidence of reactivation in these patients is comparable to our matched sibling transplants that do not receive Campath, and only receive preemptive therapy on CMV reactivation. Disclosure of Interest: None declared. P204 Poor outcome following ciclosporin-related neurotoxicity in paediatric allogeneic haematopoietic stem cell transplantation K. Straathof1,*, P. Anoop1, Z. Allwood1, J. Silva1, O. Nikolajeva1, R. Chiesa1, P. Veys1, P. Amrolia1, K. Rao1 1 Department of Blood and Marrow Transplantation, Great Ormond Street Hospital, London, United Kingdom Introduction: Ciclosporin A (CSA)-related neurotoxicity poses a significant clinical problem in allogeneic haematopoietic stem cell transplantation (HSCT) as it necessitates withdrawal of one of the most effective agents in the prevention and treatment of graft-versus-host disease (GVHD). The spectrum of CSA-related neurological complications has been well characterised in several case series of paediatric HSCT recipients, but long term follow up data are lacking.
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P205 Abstract Withdrawn
P206 Intravesical Sodium Hyaluronate and Sodium Chondroitin Sulfate As A Prophylaxis For Hemorrhagic Cystitis In Pediatric Hematopoietic Stem Cell Transplantation Patients K. Yalc¸ın1,*, F. Tayfun2, N. Eker1, S. Kaya1, S. Yu¨cel3, E. Gu¨ler1, A. Ku¨pesiz1 1 Pediatric Hematopoietic Stem Cell Transplantation Unit, Akdeniz University School of Medicine, Antalya, 2Pediatric HematologyOncology, Diyarbakır Children Hospital, Diyarbakır, 3Pediatric Urology, Acıbadem Atakent Hospital, ˙Istanbul, Turkey
Here, we report the outcome of a cohort of paediatric HSCT recipients who experience CSA-related neurotoxicity. Materials (or patients) and methods: From a cohort of 569 consecutive paediatric allogeneic HSCT recipients between 1st January 2003 and 31st December 2011 at our institution we identified those children who developed neurological complications at any time after start of conditioning whilst receiving CSA. Children with obvious alternate causes for neurotoxicity such as infectious meningoencephalitis, prior history of seizure disorder, structural brain lesions, CNS relapse of underlying disease, busulfan-induced seizures and intracranial haemorrhage were excluded. Results: During this 9-year period, 26 of 569 (4.6%) children developed CSA-related neurotoxicity. The median time from HSCT to neurotoxicity was 47.5 days (range day -1 to þ 545). Clinical symptoms included seizures (n ¼ 22), headaches (n ¼ 8), visual disturbances (n ¼ 3) and reduced sensorium (n ¼ 3). 24/26 patients had cranial imaging with MRI or CT or both; 19 had classical findings of PRES, 2 showed non-specific changes consistent with encephalopathy and 3 had normal appearances of brain. CSA and MMF was the most commonly employed GVHD prophylaxis (n ¼ 19), followed by CSA and MTX (n ¼ 4) and CSA alone (n ¼ 3). At the time of neurotoxicity, 18 had prior acute GVHD (8 grade I/II, 5 grade III, 5 grade IV). Five patients had chronic GVHD prior to developing neurotoxicity. Initial management consisted of discontinuation of CSA and supportive care. After resolution of neurological symptoms, 18 patients were re-challenged with CSA and/or FK506. In 8/18 this resulted in recurrence of neurotoxicity symptoms. Additional GVHD prophylaxis/treatment consisted of continuation or (re) introduction of MMF (n ¼ 15), corticosteroids (n ¼ 25), sirolimus (n ¼ 4), immunomodulatory antibodies (n ¼ 12), methotrexate (n ¼ 1), anti-thymocyte globulin (n ¼ 1) and/or mesenchymal stem cells (n ¼ 3). Prognosis in this cohort was poor: 14/26 (54%) died due to severe refractory GVHD and its associated complications. In contrast, during the same study period in our institution, the overall survival at 5 years following allogeneic HCST was 67.7% for haematological malignancy, 82.1% for immunodeficiency and 87.2% for metabolic disorders. Of those surviving 5/12 (42%) live with extensive chronic GVHD. Prognosis was poorest in those who did not tolerate re-introduction of a calcineurin inhibitor (1/8 (12%) survival). Conclusion: Our results suggest that the development of CSArelated neurotoxicity is a poor prognostic factor associated with high mortality and morbidity due to severe GVHD. Survival is enhanced in patients who tolerate the re-introduction of an immunosuppressive agent. Disclosure of Interest: None declared.
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Introduction: Hemorrhagic cystitis (HC) is a relatively common and potentially severe complication observed after hematopoietic stem cell transplantation (HSCT). Many prospective and retrospective studies addressed the etiology and mechanisms of HC in HSCT and a variety of risk factors have been reported in pediatric population but especially cyclophosphamide (Cy) was identified as a cause of HC several decades ago. According to literature, none of the therapeutic modalities which have been tested in the treatment of HC, proved to be effective to date. Also, hyaluronic acid and chondroitin sulfate have been used in the treatment of HC but in this study, we used intravesical hyaluronic acid and chondroitin sulfate as a prophylaxis for HC. Materials (or patients) and methods: Eighty-six children affected by malignant or non-malignant hematological disorders undergoing HSCT from January 2012 to September 2014, in Akdeniz University Pediatric Hematopoetic Stem Cell Transplantation Unit, are enrolled in this study. We focused on 38 children who have Cy in the conditioning regimen. 38 patients had been divided in two groups, 17 patients received sodium hyaluronate and sodium chondroitin sulfate (Ialurils) as a prophylaxis for hemorrhagic cystitis and 21 patients had no prophylaxis. We analyzed the possible risk factors for post-hematopoietic stem cell transplantation hemorrhagic cystitis and focused on the effects of intravesical sodium hyaluronate and sodium chondroitin sulfate prophylaxis. Results: The median age of 38 children who have Cy in the conditioning regimen was 8,2 years old. In all, 24 patients (%63) developed HC a median 23,8 days after HSCT. 16 patients had unrelated, 4 patients had haploidentic HSCT and the incidence of HC was not different according to the type of donor. The incidence of HC was significantly lower in prophylaxis group than non-prophylaxis patients (P ¼ 0.002) In all prophylaxis group only 2 patients had HC (%11), but in non-prophylaxis group 13 patients had HC (%61). Also HC degree was lower in prophylaxis group. Median degree was 1 in the prophylaxis group while it was 3 in non-prophylaxis group. Conclusion: HC is a common cause of morbidity after HSCT and may give rise to serious complications. The reported incidence varies from o10% to more than 70%. High-dose Cy is a well-known risk factor associated with the development of HC. In this study, we administrate intravesical sodium hyaluronate and sodium chondroitin sulfate as a prophylaxis for the patients who have Cy in the conditioning regimen. Lower HC and HC degree were found in patients on prophylaxis group. We consider that the results of our study are promising and the efficacy of sodium hyaluronate and sodium chondroitin sulfate in the prophylaxis of post-transplant HC should be further investigated in larger cohorts of patients. Disclosure of Interest: None declared.
Conditioning regimen I P207 Therapeutic drug monitoring (TDM) of iv busulfan (Bu) in adults undergoing allogeneic hematopoietic stem-cell transplantation (allo-HSCT) to better control the targeted exposure after a 3-day Bu-based myeloablative conditioning (MAC) A. AMIN1,*, P. BOURGET1, A. GOMEZ2, F. ADER1, A.-C. JOLY3, A.-C. MAMEZ2, E. BRISSOT2, M. T. BAYLATRY3, M. MOHTY2, M.-T. RUBIO2 1 Department of Clinical Pharmacy, University Hospital NeckerEnfants Malades, 2Department of Hematology, 3Department of Clinical Pharmacy, University Hospital Saint-Antoine, Paris, France, Paris, France Introduction: High-dose IV Bu is the backbone of different preparative regimens prior to allo-HSCT. However, Bu has a narrow therapeutic window, exhibits large pharmacokinetic (PK) variability, and its clearance is non-linearly related to body weight (BW). To optimize Bu duration/intensity exposure, we studied the contribution of reiterated TDM in patients (Pts) receiving 3-day Bu-based MAC (experimental cohort [EC]). We compared the experimental vs expected values of Bu exposure after performing 3 daily PK. Selected parameters of the EC were compared to a control cohort (CC) of non-monitored Pts. Materials (or patients) and methods: In this observational study (07/2013-10/2014), Pts with hematologic malignancies in CR receiving a 3-day Bu-based MAC in combination with Flu±Thiotepa were prospectively included. The regular Bu dosing schedule of 3 daily 3-hr infusions (72-hr exposure) was used. Bu PK was performed using non-compartmental method with area under the concentration-time curve (AUC) calculation from the 1st (PK1) to the 3rd (PK3) dose; 12 blood samples (4/PK) were assayed by LC-MS2. In the EC (n ¼ 20), dose 1 was calculated from BW (3.2 mg/kg) and corrected in 8 obese Pts using ideal BW; doses 2 and 3 were adjusted based on PK1 (TDM1) and PK2 (TDM2), respectively, with an expected AUC of 4,400-6,000 mM*min/day. AUC values calculated after each dose (AUC1 þ 2 þ 3) were compared to the expected AUC and to the theoretical AUC sum without TDM (AUC1x3) using a Wilcoxon signed-rank test. In the CC (n ¼ 20, 03/2009 09/2014), Pts received 3.2 mg/kg/day of Bu x 3 d. Parameters from the EC and CC were compared using w2, Student t, and log-rank tests. Results: EC and CC Pts had similar sex ratio, median age (56 vs 60 yrs, P ¼ 0.7), BW (68 vs 72 kg, P ¼ 0.9), follow up (263 vs 269 days, P ¼ 0.2), and were transplanted for high-risk diseases (16/ 20 vs 17/20, P ¼ 1.0). In the EC: (a) reiterated TDM showed no statistical difference between the expected AUC [13,20018,000 mM*min] vs AUC1 þ 2 þ 3 (P ¼ 0.3053), (b) AUC1 þ 2 þ 3 and total dose of Bu were significantly different from the AUC1x3 and Dose1x3 without TDM (P ¼ 0.0094 and P ¼ 0.02829, respectively), (c) EC Pts received higher Bu doses compared to CC Pts (mean total dose 717 vs 666 mg [P ¼ 0.028] and 10.2 vs 9.2 mg/kg [P ¼ 0.0014], respectively). Median time to engrafment for neutrophil and platelets were 14 vs 17 days (P ¼ 0.1) and 14 vs 12 days (P ¼ 0.6) in EC and CC Pts, respectively. Grade (Gr) 2-4 liver toxicities were observed in 20 vs 5% of EC and CC Pts (P ¼ 0.3), and renal Gr 2-4 toxicity in 25 vs 10%, respectively (P ¼ 0.4). One death in the EC was attributed to toxicity (SOS after a 2nd allo-HSCT). Grade 2-4 acute GVHD at day 100 was 30% in both groups (P ¼ 0.8). Chronic GVHD at 1 yr in the EC and CC were 31% vs 17% (P ¼ 0.4), while NRM were 20.1 and 5.8% (P ¼ 0.4), respectively. 1-yr relapse incidence, DFS, and OS were 29, 68, and 82.5% in the EC vs 65, 31, and 42.5% in the CC (P ¼ 0.1, 0.13, and 0.18). Conclusion: Reiterated TDM of Bu in a 3-day Bu-based MAC for allo-HSCT is relevant to achieve optimal Bu exposure. It led to administer higher total Bu dose without significant increased toxicity. Disclosure of Interest: A. AMIN: None declared, P. BOURGET: None declared, A. GOMEZ: None declared, F. ADER: None
declared, A.-C. JOLY: None declared, A.-C. MAMEZ: None declared, E. BRISSOT: None declared, M. T. BAYLATRY: None declared, M. MOHTY Funding from: Pierre-Fabre (lecture honoraria and research support)., M.-T. RUBIO: None declared. P208 Allogeneic bone marrow transplantation in patients with hematological malignancy using busulfan, thiotepa, fludarabine as conditioning regimen A. M. Raiola1, A. Dominietto1, R. Varaldo1, F. Gualandi1, C. Di Grazia1, T. Lamparelli1, S. Bregante1, M. T. van Lint1, F. Galaverna1, A. Ghiso1, C. Marani1, A. Bacigalupo1,* 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: In 2012 Sanz and coworkers reported a conditioning regimen containing thiotepa (5mg/kgx2), intravenous busulfan (3,2 mg/kgx3), and fludarabine (50 mg/ m^2x3) (TBF) in patients receiving transplants from single unit cord blood (Sanz et al BMT 2012). Materials (or patients) and methods: We have used the same conditioning regimen for patients with hematologic malignancies : in the present study we analyzed the outcome of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Patients: This is a unicentric retrospective analysis of 97 patients undergoing an allogeneic bone marrow transplantat for AML (n.50) or MDS (n ¼ 47) following the TBF regimen . Patients over the age of 60 or with comorbidities were given TBF with 2 days of bulsulfan (BU2) instead of the full 3 days (BU3). Fifty one patients were conditioning with BU3 and 46 with BU2 regimen. More patients in the BU2 group had advanced disease (57% vs 35%, P ¼ 0.03). BU2 patients were older: median age 50 years (range 18-65) versus 62 years (range 44 – 74) (P ¼ 0,001). Donor type was equally distributed between the two groups with a prevalence for transplant from haploidentical donor (70%). Results: The cumulative incidence of rejection was 0% for BU3 and 8% for BU2 (P ¼ 0.04 Fisher test). The CI for II-IV grade of acute GVHD and of moderate- severe chronic GVHD was 17% and 15% with no difference between the two groups. The 1-year CI of transplant-related mortality was 14% (13% for BU3 and 15% for BU2; P ¼ ns). The CI of relapse related death (RRD) was 12% (BU3) and 15% (BU2) (P ¼ 0.7). After a median followup of 328 days (100-1515) 39 of 51 patients for BU3 (77%) and 33 of 46 (70%) patients for BU2, are surviving. The 5-year actuarial overall survival for patient in CR1/CR2 and advanced disease patients was 83% and 45% in the BU3 group , versus 81% and 36% in BU2 group respectively (P ¼ ns). Also DFS was comparable. Conclusion: This analysis shows that thiotepa-busulfanfludara is an effective regimen for patients with AML and MDS, with encouraging outcomes. Patients receiving the BU2 regimen had outcome in terms of TRM, RRD, and OS , comparable to BU3, despite older age and more advanced disease. A TRM of 15% for patients with a median age of 62 is noteworthy. Disclosure of Interest: None declared. P209 TEPADINAs (thiotepa) plus Busulphan i.v as conditioning regimen before Autologous Stem Cells Transplantation (ASCT) in patients with acute myeloid leukaemia (AML) A. M. Carella1,*, E. Merla1, M. M. Greco1, G. Pisapia2, G. Palazzo2, N. Cascavilla1 1 Hematology, IRCCS CASA SOLLIEVO DELLA SOFFERENZA HOSPITAL, San Giovanni Rotondo, 2Hematology, Taranto, Taranto, Italy Introduction: From June 2010, 24 acute myeloid leukemia (AML) patients (pts) in first Complete Remission (CR) were
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included in a prospective multicenter trial to evaluate the efficacy and toxicity of a new preparative conditioning regimen based on TEPADINA and intravenous (iv) Busulphan for autologous stem cell transplantation (ASCT). Materials (or patients) and methods: This new conditioning regimen consisted of Tepadina 10 mg/kg (5 mg twice every 12 hours) with an intravenous infusion of 4 hours on day -7 and Busulfan (i.v.) 3.2 mg/kg given intravenously on day -6 to-3 (cumulative dosage: 12,8 mg/kg). Patients older than 65 years (n ¼ 6) received Tepadine at the dose of 8 mg/kg (4 mg twice every 12 hour) on day -6 and Busulfan on day-5 to 3 at cumulative dosage of 9 mg/kg. Most patients were female (n ¼ 15, 63%), and the median age at transplantation was 58 years. Results: All patients received peripheral blood stem cells (PBSC), the median CD34 þ cells doses were 5 x106/kg (range: 2,6-9,0). From day þ 2 all pts received granulocyte colony stimulating factor every day until the neutrophil count was at least 41000x109/L for 3 consecutive days. Twenty pts showed normal kariotype, the other 4 pts showed unfavourable cytogenetics. Two pts had mutation of NPM1 gene, none pts had mutation of FLT3 gene. All pts achieved leukocyte and platelets recovery after a median time of 9 (range: 9-14) and 12 (range: 9-30) days, respectively. Grade III-IV mucositis occurred in 6 (25%) pts. Only one patient died due to transplant-related causes after transplantation; in particular this patient died at day þ 26 because of a gram-negative sepsis worsened by kidney and liver failure. The relapse rate was 33% (8 pts relapsed between 3 and 31 months after autoSCT) and the cumulative treatment-related mortality was less than 1%. The average number of days of hospital stay was 20 (range: 19-35). Febrile neutropenia occurred in 14 patients (59%) with a median duration of 2 days (range: 0-10); out of these patients,11 experienced a grade I-II fever of unknown origin (FUO). Two patients developed a fever caused by grampositive infection (n ¼ 1), while 1 had gram-negative fever. Only 1 patient developed a cytomegalovirus infection. To date 15 pts (62%) are in CCR with a median follow-up of 25 months (range 3-49). Two patients (9%) died of progressive disease between 16 and 53 months after autografting. Twenty-one (87%) pts are alive. Conclusion: In conclusion our experience with this new conditioning regimen has shown a significant efficacy and a low toxicity profile. Only minor grades of treatment related nausea and vomiting were observed, also gastro intestinal and liver dysfunction were minor events in our study. Disclosure of Interest: None declared. P210 Comparison of two fludarabine-based reduced-intensity conditioning regimens in elderly or infirm patients with myeloid malignancies treated with allogeneic hematopoietic stem cell transplantation A. Czyz1,*, B. Nasilowska-Adamska2, A. Lojko-Dankowska1, M. Matuszak1, E. Bembnista1, M. Kozlowska-Skrzypczak1, K. Halaburda2, K. Warzocha2, M. Komarnicki1 1 Poznan University of Medical Sciences, Poznan, 2Institute of Hematology and Blood Transfusion, Warsaw, Poland Introduction: An optimal pre-transplant reduced-intensity conditioning (RIC) regimen for elderly or infirm patients with myeloid malignancies has not been established. Materials (or patients) and methods: We compared fludarabine (Flu) plus intravenous busulfan (Bu) regimen with Flu plus melphalan (Mel) therapy to determine long-term outcomes after allogeneic hematopoietic stem cell transplantation (alloSCT) in myeloid malignancies. A total of 90 patients (median age 59 years, range 19-70) with acute myeloid leukemia (n ¼ 61), myelodysplastic syndrome (n ¼ 6), chronic myeloid leukemia (n ¼ 17) and other myeloid neoplasms (n ¼ 6) were given peripheral blood stem cells from HLA– identical siblings (n ¼ 22) or matched unrelated donor (n ¼ 68) after conditioning with Flu combined with a total Bu dose
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ranging from 6.4 to 8.0 mg/kg (n ¼ 30) or Flu combined with a total melphalan dose of 140 mg/m2 (n ¼ 60) between 2009 and 2014. Allotransplantation was performed in advanced phase of the disease (beyond first complete remission in AML or first chronic phase in CML) in 33% of patients. Patients younger than 50 years old (n ¼ 24) were qualified to treatment with RIC regimen due to clinically significant comorbidities or prior alloSCT. Graft versus host disease (GvHD) prophylaxis consisted of CSA and methotrexate in standard dose. In addition, ATG was used in alloSCT from unrelated donors. Patients conditioned with FluBu were older (median age 59 years, range 49-70) than those treated with FluMel (median age 51 years, range 19-59) (Po0.001). The other characteristics such as type of the disease, advanced phase of the disease or type of the donor were similar between the groups. Results: Engraftment was observed in all but four patients who died within 28 days of alloHSCT from infection (n ¼ 3) or multiorgan failure (n ¼ 1). The median time to neutrophil count (0.5 G/L) and platelet count (20 G/L) recovery was shorter after FluBu in comparison with FluMel regimen (18 days vs 22 days; P ¼ 0.045 and 14 days vs 23 days; Po 0.001). Acute GvHD grade II-IV occurs in 17% of FluBu and 33% of FluMel patients (P ¼ 0.13). The respective frequency of moderate/severe chronic GvHD was 12% and 32% (P ¼ 0.078). The median follow-up of surviving patients is 23 months (range 2-70). The 3-year overall survival for patients treated with FluBu and for those conditioned with FluMel was 51% (95%CI 27-74) and 61% (95%CI 47-74) (P ¼ 0.82), respectively (Figure 1). The respective 3-year leukemia-free survival was 56% (95%CI 52-71) and 54% (95%CI 35-75) (P ¼ 0.72). Similarly, two-year non-relapse mortality [21% (95%CI 10-44) vs 21% (95%CI 12-35)] and relapse incidence [18% (95%CI 8-40) vs 20% (95%CI 11-35)] did not differ between two groups. Conclusion: In conclusion, our results confirm that FluBu and FluMel as RIC regimens for alloSCT provide similar survival for the elderly or infirm patients with myeloid malignancies. Nonrelapse mortality is acceptable after both type of RIC alloSCT. Disclosure of Interest: None declared.
P211 Reduced risk of oral mucositis among autoHSCT recipients treated with total marrow irradiation compared to either total body irradiation or chemotherapy-based conditioning. Results of a prospective, observatory study A. M. Polakiewicz-Gilowska1,*, T. Czerw1, M. Sadus´-Wojciechowska1, J. Najda1, W. Mendrek1, L. Miszczyk2, K. S´losarek2, J. Ho"owiecki1, S. Giebel1 1 Department of Bone Marrow Transplantation and OncoHematology, 2Department of Radiotherapy, Maria SklodowskaCurie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland Introduction: Oral mucositis (OM) is one of the most frequent complications of high-dose therapy and considered as a risk factor for infections, prolonged hospitalization, and the need for parenteral nutrition and analgesia. High incidence of OM has been reported after both total body irradiation (TBI) and chemotherapy-based conditioning regimens. In recent years, a new protocol including total marrow irradiation (TMI) has been introduced in clinical practice. However, its impact of the occurrence of OM has not been documented so far. The goal of this study was to prospectively evaluate the incidence of OM according to the type of conditioning, including TMI. Materials (or patients) and methods: 155 consecutive patients (median age 55, range 17-70 years) with multiple myeloma (n ¼ 84), non-Hodgkin lymphoma (n ¼ 45) and Hodgkin’s lymphoma (n ¼ 26) referred for autologous HSCT were included in the analysis. Conditioning regimen consisted of high dose melphalan or BEAM chemotherapy (n ¼ 67), TBI (12 Gy in 3 fractions) alone (n ¼ 11), TBI þ chemotherapy (n ¼ 21) or TMI (12 Gy in 3 fractions) alone (n ¼ 56). All patients had intra-oral assessment before conditioning and were thoroughly instructed in everyday oral hygiene. Oral mouth washes with saline, natrium bicarbonicum, and chlorhexidine were provided during hospitalization at least 3 times daily. In the melphlan setting cryotherapy with crushed ice was additionally used. Standard systemic anti-infectious prophylaxis (quinolones, fluconazole, acyclovir) was administered. Filgrastim 5 mg/kg/day was used to enhance neutrophil engraftment. OM assessment was made daily until discharge. Results: In the whole group, OM occurred in 34% of patients with the median duration of 7.5 days (range 2-19). Its incidence was 40% in the chemotherapy group, 64% after TBI alone, 57% after TBI þ chemotherapy and 11% in the TMI group (P ¼ 0.0001). The median time from start of conditioning to peak OM score was 14 days. Severe OM (WHO score G3 and G4) occurred in 5% of all patients without significant differences between groups. In a multivariate analysis, adjusted for other risk factors, the incidence of OM (grade 1-4) was significantly reduced for TMI (HR ¼ 0.36, 95%CI, 0.220.58, P ¼ 0.0001) together with increasing values of BMI. In contrast to some previous reports, the risk of OM was not related to age, performance status, oral hygiene status and gender. Conclusion: The use of TMI as conditioning prior to autoHSCT for patients with lymphoproliferative disorders is associated with reduced risk of OM compared to both TBI and high-dose chemotherapy. Disclosure of Interest: None declared. P212 LACE-conditioned allogeneic transplantation for high-risk haematological malignancies: treatment outcomes in 62 patients from a single centre B. Sevillano1,*, J. Pavlu1, R. Szydlo1, A. Rotolo1, A. Khoder1, M. Sever1, M. Lasa1, R. Palinacawandar1, C. Giles1, A. Chaidos1, D. MacDonald1, A. Karadimitris1, I. Gabriel1, A. Marks1, D. Milojkovik1, E. Olavarria1, A. Rahemtulla1, J. Apperley1, E. Kanfer1 1 Haematology, Hammesmith Hospital, London, United Kingdom
potential for cure in high-risk haematological malignancies, but with a high risk of non-relapse mortality (NRM), particularly in the elderly or those with significant co-morbidities. The concept of reduced intensity conditioning (RIC) SCT attempts to address this risk. 25 years ago we developed the RIC conditioning protocol (LACE) consisting of oral lomustine 200 mg/m2, (day -7), iv etoposide 1000 mg/m2 (day -7) iv cytarabine 2000 mg/m2 (day -6 and -5) and iv cyclophosphamide 1800 mg/m2 ( days -4, -3 and -2). Here we summarise outcomes of SCT with this regimen. Materials (or patients) and methods: From 1989 to June 2014, 62 patients (median age 45 years; range 13-68) underwent LACE SCT. Recipients of matched sibling allografts (n ¼ 46) received GvHD prophylaxis with Cyclosporine and Methotrexate. T-cell depletion with Campath was used in recipients of matched unrelated (n ¼ 14), haploidentical (n ¼ 1) and mismatch sibling (n ¼ 1) transplants. Patients not eligible for myeloablative conditioning due to advanced age or associated comorbidities were selected with the following diagnoses: AML (n ¼ 23, 37%), non-Hodgkin lymphoma (NHL, n ¼ 16, 26%), MDS (n ¼ 12, 19%), and CML in accelerated (n ¼ 2, 3%) and second chronic phase (n ¼ 1, 2%). All malignancies were classified as high risk for relapse. 7 patients (11%) were not in remission at the time of transplantation. Results: NRM at day 100 was 27%, with the following causes: infection (n ¼ 9), hepatic veno-occlusive disease (n ¼ 1), multi organ failure (n ¼ 1) and unknown (n ¼ 3). After day 100, mortality was mainly related to disease relapse. Acute GvHD occurred in 20 patients (33%). These were scored as grade I in 8 (13%), grade II in 5 (8%) and grade III in 7 patients (11%). None of the patients developed grade IV acute GvHD. Chronic GvHD occurred in 12 (19%) patients. It was graded as limited (localized skin involvement and/or hepatic dysfunction) in 8 and extensive in 4 patients. The probability of overall survival and progression free survival at 5 years was 40% and 37% respectively, with a median follow up of 3.6 years. Kaplan Meyer curves indicating probability of survival according to disease are shown below. Conclusion: LACE RIC showed acceptable toxicity, with a relatively low incidence of acute and chronic GvHD. A significant proportion of patients with high-risk haematological malignancies unfit for MAC achieved good long-term results with this conditioning protocol. Disclosure of Interest: None declared.
Introduction: Haematopoietic stem cell transplantation (SCT), preceded by a myeloablative-conditioning regimen provides a
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P213 Research of Iron chelator treatment in allogeneic hematopoietic stem cell transplantation C. Li1,2,*, J. Ma1, L. Ye1, X. Ye1, C. Ma1, C. Gu1, T. Wang1, Q. Zhou1, X. Chen1, D. Wu1 1 The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, 2Immunology, School of Biology & Basic Medical Science, Medical College of Soochow University, Suzhou, China Introduction: To investigate the efficacy and safety of ironchelator treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Materials (or patients) and methods: a total of 50 allo - HSCT recipients with IO were retrospectively analyzed. Patients were divided into two groups. The experimental group consisted of 23patients who did not receive any chelator treatment. The control group consisted of 27 patients who received ironchelator treatment. Results: The iron chelating and allo-HSCT treatment significantly reduced serum ferritin levels (1262, 824, min 501–max 1305 vs min 1000–max 2352) (Po0.01). In the Treatment group after HSCT the median time of grain giant nuclear reconstruction is 12±2 days and 20±8 days, 13±3 days and 25±14 days in the control group. Nuclear hematopoietic reconstruction time was significantly shortened with iron chelation therapy (P ¼ 0.049). In the treatment group , 39.1% (9/23) cases occurred infection, while 74.1% (20/27) cases occurred infection in control group. The difference was found statistically signicant (P ¼ 0. 014). 17.4% (4/23) cases were found GVHD in the treatment group and 55.6% (15/27) cases were found GVHD in control group. There was a significant reduction in the risk of GVHD (P ¼ 0. 008). The median OS in control group was 12 (min:0.3 B max:50) and 18 (min:3.5max:78) months in Treatment group. The difference was found statistically signicant (P ¼ 0.050). Conclusion: Iron chelation therapy can safely and effectively to reduce iron load of allo - HSCT patients with iron overload, and promote hematopoietic reconstruction, reduce the infection and the incidence of GVHD, prolong survival time. Disclosure of Interest: None declared. P214 Comparison between BEAM and Yttrium-90 Ibritumomab Tiuxetan (Zevalin)-BEAM as conditioning regimen before autologous stem cell transplantation in patients with nonHodgkin B-Cell Lymphoma C. CHIC ACEVEDO1,*, E. GARCIA TORRES1, M. J. LLAMAS POYATO1, G. RODRIGUEZ1, C. MARTIN1, R. ROJAS1, J. SERRANO1, S. TABARES1, J. A. VALLEJO1, J. SANCHEZ1 1 HEMATOLOGY, HOSPITAL UNIVERSITARIO REINA SOFIA, CORDOBA, Spain Introduction: Patients with chemosensitive relapsed nonHodgkin B-Cell lymphomas are candidates to intensification with autologous stem cell transplantation (ASCT). The addition of Ibritumomab Tiuxetan (Zevalin) in combination with highdose chemotherapy as BEAM (Z-BEAM) has proven to be safe and effective in patients with poor-risk features. However the precise role of Z-BEAM as conditioning regimen for ASCT is not well established. The aim of this study is to analyze the safety and efficacy of Z-BEAM as conditioning for ASCT in nonHodgkin B-lymphoma. Materials (or patients) and methods: In this retrospective study we analyze 54 patients diagnosed with non-Hodgkin B-lymphoma candidates to ASCT who received conditioning regimen with BEAM or Z-BEAM in our institution, from 2004 to 2014. 31 (57.4%) were males and 23 (42.6%) females. Median age was 50years (30-68). Zevalin was administered 7days previous to standard BEAM at dose of 0.4mCi/Kg. Patients were assigned to each arm according to lymphoma-risk features. Toxicity was measured according to CTCAEv4.1. Overall Survival (OS) and Progression free survival (PFS) were
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compared. Statistical analysis was performed using SPSS.v17 software package. Results: 27 patients received BEAM and 27 Z-BEAM. We did not find statistical differences in age, sex, IPI, FLIPI, histological grade and diagnosis between both groups. However we found significant difference in disease-status at ASCT: 70.4% (n ¼ 19) in BEAM group were in complete remission (CR) vs. only 33.3% (n ¼ 9) in Z-BEAM group (P ¼ 0.006). Significant difference was also found in the number of prior chemotherapy (CT) lines (42 lines): 14.8% (n ¼ 4) vs 40.7%(n ¼ 11); P ¼ 0.033 for BEAM and Z-BEAM respectively. No statistical differences were found in neutrophil engraftment (4500/mm3): 11.59 days (11-12.7) in BEAM group vs 11.38 (10.6-12) in Z-BEAM (P ¼ 0.747) nor in platelet engraftment (420,000/mm3): 13 days (12-14) vs 15.7 (13.7-18); (P ¼ 0.073). Significant differences were neither found in transplant-related toxicity except for the mucositis which was significantly higher in Z-BEAM: 92.6% (n ¼ 25) than in BEAM: 62.9% (n ¼ 17); P ¼ 0.009. With a median follow-up of 34months (0-104), there was no statistically significant difference either in OS or PFS between both groups. OS was 76.5% (60.7-92) in BEAM group vs 67.1% (50.6-84) in Z-BEAM; P ¼ 0.65. PFS was 71% (53.4-88.6) vs 54.2% (36.1-72.2) respectively; P ¼ 0.387. Other clinical variables such as age, FLIPI and IPI did not impact on outcomes(OS or PFS) in our series. The number of previous CT lines negatively impact PFS: 74.8% (60.4-89.3) in d 2 lines vs 39.6% (15.1-64.1) for 42 CTlines (P ¼ 0.013). Disease-status at ASCT had a significant statistical impact on outcomes:OS was 87.3% in those in CR (74.2-100) vs 51.6% (35.3-67.9) in partial response (P ¼ 0.014) and PFS was 82.6% (67.6-98) vs 33.1% (1947); P ¼ 0.002 respectively. This variable remained as the only significant variable in multivariate analysis: OR: 3.45 (1.1-11.1) P ¼ 0.027 in SG; OR: 2.96 (1.1-8.7) P ¼ 0.05 in PFS. Conclusion: In our series, patients receiving Z-BEAM for ASCT harbored significantly higher risk features than those with BEAM. However we did not find statistical differences in outcomes comparing both groups. This finding suggests that patients who did not achieve CR before ASCT could benefit of the use of Z-BEAM as conditioning regimen. Disclosure of Interest: None declared. P215 Effects of Busulfan dose and administration route on transplantation outcome in adult patients with AML or MDS - a retrospective analysis of the German transplant registry DRST C. Scheid1,*, U. Holtick2, M. Bornha¨user3, N. Kro¨ger4, A. Ganser5, R. Arnold6, P. Dreger7, G. Stuhler8, M. Theobald9, C. Peschel10, M. Hallek11, D. Niederwieser12, J. Finke13, B. Hertenstein14, D. Beelen15 on behalf of Deutsches Register Stammzelltransplantation (DRST) 1 Stem cell transplantation program, University of Cologne, Ko¨ln, 2 Stem cell transplantation program, University of Cologne, Cologne, 3BMT, University of Dresden, Dresden, 4BMT, University Hospital Eppendorf, Hamburg, 5Hematology, Hannover Medical School, Hannover, 6BMT, Charite University Hospital, Berlin, 7BMT, University of Heidelberg, Heidelberg, 8BMT, Helios Kliniken, Wiesbaden, 9Hematology, University Hospital Mainz, Mainz, 10 Hematology, Klinikum rechts der Isar, Mu¨nchen, 11Dept. I of Internal Medicine, University of Cologne, Cologne, 12Hematology, University of Leipzig, Leipzig, 13BMT, University of Freiburg, Freiburg, 14Hematology, Kliniken Bremen-Mitte, Bremen, 15BMT, University Hospital Essen, Essen, Germany Introduction: Busulfan is frequently used as part of the conditioning for allogeneic stem cell transplantation. It can be given orally or intravenously in a variety of dosages. While the advantages of the intravenous administration compared to the oral formulation is well established, the optimal dose for each situation is less clear. Materials (or patients) and methods: To assess the current use of Busulfan in terms of application form and dosage in
Germany, we collected registry data of 1844 patients with AML (n ¼ 986), MDS (n ¼ 649) or secondary AML (n ¼ 209) receiving an allogeneic stem cell transplantation after a Busulfancontaining conditioning regimen. The median age was 53 years (range 18-80), 55% were male, 45% female. Karnofsky performance status was 100 in 24.7%, 90 in 44.1%, 80 in 18.8%, 70 or less in 6.1% and missing in 6.5%. 49.8% were in complete remission at transplantation, 11.7% were transplanted upfront and 48.5% had active disease after chemotherapy. Stem cell source was PB in 94%, BM in 5.3% and others in 0.7%. Results: Busulfan was given orally in 607 patients and intravenously in 1237 patients. The dose was up to 4mg/kg in 212 patients, 4.1- 8 mg/kg in 1084 patients, 8.1 -12 mg in 82 patients and more than 12 mg/kg in 466 patients. In the age group o40 yrs most patients received 412 mg/kg (61%), while in patients between 41 and 50 yrs 50% had 412 mg/kg and 32% had 4.1 -8 mg/kg. In contrast patients between 51 and 60yrs a reduced dose of 4.1-8 mg/kg was used in 57%, increasing to 78% and 82% in age groups 61-65yrs and 465 yrs respectively. Multivariate Cox model analysis revealed age and status at transplant as significant parameters for overall survival and non-relapse mortality (NRM). To exclude status at transplant as confounding variable, a subgroup analysis of AML patients in CR1 was performed. In this subgroup cumulative incidence of NRM at 24 months was 15.7% in patients o40 yrs, 17.6% in 41-50 yrs, 23.7% in 51-60 yrs, 31.8% in 61-65 yrs and 35.7% in patients 4 65 yrs. Overall survival at 24 months was 72%, 68%, 57%, 53% and 43% respectively. Conclusion: Busulfan can be applied in a wide dose range and adapted to age and clinical situation. Our data from the German stem cell transplantation registry indicate that most patients 450 yrs receive a reduced dose of up to 8mg/kg. With this age-adjusted dosing there was only a moderate increase of NRM between the different age groups 450 yrs, amounting to 35.7% in patients 465 yrs with AML in CR1 leading to an overall survival of 43% at 2 years. Disclosure of Interest: C. Scheid Funding from: Pierre Fabre, Amgen, Novartis, Janssen, Celgene, U. Holtick: None declared, M. Bornha¨user: None declared, N. Kro¨ger: None declared, A. Ganser: None declared, R. Arnold: None declared, P. Dreger: None declared, G. Stuhler: None declared, M. Theobald: None declared, C. Peschel: None declared, M. Hallek: None declared, D. Niederwieser: None declared, J. Finke: None declared, B. Hertenstein: None declared, D. Beelen: None declared P216 Haploidentical hematopoietic stem cell transplantation using a reduced-intensity conditioning regimen which consist rituximab and mesencymal stem cell infusion in children; preliminary results D. Atay1,*, F. Erbey1, A. Akcay1, M. Akbiyik1, O. Harman1, E. Ovali2, G. Ozturk1 1 PEDI˙ATRI˙C HEMATOLOGY ONCOLOGY BONE MARROW TRANSPLANTATI˙ON UNIT, 2Acibadem Labcell, ACIBADEM UNIVERSITY MEDICINE FACULTY, Istanbul, Turkey Introduction: Haploidentical stem cell transplantation (haploHSCT) has been developed as an alternative transplant strategy for children with hematological disorders without a HLA matched donor. Reduced-intensity conditioning (RIC) regimens allow haplo-HSCT with less toxicity, but graft versus host disease (GVHD) remains still an actual problem. T cell receptor (TCR) a and b/CD3 depletion for the prophylaxis of GVHD achieved a significant success in haplo-HSCT. The aim of our study is to investigate the efficacy of rituximab (RTX) and mesencymal stem cell (MSC) administration for GVHD prevention in haplo-HSCT. Materials (or patients) and methods: We analyzed the outcome of eleven pediatric patients (median:8.6 years) with various hematological diseases who underwent haplo-HSCT using a RIC regimen [ATG (days –13 to –9), fludarabine (days – 8 to –5) thiotepa (day –4) and melphalan (days –3 and –2)] and TCRab /CD3 depletion at our institution from January, 2014 to
October, 2014. To prevent EBV related posttransplant lymphoproliferative disease (PTLD) and GVHD, RTX 375mg/m2 was added into conditioning regimen on day -2. MSC was infused at day -1 to suppress alloreactive donor anti-host T-cell responses and to use their ability to promote angiogenesis and support micro-environment, which facilitate engraftment. Mycophenolat mofetil was used for GVHD prophylaxis. PCR screening for BK, Adeno, EBV and CMV viruses were performed routinely weekly. Analysis on immune reconstitution was performed at day 30, 60, 90, 120 and 180. Results: Reducing a/b of the graft was 99.9%. The grafts contained a median of 36.08x106 (range: 14.27-281.13) CD34 þ cells. Engraftment of neutrophils and platelets was achieved at a median of 12.5 (8-17) days and 14.5 (9-22) days, respectively. Lymphocyte count 41000/mm3 was seen at a median of 24 days (13-186). Seven patients engrafted with full donor chimerism. Two patients were mix chimeric. Graft rejection occurred in two patients, one of them was successfully rescued by CD34 infusion from same donor. The regimen was generally well tolerated. Two patients developed grade 3-4 acute GVHD. One patient died due to massive gastrointestinal system bleeding. A number of 2/11 patients suffered from CMV-reactivation at day 13 and 35 without clinical or radiological involvement, with prompt response to specific antiviral therapy. Adeno virus reactivation was seen in three patients at day 33, 66, 82; they didn’t need any therapy. BK virus reactivation was seen in four patients at day 30,12,10,11; they were only treated with hydration. None of the patients developed EBV infection or severe bacterial infection during or after HSCT. Transplant related mortality (TRM) at day 30 and at day 100 was 0%. Median discharge day was þ 27. Our data at þ 30, þ 60 and þ 90 from engraftment showed delayed immune reconstitution. Conclusion: These data indicate that a selective graft manipulation and MSC results into effective prevention of acute GVHD, rapid recovery of neutrophil and platelet counts without severe complication at acute phase of HSCT and low TRM. Rituximab is less expensive then hardware-based depletion and more applicable. But the median observation time is limited. Following investigation of advantages and risks of Rituximab and MSC application are required. Disclosure of Interest: None declared. P217 Clinical Comparison of Weight and Age Based Strategy of Dose Administration in Children Receiving Intravenous Busulfan for Hematopoietic Stem Cell Transplantation D. Gurlek Gokcebay1,*, F. Azik1, N. Ozbek1, P. Isik1, Z. Avci1, B. Tavil1, B. Tunc1 1 Pediatric Hematology, Ankara Children’s Hematology and Oncology hospital, Ankara, Turkey Introduction: Busulfan (Bu), combined with therapeutic drug monitoring-guided (TDM) dosing, is associated with fewer graft failure/relapse and lower toxicity in children who had hematopoietic stem cell transplantation (HSCT). Because TDM of Bu cannot be performed in Turkey formerly, based on previous pharmacokinetic analyses, we planned this retrospective study for comparison of weight and age based dosing in terms of clinical outcomes. Materials (or patients) and methods: Patients underwent HSCT from April 2010 to February 2013 and completed 100 days after transplantation in our Hospital were enrolled in this study. Intravenous Bu was administrated according to age in Group 1 that includes 37 patients. The dose of Bu was 1 mg/kg foro4 years and 0.8 mg/kg for Z4 years. In Group 2 that includes 24 patients Bu doses were adjusted according to actual body weight; patients o9 kg received 1 mg/kg, 9-16 kg 1.2 mg/kg, 1623 kg 1.1 mg/kg, 23-34 kg 0.95 mg/kg, and 434 kg 0.8 mg/kg. Results: A total of 61 patients (median age 8.2 years, range 0.54-16.8) received allogeneic HSCT. The weight based dosing group received higher amount of Bu, and this group was significantly prone to develop sinusoidal obstruction
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syndrome (SOS) (P ¼ 0.001). A statistically significant but mild correlation was found between Bu dose and the incidence of SOS (r:0.26, P ¼ 0.04). There was no relation between high levels of serum aspartate aminotransferase or ferritin and SOS. Although univariate analysis revealed association between SOS and conditioning regimen, weight or age based Bu dosing strategy, total amount of Bu and cyclophosphamide, and use of Pesaro protocol 26 as conditioning regimen (Po0.05); multivariate analysis revealed that only weight based dosing of Bu was a significant predictor of SOS (HR: 9.46; P ¼ 0.009). Febrile neutropenia, hypoxia and dyspnea occurred more frequently in the weight based dosing group, and this group also had higher bilirubin concentration, and longer duration of hospitalization (Po0.05). However, no relation was found between two groups in terms of hemorrhagic cystitis, engraftment syndrome, acute or chronic graft-versus-host disease (GvHD), time to engraftment, chimerism, transplant related mortality, survival rates. Conclusion: Therapeutic drug monitoring of Bu is essential for improvement of efficacy and reduction of toxicity. We suggest use of age based dosing strategy for Bu administration in countries where TDM cannot be performed. References: 1. Tran HT, Madden T, Petropoulos D, et al. Individualizing high-dose oral busulfan: prospective dose adjustment in a pediatric population undergoing allogeneic stem cell transplantation for advanced hematologic malignancies. Bone Marrow Transplant 2000; 26: 463–470. 2. Nguyen L, Fuller D, Lennon s, et al. Iv Busulfan in pediatrics: a novel dosing to improve safety/efficacy for hematopoietic progenitor cell transplantation recipients. Bone Marrow Transplant 2004; 33: 979–987. 3. Zwaveling J, Bredius RGM, Cremers SC, et al. Intravenous busulfan in children prior to stem cell transplantation: study of pharmacokinetics in association with early clinical outcome and toxicity. Bone Marrow Transplant 2005; 35: 17–23. Disclosure of Interest: None declared. P218 Once daily intravenous infusion Busulphan plus Fludarabine as conditioning for Acute Myeloid Leukemia and MDS allogeneic transplantation. A single Centre experience D. Baronciani1,*, C. Depau1, C. Targhetta1, A. A. DiTucci1, E. Angelucci1 1 Ospedale Oncologico di Riferimento Regionale ‘‘A. Businco’’, Cagliari, Italy., U.O. Ematologia-CTMO, Cagliari, Italy Introduction: Allogeneic HSCT is the treatment of choice for higher risk AML and MDS patients. The traditional preparative regimens TBI-CY and BU-CY have been widely used but extra hematologic toxicity remains a significant concern. Even if the combination of intravenous formulation Busulphan/CY has shown lower toxicity and suggests favorable safety and efficacy new drugs associations are continuously explored. Recently the association Busulphan –Fludarabine has replaced the Bu-Cy regimen with the aim to reduce toxicity. Materials (or patients) and methods: In our Institution we started to use this regimen as preparation for allogeneic HSCT in patients with AML and MDS in October 2008. Primary objectives were evaluation of overall survival, disease free survival, transplant related mortality and relapse. Since October 2008 up to October 2014, 28 consecutive (21 males, 7 females) patients entered this study. Median age was 55 years (range 3563). Underlying diseases were: AML (23 patients: CR1 ¼ 18, advanced disease ¼ 5) and MDS (5 IPSS Intetermediate II patients). Conditioning consisted of iv Busulphan (Busilvexs 9.6 -12.8 mg/kg) given once daily in a 4 hours infusion together with Fludarabine (160 mg/m2) for 4 consecutive days. The association CSA þ short MTX was used as GVHD prophylaxis and anti-lymphocyte globulins (Thymoglobulins or ATG Freseniuss) were used in case of MUD transplants. Sixteen patients received HSCs from an HLA identical siblings, 1 from a mismatched family donor, 11from unrelated donors. Source of stem cells was bone
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marrow in 14 patients, peripheral blood stem cells in 13 patients and both in 1 patient. Results: All patients regularly engrafted (median time of ANC 4500 was 13 days (range 11-15 ). One patient presented late rejection in aplasia 2 months after a unrelated transplant. She received rescue with an haploidentical family donor transplant. Five patients presented a pattern of mixed chimerism in a state of complete remission. None of them received donor lymphocyte infusion; all recovered to full donor status with immunosuppression tapering. Nine patients experienced gastro-intestinal toxicity; three patients had grade II cystitis. No other extra-hematologic toxicity occurred. Acute grade III gastro-intestinal GVHD occurred in one patient; 13 patients presented grade I skin GVHD, associated with grade II gastrointestinal in one. Cr.GVHD occurred in 6 over 20 evaluable patients (it was mild in 5 and moderate in 1). One patient presented early infection (pneumonia), four late infections (3 fungal infections, 1 Pneumocystis Jirovecii pneumonia); Cytomegalovirus reactivation occurred in 18 over 28 evaluable patients (64%). Nineteen patients are alive (68%), 18 in complete remission with a median follow-up of 12 months (range 1-41). Nine patients died (32%): eight by recurrent disease and one by acute GVHD. Among the 18 patients transplanted in first complete remission, 14 are alive and well. Four patients have died for high risk leukemia recurrence. Conclusion: This single Centre report, even if with limited number of patients, underlines the association iv Busulphan– Fludarabine is a well-tolerated and effective conditioning regimen in AML/MDS patients. Intravenous Busulphan administered once daily is convenient, effective and well tolerated. Disclosure of Interest: None declared. P219 Therapeutic drug monitoring of oral and intravenous busulfan in paediatric patients E. Fernandez De Gamarra1,*, M. Torrent2, L. Sisinni2, E. Zapico3, T. Olive4, C. Dı´az de Heredia4, I. Badell2 1 PHARMACY DEPARTMENT, 2PEDIATRIC BONE MARROW TRANSPLANTATION UNIT, 3BIOCHEMISTRY DEPARTMENT, HOSPITAL SANTA CREU I SANT PAU, 4PEDIATRIC BONE MARROW TRANSPLANTATION UNIT, HOSPITAL VALL D’HEBRON, BARCELONA, Spain Introduction: Busulfan is commonly used in preparative regimens for hematopoietic stem-cell transplantation in adults and children for a variety of malignant and non-malignant diseases. It has a narrow therapeutic range and a large pharmacokinetic variability that could have consequences in treatment outcomes and toxicity. It is available for oral and intravenous administration. The aim of this study is to assess the benefits of therapeutic drug monitoring (TDM) to guide busulfan dosing and to review efficacy and safety of busulfan. Materials (or patients) and methods: Data from 36 paediatric patients treated with busulfan between 2010 and 2014 in two bone marrow transplantation units was collected. Protocols were chosen according to the underlying disease. We included children treated with oral or intravenous busulfan who underwent TDM. We applied a non-linear regression method and used ID3 software for pharmacokinetic studies. Area under the curve target was 55000-95000 ng/ml.h, depending on the conditioning protocol (reduced intensity or myeloablative). Graft failure and incidence of veno-occlusive disease (VOD) were recorded to assess efficacy and safety. Results: Thirty-six patients with ages ranging from three months to 18 years were included in the study. Twenty-five of them received IV busulfan and 11 patients received oral busulfan. Allogeneic or autologous transplantations were performed in 33 and three patients respectively. Baseline diseases in the allogeneic group were: 27 malignant and 16 non-malignant haematological diseases; and in the autologous group: 2 neuroblastoma and 1 Ewing’s sarcoma. Regarding the conditioning regimen, 26/36 were myeloablative and 10/36 non-myeloablative. Busulfan initial doses ranged from 2.8 and
5.1 mg/kg/day (related to adjusted body weight), according to the protocol and the weight band. Oral busulfan was administered in four divided doses (every six hours) and daily IV busulfan dose was either administered in one or four doses. All patients received seizures prophylaxis with phenytoin. TDM and follow-up results for oral treatments (N ¼ 11): all treatments were myeloablative. Ten patients needed a dose adjustment (four reductions and six increases) to guarantee a drug exposure within the desired range. Any of them experienced graft failure or VOD. TDM and follow-up results for intravenous treatments (N ¼ 25): - Myeloablative regimens (N ¼ 15): 13 patients needed a dose adjustment (nine reductions and four increases) to guarantee a drug exposure within the desired range. Any of them experienced graft failure. - Non-myeloablative regimens (N ¼ 10): all patients needed a dose adjustment (four reductions and six increases) to guarantee a drug exposure within the desired range. Secondary graft failure was observed in four patients (in three of them it was recovered). These four patients had a nonmalignant baseline disease. Any VOD was reported for this group of intravenous treatments. Globally, considering all 36 patients, 33 needed a dose variation: 17 reductions (average -18.6%) and 16 increases (average þ 21.4%). Conclusion: TDM of oral and IV busulfan in paediatric patients is a valuable tool to guide drug dosing. It should be strongly recommended even for intravenous treatments, in spite of its theoretical advantage of less variability compared to the oral form. TDM might provide improvements in efficacy and safety. Disclosure of Interest: None declared. P220 Final results of a prospective multicenter phase II study using Busulfan AUC intensification in AML patients undergoing allogeneic HSCT in first complete remission F. Suarez1,*, P. Bourget2, E. P. Alessandrino3, A. Bartoli4, G. Socie´5, M. Michallet6, J. Bourhis7, A. Paci8, D. Blaise9, A. Berceanu10, V. Kemmel11, J. Sierra12, M. E. Moreno Martinez13, A. Amin2, A. A. Colombo14, A. Xhaard5, F.-E. Nicolini6, X. Thomas6, S. Fu¨rst15, B. Lioure16, R. Martino17, C. Levrault18, A. Pe´tain19, C. Ta Thanh Minh20 1 Hematology, 2Pharmacy, Hoˆpital Necker-Enfants Malades, Paris, France, 3Hematology and Oncology, Fondazione IRCCS Policlinico San Matteo and University of Pavia, 4Clinical Pharmacokinetic Laboratory, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 5Hematology and Bone marrow Transplantation, Hoˆpital Saint-Louis, Paris, 6Hematology and Bone Marrow Transplantation, CHU Lyon Sud, Lyon, 7Hematology, 8Service de Pharmacologie & d’Analyse du Me´dicament (SIPAM), Institut Gustave Roussy, Villejuif, 9Hematology and Bone Marrow Transplantation department, Institut Paoli-Calmettes, Marseille, 10 Hematology, CHU Besanc¸on, Besanc¸on, 11Service de Biochimie, CHRU Strasbourg, Strasbourg, France, 12Hematology and Bone Marrow Transplantation, 13Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 14Hematology and Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 15Hematology and Bone Marrow Transplantation, Institut Paoli-Calm16 Hematology and Bone Marrow ettes, Marseille, Transplantation, CHRU Strasbourg, Strasbourg, France, 17Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 18Institut de Recherche Pierre Fabre, Boulogne, 19De´partement de Pharmacocine´tique, Pierre Fabre Me´dicament, Toulouse, 20PIERRE FABRE MEDICAMENT, PIERRE FABRE MEDICAMENT, Boulogne Billancourt, France Introduction: Despite major advances in the risk-stratification and management of acute myelogenous leukaemia (AML) based on cytogenetics and molecular biology, the relapse rate remains as high as 25%. Therefore, a prospective phase II study aimed to assess a strategy based on Busulfan (Bu) AUC (area under the curve) intensification in AML patients was designed. The
administration of IV Bu in once-daily infusion over 3 hours combined with Fludarabine prior to haematopoietic stem cell transplantation (HSCT) was approved recently in adult patients who are candidates for a reduced-intensity conditioning regimen. Since limited experiences were available with the combination IV BuCy2, in this protocol, IV Bu was infused in a single daily infusion during 4 days, followed by Cyclophosphamide. Materials (or patients) and methods: The primary objective was to target the upper range of the Bu TW (therapeutic window) [4400 - 6000 mmol/L.min]. A daily pharmacokinetic (PK) guided adjustment from Dose 2 to Dose 4 of Bu treatment was expected to enable the same targeting performance (Z70 to 80% of patients within the TW) as that achieved for the common large TW[3600-6000 mmol/L.min] without adjustment. The secondary objective was to determine the clinical outcomes at 2 years. The eligible patients were required to have AML in first complete remission (CR1) and eligible for an allogeneic HSCT following a myeloablative regimen. The graftversus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitor and IV methotrexate. The addition of antithymoglobulin for unrelated donors was decided by each centre based on their institutional guidelines. Results: Thirty adult patients with a median age of 43.5 years were treated. Ninety percent of patients reached the cumulated therapeutic window of AUC [17600-24400 mmol.L/ min]. Clearance after once daily administration was similar to clearance with a quarter in die regimen demonstrating the linearity of Bu pharmacokinetic from 0.8 mg/kg to 3.2 mg/kg. Overall, the cumulative incidence of acute graft-versus-host disease (aGVHD) grade II-IV at 100 days was 26.7% [23.7-29.6]. The cumulative incidence of aGVHD grade III-IV was 6.7% [5.0; 8.3]. None of the patients developed grade IV aGVHD. Five patients died of late non relapse mortality (NRM) between day þ 197 and day þ 391 (2-year cumulative incidence of NRM was 16.7%). No patient died before day þ 100. The causes of late NRM were GVHD (2 patients), hemorrhage (1 patient), non-infectious interstitial pneumonitis (1 patient), vasculitis (1 patient). No death due to VOD was observed. The cumulative incidence of relapse at 2 years was 20% [17.3 – 22.7]. The 2year overall survival (OS) was 63.2% [43.4-77.6]. This study proved the feasibility of a daily adjustment according to PK measurement. The 2-year OS was satisfactory with a low relapse rate; these values are comparable with previous publications reporting experiences with IV BuCy2 without AUC intensification. The 2-year NRM was comparable with the previously published safety profile of a myeloablative BuCy2. Conclusion: The clinical outcomes (OS, Relapse rate) were good in this homogeneous cohort of high-risk AML patients allografted in CR1. However, compared to the literature, no additional benefit could be demonstrated using Bu AUC intensification. Disclosure of Interest: F. Suarez: None declared, P. Bourget Funding from: Pierre Fabre Medicament, Conflict with: Travel grants –Pierre Fabre Medicament, E. P. Alessandrino: None declared, A. Bartoli: None declared, G. Socie´: None declared, M. Michallet Conflict with: Fundings and Personal Financial interest : Advisory boards and consultancy BMS, Novartis, Genzyme, MSD, Hospira, GILEAD, J. Bourhis: None declared, A. Paci Funding from: Pierre Fabre Me´dicament, Personal Interest: Pierre Fabre Me´dicament, D. Blaise Funding from: Pierre Fabre Medicament, Personal Interest: Pierre Fabre Medicament, A. Berceanu: None declared, V. Kemmel: None declared, J. Sierra: None declared, M. E. Moreno Martinez: None declared, A. Amin: None declared, A. A. Colombo: None declared, A. Xhaard: None declared, F.-E. Nicolini Funding from: Novartis Pharma, Personal Interest: Novartis Pharma, Conflict with: Advisory boards; consultancy, speakers bureau; BMS: consultancy, advisory boards, speakers bureau; ARIAD: Advisory board, speakers bureau, X. Thomas: None declared, S. Fu¨rst: None declared, B. Lioure: None declared, R. Martino: None declared, C. Levrault Employee of: Pierre Fabre Me´dicament, A. Pe´tain Employee of: Pierre Fabre Me´dicament, C. Ta Thanh Minh Employee of: PIERRE FABRE MEDICAMENT.
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P221 Comparison of two conditioning regimen for autologous stem-cell transplantation for lymphoma. FEAM vs TEAM A. Gravetti1, R. Della Pepa1, S. Avilia1, N. Pugliese1, F. Pane1, A. M. Risitano1, G. De Rosa1,* 1 Hematology, Federico II University, Naples, Italy Introduction: The treatment of choice for relapsed/refractory non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL) consists of high-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT). Several HDC regimens with different drug combinations have been in use, however, no randomized data is available to demonstrate superiority of any one regimen and retrospective comparative reports are sparse. BEAM (BCNU, etoposide, cytarabine, melphalan) is a widely adopted conditioning regimen for autologous hemopoietic SCT for lymphoma transplants, and has an acceptable toxicity and high efficacy. Recently, there was a shortage of BCNU globally, to overcome this situation and the increased pulmonary toxicity associated with BCNU we instead used two modified regimens, FEAM or TEAM, where BCNU was replaced with Fotemustine or Thiotepa, respectively. Materials (or patients) and methods: In this study we analyzed these alternative drug schedules and compared them, focusing on early toxicities, infectious complications and hemopoietic engraftment. We studied 35 patients with relapsed or refractory lymphoma (18 NHL-17 HL) who were treated with HDC followed by ASCT in the Centro Trapianti Midollo Osseo–Federico II between October 2010 and April 2014. HDC regimen administered was FEAM in 17 patients and TEAM in 18 patients. FEAM consisted of Fotemustine 150 mg/m2 on days –8, –7, etoposide 200 mg/m2 and aracytin 400 mg/m2 on days –6 to –3 and melphalan 140 mg/m2 on day –2. TEAM consisted of Thiotepa 10 mg/kg on day -8, -7, etoposide 200 mg/m2 and aracytin 200 mg/m2 on days –6 to –3 and melphalan 140 mg/m2 on day –2. The median age of the study group was 46 years (range 17–66 years). The evaluation of the toxicity and tolerability profile were performed by comparing the two groups on the following variables: time to haemopoietic recovery (plt420.000/109 dL, neutrophils41500/109 dL), transfusion support therapy, presence of fever, days on intravenous antibiotic therapy, appearance and degree of mucositis, onset of hepatic and renal abnormalities. Results: We observed no significant difference in the peritransplant outcomes in terms of engraftment, febrile complications, regimen related organ toxicities. The time to neutrophil engraftment and to platelet recovery are the same in the two groups (10 and 14 days respectively). 8 patients (47%) in the FEAM group and 9 patients (50%) in the TEAM group need transfusion support therapy with RBC units; 16 patients (94.1%) in the FEAM group and 18/18 in the TEAM group need transfusion support therapy with platelet unit. 13 patients (76%) in the FEAM group and 16 patients (88%) in the TEAM group developed neutropenic fever. The median duration of intravenous antibiotic use are 7 and 6 days for FEAM and TEAM group respectively. The most common grade 3 or 4 organ toxicity is oral mucositis and is observed in 6 patients (35%) in FEAM group and in 4 patients (22%) in TEAM group, while G2 diarrhea is observed in 3 (17%) and 7 (38%) patients, respectively. No severe hepatic or renal toxicity is detected. Conclusion: In conclusion, we would like to document that Fotemustine and Thiotepa could be a readily available alternative to BCNU. According to preliminary comparative results we can conclude that the two regimens, FEAM and TEAM are equivalent for safe and feasible, but longer follow-up and a wider sample are needed to evaluate fully their efficacy and safety. Disclosure of Interest: A. Gravetti: None declared, R. Della Pepa: None declared, S. Avilia: None declared, N. Pugliese: None declared, F. Pane: None declared, A. M. Risitano: None declared, G. De Rosa Funding from: no, Employee of: no, Personal Interest: no, Conflict with: no.
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P222 Different Doses of Anti-Thymocyte Globulin (ATG) in the Conditioning Regimen for Allogeneic Hematopoetic Stem Cell Transplantation G. Pekcan1,*, B. Atesagaoglu1, M. Merter1, P. Topcuoglu1, S. Civriz Bozdag1, S. K. Toprak1, M. Kurt Yuksel1, O. Ates1, M. Ozcan1, T. Demirer1, O. Ilhan1, H. Akan1, M. Beksac1, N. Konuk1, G. Gurman1 1 HEMATOLOGY, ANKARA UNIVERSITY DEPARTMENT OF HEMATOLOGY, ankara, Turkey Introduction: ATG as a part of conditioning regimens has been known to reduce the incidence and severity of acute and chronic graft versus host disease (GvHD). We retrospectively evaluated the impact of ATG at two different doses (r45mg/kg-low vs 445 mg/ kg-high) on the early and late complications and the probability of progression free- or overall survival. Materials (or patients) and methods: Between July 2003 and October 2014, 102 adult patients (61M;41F) with hematological malignancies underwent allo-HSCT from an HLA matched or mismatched donor. Median age was 39 years (18-62 ys). Their diagnoses were acute leukemia (n ¼ 71), chronic myeloprolipherative neoplasia (n ¼ 14), MDS/CMML (n ¼ 10), lymphoma, (n ¼ 6) and myeloma (n ¼ 1). Stem cell sources were peripheral blood (PB, n ¼ 94) and bone marrow (BM, n ¼ 8). Most patients were transplanted from an unrelated donor (83.3%). The ratio of HLA matched:mismatched donor was 55:47. Eighty-one patients (79.4%) received a myeloablative conditioning regimen. While most of the patients (n ¼ 78) received less than 45 mg/kg of ATG-Fresenius (F) in totally (7-45 mg/kg), more than a total of 45 mg/kg was given to 24 patients. (Max: 60 mg/kg/day). ATG-F was infused to the patients in equal divided doses from day minus 3 to 1. Results: The patients’ age and gender was similar between two groups (P40.05). Most of the patients in two groups had acute leukemia (69.2% in low-dose ATG vs 78.8% in high-dose ATG). High dose ATG-F was frequently used in patients with HLA mismatched and/or unrelated donors (Table). The intensity of conditioning regimen and stem cell source were similar distributions between two groups. Transplant outcomes including acute or chronic GvHD, transplant-related mortality and the probability progression-free or overall survival were not affected by ATG-F doses (Table). When we repeated the similar analysis in patients with an HLA-mismatched donor or in those with an unrelated donor, the statistical results did not show any differences.
Table.
Patients’ features and transplant outcome
Variables /ATG Fresenius HLA mismatched donor, present (n ¼ 55) Unrelated donor, present (n ¼ 85) Engraftment kinetics Graft failure Neutrophil (0.5x10e9/L) Platelet (20x10e9/ L) CMV reactivation or infection Acute GvHD, present Gr0-I vs GrII-IV cGvHD TRM Relapse 2-year PFS 2-year OS
r 45 mg/ kg (n ¼ 78)
445 mg/kg (n ¼ 24)
P
41.0% (n ¼ 32)
95.8% (n ¼ 23)
o.0001*
78.2 % (n ¼ 61)
100 % (n ¼ 24)
0.01*
7.0% 17.5 (1028) 19 (7-150)
0.5% 17 (10-26)
1.0 0.67
17.5 (0-40)
0.60
94.7 %
0.60
45.0 % 70% vs 30%
0.78 0.64
35.6% 38.5% 33.3% 39.7±11.9% 27.9±1.51%
0.96 0.23 0.12 0.89 0.71
91.0 % 48.6 % 64.3% vs 35.7% 35.0% 25% 16.7% 31.0±5.4% 40.7±5.8%
Conclusion: In conclusion, based on these result lower doses of ATG might be as effective as higher doses. Therefore we need to find the lowest efficient doses of ATG Fresenius by randomized prospective trials. Disclosure of Interest: None declared. P223 EBMT metaanalysis of treosulfan conditioning before allogeneic hematopoietic stem cell transplantation in paediatric patients with haematological malignancies H. Boztug1,*, E. Glogova1, U. Po¨tschger1, M. Zecca2, P. Veys3, A. Lankester4, A. Cant5, R. Skinner6, M. Slatter7, J. Wachowiak8, K.-W. Sykora9, C. Peters1 on behalf of on behalf of the EBMT Pediatric Diseases Working Party 1 St. Anna Kinderspital and Children´s Cancer Research Institute, Department of Paediatrics , Medical University of Vienna, Vienna, Austria, 2Paediatric Haematology / Oncology, Fondazione IRCCS, Policlinico San Matteo Foundation, Pavia, Italy, 3Great Ormond Street Hospital for Children National Health Service Trust, London, United Kingdom, 4Department of Paediatrics, Leiden University Medical Center, Leiden, Netherlands, 5Department of Pediatric Immunology, Great North Children’s Hospital, 6Department of Paediatric and Adolescent Haematology and Oncology, and Children’s HSCT Unit, Great North Children’s Hospital, Royal Victoria Infirmary, 7Paediatric Immunology Department, Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom, 8 Department of Paediatric Haematology, Oncology, and Haematopoietic Stem Cell Transplantation, University of Medical Sciences, Poznan´, Poland, 9Department of Paediatric Hematology/Oncology, Hannover Medical School, Hannover, Germany Introduction: Standard myeloablative conditioning regimens for allogeneic haematopoietic stem cell transplantation (HSCT) in children with haematological malignancies are mainly based on total body irradiation or busulfan. Their profound short and longterm side effects have necessitated the exploration of less toxic alternatives. Treosulfan is increasingly used in view of its potent immunosuppressive and cytotoxic effects and low toxicity. Materials (or patients) and methods: To further investigate the role of treosulfan in children, the EBMT paediatric diseases working party performed a retrospective analyses of 193 children with haematological malignancies (ALL n ¼ 71, AML n ¼ 47, MDS/ MPS n ¼ 40, other leukaemia/lymphoma n ¼ 25) undergoing allogeneic HSCT following treosulfan conditioning.
Results: Early regimen-related toxicity was low and mainly gastrointestinal. There was no association of toxicity with type of disease or higher treosulfan dose (43x13 g/sqm). High grade early toxicity was not higher in infants (n ¼ 11) or patients undergoing second or later transplantation (n ¼ 51). Treatment-related mortality was low at 14%. Three-year event-free survival was 45±4% and not significantly influenced by number of HSCT (first HSCT 46±4%, 4first HSCT 41±7%, P ¼ 0.32), however it was significantly better in infants (P ¼ 0.022). When compared to treosulfan plus fludarabine, the combination of treosulfan, fludarabine and an alkylator (either thiotepa or melphalan) resulted in significantly better overall survival (OS, P ¼ 0.048) and a trend towards better EFS. In multivariate analysis, OS and EFS were significantly worse for patients transplanted not in remission (P ¼ 0.033 and 0.003). Conclusion: In conclusion, treosulfan based conditioning is a safe and efficacious approach for paediatric haematological malignancies, and especially well tolerated by patients undergoing second or later transplantation and infants. Disclosure of Interest: H. Boztug Funding from: HB received a travel grant from Pierre Fabre, E. Glogova: None declared, U. Po¨tschger: None declared, M. Zecca: None declared, P. Veys: None declared, A. Lankester: None declared, A. Cant: None declared, R. Skinner: None declared, M. Slatter: None declared, J. Wachowiak: None declared, K.-W. Sykora Funding from: KWS received travel grants and study support from medac., C. Peters Funding from: CP received travel grants and study support from medac, Pierre Fabre, Sanofi and Novartis. P224 Allogenic Hematopoietic Stemcell Transplantation Results of Treosulphan Based Conditioning Regimens in Hematological Malignancies: Seven Case Reports From a Single Center ¨ zgu¨r2, O. Gu¨rsel1, M. Yıldırım2, O. Babacan1, I. Eker1,*, G. O 2 S. Sayın , F. Avcu2, A. E. Ku¨rekc¸i1 1 Pediatric Hematology, 2Adult Hematology, Gulhane Military Medical Faculty, Ankara, Turkey Introduction: Treosulphan (Treo), which is structural analog of Busulphan (Bu) is an alkylating agent that widely used in the treatment of hematologic malignancies and solid tumors. Recent years, Treo has become an important component of the conditioning regimens due to its predictable pharmacokinetics,
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sufficient immunosuppressive activity for engraftment and high antileukemic effect. Here we report allogenic hematopoietic stemcell transplantation results (HSCT) of adults and children with hematological malignancies, who had undergone HSCT in Gulhane Military Medical Faculty HSCT Center. Materials (or patients) and methods: Between September 2012 and January 2014, HSCT with treosulphan based conditioning regimen, performed for 7 high-risk leukemia (6 with AML, 1 with ALL) patients between the ages of 4-40 years. The characteristics of the patients and HSCT are summarized in Table 1. Three patients were in first complete remission, while other patients were in Z second complete remission. Two cases were treated with HSCT before, with a TBI based regimen and the other two cases received intensive antileukemic treatment, while the other three cases had no risk factors. Three of the transplantations were performed with peripheral derived stem cells, while the others were performed with bone marrow derived stem cells. In five of the transplantations, donors were full matched siblings (one syngenic), while haploidentical HSCT was performed in one patient. Conditioning regimen of all patients were including 36g/m2 Treo. Results: Full engraftment was achieved in all patients. The mean discharge day of the patients was þ 38 (35-42). While hemorrhagic cystitis, sepsis and HBV reactivation developed in two cases, no complications were observed in other five patients. Primary disease relapsed after HSCT in three cases, within the time periods ranging from 4-9 months. Other four cases didn’t relapsed during follow up. Three of the seven cases (all of them were pediatric cases) are in complete remission without complications for 10, 15 and 20 months follow up period. Two cases died due to primary disease relapse and two cases died due to complicaitons, though they were in complete remission. Conclusion: In the literature it is reported that especially in pediatric myeloid malignancies and in second HSCT, Treo has same myeloablative, immunosuppressive and cytotoxic activity with Bu, but it is reported that it is better than Bu in splenic B and T cell depletion. Treosulphan is associated with much more lower risk of seizures, severe mucositis or hepatotoxicity than Bu, our experiences also supported this. Treo is therefore increasingly replacing Busulphan in radiation free conditioning regimens, especially in pediatric cases. Further safety and efficacy studies are needed especially, for the useage of Treo in HSCTs for adult hematologic malignancies. Disclosure of Interest: None declared. P225 Low dose of ATG-Fresenius is effective to reduce severe Graft versus host disease with low non relapse mortality in high risk allogenic hematopoietic stem cell transplantation I. Ormazabal Velez1,*, A. Bermu´dez1, L. Ya´n˜ez1, B. Lo´pez-Pereira1, G. Martin2, E. Conde1 1 Hospital Universitario Marques de Valdecilla, Hospital Universitario Marques de Valdecilla, 2Banco de Sangre y Tejidos de Cantabria, Banco de Sangre y Tejidos de Cantabria , Santander, Spain Introduction: Graft versus host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). ATG-Fresenius (ATG-F) has demonstrated its efficacy in reducing the risk of acute GvHD
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(aGvHD)1 and chronic GvHD (cGvHD)2 at a dose of 60mg/kg, and at a dose of 30mg/kg3. We analysed the incidence of aGvHD grade I-IV, limited/extense cGvHD, early and late infections including CMV and EBV-PTLD, overall survival (OS), disease free survival (DSF) and non relapse mortality (NRM) in a cohort of patients who received low dose of ATG-F (2132mg/kg). We compare these data with the literature (Ju¨rgen Finke1, Gerard Socie´2). Materials (or patients) and methods: Between 2012 and 2014, 20 patients received ATG-F in our hospital. We used ATGF at a dose of 21 mg/kg (7 mg/Kg days –3,-2,-1) in unrelated peripheral blood donor and in mismatched donor or ATG-F at a dose of 28-32 mg/kg (7-8mg/kg days -4 to –1) in aplasia. Median age was 53 years (9-68) and sixteen patients (80%) received myeloablative conditioning regimen. The diagnosis were 5 acute myeloid leukaemia (25%), 2 acute lymphoid leukaemia (10%), 3 myelodisplastic syndrome, 2 non Hodgkin lymphoma (10%), 1 chronic myeloid leukaemia (5%), 3 aplasia (15%), 1 myelofibrosis (5%) and 3 other (15%) (chronic myelomonocitic leukaemia, blastic plasmacytoid dendritic cell neoplasm and secondary engraftment failure). There were nine patients (45%) in first complete remission at the moment of undergoing HSCT. Results: With a median follow up of 11 months (1-24), the cumulative incidence of aGvHD grade I-IV and grade III-IV were similar (13.6% and 45%) to described by Ju¨rgen Finke (11.7% and 56,3%1). The incidence of CMV infection was higher (70% vs. 54%1 ) which might be due to a higher rate of seropositive patients (85% vs. 62%1) being of high risk (receptor þ /donor -) the 25% of them. Four patients (3 receptor þ /donor -) developed recurrent CMV Three patients (15%) had EBV viremia and one patient developed EBV-PTLD (5% vs. 4,8%1). Five patients suffered JC/BK cystitis o100 days. The percentage of patients who experienced at least one early (o100 days) bacterial and fungal infection were 60% and 5% (vs. 62,3%1 and 33%1), respectively. Fourteen patients were valuable for cGvHD. Eight patients developed cGvHD (57%) and three were extensive (21,4% vs. 12,2%2 ). Late infection (4 100 days) appear in 8/14 patients so that the percentage of bacterial, viral (excluding CMV and EBV) and fungal infection were 21%, 36% and 0%, respectively (viral and fungal infection were not mentioned in the referenced article2). The OS and DFS rates were 75% and 70%. Relapse occurred in 3 patients. The incidence of NRM in the first 100 days was lower (15% vs. 19,6%1,2) being the death causes EBV-PTLD, aGVHD grade IV and multiresistant pseudomonas pneumonia. Conclusion: In our cohort that includes older patients than other published low dose of ATG-F is effective to reduce the incidence of severe GvHD with low early NRM and high OS rate, though a longer follow up is necessary to confirm this results. References: 1. Ju¨rgen Finke, Lancet Oncology, 2009. 2. Ge`rard Socie`, Blood, 2011. 3. Ayuk F, Biol bone marrow transplant, 2008. Disclosure of Interest: None declared.
P226 Comparable outcomes of hematopoietic stem cell transplantation with myeloablative and reduced intensity conditioning in patients with acute myeloid leukemia – a single centre study J. Vydra1,*, V. Valkova1, M. Markova1, L. Novakova1, J. Schwarz1, C. Salek1, P. Cetkovsky1, A. Vitek1 1 Institute of Hematology and Blood Transfusion, Prague, Czech Republic Introduction: We analyzed retrospectively outcomes of allogeneic hematopoietic cell transplantation for acute myeloid leukemia performed at our centre with respect to the type of conditioning regimen. Materials (or patients) and methods: Patients treated with allogeneic hematopoietic cell transplantation (HCT) for acute myeloid leukemia at Institute of Hematology and Blood Transfusion in Prague between 1987 and 2014 were included in the study. Results: 225 patients were identified, 109 males and 116 females. Median age was 46 years (range; 19 – 67). 184 patients received myeloablative conditioning (MA group) and 41 reduced intensity conditioning (RIC group). Myeloablative conditiong regimens used were TBI/Cy (n ¼ 52), Bu/Cy (n ¼ 22), Bu/Flu(n ¼ 27), TBI/Flu/Ida (n ¼ 24) and TBI/Flu/AraC/ Ida(n ¼ 59). Reduced intensity conditioning regimen was Flu/Mel (n ¼ 32) or Bu8/Flu (n ¼ 9). In the MA group, 71 (39%) of patients received graft from matched sibling donor, 58 (32%) from 10/10 matched unrelated donor and 55 (29%) from mismatched unrelated donor (8/10 or 9/10). In the RIC group, 10 (24%) of patients received graft from matched sibling donor, 21 (51%) from 10/10 matched unrelated donor and 10 (25%) from mismatched unrelated donor (8/10 or 9/10), the difference in donor type was statistically significant between the groups (P ¼ 0.025). Prophylaxis of GvHD was cyclosporine and mycophenolate mofetil in RIC patients and in 56% of MA patients, other MA patients received cyclosporine with methothrexate. ATG-Fressenius 20 – 40mg/kg was given to patients with unrelated donors. Patients in the RIC group were older, with median age 60 years (range 23 – 67) compared to 43 (19 – 64) in the MA group (P ¼ 1e-9). Cytogenetic risk group, disease status, CMV serology and AB0 matching were not significantly different between the groups Median OS in the MA group was 2.31 years and 2.08 years in the RIC group (P ¼ 0.281). Cumulative incidence of relapse was 23% and 24% in the MA and RIC groups, respectively. In multivariate analysis of the effect of pretransplant and transplant variables on overall survival, only HLA mismatch, CMV status of the donor and graft type had statistically significant effect on overall survival. HLA mismatch led to decreased OS with HR of mortality of 4.33 and 3.16 for 8/10 and 9/10 mismatch, respectively. Age, gender match, cytogenetic risk group, AB0 match and type of conditioning used were not statistically significant. The overall survival was not different between MA and RIC groups even after adjustment for other transplant characteristics using the multivariate Cox proportional hazards model. Conclusion: In our retrospective single centre analysis, outcome of RIC based allogeneic transplantation for AML was comparable to myeloablative transplantation despite older patient population. Disclosure of Interest: None declared.
P227 Reduced-toxicity conditioning with 4 days of once-daily 100mg/m2 intravenous Busulfan associated with fludarabine and antithymocyte globulins prior to allogeneic stem cell transplantation in patients with high risk hematological malignancies J. Elcheikh1,*, A. wanquet1, R. crocchiolo1, S. furst1, A. granata1, C. faucher1, R. devillier1, S. harbi1, C. lemarie1, B. calmels1, R. bouabdallah2, N. vey2, P. J. weiller2, C. chabannon2, L. castagna1, D. blaise1 1 transplantation, 2hematology, institut paoli calmettes, marseille, France Introduction: The optimal intensity of myeloablation delivered as part of a reduced-intensity/toxicity conditioning (RIC/ RTC) regimen to decrease the recurrence rate, without increasing non relapse mortality (NRM), remains to be established and the disease control remains a major challenge. The introduction of RTC regimens has allowed allogeneic hematopoietic cell transplantation to be performed in patients who were previously considered too old or otherwise unfit. When busulfan Pharmacokinetic is not available the optimal dose is difficult to determine. In this perspective we made the hypothesis that decreasing the daily dose can be safer and efficient in high risk patients. Materials (or patients) and methods: We studied the outcome of 27 patients (median age, 50 years; range, 21-65 years) with hematological malignancies were included. The conditioning regimen based on busulfan at a dose of 100 mg/m2/day intravenously for 4 days, fludarabine at a dose of 30 mg/m2/day for 5 days, and antithymocyte globulins at a dose of 2.5 mg/kg/day for 2 days. Patient, disease and transplant characteristics are shown in Table 1. Results: No patients experienced graft rejection. The median HCT comorbidity index score was 2 (range, 0 to 5). With a median follow-up of 13 months (range, 3-16months), the cumulative incidences of grade 2 to 4 acute graft-versus-host disease (GVHD) and chronic GVHD (all grades) were 43% (95% CI, 26%460%) and 44% (95% CI, 20%468%), respectively. The Kaplan-Meier estimates of overall and disease-free survival at 1 year were 63% (95% confidence interval [95% CI], 42%484%) and 49% (95% CI, 27%471%), respectively. At 1 year, the cumulative incidence of recurrence/disease progression was 32% (95% CI, 12%452%). Non relapse mortality (NRM) was 4% and 19% at day 100 and at 1 year respectively. Patient age, diagnosis, donor type, sex, presence of comorbidities, and the Hematopoietic cell transplantation-specific comorbidities index did not appear to have any statistically significant impact on NRM, recurrence/disease progression, disease-free survival, or overall survival. Conclusion: This well-tolerated conditioning platform can lead to long-term disease control The RTC regimen used in the current study appeared to be safe, with a low NRM rate at 1 year noted among high-risk patients, and efficient disease control, warranting prospective phase 3 trials. Disclosure of Interest: None declared. P228 Application of global metabolomics to predict busulfan pharmacokinetics J. W. Lee1,*, B. Kim2, H. J. Kang1, C. R. Hong1, H. Y. Ju1, J. Y. Choi1, H. Kim1, K.-S. Yu2, I.-J. Jang2, J.-Y. Cho2, K. D. Park1, H. Y. Shin1, H. S. Ahn1 1 Department of pediatrics, Cancer Research Institute, 2Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine, Seoul, Korea, Republic Of Introduction: Busulfan shows narrow therapeutic range. High exposure is associated with systemic toxicity such as venoocclusive disease (VOD) and underexposure is associated with graft failure or relapse. In children, inter-patient pharmacokinetic variability was found to be high even after the use of intravenous (IV) busulfan. In this study, we identified
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biomarkers to predict busulfan pharmacokinetics using global metabolomics. Materials (or patients) and methods: Therapeutic drug monitoring (TDM) was done for pediatric patients who underwent busulfan-based hematopoietic stem cell transplantation (HSCT). The 6-hours interval urine samples were collected before busulfan administration in 59 patients. Patients were divided into 2 groups according to the area under the curve (AUC) of busulfan at the 1st day of busulfan administration; group 1 (n ¼ 50), AUC o25,000 mg*h/L and group 2 (n ¼ 9), AUC Z25,000 mg*h/L. The urinary metabolite profiling was performed by ultra performance liquid chromatography time of flight mass spectrometry (UPLC-TOF-MS) in conjugation with multivariate statistical analysis. Results: Multivariate analysis was performed between group 1 and group 2. Principle components analysis (PCA) of urinary profiling was not separated between group 1 and group 2 clearly, but group 2 tended to cluster together. The urinary metabolites associated with high level of busulfan AUC were identified as deferoxamine metabolites. Because deferoxamine was used for patients with high ferritin level before HSCT, we analyzed the association between ferritin level and the 1st day AUC in 136 pediatric patients. The 1st day AUC/dose (mg/m2) was higher in patients whose ferritin level Z1,000 ng/mL compared to that of the other patients (192.5±56.5 mg*h/L vs 164.4±40.0 mg*h/L, P ¼ 0.006). The optimal busulfan dose to meet the target AUC of 18,750 mg*h/L/day was 120.7±28.1 mg/m2 in patients with ferritin o1,000 mg/mL and 107.6±28.0 mg/m2 in patients with ferritin Z1,000 ng/mL (P ¼ 0.025). Conclusion: This study suggested the possibility of decreased busulfan metabolism in patients with iron overload or a possibility of drug interaction between busulfan and deferoxamine. Further study is needed to identify the exact mechanism of these results. Disclosure of Interest: None declared. P229 Impact of Anti-Thymocyte Globulin (ATG) on overall survival depends on HLA-C group KIR ligand status – Significant benefit in C1 homozygous HSCT recipients J. Clausen1,*, A. Bo¨hm1, V. Buxhofer-Ausch1, S. MachherndlSpandl1, D. Nachbaur2 1 Elisabethinen Hospital Linz, Linz, 2Internal Medicine V, University Hospital Innsbruck, Innsbruck, Austria Introduction: Anti-thymocyte globuline (ATG) is commonly used to decrease the risk for severe acute and chronic GVHD following allogeneic hematopoietic stem cell transplantation (HSCT). However, ATG has not been shown to significantly improve overall survival in randomized studies. Natural killer cell development after SCT is affected by T-cells, and both graft versus host (GVH) and graft versus leukemia (GVL) effects are influenced by killer immunoglobuline-like receptor (KIR) genotype and KIR ligands. Thus, in-vivo T-cell depletion by ATG may have different effects in subgroups defined by the recipient’s HLA-C KIR ligand status (C1/1, C1/2, or C2/2). Materials (or patients) and methods: We analyze retrospectively a two-center cohort of 553 consecutive, HLAmatched, related (n ¼ 361) or unrelated (n ¼ 192) HSCT. Graft source was peripheral blood (n ¼ 434), or bone marrow (n ¼ 110). HSCT were performed between 11/1988 and 1/ 2014. Anti-T-cell serotherapy was applied during conditioning in 178 transplants (mainly ATG-Fresenius (n ¼ 128), median dose 25mg/kg, range 10-90; or Thymoglobuline (n ¼ 44), median dose 5mg/kg, range 1.75-7.5; or Campath (n ¼ 6), 50100mg). Serotherapy was used for an unrelated donor in most instances (143), whereas some matched related transplants (35) included ATG (e.g., per protocol). The predominant indication for SCT was a hematologic malignancy, in an advanced phase in 313 cases.
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Results: In C1/1 homozygous patients (n ¼ 215), cumulative incidence of non-relapse mortality (NRM) at 4y was significantly lower following ATG- versus non-ATG based transplants (18% vs 39%; P ¼ 0.003), resulting in a trend towards better 4y overall survival (OS; 50% vs 39%, P ¼ 0.05). This effect of ATG was not found in C1/2 patients (n ¼ 249) or in C2/2 patients (n ¼ 89). By multivariate analysis considering major confounding factors (recipient age, donor age, disease phase, donor relationship, conditioning type, sex mismatch female-to-male), C1/1, but not C1/2 or C2/2 recipients significantly benefited from ATG in terms of overall mortality (RR, 0.50; P ¼ 0.01), treatment failure (progression or death) (RR, 0.67; P ¼ 0.04) and NRM (RR, 0.31; P ¼ 0.003). In contrast, survival of C2/2 recipients was inferior after ATG-based transplants (RR, 2.4; P ¼ 0.009). Conclusion: We show for the first time that the impact of ATG on HSCT outcomes depends on the recipient’s HLA-C KIR ligand status. In C1/1 homozygous recipients, ATG significantly reduced overall and non-relapse mortality, while in C2/2 recipients, ATG had the opposite effect. In the era of high-resolution HLA-typing, immunological determinants as the KIR ligand status may become more important for the decision for or against ATG, than the donor-recipient family relationship. Disclosure of Interest: None declared. P230 Low incidence of veno-occlusive disease after using intravenous busulfan in allogenic hematpoietic stem cell transplantation for acute lymphoblastic leukemia M. Ramzi1,*, M. Dehghani1, R. Vojdani1, M. Karimi1 1 department of hematology and oncology&stem cell transplantation, Shiraz university of medical sciences, Shiraz, Iran, Islamic Republic Of Introduction: BU and CY is a common conditioning regimen for allogeneic hematopoietic stem cell transplantation (HSCT).The use of i.v. busulfan instead of the oral busulfan can improve outcomes in patients undergoing HSCT by reducing toxicity and transplantation related mortality. Materials (or patients) and methods: We prospectively treated 42 Acute Lymphoblastic Leukemia( ALL) patients who underwent allogeneic HSCT at our center with BU/CY using i.v. BU without dose adjustment. IV BU was administered at a dosage of 0.8 mg/kg through a central venous catheter for 2 hours every 6 hours at a total of 16 doses for 4 days (On days -8 to -5). CY 40 my/kg /day was administered through a central venous catheter for 3 days on days -4 and -2. After one day rest (-1), GCSF-mobilized peripheral blood stem cell (PBSC) without T-cell depletion was infused on day 0. Results: All 42 patients achieved hematopoietic engraftment at a median 14days (range 9-23). All adverse events were those that are commonly observed in HSCT and no specific events related to i.v. BU were observed. The transplant related mortality for were 14.2%. The 1- and 2-year disease free survival (DFS) for i.v. BU group was 59.5%and 54.7% respectively. Disease relapse was the most common cause of death. We found also incidence of veno occulusive disease was very low with i.v. BU (4.8%). Conclusion: Our study demonstrates that i.v. BU conditioning regimen was associated with lower toxicity and promising survival. We found also lower incidence of veno occulusive disease after using i.v. BU in comparison with previous reported data and concluded i.v. BU/CY regimen is less toxic, safe, well tolerated and effective in our ALL patients. Disclosure of Interest: None declared.
Minimal residual disease, tolerance, chimerism and immune reconstitution I P231 Mixed chimerism after allogeneic hematopoietic stem cell transplantation in sickle cell disease: preliminary results after chimerism analysis on peripheral blood sorted cells and erythroid progenitors A. Magnani1,2,*, J. Magalon1,2, F. Touzot1,2, C. Cuzin1, J.-A. Ribeil1,2, F. Bernaudin3, D. Bories4, M. Cavazzana1,2 1 De´partement de biothe´rapie, Hopital Necker-Enfants malades, 2 Institut Imagine, Paris, 3Centre de re´fe´rence de dre´panocytose, CHIC Centre Hospitalier Intercommunal de Cre´teil, 4He´matologie Mole´culaire, Hoˆpital Henri Mondor, UPEC, Cre´teil, France Introduction: A small proportion of patients following hematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD) develop a persistent mixed chimerism (MC) over time associated with clinical control of the disease despite a seemingly low donor engraftment. In order to investigate this condition we analyzed the chimerism in sorted myeloid and lymphoid subpopulations, and erythroid progenitors. Materials (or patients) and methods: Between May 1990 and November 2012, 108 SCD patients followed in the pediatric Cre´teil center underwent allogeneic HSCT in different centers. Myeloablative conditioning consisted of busulfan (16 mg/Kg), cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (20 mg/kg). Among the 104 patients with available chimerism at 2 years, 3 had total autologous reconstitution, 54 (51.9%) had a MC, i.e. o95% of cells from donor origin on total white blood cells (WBCs), the majority ((n ¼ 50) of whom with MC 450%. These patients displayed the same hemoglobin (Hb) electrophoresis than their donors and were free of any SCD symptoms. We analyzed the chimerism in CD14 þ , CD15 þ , CD3 þ , CD19 þ and erythroid burst-forming units (BFU-E) in patients presenting with: (a) donor MCo70% on WBCs, (b) follow-up Z1 year after HSCT and (c) the same Hb electrophoresis as their donors. Results: Preliminary results obtained from 9 patients reveals heterogeneous pattern of subpopulation chimerism (Table 1). The mean follow-up duration is 88.6 months (range 21-155). Patients present a stable Hb comparable to the donor’s. A discrepancy in donor chimerism measured in total WBCs compared to the subpopulations or the erythroid precursors is observed. Patients were divided into two groups according to the donor chimerism in the CD3 þ and in the myeloid compartment. The first group includes patients with donor chimerism in CD3 þ cells lower than in the myeloid compartment. This could be explained by the expansion of recipient’s memory T cells after transplantation and/or a lower T-cell progenitor engraftment in these patients. The second group includes patients with a donor chimerism higher in the CD3 þ than in the myeloid compartment. Interestingly, there is a trend to an higher percentage of donor chimerism in the BFU-E as compared to the myeloid compartment suggesting a selective advantage of donors cells during erythropoiesis.
Donorpheno type
Hb (g/dl)
HbA (%)
HbS Donor chimerism (%) (%) TotalCD3 þ CD14 þ CD15 þ CD19 þ BFUE
AA AA AS AS AS AA AS AS AA
14.2 11.8 13.5 10.6 8.4 10.5 9.7 9.5 10.5
84.5 79.7 50.2 54 24 78.3 31.7 43.4 79.8
0 5.6 37.9 33.3 68.1 7.6 60.6 47.5 4.3
66 19 58 53 21 44 16 18 30
40.5 12 34 44 70 46 31 30 63
98.5 23 71 44 9.4 41 12 26 17
495 31 na 46 5.4 na 10 16 18
90 13 49 58 31 50 13 11 23
Conclusion: These data show that the analysis of chimerism on total WBCs is a poor indicator of the myeloid and erythroid engraftment in SCD patients. Analysis of chimerism on sorted subsets and erythroid progenitors allows a more detailed investigation of this population. Final analysis will include about twenty cases of MC and further investigation on memory/naive T cells compartment as well as on granulocyte monocyte colony forming units (CFU-GM) are necessary to complete these informations. This study may have remarkable implications not only in the setting of HSCT but also for gene therapy trials based on the autologous transplantation of genetically modified CD34 þ cells. Disclosure of Interest: None declared. P232 Delayed CD4 þ T Lymphocyte reconstitution in Haploidentical T Cell Repleted Transplantation with Postransplant Cyclophosphamide compared with HLA Identical Sibling Donor Transplantation in the setting of reduced intensity conditioning regimen A. M. Pe´rez-Corral1,*, D. Champ1, A. C. Franco1, J. Gayoso1, C. Pascual1, J. Anguita1, M. Bastos1, M. Kwon1, I. Gonzalez1, C. Mun˜oz1, D. Serrano1, J. L. Dı´ez-Martı´n1 1 Hematology, Gregorio Maran˜on Hospital, Madrid, Spain Introduction: Haploidentical T Cell Repleted Transplantation(Haplo HSCT) with Postransplant Cyclophosphamide (PTCy) has become a therapeutic option for high risk patients without HLA identical sibiling donor. As previously described, immune reconstitution seems to be appropriate in these patients but few publications comparing with conventional HLA identical sibling transplantation (HLAid HSCT) have been reported to date. Materials (or patients) and methods: We retrospectively recorded 28 pts who received RIC Haplo HSCT with PTCy and 13 pts who received RIC HLAid HSCT between 2007-2014 in our institution. Conditioning included fludarabine (Flu), cyclophosphamide (Cy) and busulfan (Bux) for Haplo and Flu/Bux/melphalan for HLAid. Prophylaxis for GVHD consisted of high dose Cy on þ 3/ þ 4, cyclosporineA (CsA) and
na 52 76 25 12.5 61 17 34 31
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mycophenolate for Haploid and CsA/methotrexate for HLAid. Stem cell source was mobilized peripheral blood in all cases. Immune reconstitution was assessed in terms of lymphoid subpopulations quantification by 5 colour flow-cytometry on FC500 or Navios (Beckman Coulters) on months þ 1, þ 3, þ 6, þ 9, þ 12. X2 and Mann–Whitney test were used for comparison. Results: No statistical differences between patients characteristics in the two groups were found regarding sex, disease status at transplantation, CD34 þ cells infused and acute GVHD incidence. Some differences were shown in diagnosis with more non Hodgkin Lymphoma in HLAid group and more Hodgkin Lymphoma in Haploid group (p0.04), and in median age (38 for Haploid and 52 for HLAid, p 0.006). Table 1 shows the median lymphocyte counts in both groups. In general terms Haploid group reconstitution was good. No differences were recorded in NK cell total counts on any of the studied time points. Total T cell counts, CD4 þ cells , CD8 þ cells were significantly lower in the first three months (time points þ 1 month and þ 3 months) in the Haploid group (Po0.01). On þ 6, þ 9 and þ 12 months CD4 þ cells remained at slightly lower levels in Haploid group without statistically significant differences, while total T cells and CD8 þ lymphocytes were similar in both groups. B cells were practically absent in the first month in Haploid group, but reconstituted from the third month on, with no significantly differences on þ 3, þ 6, þ 9 and þ 12 between both groups. Conclusion: As previously shown, immune reconstitution in Haploid HSCT pts is good. Despite the heterogeneity in initial diagnosis, we observe worse immune reconstitution in our Haploid HSCT group in the first month and a tendency for more delayed CD4 þ cells reconstitution compared with this HLA id HSCT patients. More studies are needed to confirm these findings and determine its clinical implications. Disclosure of Interest: None declared. P233 Immune reconstitution after allogeneic stem cell transplantation: Impact of reduced conditioning regimen, alemtuzumab, anti-thymoglobulin and CMV infection A.-S. Hartjen1, A. Krumbholz2, F. Thieme3, M. Bulduk1, N. Schub1, A. Humpe1, M. Gramatzki1, A. Guenther1,* 1 Division of Stem Cell Transplantation and Immunotherapy, 2nd Medical Department, Christian-Albrecht University of Kiel, 2 Institute for Infection Medicine, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein Campus Kiel, 3Central laboratory, University Medical Center SchleswigHolstein Campus Kiel, Kiel, Germany Introduction: The renewal of the immune system after allogeneic stem cell transplantation is a complex process and not fully understood. Recently, the use of reduced intensity conditioning (RIC) regimens, new anti-viral drugs and intensified GvHD treatment changed the landscape of allogeneic transplantation. However, establishment of a functional immune response against the underlying disease as well as control of infections is still crucial for the long-term outcome. Here, the influence of several parameters on the development of the cellular and humoral immunity of 136 patients consecutively transplanted in a single center was analyzed. Materials (or patients) and methods: All adult patients (81 male, 55 female, median age 52 years) transplanted within three years starting in September 2010 were analyzed. Retrospectively immunoglobulin levels (turbidimetric method) and lymphocyte subset counts by FACS staining were evaluated. In 91 % of the patients a reduced or intermediate conditioning regimen was used; nearly all patients received either anti-thymoglobulin (ATG) or alemtuzumab (25/136 matched related donors) leading to a relatively low rate of chronic GvHD (40%). The immune parameters achieved one year after stem cell transplantation were correlated with the conditioning regimen, diagnosis, relapse, age, sex and history
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of CMV infection, acute and chronic GvHD using Pearson correlation, t-test and regression analysis (SPSS). Results: A normal CD4/CD8 ratio at day þ 360 was only seen in 14% of available patients (3% elevated, 83% reduced) mostly caused by impaired CD4 cell count (90%onormal) while CD8 cell count was increased in 42%. The IgG level was normal in 51%, reduced in 31% and increased in 17 % of tested patients. The CD8 cell count was significantly higher in patients after CMV infection (Po 0.001) and without alemtuzumab (P ¼ 0.017) in the conditioning regimen; the IgG level correlated with history of CMV infection (P ¼ 0.002), CD4, NK (both P ¼ 0.002) and CD8 (P ¼ 0.025) cell count; age, sex, diagnosis, relapse, use of reduced intensity conditioning, acute and chronic GvHD had no significant impact on immune system one year after stem cell transplantation Conclusion: Despite using frequently RIC regimens, one year after allogeneic stem cell transplantation nearly all patients showed a compromised immune system mainly characterized by a reduced CD4 cell count. In contrast, the CD8 cell count is often elevated, in part probably reflecting response to viral infections. Interestingly, the immunoglobulin levels are highly variable ranging from antibody deficiency to hyper-immunoglobulinemia not fully explained by viral triggers. Surprisingly, GvHD had only a minor impact on the immune reconstitution what may be explained by the frequent use of alemtuzumab and ATG in the investigated patient cohort. Disclosure of Interest: None declared. P234 Integrative Profiling of Natural Killer Cell Repertoires Reveal a Role for Less Differentiated NK Cells in Protection from Leukemia Relapse A. T. Bjo¨rklund1,2,*, T. Clancy3, M. Schaffer4, P. Ljungman1, K.-J. Malmberg3 1 Hematology Center Karolinska University Hospital, 2Center for Infectious Medicine, Karolinska Institute, Stockholm, Sweden, 3 Dep of immunology, Radiumhospitalet, Oslo, Norway, 4Dep of Immunology, Karolinska University Hospital, Stockholm, Sweden Introduction: Reactivation of cytomegalovirus (CMV) following transplantation leads to profound changes in the recipient natural killer (NK) cell repertoire with expansion and differentiation of educated NK cells expressing self-specific inhibitory killer cell immunoglobulin-like receptors (KIRs). It has been postulated that such adaptive-like NK cell responses may contribute to anti-leukemic activity thereby protecting from relapse. Materials (or patients) and methods: To address this possibility, we examined donor NK cell repertoires in 106 peripheral stem cell donors and the dynamic reconstitution in 65 donor-recipient pairs. Hierarchical clustering of key parameters (frequencies of CD56bright, CD57 þ , NKG2A þ , NKG2C þ , educated NK cells) that define the heterogenic NK cell compartment, linked clusters with distinct NK cell repertoires, to clinical outcomes. Results: Clusters defining naı¨ve NK cell repertoires in the donor were associated with decreased risk for relapse; HR 0.15, 95% CI (0.14-0.93), P ¼ .04. Corroborating this finding, recipients with naı¨ve NK cell repertoires at 9-12 months and those that displayed resetting of donor repertoires to less differentiated repertoires had lower risk for leukemic relapse; HR 0.18, 95% CI (0.05-0.71), P ¼ .01, and better overall survival; HR 0.11, 95% CI (0.03-0.65), P ¼ .01. Conclusion: In contrast to current beliefs our data suggests that maintenance of a naı¨ve NK cell repertoire rather than an expansion and differentiation of memory-like NK cells is associated with protection against leukemia relapse after allogeneic stem cell transplantation. Disclosure of Interest: None declared.
P235 Donor KIR A haplotype is associated with an increased in T cell recovery after DLI and better overall survival after HLA match sibling pediatric stem cell transplantation A. Pe´rez Martı´nez1,*, I. Romera2,3, J. Valentı´n4, L. Fernandez5,
J. L. Vicario6, M. A´. Dı´az7 1
Servicio de Hemato-Oncologı´a Infantil. Hospital La Paz., Universidad Auto´noma de Madrid, 3Servicio de Hematooncologia infantil, Hospital Monteprı´ncipe, 4IdiPAZ, 5Centro nacional de investigaciones oncolo´gicas (CNIO), 6Servicio de tipaje y HLA, Centro regional de transfusiones de la Comunidad de Madrid, 7Servicio de Hemato-Oncologı´a Infantil., Hospital Nin˜o Jesu´s, Madrid, Spain
P236 CD4 þ FoxP3 þ Regulatory T Cells Induce Tolerance to Bone Marrow Grafts and Promote B Cell Differentiation A. Pierini1,*, H. Nishikii1, Y. Pan1, J. Baker1, D. Schneidawind1, M. Alvarez-Rodriguez1, D. Leveson-Gower1, M. Florek1, R. Negrin1 1 Department of Medicine, Division of Blood and Marrow Transplantation, Stanford University, Stanford, United States
2
Introduction: Graft versus leukemia (GvL) effect after hemotopoietic stem cell transplantation (HSCT) is mediated by donor immune cells recovery. Natural Killer (NK) cell alloreactivity is controlled by the interaction of activatory receptors and inhibitory killer-immunoglobulin-like receptors (KIRs) with major histocompatibility locus class I antigens on the leukemia cells. Haploidentical setting is a plattform for NK cell alloreactivity however HLA identical setting remains unclear. Materials (or patients) and methods: We performed KIRgenotyping of HLA-identical sibling donors in 35 pediatric CD34 selection peripheral blood stem cell transplantations with donor lymphocyte infusion (DLI) at day þ 30 to identify genetic factors affecting leukemia relapse and overall survival. Univariate analysis of leukemia relapse and KIR genotyping was performed in order to identify independent variables predictive of outocome for pediatric acute lymphoblastic leukemia (ALL), (n ¼ 20) and acute myeloblastic leukemia (AML), (n ¼ 15). Results: Donor B haplotype was observed in 21 cases (60%). Statistical analysis shown that donor B haplotype was associated with significantly more relapse in leukemia pediatric patients (38% vs. 0%) and worse overall survival (40% vs. 7%). Further analysis revealed that 2DL5a, 2DS3 and 2DS5 were associated with an increased rate of leukemia relapse (47% vs. 6%, 54% vs. 10% and 58% vs. 13%, respectively) and 2DL5a, 2DS1 and 3DS1 were associated with a worse overall survival (48% vs. 6%, 64% vs. 10% and 58% vs. 13%, respectively). No difference was observed in patients KIR haplotype or KIR donor-recipient KIR haplotype mismatch. We also observed a positive impact of DLI on immune reconstitution, specifically in KIR A donors. T-cell reconstitution was increased on KIR A donors with a median of 1282 vs 477 CD3 þ cells/ml, 205 vs 146 CD3 þ CD4 þ cells/ml and 1002 vs 294 CD3 þ CD8 þ cells/ml on day þ 60, respectively. NK cell reconstitution was impired on KIR A donors with a median of 182 vs 242 CD56 þ cells/ml. Conclusion: In our study, which included only reduced intensity conditioning without antithymocyte globulin followed by related peripheral blood stem cell transplantation and DLI, we found a significant better impact of donor KIR A haplotype in stem cell transplantation outcome. Our results suggest that donor KIR A haplotype had a better T cell expansion after DLI and could decrease leukemia relapse in HLA-identical sibling HSCT. In this setting, T cell alloreactivity dominates NK alloreactivity. References: Kro¨ger N1, Binder T, Zabelina T, Wolschke C, Schieder H, Renges H, Ayuk F, Dahlke J, Eiermann T, Zander A. Low number of donor activating killer immunoglobulin-like receptors (KIR) genes but not KIR-ligand mismatch prevents relapse and improves disease-free survival in leukemia patients after in vivo T-cell depleted unrelated stem cell transplantation. Transplantation. 2006 Oct 27; 82(8): 10241030. Disclosure of Interest: None declared.
Introduction: CD4 þ FoxP3 þ regulatory T cell (Treg) adoptive transfer has proved to prevent GVHD while Treg impact on immune reconstitution and engraftment has been less well studied. Materials (or patients) and methods: We tested the impact of Diphtheria Toxin (DT) induced Treg ablation in C57BL/6 FoxP3DTR mice in different transplantation models. We used mass cytometry (Cytof) and FACS analysis for studying bone marrow populations. Cytokines were quantified by ELISA assays. Bone marrow cell localization has been explored with confocal microscopy. Sorted purified CD4 þ CD25 þ FoxP3 þ Treg from lymph nodes and spleen were adoptively transferred for several chimerism and reconstitution studies as reported. Results: Transplantation of lethally irradiated (TBI 10 Gy) Treg depleted mice with allogeneic (BALB/C or FVB/N) T-cell depleted bone marrow (TCD BM) or purified hematopoietic stem cells (HSCs) resulted in donor rejection or mixed chimerism (Po0.01). Treg depletion favored host CD4 þ (Po0.001), CD8 þ (Po0.01) and GR1 þ cell (Po0.01) persistence and delayed B cell reconstitution (Po0.001). Treg transfer to DT treated mice rescued engraftment (Po0.01) and boosted B cell reconstitution (Po0.001). Syngeneic transplanted Treg depleted mice engrafted but had markedly delayed B cell reconstitution (Po0.01) thus Treg promote donor B cell differentiation in manner not dependent upon alloreactivity. These mice had higher numbers of donor Lin-Sca1 þ cKit þ HSCs (Po0.05) and Lin-Sca1 þ cKit þ Flt3 þ lymphoid progenitors (Po0.05) while B220 þ IgM-CD19 þ cKit þ Pro-B cells (Po0.05) and total CD19 þ cells (Po0.01) were reduced showing a block of maturation in the early phases of B cell differentiation. IL-7 production was reduced after Treg depletion (Po0.05) and IL-7 administration rescued B cell reconstitution (Po0.05) in syngeneic transplanted Treg depleted mice demonstrating that Treg control B cell differentiation through an IL-7 dependent mechanism. We found that Treg localize near the endosteum (Po0.0001) co-located with donor B220 þ B cell clusters and gather in the epiphyseal areas where donor HSCs were mainly detectable suggesting that Treg act as an immunological barrier for HSCs and B cell progenitors, providing a protective niche. Treg transfer induced B cell reconstitution (Po0.01) in non irradiated immune deficient BALB/C rag2-/-gc-/- mice that received allogeneic TCD BM. These mice had higher numbers of Pro-B (Po0.05) and total CD19 þ cells (Po0.05) with reduced levels of inflammatory cytokines such as INFg and IL12 and increased IL-7 and VEGF. In a non myeloablative (TBI 5.5 Gy) allogeneic (C57BL/6 -4 BALB/C) model of rejection, host Treg adoptive transfer induced persistent full donor chimerism if Treg were injected together with a sublethal dose of donor T cells (P ¼ 0.016) and enhanced donor chimerism if Treg were injected with low dose IL-2 (Po0.001) without impacting on GvHD. Conclusion: Our findings indicate that Treg act as a key regulator of B cell differentiation promoting production of mature B cells from lymphoid precursors through an IL-7 mediated mechanism not dependent on alloantigen recognition. Our data suggest that Treg play a role in building the donor HSC and B cell precursor niche. Finally, Treg adoptive transfer induces immune reconstitution and tolerance to bone marrow grafts even in the absence of conditioning providing a new tool for clinical translation especially in children with SCID or hemoglobinopathies. Disclosure of Interest: None declared.
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P237 Immunogenetic Cross-Talk in Patients Transplanted for AML: CMV Reactivation Is Not a Strong Stimulus for Immune Response Against Leukemia A. Papalexandri1,* on behalf of Hematology Department and BMT unit, George Papanicolaou Hospital, Z. Boussiou1, C. Apostolou1, S. Charalampidou1, P. Zerva1, T. Touloumenidou1, V. Konstantinou1, D. Mallouri1, I. Batsis1, A. Anagnostopoulos1, I. Sakellari1 1 Hematology Department and BMT Unit, G.PAPANIKOLAOU HOSPITAL, THESSALONIKI, Greece Introduction: Early cytomegalovirus (CMV) re-activation after allogeneic hematopoetic stem cell transplantation (allo-HCT) for AML has been correlated with enhanced graft versus leukemia (GvL) effect, thus implying an immunogenetic cross- talk between donor-derived cytotoxic response against both CMV and leukemia. Data on this are conflicting and correlation could not be confirmed for special group populations. Materials (or patients) and methods: In this study we explore whether CMV re-activation favors the outcome in a group of 49 AML patients consecutively allo-transplanted between 2008-2012. Forty-nine patients (29 male, 20 female), aged 14-63 years (median 42) underwent allo-HCT with myeloablative (42/49) or reduced-intensity conditioning (7/49), receiving graft from an HLA- identical sibling (25/49), matched volunteer (23/49) or haplo-identical (1) donor. Most patients were transplanted in complete remmision (CR), 22/49 in CR1 and 11/49 in CR2 and 16/49 patients had refractory disease. Monitoring of CMV viral load was performed weekly for 3 months with real-time PCR, or more if patients were treated for GvHD. Upon clinical suspicion, other samples were tested. Results: Overall, 19/49 patients (39%) presented with CMV reactivation {viremia (4 ¼ 1000 copies/ml) in 16/49, gastritis in 2/49, cystitis in 1) at a median of 46 days post-tranplant (13402). Leukemia relapse occured in 7/19 patients with CMV at a median of 189 days (2-512), versus 7/30 CMV-free patients (37% vs 23%, P ¼ ns). Disease free survival (DFS) was 591 days for CMV patients (95% CI: 417-939) and 609 for CMV-free (95% CI: 474-871, P ¼ ns). Overal survival (OS) was 746 (95% CI:472963) and 625 (95% CI:504-899) days, respectively, P ¼ ns. Only acute and chronic GvHD were correlated with longer DFS and OS. Sixteen out of 19 CMV (84%) patients presented with increasing/full donor chimerism 30 days after reactivation, due to expansion of donor cytotoxic lymphocytes; however, this did not protect from imminent relapse. No particular clinical correlations could be found even for 14/19 patients with early (o100 days) CMV reactivation (RR: 28% vs 28% for all other patients, DFS: 714 vs 503 days, OS: 746 vs 625 days, P ¼ ns for all comparisons). Conclusion: In conclusion, CMV reactivation in AML transplanted patients is not related to graft allo-reactivity. Late relapses in this cohort of AML patients, transplanted often in remission, are not influenced by immunogenetic dynamics at the first semester, when CMV occurs. Regardless CMV reactivation, OS was comparable in all patients, providing evidence on a succesfull pre-emptive treatment of CMV. Larger prospective studies are strongly warranted in order to explore possible immunogenetic interactions. Disclosure of Interest: None declared.
P238 Long-term stable mixed chimerism in patients undergoing HSCT for non-malignant disorders A. Stikvoort1,2,*, J. Mattsson1,2, M. Sundin3,4, M. Uhlin1,2, B. Sundberg1, M. Uzunel5 1 Oncology-Pathology, Karolinska Institutet, Stockholm, 2Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Huddinge, 3Clinical sciences, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, 4 Hematology/Immunology section, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Huddinge, 5Therapeutic Immunology, Karolinska Institutet, Stockholm, Sweden Introduction: Long-term stable mixed chimerism (MC) is rare after allogeneic hematopoietic stem cell transplantation (HSCT). MC is defined as 5-95% residual recipient hematopoietic cells beyond one-year post-HSCT. The mechanisms directing hematopoietic recovery to MC in some patients and to full donor chimerism (DC) in others are poorly understood. Materials (or patients) and methods: We compared patients with DC and long-term stable MC for a median of 9.5 years postHSCT. Both the clinical situation and the immune system (functional and phenotypic properties) were investigated by questionnaires, western blot, multiplex bead-based immunoassay, ELISA and flow cytometry. Blood samples were drawn at 2 weeks and 45 years post-HSCT. Additionally, lineage-specific chimerism status in the MC patients was determined. The results were analyzed by the Mann-Whitney U test, Fisher’s exact test, Spearman’s rank correlation coefficient, ordinary fit least squares linear regression and robust fit linear regression. Results: At 2 weeks post-HSCT cytokine levels did not differ between the groups. Neither did the groups differ in long-term outcome in terms of general well being, immunoglobulin response to vaccinations and in the most central phenotypic features of the immune system (e.g., differentiation status, CD4/ CD8 ratio, B and NK cell frequency). More than 5 years post-HSCT, MC patients had significantly higher NKT cell levels (CD94 þ CD8 þ and CD56 þ CD8 þ T cells) than DC patients. In depth analysis of the MC patients revealed that recipient chimerism could be observed in a large variety of immune cell lineages (i.e., total, CD4 þ , CD8 þ and gd þ T cells; B cells; NK cells; myeloid cells; and cytokine producing cells). Furthermore, the percentages of recipient chimerism in different cell lineages were positively correlated with each other, e.g., an increase in CD3 chimerism was associated to an increase in CD56 chimerism. Both recipient and donor derived cells in MC patients produced cytokines to the same degree in response to mitogen stimulation. No difference in mitogen response was observed between the groups. Conclusion: Long-term MC does not appear to negatively affect the well being of the patients. It could even be argued that the recipient system has a more differentiated phenotype than the donor system, indicating that MC might be of benefit, protecting against infections. The recipient immune system may still be functional in MC patients, as both recipient and donor derived cells responded to mitogens. Two scenarios could possibly explain such a symbiotic relationship between donor and recipient system: a superfluous duplicity of the entire immune system or an adjuvant role by the recipient system to the donor system. Disclosure of Interest: None declared. P239 Early post-allogeneic transplantation WT1 transcript positivity predicts AML relapse C. Messina1,*, E. Sala1, M. Carrabba1, F. Pavesi1, L. Vago1, B. Gentner1, C. Tresoldi1, M. Tassara1, E. Xue1, C. Corti1, J. Peccatori1, F. Ciceri1, M. Bernardi1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Patients (pts) with refractory or relapsed acute myeloid leukemia (AML) or high risk myelodysplastic syndrome (MDS) receiving allogeneic hematopoietic stem cell transplantation (HSCT) with active disease have a poor outcome. Thus,
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HSCT and post transplant immunotherapy need to be further optimized to treat pts with persistent disease at HSCT. When complete remission (CR) is achieved after myeloablative transplant conditioning, reliable approaches are essential to detect minimal residual disease (MRD) and predict the risk of relapse. The Wilms’ tumor gene 1 (WT1) is over-expressed in the bone marrow (BM) of 480% of AML and advanced MDS pts, and is considered a promising marker for MRD detection. We retrospectively evaluated data from BM of pts with active AML or MDS transplanted at our Institute and in CR after HSCT, to evaluate the sensitivity and specificity of quantitative WT1 monitoring to predict disease relapse. Aim of the study was to evaluate if early post-HSCT WT1 positivity in BM of AML/MDS pts in CR correlates with subsequent overt relapse. Materials (or patients) and methods: We reviewed our institutional database in order to select AML/MDS pts receiving HSCT for active WT1 overexpressing disease. Between 1/2009 and 11/2013, 38 consecutive pts: 22 AML, 4 sAML, 3 tAML, 1 AML-GS and 8 MDS; source of HSCT: 7 MRD, 2 MUD, 27 MMRD, 2 CB. During post-transplant follow-up (FU), BM WT1 levels were quantified using the ELN WT1 assay with a cut-off level of positivity of 250 copy numbers [1], from 34 evaluable pts. We analysed BM data from the three earliest time points of FU after HSCT (day þ 30, þ 60 and þ 90) and latest FU available for each patient. Results: At day þ 30 after HSCT 33 out of 34 pts (97%) achieved CR. One patient experienced a WT1-negative relapse and 4 pts were excluded because they lacked complete molecular data for the time points considered in the present analysis. Thus, 28 pts were evaluable for early WT1 monitoring after HSCT. Overall, at a median follow-up (FU) of 472 days (range 55-2110), 16 pts (60%) had relapsed while 12 pts (40%) were still in CR. Of the relapsed pts, 12 (75%) showed at least one early BM WT1 positivity while in haematological CR and 4 (25%) had WT1 levels4250 during all time points. On the contrary, only 3/12 (33%) of pts who did not relapse had a positive WT1 at Z1 timepoint while 9/12 (67%) had WT1 levels below the threshold of 250 during all time points. We observed a significant correlation between positive early BM WT1 levels and subsequent haematological relapse and between negative early BM WT1 levels and persistence of CR (Fisher’s test P ¼ 0.02). BM WT1 positivity anticipated the haematological relapse of 158 days (range, 20-360). Conclusion: Early post HSCT BM WT1 positivity is a promising tool to predict relapse in AML/MDS pts transplanted with active disease that achieved haematological CR after HSCT. A single quantitative level above the threshold of MRD positivity in the early post HSCT phase is predictive of subsequent overt disease relapse, with a median time to relapse of 158 days. Our results suggest that quantitative monthly monitoring of BM WT1 in the first 3 months after HSCT is useful to predict relapse and to drive proper post transplant immunotherapy (e.g. immunosuppression withdrawal and/or donor lymphocyte infusion) to induce an anti-leukemia effect at an early stage. References: 1. Cilloni et al. J Clin Oncol. 2009 Nov 1;27(31):5195-201. Disclosure of Interest: None declared. P240 Droplet Digital PCR for DNMT3A and IDH1/2 Mutations to Improve Early Diagnosis of Acute Myeloid Leukemia Relapse after Allogeneic HSCT C. Brambati1,*, C. Toffalori1, E. Xue1,2, L. Crucitti2, R. Greco2, A. Crippa3, L. Chiesa1, B. Mazzi1, C. Tresoldi3, S. Galbiati4, N. Soriani4, J. Peccatori2, M. G. Carrabba2, M. Bernardi2, M. Ferrari4, V. Lampasona4, L. Vago1,2, F. Ciceri2 1 Unit of Molecular and Functional Immunogenetics, 2Unit of Hematology and Bone Marrow Transplantation, 3Molecular Hematology Laboratory, 4Genomics for the Diagnosis of Human Pathologies Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Leukemia relapse after allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) represents a major
unsolved issue, and efforts are aimed to anticipate relapse detection and treatment to the Minimal Residual Disease (MRD) stage. Mutations in DNMT3A and IDH1/2 occur early during the step-wise process of Acute Myeloid Leukemia (AML) tumorigenesis, possibly representing disease founder mutations (Shlush et al., 2014) and thus putatively optimal markers for MRD monitoring. Materials (or patients) and methods: By conventional Sanger sequencing, we screened for presence of the mutations of interest (DNMT3A R882H, IDH1 R132C, IDH1 R132H, IDH2 R140Q and IDH2 R172K) 67 AML samples collected at diagnosis from patients who underwent allo-HSCT at our center. We selected 19/67 patients who carried at least one of the mutations and tested a total of 79 bone marrow samples collected longitudinally over time by ultra-sensitive droplet digital PCR (ddPCR) assays. As controls, we tested diagnosis bone marrow samples from 10 patients typing negative for the mutations, and peripheral blood samples from 15 healthy individuals. ddPCR assays were performed using the Bio-Rad QX100 system: each sample was tested in duplicates, employing 25 ng of genomic DNA in each reaction well. Samples with a mutant-to-wild-type ratio above 0.1% were considered positive. ddPCR results were compared to those obtained testing the same samples by quantitative PCR (qPCR) assessment of the WT1 gene transcript and of hematopoietic chimerism. Results: All the samples which resulted positive by conventional sequencing were confirmed by ddPCR, and in all of them the population carrying the mutant allele, quantified by ddPCR, consistently exceeded the morphological count of leukemic blasts, suggesting the presence of the mutation also in apparently normal hemopoietic cells. All the samples tested at post-transplantation relapse resulted positive for the mutations present at diagnosis, except for one case, originally carrying both DNMT3A and IDH2 mutations and typing negative for the latter at relapse. When post-transplantation remission samples were tested, 41/42 (98%) of those harvested from 5 patients who remained long-term leukemia-free (median follow-up after allo-HSCT: 20 months) resulted negative for the mutations of interest. 4/5 patients who subsequently relapsed presented at least one sample harvested during apparent disease remission which resulted positive by ddPCR, 3 of which immediately preceeding relapse. Of notice, only 2 of those 5 patients displayed WT1 transcript overexpression before relapse and only one displayed host chimerism above the 1% threshold. Conclusion: Although the very small number of patients included in this preliminary analysis warrants for caution, ddPCR for DNMT3A and IDH1/2 mutations appears extremely promising, displaying very high specificity and sensitivity in relapse prediction, and comparing favorably with our present and historical results obtained by qPCR-based post-transplantation monitoring techniques. References: Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia (Shlush et al., Nature, 2014). The origin and evolution of mutations in acute myeloid leukemia (Welch et al., Cell, 2012). Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia (Kro¨nke et al., Blood, 2013). DNMT3A Mutations in Acute Myeloid Leukemia (Ley et al., NEJM, 2010). Disclosure of Interest: None declared. P241 Automated workflow for microchimerism analyses A. Wijngaard1, B. Luiken1, E. Torun2, N. Westerink1, J. Geerligs1, E. Rozemuller1,3, D. Bost3,* 1 Research and Development, GenDx, Utrecht, Netherlands, 2ATQ Biyoteknoloji, Ankara, Turkey, 3Research and Development, KimerDx BV, Utrecht, Netherlands Introduction: In an effort to enhance qPCR-based microchimerism analysis, we are investigating the ability to
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automate microchimerism analysis workflow using robotic pipetting stations and the Rotor-Genes qPCR platform. The use of robotic systems with our proprietary reagents and software will likely automate the process of post-transplant monitoring almost completely. The robotic station prepares and dispenses the reaction mixes, while our software provides reaction set up and analysis tools. With its rapid software analysis and not needing post-PCR sample processing, our microchimerism analysis workflow may become the most automated to date. Materials (or patients) and methods: We currently utilize analysis software and a panel of 30 qPCR assays to comparatively genotype and quantify DNA samples. The assays are designed and validated to detect INDEL and copy number variants between individuals on 20 different chromosomes. These differences are then used to track an individual’s genomic material in a mixed sample. Results: The genotyping is performed using 10 mixtures, each containing 3 qPCR assays multiplexed in a single well. We program the robot to set up the genotyping experiment and load the samples into the 72-well ring. Our software, combined with the RGQ 72-well ring configuration, enables simultaneous comparative genotyping of up to six samples. By tracking the relationships between samples, the software automatically selects informative markers between 2 or more individuals. The results from the genotyping are stored by the software for recall during subsequent monitoring. For monitoring an individual in a mixture, informative markers are used in a singleplex configuration, with each marker validated to a sensitivity of 0.05% of a genomic mixture. Reports from a monitoring test may be generated from a single time point or longitudinally, using the data collected from the individual over time. Longitudinal reports provide a temporal view for better understanding of rejection or relapse kinetics. Conclusion: With the development of our software, reagents and automated platform approach, our workflow reduces manual calculations, increases throughput and helps to minimize errors in test execution, potentially making it a fully-automated solution for microchimerism analysis. Disclosure of Interest: A. Wijngaard Employee of: Employee, B. Luiken Employee of: Employee, E. Torun Employee of: Employee, N. Westerink Employee of: Employee, J. Geerligs: None declared, E. Rozemuller Employee of: Employee, Conflict with: Shareholder, D. Bost Employee of: Employee, Conflict with: Shareholder. P242 Nucleophosmin 1 Mutation Monitoring after Allogeneic Hematopoietic Stem Cell Transplantation to Predict Relapse and Guide Preemptive Therapy E. Xue1,2,*, E. Sala1, C. Tresoldi3, L. Crucitti1,2, R. Greco1, C. Messina1, M. Carrabba1, L. Vago1,2, M. Bernardi1, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, 2Unit of Molecular and Functional Immunogenetics, 3Molecular Hematology Laboratory, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Nucleophosmin 1 (NPM1) mutations represent the most frequent genetic alterations in adult patients with acute myeloid leukemia (AML). In addition, NPM1 mutations are considered early events in leukemogenesis, shared by the majority of leukemic subclones in a given patient and remaining stable throughout the disease course. For these reasons, they represent promising minimal residual disease (MRD) markers for the longitudinal monitoring of AML patients. In the present study we investigated the potential value of NPM1 mutation monitoring in the context of allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT). Materials (or patients) and methods: Between January 2011 and June 2014, we performed 135 alloHSCTs for AML, and in 25/135 (18.5%) we could monitor NPM1 mutations during the
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post-transplantation follow-up. Among these 25 patients, 7 were transplanted in active disease (AD), while 18 had achieved complete remission (CR) before the start of conditioning; 20 patients were treated with full dose treosulfan-based conditioning, while 5 patients were treated with busulfan-based myeloablative conditioning. To monitor NPM1 mutations we employed Real Time quantitative PCR (RTqPCR), using as reference the ABL gene and as template genomic DNA extracted from bone marrow (BM) samples harvested monthly during the post-transplantation follow up, starting from day þ 30 after alloHSCT. Results: The median follow up in this cohort was 255 days (range 68-1332); 9/25 patients experienced leukemic relapse after alloHSCT with a median time to relapse of 136 days (range 68-384), whereas median follow-up for the 16 nonrelapsed patients was 556 days (range 126-1332). Out of the 9 patients who experienced relapse, 8 remained positive for NPM1 mutation at time of relapse, whereas 1 was persistently negative for NPM1 during all his follow up and ultimately experienced a NPM1-negative relapse. Among the 8 patients with NPM1-positive relapses, each of them showed at least one detectable NPM1 value during follow up, 6/8 showed two consecutive positive values and 5/8 showed an increasing value of NPM1 quantification. 4/8 patients never displayed negative values. The average time from molecular conversion to morphological and clinical relapse was 127 days. Among the 16 patients who remained in LT CR, 8/16 of them showed persistent negativity for NPM1 during all the follow up, whereas 8/16 patients showed at least one sample with detectable NPM1. Notably in 4 out of these eight patients the positive samples were those early after alloHSCT, and NPM1 became subsequently negative. Only 2/16 non-relapsed patients showed two consecutive positive values, and only one displayed increasing values. Conclusion: In line with the biological notion that NPM1 mutation is an early and stable event in AML, in our analysis its monitoring could predict relapse in 8/9 patients with a useful time of advance, confirming its usefulness as MRD marker also in the transplantation setting. In particular, increasing values in consecutive BM samples are highly suggestive of impending relapse. Although this study was performed on a small patient cohort, it suggest that NPM1 is a useful tool to identify those patients that will experience relapse and to timely carry out a preemptive therapy. Disclosure of Interest: None declared. P243 Impact of pretransplant minimal residual disease detection by flow cytometry in acute myeloid leukemia E. Perez1,*, O. Lo´pez-Godino1, M. Cabrero1, J. Labrador1, S. Alonso1, J. J. Perez1, L. Lo´pez-Corral1, L. Vazquez2, M. Gonzalez1, J. M. Hernadez1, N. Puig1, M. Diez-Campelo1, A. Martin1, M. V. Mateos1, B. Paiva2, M. C. del Can˜izo1, J. San Miguel1, M. D. Caballero1, M. B. Vidriales1 1 HOSPITAL UNIVERSITY OF SALAMANCA, 2HOSPITAL UNIVERSITARIO DE SALAMANCA, Salamanca, Spain Introduction: The value of minimal residual disease (MRD) analysis by flow cytometry (FC) in acute myeloid leukemia (AML) patients early during treatment have been reported by several groups, including our own. However, few data is available about the impact of MRD detection by FC before stem cell transplantation (SCT). The purpose of the present study is evaluate the impact of pre-transplant MRD detection by FC in terms of disease free survival (DFS) in AML patients. Materials (or patients) and methods: We included 122 ‘‘de novo’’ non APL AML patients, who achieved complete remission (CR) after induction chemotherapy and who further underwent an allogeneic (n ¼ 62) or autologous (n ¼ 60) SCT, all of them having immunophenotypic aberrancies detected in blast cells at diagnosis, suitable for MRD monitoring during follow-up. Bone marrow samples were analysed by four-colour multiparametric FC.
Results: With a median follow-up of 61 months, we demonstrated that pre-transplant MRD allowed to stratify relapse risk when considering both the whole group of 122 patients (5 years DFS: 51% vs 71% for patients with MRDo10-4 (n ¼ 43) or MRD4 ¼ 10-4 (n ¼ 79) respectively; P ¼ 0.004) or the autologous stem cell transplant group (5 years DFS: 66% vs 40% for patients with MRDo10-4 (n ¼ 18) or MRD4 ¼ 10-4 (n ¼ 42) respectively; P ¼ 0.05). However, in the subanalysis performed in patients who underwent allogeneic SCT, no statistical differences in DFS were detected according with the MRD level (MRDo10-4 or MRD4 ¼ 10-4 76% vs 66%; P ¼ 0.37),that could suggests that allogeneic SCT can overcome the adverse impact of high MRD level. In addition, considering those patients with higher MRD levels (410-4), allogeneic SCT as a consolidation treatment (n ¼ 37) was associated with a better outcome compared to autologous SCT (n ¼ 42), with a 5y-DFS of 66% vs 40% respectively; P ¼ 0.10), while no differences were detected according with the type od SCT in low MRD level (o10-4) patients. In the multivariate analysis performed in the whole group of patients, consolidation with allogeneic SCT (P ¼ 0.04) and MRD levels o10-4 (P ¼ 0.01) were independent factors associated with DFS, together with cytogenetic at the diagnosis (P ¼ 0.02). When considering only the allogeneic SCT group, development of chronic graft versus host disease (P ¼ 0.006) and cytogenetic at the diagnosis (P ¼ 0.02) were the only factors with independent impact on DFS, while pre-transplant MRD lost its significant impact in this group. Conclusion: These results support the role of pre-transplant MRD detection by FC in AML patients, which could be useful to decide therapeutic strategy early after transplant. In addition, according to our results, allogeneic SCT could overcome adverse prognosis associated with higher MRD levels confirming the existence of a graft versus leukemia effect. Disclosure of Interest: None declared. P244 Time-points for minimal residual disease monitoring in patients with acute myeloid leukemia who underwent allogeneic stem cells transplantation: a comparison between multiparameter flow cytometry and Wilm’s tumor 1 expression G. Rossi1,*, A. M. Carella1, M. M. Minervini1, F. di Nardo2, M. M. Greco1, E. Merla1, L. Savino1, G. P. de Cillis1, N. Di Renzo3, A. Melpignano4, S. Capalbo5, G. Pisapia6, N. Cascavilla1 1 Department of Hematology, IRCCS "Casa Sollievo della Sofferenza", San Giovanni Rotondo, 2Department of Hygiene, Catholic University of Sacred Heart, Rome, 3Department of Hematology, "Vito Fazzi’’ Hospital, Lecce, 4Department of Hematology, ‘‘Perrino’’ Hospital, Brindisi, 5Department of Hematology, ‘‘Riuniti’’ Hospital, Foggia, 6Department of Hematology, ‘‘San Giuseppe Moscati’’ Hospital, Taranto, Italy Introduction: Allogeneic stem cell transplantation (allo-SCT) represents the only effective therapy for high risk patients with acute myeloid leukemia (AML). Nevertheless, relapse remains a crucial issue in this setting and new methods able to prevent it are needed. The detection of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) and WT1 expression showed to be a powerful predictor of prognosis Anyway, the most sensitive method in predicting the relapse as well as the optimal time point to evaluate MRD in allografts remain to be determined yet. Thus, with this purpose, we studied technical performance and prognostic impact of MRD assessed at the optimal cut-off by MFC and WT1 expression, before and after transplant Materials (or patients) and methods: Between June 2010 and January 2014, 30 adult AML patients achieving a complete remission (CR) underwent allo-SCT at the Hematology Department of IRCCS ‘‘Casa Sollievo della Sofferenza’’ Hospital. Samples for MRD analysis were collected before (-10 days), at day þ 30 and þ 90 after transplant. Samples were investigated by 6-color MFC and quantitative analysis of WT1. All patients received a myeloablative conditioning regimen.
Results: When MFC and WT1 were compared at the optimal cut offs before transplant MRD identified by MFC showed better values of sensitivity (71.4% vs 57.1%), specificity (87.0% vs 72.7%), positive predictive value (PPV) (62.5% vs 40.0%) and negative predictive value (NPV) (90.9% vs 84.2%) than WT1. At day þ 30 post-transplant, MFC displayed a higher sensitivity (85.7% vs 60.0%) but a lower specificity (68.2% vs 90.9%), PPV (46.7% vs 60.0%) and similar NPV (93.8% vs 90.9%) compared to WT1. Finally, at day þ 90 diagnostic performance of WT1 was better than MFC. Combining the methods by considering as positive the patients who were positive by both MFC and WT1, pre and post-transplant specificity improved (91.3%, 95.8% and 87.0%, respectively at each check-point). Patients with pre-transplant MRD values greater than 0.1% by MFC showed a shorter DFS compared to others with lower values (P ¼ 0.006). Only a trend to significance was found between ptients having more and less than 64 copies WT1/104 ABL (P ¼ 0.081). One month after transplant, patients having MRD higher than 0.05% by MFC and 138.0 copies WT1/104 ABL by WT1 showed a poorer DFS (P ¼ 0.005 and P ¼ 0.010, respectively). Multivariate analysis confirmed results obtained from univariate analysis. At day þ 90, only MRD by WT1 higher than 103 copies WT1/104 ABL seems to identify pts with high risk to experience a recurrence (P ¼ 0.085). Conclusion: MFC may be used indifferently before and thirty days after the transplant to detect MRD, while WT1 expression should be preferentially measured after the transplant, based on different diagnostic performance and prognostic impact reported in our series. At day þ 30 post- transplant we recommend that minimal residual disease should be investigated by either or both methods, as it has a strongly predictive role. MRD measurements appear to be less predictive at later time points. The combination of MFC and WT1 may be preferred to a single one when further treatments should be administered to prevent the relapse. In fact, only in this case patients have a high probability of experiencing a recurrence. Disclosure of Interest: None declared. P245 Immune Reconstitution after HLA-Haploidentical and HLA-Matched Allogeneic Transplantation (BMT) Utilizing Posttransplantation Cyclophosphamide G. Cengiz-Seval1,*, A. Vulic1, S. McCurdy1, C. Kanakry1, R. Varadhan2, H. Symons1, L. Luznik1 1 Oncology, SKCCC at Johns Hopkins, Baltimore, MD, 2Oncology and Biostatistics, SKCCC at Johns Hopkins, Baltimore, United States Introduction: Posttransplantation cyclophosphamide (PTCy) limits severe acute and chronic graft-versus-host disease (GVHD) and facilitates engraftment, permitting HLA-matched donor (MD) BMT without additional GVHD prophylaxis and the safe performance of HLA-haploidentical (haplo) BMT. Low rates of transplant related mortality, infection, and posttransplant lymphoproliferative disorders (PTLD) support that PTCy selectively depletes proliferating alloreactive T-cells, while preserving resting memory T-cells essential for immunologic recovery. However there is limited data regarding immune reconstitution after haplo or MD BMT utilizing PTCy. Materials (or patients) and methods: We assessed immune reconstitution in 71 patients undergoing myeloablative (MA) haplo BMT with PTCy (50mg/kg on days þ 3 and þ 4), mycophenolate mofetil (MMF) (days þ 5-35), and tacrolimus (days þ 5-180) as GVHD prophylaxis and 73 patients undergoing MA MD BMT with PTCy (50mg/kg on days þ 3 and þ 4) as sole GVHD prophylaxis. Flow cytometric immunophenotyping was performed on peripheral blood samples collected serially at predetermined time points. Results: Immune Reconstitution was not statistically different after HLA-matched related and HLA-matched unrelated BMT; therefore these groups are reported together. In all groups NK cells recovered to normal donor counts by 6 months. In patients without GVHD, NK recovery was more rapid with no
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significant difference by 2-3 months. By 1 year following haplo and MD BMT, median ALCs were in the normal range (11004800 cells/ml) and B-cell counts were higher than those in normal donors. Results were similar after MD and haplo BMT. However, CD4 þ and CD8 þ T-cell counts at 1 month were statistically significantly lower after haplo BMT (both p’so0.0001). Median CD4 þ T-cell counts at 1 year were significantly lower after both MD and haplo BMT compared with normal donors, but higher after haplo compared with MD BMT. At 6 months and 1 year there was no significant difference in CD8 þ T-cells after haplo or MD BMT compared with normal donors. There was a trend towards lower total CD8 þ T-cell counts at all time-points after haplo compared with MD BMT. Phenotypic effector memory (EM) and terminally differentiated effector memory T-cells (TEMRAs) recovered rapidly after BMT, particularly within the CD8 þ fraction after both haplo and MD BMT. However, phenotypically naı¨ve cells remained low throughout the first post-BMT year. Consistent with our prior reports, Tregs comprised a larger portion of CD4 þ T-cells after MD or haplo BMT compared with donors (Po0.0001). The statisticaly significant difference in expression of PD-1 and TIM3 but not CD160, 2B4 and BTLA on CD4 þ and CD8 þ T was noted beetwen patients that received MD vs haplo BMT. Conclusion: Haplo and MD BMT lead to comparable reconstitution of NK and B-cells, however CD4 þ and CD8 þ T-cell early recovery lags after haplo BMT. This delay is likely attributable to the addition of MMF and tacrolimus, which may be mitigated by discontinuation of MMF at Day 35, resulting in equivalent CD4 þ T-cell and CD8 þ T-cell numbers by 3 and 6 months respectively. Further deciphering of co-inhibitory molecules expression is needed. Excellent immune reconstitution by 1 year supports the low rates of PTLD and infectious deaths after haplo or MD transplantation with PTCy. Disclosure of Interest: None declared. P246 Evaluating of posttransplant relapse risk in patients with acute leukemia using the gene expression markers A. Shakirova1, I. Barkhatov1,*, O. Shakirova2, V. Teplyashina1, A. Evdokimov1, Y. Gudozhnikova1, S. Bondarenko1, L. Zubarovskaya1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, First Pavlov Saint Petersburg State Medical University, 2St.Petersburg State Polytechnic University, St.Petersburg, Russian Federation Introduction: It is known that up to 50 percent of cases of acute myeloid leukemia (AML) does not have informative genetic markers. At the same time, the studies of donor chimerism do not fully indicate the degree of tumor cells elimination and not always allows to estimate the risk of posttransplant relapse. Thus, finding of universal markers allowing for adequate therapy in post-transplant period, is quite important. WT1, BAALC, EVI1 and PRAME gene expression analysis is one of the possible approaches is this field. Materials (or patients) and methods: Our study included 63 patients with AML (1 to 60 years old) (M0-2pts, M1-11pts, M213pts, M3-2pts, M4-21pts, M5-11pts, M6-1pt, M7-2pts ) who underwent allogeneic transplantation of hematopoietic stem cells (allo-HSCT). In 24 patients myeloablative conditioning regimen were used, 39 – received reduced toxicity protocols. Assessing the levels of WT1, BAALC, EVI1, PRAME gene expression and the level of chimeric transcripts was performed by means of RQ-PCR with normalization for ABL gene expression. For the donor chimerism monitoring a panel of STR-markers was used. Results: As based on gene expression in healthy donors, we have established cut-off overexpression (gene exp. / ABL exp. X100) values for the genes: WT1 – 250, EVI1-10, BAALC – 20, PRAME - 200. For the present patient setting, we found no statistically significant differences in WT1, BAALC, EVI1, PRAME genes expression between the patients with different FAB variants of AML. However we were able to identify a trend to
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higher values of PRAME and BAALC gene expression in patients with M1 variant, and WT1 gene values among patients with M4 variant of AML. In patients who underwent transplantation in relapse state, we have noted a significant overexpression of EVI1 (P ¼ 0.006), WT1 (Po0.001), BAALC (Po0.001). A similar trend was observed for PRAME gene (P ¼ 0.08). EVI1 gene overexpression was revealed for 6 patients (33%), WT1 in 13 cases (72%), BAALC in 10 patients (55%) and PRAME in 4 patients (22%). When comparing the data on chimeric transcripts expression, we detected a correlation between expression levels of the studied genes and chimeric transcripts (PML-RARa and RUNX1-RUNX1T1, Po0.05) as well as with donor chimerism levels. At the same time, we did not find this relationship, when comparing with data about of expression of a CBFB-MYH11 chimeric gene. When evaluating the test sensitivity and specificity analysis we revealed a bellow-cutoff value of the expressed genes in presence of the chimeric transcript less than 2%. Conclusion: Evaluation of universal gene marker expression is an attractive approach for assessing efficiency of therapy in patients with AML. Thus, their use in early diagnostics of relapse in post-transplant period is quite promising. However, due to low specificity caused by basal expression in normal cells, futrher application of these markers is limited, when detecting minimal residual disease levels. Disclosure of Interest: None declared. P247 Analysis of donor chimerism kinetics to assess relapse in allogeneic hematopoietic stem cell transplantation in patients with hematologic malignancies I. Barkhatov1,*, A. Evdokimov1, A. Shakirova1, V. Teplyashina1, S. Bondarenko1, E. Semenova1, A. Chukhlovin1, L. Zubarovskaya1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, First Pavlov Saint Petersburg State Medical University, St.Petersburg, Russian Federation Introduction: Allogeneic hematopoietic stem cell transplantation is an effective treatment for different hematological malignances. Efficiency of donor cell engraftment is determined by donor chimerism evaluation, i.e. determining of genetically different cell populations coexistence in patient. We assume that the kinetics of chimerism in patients with hematologic malignancies, in turn, may be defined in terms of relapse probability, and it could be used as a marker of minimal residual disease (MRD) after allogeneic transplantation of HSCs (allo-HSCT). Materials (or patients) and methods: We have analyzed 440 allo-HSCT’s in patients with various hematologic malignancies. For the donor chimerism monitoring we used standard panels of human DNA markers based on STR variability (short tandem repeats, microsatellites). The ratios of donor and recipient cells were calculated according to the data derived from fragment analysis, using the Genemarker program. The study was performed on days þ 15, þ 30, þ 45, þ 60 and þ 100 after transplantation. Results: Achieving of full donor chimerism (95%) significantly increases the 5-year survival (P ¼ 0.001) and reduces the probability of disease recurrence (P ¼ 0.01). Among patients with mixed chimerism the stability of donor chimerism level is most important, being associated with lower risk of relapse (P ¼ 0.013). Thus, in patients with stable mixed chimerism, the markers molecular markers of MRD had no significant effect either on overall survival, or the likelihood of relapse (P ¼ 0.55 and P ¼ 0.34 respectively). Meanwhile, we had found an inverse correlation between the level of donor chimerism and expression of relapse markers, i.e. WT1 (R ¼ -0.4, P ¼ 0.0001) and EVI1 (R ¼ -0.16, P ¼ 0.03). Similar data were obtained when analyzing expression of the chimeric transcripts: BCR-ABL (R ¼ -0.26, P ¼ 0.0001), MLL-AF4 (R ¼ -0.36, P ¼ 0.003), TEL-AML (R ¼ -0.37, P ¼ 0.003). Hovewer, this dependence with other markers (CBFB-MYH11, PML-RARa, RUNX1-RUNX1T1) could be also obtained.
Conclusion: Monitoring of donor chimerism after allo-HSCT adequately reflects the dynamics of donor cells engraftment and may be used as an adequate method for predicting recurrence of the disease, but it can not be used as a single method in MRD analysis. Disclosure of Interest: None declared. P248 Fetal microchimerism in development of acute GVHD after haploidentical BMT I. Barkhatov1,*, A. Shakirova1, O. Smykova1, V. Teplyashina1, A. Evdokimov1, A. Chukhlovin1, L. Zubarovskaya1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, First Pavlov Saint Petersburg State Medical University, St.Petersburg, Russian Federation Introduction: Haploidentical transplantation of hematopoietic stem cells (haplo-HSCT) is a feasible therapeutic approach in patients lacking an HLA-identical donor. However, high risk of graft-versus-host disease (GVHD) is among major complications in haplo-BMT. Currently, there exist some data concerning associations between maternal microchimerism levels (detection of maternal cells in recipient’s blood) and increased risk for GVHD development. Meanwhile, the effects of microchimerism upon GVHD probability are still controversial. The aim of our study was to adapt a technique for quantitative evaluation of fetal (recipient-cell) and mathernal (donor-cell) microchimerism and investigation of its effects upon actual risk of common complications after HSCT. Materials (or patients) and methods: Determination of microchimerism was carried out by real-time alle-specific polymerase chain reaction (AS RQ-PCR). The analytic panel included 20 SNP markers located at different chromosomes. Evaluation of PCR sensitivity was carried out using DNA mixtures from several established cell lines (HL60, K562, 293T, MOLT3, A549) at serial dilutions. The samples from 20 donorrecipient pairs were analyzed. The patients’ age ranged from 2 to 27 years. Results: The sensitivity thresholds for the SNP-based test panel ranged from 10^-4 to 10^-5. We have found out that detection of fetal microchimerism in the donor organism is associated with lower risk, or lower degree of acute GVHD (P ¼ 0.01). Moreover, we observed a trend to higher timeframe for transplant engraftment, and lower probability of full donor chimerism achievement, if the donor exhibited fetal microchimerism (P ¼ 0.12). The patients in our setting transplanted from donors with detectable fetal microchimerism had a tendency to a higher overall survival (P ¼ 0.14). Hovewer, we did not reveal any significant association between microchimerism levels (fetal and maternal), and higher probability of chronic GVHD development, as well as any differences in other posttransplant complications for recipients with maternal microchimerism. Conclusion: Evaluation of fetal microchimerism may be considered a useful and informative approach to selection of potential donors, or a method of GVHD prediction in haploidentical HSCT. Disclosure of Interest: None declared. P249 B-cell intrinsic and extrinsic defects in apoptosis, migration and T-cell communication impede the germinalcenter formation of memory B cells after allogeneic-HSCT A. Mensen1, Y. Oh1, S. Demski1, P. Hemmati2, C. Jehn2, J. Westermann2, G. L. Vuong2, B. Do¨rken2,3, C. Scheibenbogen1, R. Arnold2, I.-K. Na1,2,4,* 1 Institute for Medical Immunology, Charite´ Berlin, 2Department of Hematology, Oncology and Tumorimmunology, Charite´ Berlin, 3 Max-Delbru¨ck Center (MDC) for Molecular Medicine, 4Experimental and Clinical Research Center (ECRC), Berlin, Germany Introduction: Sustained CD27 þ memory B-cell deficiency despite normalized lymphocyte counts is associated with
prolonged susceptibility to infections and poor vaccination responses within one year after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Little is known about mechanisms contributing to defects in the germinal-center (GC) formation of memory B cells in allo-HSCT patients. Materials (or patients) and methods: We have analyzed the peripheral B and T-cell subset reconstitution and their expression of functional markers for 37 adult acute leukemic patients at day (D) 90, D180 and D360 after allo-HSCT. In addition, efflux capacity of naı¨ve B cells as an intrinsic contributor to B-cell survival as well as B-cell apoptosis after T-cell dependent B-cell activation have been investigated. Quantification of cytokines and chemokines in plasma at D180 was performed with the luminex multiplex technology. Results: We could confirm a sustained CD27 þ memory B and CD4 þ T-cell deficiency at one year after allo-HSCT. Additionally, we found significantly decreased percentages of CXCR5expressing memory T follicular helper (TFH) cells in patients when compared to age-matched healthy controls (Mean %±SEM: HC 7±1, alloHSCT 2±1; pr0.05). Moreover, CD27-IgD- double negative B cells of putative GC-independent origin, were fully recovered. This suggested defects in the GC reaction, which was further supported by significant lower CXCR5 expression on naı¨ve B cells (MFI±SEM: HC 5899±975, alloHSCT 3494±554; pr0.001), important for the entry into B-cell follicles. CCR7, mediating the migration into the T-cell zone, was only slighty decreased. Lower HLA-DR expression on naı¨ve B cells further could impair antigen-presentation and thus maintenance of TFH cells in patients (HC 20446±4742, alloHSCT 9150±2386). B and CD4 þ T cells exhibited an increased Fas expression ex vivo, indicating an apoptoticprone/activated state (Mean %±SEM: naı¨ve B, HC 4±1, alloHSCT 13±2, pr0.001; CD4 þ T, HC 19±5, alloHSCT 63±8; pr0.05). This might result from an inflammatory milieu in patients as expressed by increased plasma level of G-CSF, GMCSF, IFNa, TNFa, IP-10, MCP-1, MIP-1b and Eotaxin and increased B-cell subset expression of CD86 and CXCR3. We also observed a significantly reduced capacity of naı¨ve B cells from Cyclosporin A-treated patients to efflux the dye rhodamine 123, which further could increase apoptosis susceptibility (Mean % effluxing naı¨ve B cells±SEM: HC 51±6, alloHSCT 28±7, pr0.05). Upon T-cell dependent stimulation with CpG/CD40L/PWM/ionomycin, patient B cells up-regulated Fas, which was paralleled by significantly increased Fas-L expression on CD4 þ and CD8 þ T-cell subsets and associated with significantly increased B-cell apoptosis for patients than for HC (Mean % cells±SEM: HC 15±2; alloHSCT D360 60±6, pr0.001). Conclusion: We conclude that B-cell intrinsic and extrinsic survival defects in the T-cell dependent B-cell activation impede the GC formation of memory B cells up to one year after allo-HSCT. This emphasizes the importance to consider prolonged infectious prophylaxis, to control cellular vaccination responses and to evaluate the adoptive transfer of memory B cells as a new treatment option to improve B-cell immune function after alloHSCT. Disclosure of Interest: None declared. P250 Minimal residual disease analysis by flow cytometry patients with acute myeloid leukemia before and after allogeneic hematopoietic stem cell transplantation I. Kozanoglu1,*, G. Unver1, M. Yeral2, C. Gereklioglu2, A. P. Korur2, N. Buyukkurt2, S. Solmaz2, M. Kasar2, C. Boga2, H. Ozdogu2 1 Cell Processing Unit, 2Clinical Unit, Baskent University Adana Adult Bone Marrow Transplantation Center, Adana, Turkey Introduction: Allogeneic stem cell transplantation has become a treatment choice in patients with acute myeloid leukemia (AML). Monitoring of minimal residual disease (MRD) by multiparametric flow cytometry (MFC) has the advantage of being applicable in most patients. The aim of the present
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study was to evaluate the efficacy of monitoring of MRD using 8-color MFC both before and after transplantation to predict relapse in allo-SCT patients with AML. Materials (or patients) and methods: We identified 24 consecutive AML patients who had undergone allo-SCT between March 2011 and December 2014. To be included in the study, patients had to have at least one leukemiaassociated immunophenotypes (LAIPs) at diagnosis. The study was performed on erythrocyte-lysed whole bone marrow samples, after staining with directly conjugated monoclonal antibodies. Samples were analyzed using 8-color MFC (FACS Canto II; BD, Bioscience), and FACS DIVA was used for data analysis. When possible, the same monoclonal antibody and fluorochrome were used at diagnosis and for subsequent monitoring of MRD. Positive MRD was defined as the observation of a cell population with 40.1% LAIPs at diagnosis in bone marrow samples. Minimal residual disease testing was performed before transplantation (immediately before initiation of the conditioning regimen) and at months 1, 3, 6, and 12 after allo-SCT. Results: Sixteen patients who were shown to be MRDnegative by MFC at transplantation and underwent allo-SCT had lower rates of relapse (18.75% vs. 33.4%, P ¼ 0.04), better overall 1-yr survival (85% vs. 34%, P ¼ 0.01) and a lower cumulative incidence of relapse (P ¼ 0.032) than patients who were MRD-positive (40.1%). All post-transplant MRDpositive patients underwent a therapeutic intervention after transplant (tapering of immunosuppression, donor lymphocyte infusion, or re-transplant) with the intention of preventing relapse. Disease was controlled and MRD disappeared in four of these patients. Disease recurred in the other four patients Conclusion: We can conclude that follow-up with MFC for the detection of MRD in AML before and after SCT is useful for predicting relapse. In the post-transplant setting, monitoring of MRD by MFC could be a key preemptive intervention. References: 1.Walter RB, Gyurkocza B, Storer BE, Godwin CD, Pagel JM, Buckley SA, Sorror ML, Wood BL, Storb R, Appelbaum FR, Sandmaier BM. Comparison of minimal residual disease as outcome predictor for AML patients in first complete remission undergoing myeloablative or nonmyeloablative allogeneic hematopoietic cell transplantation. Leukemia. 2014 Jun 3. 2. Bastos-Oreiro M, Perez-Corral A, Martı´nez-Laperche Prognostic impact of minimal residual disease analysis by flow cytometry in patients with acute myeloid leukemia before and after allogeneic hemopoietic stem cell transplantation. Eur J Haematol. 2014 Sep;93(3):239-46. Disclosure of Interest: None declared.
Paediatric issues I P251 The outcomes of haploidentical hematopoetic stem cell transplantation performed with unmanipulated donor stem cells and post-transplant cyclophosphamide in high risk children: a single center experience A. Yes¸ilipek1,*, V. Uygun1, G. Karasu1, H. Dalog˘lu1 1 Pediatric Bone Marrow Transplantation Unit, Bahc¸es¸ehir University, School Of Medicine, Istanbul, Turkey Introduction: In recent years, some of haploidentical transplantations are performed with unmanipulated bone marrow or peripheral blood stem cells which the GVHD prophylaxis includes a more intense chemoterapy approach. This approach is now more popular by post-transplant high dose cyclophosphamide. In this study, we present the children who transplanted haploidentically in our center, with unmanipulated bone marrow or peripheral blood stem cells
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and GVHD prophylaxis with post-transplant high dose cyclophosphamide. Materials (or patients) and methods: We assessed 16 HSCT in 15 patients retrospectively, who underwent haploidentical related HSCT with unmanipulated bone marrow or peripheral blood stem cells and used post-transplant high dose cyclophosphamide for GVHD prophylaxis. Five of patients were male and 10 were female between 3.5-17 years old, median 9 years old. One patient was AML developed after Ewing Sarcoma and one was ALL progressed from CML. Another patient was transplanted for aplasia developed after a haploidentical transplantation from his mother which had been performed with CD34 positive selection. One patient underwent second haploidentical transplantation because of the relapse. Four patients were in the first complete remission, five were in the second and three were in the third in the transplantation date. Donors were their mother in ten transplantation, the father in five, and the sibling in one patient. For the conditioning regimen BU þ FLU þ Etoposid was used for seven patients and two patients with BU þ FLU þ ATG/, BU þ CY þ Mel, /BU þ FLU þ ATG and FLU þ CY þ Mel, and one patient with BCNU þ Etoposid þ ARA-C þ Mel. Fifty mg/kg CY on the 3rd and 5th day and CsA or tacrolimus with MMF or MP were also used for GVHD profilaxis. The source of stem cells was bone marrow in six transplantation while bone marrow and peripheral blood stem cell were used in ten transplantation. The median count of TNC, MNC, CD34 þ and CD3 þ was 12,9x108 (4,1-39), 8,8x108 (4,8-14,3), 5,9 x106 (2,5-16) and 2,9 x108 (1,1-7,8), respectively. Results: All patients engrafted with a median of 16 days (1623) and 18 days (11-37) for neutrophil and thrombocyte recovery, respectively. Grade 1 acute GVHD developed in one patient, and grade 2 in 4 and 3 in 3 patients, while limited chronic GVHD developed in two patients. Three patients had hemorrhagic cystitis and four developed VOD. Two patients were lost in the first 100 days because of sepsis (TRM 12, 5%). After 100 days, a patient died of progression of the primary disease and one patient was lost because of pneumonia. One patient with diagnosis of Hurler showed graft rejection. The rest ten patients (66.6%) are disease-free (OS is 73.3%) and on the follow up of median 10 months (between 4-20 months). Conclusion: The advantages of using unmanipulated hematopoetic stem cells are low laboratory costs, no worry for waiting for a suitable donor, and less risk for the graft failure or T cell deficiency. Our results are consistent with these circumstances in our high risk patients and encouraging for haploidentical HSCTs. However our results should be accepted as a preliminary report with these few patients. Disclosure of Interest: None declared. P252 Chronic Granulomatous Disease (GCD): diagnosis and therapy A. Beghin1,*, M. Zucchi1, R. Baffelli1, F. Bolda1, F. Porta2, A. Lanfranchi1 1 Lab Cell Stam,Children’s Hospital, Spedali Civili di Brescia, 2 Pediatric Oncohematology and BMT Unit, Brescia, Italy Introduction: Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by early onset of recurrent and severe infections. The underlying pathophysiological mechanism in CGD is a defective respiratory burst associated with an impaired microbicidal function of phagocytes. The molecular defects causing CGD are generally because of the absence, low expression or malfunctioning of one of the NADPH oxidase components. The X-linked form of the disease is caused by defects in the heavy chain of the cytochrome b588 component (gp91phox coding by CYBB gene) and accounts approximately 60% of the cases. The autosomal recessive forms (AR-CGD) are caused by defects in one of the cytosolic components of the NADPH oxidase: p47phox coding by NCF1 gene (approximately 30% of the cases), p67phox, p40phox, or
even the cytochrome b588 light chain component p22phox coding respectively by NCF2, NCF4 and CYBA genes (all together the remaining 10% of the cases).Screening for CGD is usually performed by functional assays such as the neutrophil respiratory burst assay, performed by flow cytometry (DHR123). Genetic analysis is critical for diagnostic confirmation, genetic counseling, prenatal diagnosis and gene therapy. Materials (or patients) and methods: All patients (pts) were diagnosed by dihydrorhodamine 123 (DHR123).We indentified 13 pts with impaired phagocytic functions by DHR123 assays: 10 males and 3 females. We perfomed molecular analysis in 12 of 13 pts. 4 males were related members: 2 brothers and an uncle and his grandson. Results: We investigated 7 males for CYBB mutationIn 2 Pts, no alteration in the coding region were found, in 2 pts splice site mutation was identified. For 3 a frameshift was detected and for 1 pts it was identified a missense mutation while it was found a nonsense mutations in 1 pt. For one patient no DNA was available for analysis.Moreover we analyzed genes for ARCGD in 3 females.Only 1 pt shows mutation in NCF1 gene, while the other 2 have only polymorphisms.8 pts, all males, underwent transplantation: 5 MUD (pt 8 undergoing two HSCT) and 3MRD (Matched Unrelated Donor), the mean number of CD 34 þ infused was 9.58x106/kg and the mean number of CD 3 þ infused was 267x105/kg. 30 days later HSCT, they showed 100% donor chimerism for 4 patients and a detection of mixed chimerism for 4 patients, while pt 8 underwent a second transplantation. Conclusion: CYBB was the most frequently mutated gene in our cohort of pts with an heterogeneity of mutations. For the 2 brothers in which the coding region results wild type, we hypothesized that it will be mutation in promoter region or will be a deletion in intronic region not yet analyzed. Moreover these preliminary data show a characteristic cluster of snp in AR-CGD patients. Particularly all share the same Arg90His snp in heterozigousis, that Olsonn et al. dimostrated significantly reduced reactive oxygen species production. 2 patients have no mutation but share the same snp in CYBA and NCF1 gene.In our setting of pts (5/8) have a good outcome. No severe or chronic was observed: GvHD I grade in 2 of 8. Disclosure of Interest: None declared. P253 Outcome of hematopoietic stem cell transplantation using full matched grandparents in pediatric patients lacking an HLA identical sibling A. A. Hamidieh1,*, M. Behfar1, M. Ostadali1, S. Alizadeh1, A. Kasaeian1, A. Ghavamzadeh1 1 Hematology, Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic Of Introduction: The extended family search is a more practical method to find full matched other relative donors in regions where consanguineous marriage is more frequently practiced. However, few studies showed that searching extended families such as parents, grandparents, aunts and uncles for an appropriate donor is useful. The purpose of this retrospective study was to investigate the outcome of HSCT using grandparents as hematopoietic stem cells donors. Materials (or patients) and methods: Twenty one patients (11 female, 10 male) aged r15 years old who had received HSCT from their grandparents since 2007 to 2014 were enrolled. We compared the result obtain in 12 patients who had less than or equal to 55-year age difference with their donor (group 1) with 9 patients who had greater than 55-year age difference with their donor (group 2). Results: Indications for HSCT were Fanconi Anemia (n ¼ 6), Thalassemia Major (n ¼ 5), Acute Leukemia (n ¼ 4), Primary immunodeficiency (n ¼ 4) and Mucopolysaccharidosis (n ¼ 2).
The median age of patients was 6 years (range: 0.25-11 years) and it was 60 years for donors (range: 45-75 years). The mean age difference was 54.5 years (range: 36.5-72.9 years). All the donors were completely full matched. Regarding graft versus host disease (GvHD) occurrence, Although Acute GvHD was significantly higher in group1 (P-value ¼ 0.008), there was no significant difference in chronic GvHD between two groups. With the median fallow up 3.17 years (range: 1.4- 4.6 years), overall survival (OS) and disease free survival (DFS) was 80.9% and 76.15, respectively. Also there were no significant differences in OS and DFS in two group (P-value o0.66 & P-value o0.33). Conclusion: Although searching the extended family for HLAmatched donors especially among grandparents seems very uncommon, this can increase the chance of well-tolerated and success transplantation especially in regions where consanguinity is common. Disclosure of Interest: None declared. P254 Allogeneic HSCT for HR-ALL in I CR in children treated according ALL IC-BFM 2002 program - the Polish Pediatric Group for Hematopoietic Stem Cell Transplantation report A. Pieczonka1,*, O. Zajac-Spychala1, A. Chybicka2, J. Owoc-Lempach2, M. Wysocki3, R. Debski3, J. R. Kowalczyk4, K. Drabko4, J. Gozdzik5, J. Wachowiak1 1 Department of Oncology, Hematology and Pediatric Transplantology, University of Medical Sciences, Poznan, 2Department of Pediatric BMT Hematology and Oncology, Wroclaw Medical University, Wroclaw, 3Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, 4Department of Pediatric Hematology, Oncology and Transplantology, Medical University of Lublin, Lublin, 5Jagiellonian University Colegium Medicum, Krakow, Poland Introduction: We present results of allo-HSCT in pediatric patients with high risk group acute lymphoblastic leukemia in first complete remission (HR ALL I CR) who were prospectively qualified and uniformly treated according the same therapeutic protocol ALL IC-BFM 2002. Materials (or patients) and methods: Between 2003-2013, 112 children (41 girls, 71 boys), median age 8.5 years (1-17.8 years) were transplanted from matched sibling donor (MSD) (n ¼ 49/112; 43.7%) or matched unrelated donor (MUD) (n ¼ 63/112; 56.3%). Stem cells source were: bone marrow 61.6% (69/112) or peripheral blood in 37.5% (42/112) or cord blood in 0.9% (1/112). The majority of patients (75.9%; 85/112) received conditioning with FTBI, remaining Busulfan-based 20.5% (23/112) or Treosulfan-based 3.6% (4/112). Cyclosporine A was used as a basic drug of graft versus host disease prophylaxis (GvHD) 100% (112/112), children prepared for MUD transplantation received additionally anti-thymocyte globulin and ‘‘short’’ MTX. Results: Engraftment was achieved in 111 patients (99.1%). Two boys were transplanted twice from MUD (1- no graft take; 1- secondary graft insufficiency). Acute GvHD grade II-IV or cGvHD was observed in 46.9% (23/49) and 16.3% (8/49) children after MSD and in 54% (34/63) and 20.6% (13/63) patients who received transplantation from MUD. Cumulative incidence of transplant related mortality after 1 year was 0.063±0.035 and 0.179±0.049 for children transplanted from MSD (3/49; 1- aGvHD; 2 infection in patients with cGvHD) or MUD (11/63; 9- GvHD-related; 1-lack information; 1- sepsis) respectively. Relapse rate was 6.1% (3/49) and 9.5% (6/63) after MSD or MUD transplantation respectively. After 5 years the probability of leukemia-free survival for MSD or MUD transplantation were 0.929±0.040 and 0.872±0.050 respectively, while probability of event-free survival 0.813±0.056 and 0.720±0.059, respectively. Forty children transplanted from MSD and 46 from MUD are alive in I CR. Overall survival at 5 years for MSD was 0,832±0.054, while for MUD 0,752±0,057 for MUD. Forty one (83.6%) patients transplanted
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from MSD alive 12-138 (median 86) months whereas 48 (76.2%) children after MUD alive 13-132 (median 68) months. Conclusion: In children with ALL in ICR treated according ALL IC BFM 2002 - 1. Allo-HSCT demonstrated excellent antileukemic effect. 2 TRM after transplantation from MSD was lower than after MUD. 3. The GvHD was the most common cause of TRM, indicating the need of further optimization of donor selection, appropriate cells source choice and GvHD prophylaxis. Disclosure of Interest: None declared. P255 Successful allogeneic HSCT after treosulfan-based conditioning in two children with PNH: single-center experience A. Pieczonka1,*, J. Skalska-Sadowska1, A. Sobkowiak-Sobierajska1, K. Wachowiak-Szajdak1, R. Tomaszewska2, T. Szczepanski2, J. Wachowiak1 1 Department of Oncology, Hematology and Pediatric Transplantology, University of Medical Sciences, Poznan, 2Department of Pediatric Hematology and Oncology, Zabrze, Medical University of Silesia, Katowice, Poland Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a complex hematologic disorder of the hematopoietic stem cells. Patients present with recurrent intravascular haemolysis, venous thrombosis and hematopoietic failure. In children, PNH is very rare, clinical course is similar to that in adults, but the majority of children present with bone marrow failure. Nowadays, apart from symptomatic treatment two options of treatment are used: eculizumab (humanized monoclonal antibody against complement protein C5) or allogeneic HSCT (allo-HSCT). However, little information is available in children regarding efficacy and safety of either eculizumab or allo-HSCT and the risk of VOD is very high. Materials (or patients) and methods: We report two children with documented PNH and treated with allo-HSCT after treosulfan-based preparative regimen. Results: Patient 1. Twelve year-old boy with leucopenia, anemia, recurrent infections and hemolysis, hemoglobinuria, hepatopathy, thrombosis and with proven presence of GPInegative neutrophils and erythrocytes in peripheral blood was transplanted from MUD (10/10) in 2010. Preparative regimen consisted of treosulfan (3 x 14g/m2), fludarabine (5 x 30 mg/ m2) and thymoglobulin (7.5 mg/kg) was used. He received 6.0 x 106/kg CD34 þ cells from peripheral blood. For GvHDprophylaxis CsA and ‘‘short’’ MTX course 10 mg/m2 were given. Reticulocytes appeared on day þ 7, granulocytes 40.5x109G/L on þ 14, platelets 4 20 G/L on þ 13. Full donor chimerism was proven þ 28. On day þ 14 engraftment syndrome was diagnosed and treated with steroids. Since this time patients suffered from acute (II) and chronic extensive skin GvHD. He was treated with corticosteroids (CS) and ECP. Currently GvHD seems to be resolved, CS were withdrawn in July 2014. Patient general condition is good, without any PNH symptoms with full donor chimerism, however, he suffered vertebral fracture. No liver toxicity or thrombosis occurred. Patient 2. Eleven-year-old girl with pancytopenia, chronic bacterial eyelid infection, recurrent hemolysis, red blood cells and platelet transfusion-dependent and PNH granulocytes and erythrocytes clones proved in 2013 was transplanted from UD (9/10) in June 2014. Treosulfan-based conditioning was the same as in Patient 1. She received 2.55x106/kg bone marrow derived CD34 þ cells. GvHD prophylaxis consisted of CsA with MFF (-1 to þ 28 day). Reticulocytes appeared on þ 18 day, granulocytes 40.5 x 109 G/L on þ 28, platelets 420 G/L on þ 20 was present. Full donor chimerism was proven on day þ 28. Liver toxicity and thrombosis were not observed. Nowadays, 6 months after transplantation her general condition is excellent, with normal peripheral blood and bone marrow counts and full donor chimerism.
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Conclusion: Questions about the most effective treatment of PNH remain open not only in children. Eculizumab seems to be the best option for patients with hemolysis, but it’s expensive, ineffective in about one-third of PNH patient and induce chronic inhibition of C3 activation, which increases susceptibility to infection especially with Neisseria meningitidis. Allo-HSCT is the only curative treatment for patients with PNH and bone marrow failure but information about optimal conditioning regimen are scarce. According to our experience, in children with PNH the treosulfan-based conditioning demonstrates low organ toxicity and excellent long-lasting engraftment. Disclosure of Interest: None declared. P256 Safety of peritoneal and pleural drain placement in pediatric stem cell transplant recipients with severe veno-occlusive disease A. L. Madenci1,2,*, A. Stetson3, C. Weldon1,3, L. Lehmann3,4 1 Department of Surgery, Boston Children’s Hospital, 2Department of Surgery, Brigham and Women’s Hospital, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, 4Division of Hematology/Oncology, Department of Medicine, Boston Children’s Hospital, Boston, United States Introduction: Veno-occlusive disease (VOD) of the liver is a serious complication of hematopoietic stem cell transplantation (HSCT) that has historically been associated with high mortality. The mechanism of VOD involves hepatic endothelial injury resulting in a clinical syndrome of ascites, hyperbilirubinemia, and painful hepatomegaly. Severe cases have lead to hepatorenal syndrome, multiorgan failure, and death. Defibrotide, a polydeoxyribonucleotide-derived medication that enhances the fibrinolytic pathway, is one of limited medical treatment options although still an investigational agent in the United States. Drainage of ascites via peritoneal catheters can improve respiratory excursion and mitigate renal injury from intra-abdominal hypertension. Drainage of pleural fluid can improve both ventilation and oxygenation. However, there is often concern regarding surgical complications or infections occurring as a consequence of peritoneal or pleural drain placement in critically ill, immunocompromised children. Materials (or patients) and methods: We conducted an IRB-approved retrospective review of all HSCT recipients at a single pediatric center who developed VOD (defined using Bearman criteria) of sufficient severity to be eligible for one of two clinical research protocols using defibrotide between 2000 and 2012. The primary outcome was complication from peritoneal or pleural drain placement. Secondary outcomes included mortality and infectious complications. Results: Thirty-two patients were treated with defibrotide for severe VOD. Median age at transplantation was 2.9 years (interquartile range [IQR], 1.3–5.8 years). Defibrotide treatment began at a median (IQR) 15.5 (12–21) days after transplant and lasted for median (IQR) 22 (17–30) days. Overall mortality was 47% (n ¼ 15). Six (19%) patients required renal replacement therapy, which was associated with mortality (n ¼ 6, 100% vs. n ¼ 9, 35%, P ¼ 0.004). Twenty-four patients (75%) had peritoneal drains inserted (median [IQR] ¼ 16.5 [13-25] drain days). Interventional radiologists placed 92% of drains (13% at bedside), while critical care physicians placed the remainder at bedside. Median (IQR) absolute neutrophil count at the time of peritoneal drain placement was 0.6 (0.01–4.9) K/uL. The procedure was safe and not associated with increased mortality (P40.99). Eight (25%) peritoneal drains were exchanged due to poor functioning. Seven (23%) patients had undergone prior abdominal surgery, but there were no instances of visceral perforation or hemorrhage with drain placement. Among the 24 patients with peritoneal drains, only one (4%) patient had a positive culture from ascitic fluid (Staphylococcus non-aureus
species). At the time peritoneal drains were removed, median creatinine and total bilirubin were decreased by 0.1 (0–0.3) and 1.9 (0.3–6.8) mg/dL, respectively, compared to the time of placement. Eight (25%) patients underwent pleural drain placement (median [IQR] ¼ 9 [6–35] drain days). No pleural drain-related surgical complications or infections occurred. Four (50%) patients with pleural drains had de-escalation in oxygen requirement at the time of drain removal, compared to the time of placement. Conclusion: VOD remains a devastating complication of HSCT, with a 47% mortality rate among patients with severe disease. Drain placement in the peritoneal or pleural space is a safe treatment modality for children with severe VOD. Disclosure of Interest: None declared. P257 First Report of Pediatric Hematopoietic Stem Cell Transplantation Activities in the Eastern Mediterranean Region, 1984-2012: On Behalf of Pediatric Cancer Working Committee of the Eastern Mediterranean Blood and Marrow Transplantation (EMBMT) Group A. A. Hussein1,*, A. Hamidieh2, A. Elhaddad3, M. Ramzi4, T. Ben Othman5, F. Hussain6, D. Dennison7, P. Ahmed8, M. Abood9, A. Al-Ahmari6, A. Wahadneh10, J. Fathy3, M.-A. Bekadja11, S. Al-Kindi7, S. Benchekroun12, A. Ibrahim13, M. Behfar2, M. Samra3, S. Ladeb5, S. Adil14, H. El-Solh6, M. Ayas6, A. Ghavamzadeh2, A. Al-Seraihy6 on behalf of Pediatric Cancer Working Committee of the Eastern Mediterranean Blood and Marrow Transplantation (EMBMT) Group 1 Bone Marrow and Stem Cell Transplantation Program, King Hussein Cancer Center, Amman, Jordan, 2Tehran University of Medical Sciences, Hematology, Oncology and SCT Research Center, Tahran, Iran, Islamic Republic Of, 3National Cancer Institute, Cairo University, Cario, Egypt, 4Shiraz University of Medical Science, Shiraz, Iran, Shiraz, Iran, Islamic Republic Of, 5Center National de Greffe de Moelle Osseuse de Tunis, Tunis, Tunisia, 6King Faisal Specialist Hospital and Research Centre, Riyadh, Riyadh, Saudi Arabia, 7Sultan Qaboos University Hospital, Muscat, Oman, 8Armed Forces Bone Marrow Transplant Center, Rawalpindi, Pakistan, 9 American University Beirut Medical Center, Beirut, Lebanon, 10Queen Rania Al-Abdulla Hospital, Amman, Jordan, 11Oran University-1st November Hospital, Oran, Algeria, 12 Service d’Hematologie et Oncologie Pediatrique, Casablanca, Morocco, 13Makassed General Hospital, Beirut, Lebanon, 14 Agha Khan University Hospital, Karachi, Pakistan Introduction: There is no available data in the literatures describing the state of hematopoietic stem cell transplantation (HSCT) activities for children in the Eastern Mediterranean (EM) region. Materials (or patients) and methods: Data on transplants performed for children less than 18 year of age between 1984 and 2012 in eight EM countries (Saudi Arabia, Jordan, Tunisia, Iran, Oman, Pakistan, Egypt, and Lebanon) were collected using survey sheets. The data were collected according to primary disease categories, indication, donor type, stem cell source, intensity of conditioning regimen (myeloablative versus reduced intensity), and year of transplantation. Results: The first HSCT for children in the EM region was performed in 1984. Between 1984 and 2012, 5187 transplants were performed, 4513 (87 %) allogeneic, and 674 (13%) autologous. In all countries, the number of allogeneic HSCT was significantly larger than autologous. The largest number of transplants were performed in Saudi Arabia with a total of 1977 transplants (38.2%), followed by 1197 in Iran (23.1%), 811 in Egypt (15.6%), 361 in Jordan (7%), 325 in Pakistan (6.2%), 249 in Tunisia (4.8%), 162 in Oman (3.1%), and 105 in Lebanon (2%). Lymphoma was the main indication for autologous HSCT (43.2%), followed by solid tumors (36.8%), and leukemia (20%). In the allogeneic HSCT group, leukemia was the main indication (37.5%), followed by hemoglobinopathy (24%), bone marrow failure (21%), primary immunodeficiency
disorders (13.3%) and inherited metabolic diseases (2.9%). Overall, the indication for transplantation was malignant diseases in 1736 patients (38.5%) and non-malignant in 2777 cases (61.5%). A myeloablative conditioning regimens were used in 88% of the allogeneic HSCT. Bone marrow (BM) was the most common stem cell source (56.2%), though an increasing use of peripheral blood stem cell (PBSC) was observed in the last decade. The stem cell source of autologous HSCT has shifted from BM to PBSC, and 80.9% of autologous transplants were from PBSC. Among the recipients of allogeneic transplants, 94.5% were from related donors, while 5.5% were from an unrelated source, mainly cord blood. There was a significant increase in the number of HSCT over the study period. The number of transplants increased from 132 transplants in the first decade (1984-1993) to 1300 transplants between 1994-2002 to 3755 transplants between 2003-2012. Conclusion: This is the first report on the state of HSC activities for children in the EM region. There is an overall increased rate of HSCT for children in the last 2 decades. Unlike western countries; the non-malignant disorders are the main indications for allogeneic transplantation. Unrelated transplants are still markedly underutilized. Development of regional cord blood donor banks and registries might improve the access to unrelated donor sources. Further studies on detailed clinical outcome data are planned. Disclosure of Interest: None declared. P258 Hematopoietic stem cell transplantation in Fanconi anemia and acquired aplastic anemia in children: one center experience B. Kuskonmaz1,*, T. Bayhan2, S. Aytac¸2, S. Unal2, B. Tavil2, F. Gumruk2, M. Cetin2, D. Uckan Çetinkaya1 1 Department of Pediatrics, Divison of Pediatric Bone Marrow Transplantation Unit, 2Department of Pediatrics, Divison of Hematology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) from HLA matched sibling donor is a curative therapy for patients with severe aplastic anemia either acquired (AAA) or inherited. Fludarabine based conditioning regimen is preferred in patients with FA in recent years. Here we report the results of HSCT in two groups of patients; patients with FA and patients with AA. Materials (or patients) and methods: Thirty six patients with FA and 21 patients with AA underwent HSCT between February 1995 and May 2014. The median age of the patients was 10.5±3.4 y (5.6-17.6 y) and 26 of them (72.2%) were males in FA group; median age was 9.5±3.2 y (4.3-14.3 y) 15 of them (66.7%) were male in AAA group. One patient in FA group and one patient in AA group underwent second HSCT due to engraftment failure. Fludarabine based conditioning regimen had been used in 27 of the 28 patients with FA who underwent HSCT since April 2004 as shown on Table 1. Two of the FA patients developed AML before transplantation. One of them received fludarabine based conditioning regimen. Results: Engraftment was achieved in 35 of 36 patients in FA group (97.2%) and 20 of 21 patients in AAA group (95.2%). Transplant related events were shown on Table 1. Survival rates were 82.9% (29/35) in patients with FA and 95.2% (20/21) in patients with AAA. When patients with FA who received fludarabine based regimen were evaluated separately, survival rate was found to be 92.9% (25/27). One patient with FA underwent second HSCT due to poor graft function and died with sepsis. The two patients with AML among the FA group died after HSCT due to AML relapse and acute GVHD, respectively. Conclusion: In FA patients survival rate is reported to be about 75% after HSCT from HLA identical sibling in experienced centers. In our study, survival rate is found as 82.9% in FA patients after HSCT. The survival rates increased to
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Table 1.
Transplant charactersitics and results of trasplantation Fanconi anemia
Donor characteristics HLA matched sibling HLA matched other relatives HLA 1 ag mismatched relatives Conditioning regimen
GVHD prophylaxis
Stem cell source BM PB BM þ CB Engraftment Neutrophil engraftment day Acute GVHD (Zgrade 2) Chronic GVHD VOD1 Survival/median follow-up
1
Acquired aplastic anemia
26 (72.2%)
16 (76.2%)
7 (19.4)
2 (9.5%)
3 (8.3%)
3 (14.3%) Cy þ ATG: 18 (85.7%) Bu þ Cy þ ATG: 2 (9.5%) Flu þ Cy þ ATG: 1 (4.8%)
27 (75.0%) 8 (22.2%) 1 (2.8%) 35 (97.2%) 14.2±2.8 d (9-19 d)
20 (95.2%) 1 (4.2%) 20 (95.2%) 16.7±3.4 d (1022 d) 2 (9.5%)
2/33 (6.1%) 8 (22.2%) Total patients: 29/36 (80.6%) (53 mo) Flu based regimen: 25/ 27 (92.6%) (49 mo)
Conditioning regimen Bu þ Cy1 Bu þ Flu þ ATG2 Bu þ Cy þ ATG1
Flu þ Cy þ ATG: 27 (75.0%) Cy þ TBI±ATG: 4 (11.1%) Bu þ Cy: 3 (8.3%) Cy þ ATG: 1 (2.8%) Cy þ TBI þ ATG þ Bu: 1 (2.8%) CsA þ MTX: 27 (75.0%) CsA/CsA þ MPZ: 9 (25.0%)
7/35 (20.0%)
Table 1. Characteristics of patients and results of transplantation
CsA þ MTX: 21 (100%)
1 (4.5%) (-) 20 (95.2%) (97mo)
Seven of them mild-moderate and one is severe VOD.
92.9% in FA patients who received fludarabine based regimen as expected. Allogeneic HSCT is a preferred therapy for young patients with severe AAA who have a HLA matched sibling donor with long-term survival rates reaching 80–95%. In this study, although 23.8% donors were not HLA identical siblings, disease free survival was achieved in 95.2% of the patients. Disclosure of Interest: None declared. P259 Hematopoietic stem cell transplantation in children with chronic myeloid leukemia B. Kuskonmaz1,*, T. Bayhan2, S. Aytac¸2, S. Unal2, B. Tavil2, F. Gumruk2, M. Cetin2, D. Uckan Çetinkaya1 1 Department of Pediatrics, Divison of Pediatric Bone Marrow Transplantation Unit, 2Department of Pediatrics, Divison of Hematology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Introduction: Chronic myeloid leukemia (CML) is composed of 3% of pediatric leukemias. This makes it difficult to perform clinical trials that can define the best therapeutic option, when considering the impact of tyrosine kinase inhibitors (TKIs) versus the established approach of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report the results of the 17 CML patients who underwent HSCT. Materials (or patients) and methods: Seventeen patients with CML who underwent HSCT between October 1997 and December 2011 were included in the study. The mean age of the patients was 11.5±3.7 y (5.0-16.1 y) and 11 of them were males (64.7%). Three of the 17 patients underwent second HSCT due to relapse of CML. Before HSCT 12 patients had chronic phase CML and 5 patients had accelerated phase CML. Donor characteristics were as follows: HLA identical sibling 14 (82.4%), HLA 1 ag mismatched family donor 2 (11.8) HLA identical father 1 (5.9%). Sources of stem cells was bone
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GVHD prophylaxis CsA þ MTX/CsA Engraftment/Neutrophil engraftment day Acute GVHD (Z grade 2) Chronic GVHD VOD (moderate/severe) Survival/median follow up
14 (82.4%) 2 (11.8%) 1 (5.9%) 16 (94.1%)/1 (5.9%) 17 (100%)/17.1±2.6 day (1222 day) 5 (29.4%) 3 (17.6%) 1 (5.9%) 13 (76.5%)/135 months
1
Myeloablative conditioning regimen, 2Reduced intensity conditioning regimen.
marrow (BM) in 13 (76.5%), peripheral blood (PB) in three (17.6%) and BM þ PB in one (5.9%) of the patients. Before use of imatinib in pediatric CML, patients were treated with various chemotherapeutic agents, hydroxyurea, interferon alpha before HSCT. Imatinib was used in 5 patients; solely in two patients and in combination with other agents in three patients. Imatinib was switched to dasatinib in one patient due to failure of molecular remission. Results: The results of HSCT were shown on Table 1. Engraftment was achieved in all patients. Relapse of CML developed in 5 patients (29.4%). Relapse was observed in only one of the 12 patients (8.3%) who underwent HSCT at chronic phase and four of the five patients (80.0%) at accelerated phase. Engraftment and survival were achieved in all three patients who underwent second HSCT due to disease relapse. One of them received imatinib and the other received imatinib and donor lymphocyte infusion due to molecular t(9;22) positivity. Survival rate was 91.7% (11/12) in patients who underwent HSCT at chronic phase and 40.0% (2/5) in patients who underwent HSCT at accelerated phase. Conclusion: Because there have been no randomized controlled trials comparing transplant and imatinib in pediatrics, the decision on how to treat has been individualized. Consistent with these recommendations the number of HSCT has decreased in recent years at our center as well. In a registry report from the EBMT, the overall survival (OS) at 3 years for children transplanted in first chronic phase from sibling donors or unrelated donors was 75%, and 65%, respectively. Outcome for patients who received transplants in advanced stage was significantly worse. In our study, the OS was 76.5% for all patients, this ratio was found as 91.7% in patients who underwent HSCT at chronic phase of CML. Disclosure of Interest: None declared. P260 Hematopoeitic stem cell transplantation in patients with genetic hemophagocytic lymphohistiocytosis B. Kuskonmaz1,*, S. Aytac¸2, D. Ayvaz3, T. Bayhan2, B. Tavil2, S. Unal2, M. Cetin2, F. Gumruk2, I. Tezcan3, D. Uckan Çertinkaya1 1 Department of Pediatrics, Divison of Pediatric Bone Marrow Transplantation Unit, 2Department of Pediatrics, Divison of Pediatric Hematology, 3Department of Pediatrics, Divison of Pediatric Immunology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Introduction: The genetic form of hemophagocytic lymphohistiocytosis (HLH) can be divided into 2 subgroups: familial HLH (FHL) and those associated with primary immunodeficiency syndrome including Griscelli syndrome 2 (GS2), Chediak-Higashi syndrome (CHS), X linked lymphoproliferative disorder and other rare immunodeficiency syndromes. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment modality in genetic HLH.
Table 1. Characteristics of patients and results of transplantation Donor characteristics HLA identical sibling HLA identical other relatives HLA 1 ag mismatched relatives Conditioning regimen Engraftment Acute GVHD (4grade 2) Chronic GVHD VOD (moderate-severe) Survival rate for all patients Survival rate at CR1
16 (64.0%) 5 (20.8%) 4 (16.0%) Bu þ Cy±vepesid/ATG: 20 (80%) Bu þ Flu: 4 (16.0%) Flu þ Mel þ alemtuzumab: 1 (4.0%) 24 (92.3%) 2/23 (8.7%) 3/22 (13.6%) 6 (24.0%) 18 (72%) (median follow up 67 months) 16/19 (84.2%) (median follow up 60 months)
Materials (or patients) and methods: Twenty five children with HLH who underwent HSCT between October 1997 and September 2014 are included in the study. The mean age of the patients was 4.8±5.3 (5 mo-16 y), 15 of them (60.0%) were females. Fourteen patients had familial HLH and 10 had GS2 associated HLH, one had CHS associated HLH. At the time of HSCT, 19 patients had complete remission 1 (CR), three patients had CR2, two patients had active disease, one had CR3. Source of stem cells was bone marrow (BM) in 15 patients (60%), peripheral blood in 9 patients (36%) and BM þ cord blood in one (4%) patient. The other characteristics of the patients and transplantation were shown on Table 1. Three patients underwent second HSCT and two of them underwent third HSCT due to primary or secondary engraftment failure. Results: Engraftment was achieved in 24 patients (92.3%) but one of them developed secondary graft failure. The mean engraftment day was 13.5±2.6 day (10-21 day). The survival rate for all 25 patients was 72% (16/26); when the patients who underwent HSCT at CR1 were evaluated separately, survival rate was found as 84.2% (16/19) (Table 1). In four patients in whom bu and flu were used as conditioning regimen survival was achieved. All patients who survived are disease free at present. Conclusion: The survival rate of HSCT with myeloablative conditioning was reported as 53–71%. The advent of reducedintensity conditioning with lower toxicity improved survival of HSCT. In this study survival rate for all patients was 72%, in patients transplanted at CR1 survival rate increased to 84.2%. Disclosure of Interest: None declared. P261 Infectious profile and infection-related mortality of allogeneic stem cell transplantation in children: A single center experience C. De Miguel Sanchez1,*, M. Lopez Duarte1, A. Bermudez1, M. Colorado1, B. Gonzalez-Mesones1, P. Ibarrondo1, E. Conde1 1 Hematology, HOSPITAL UNIVERSITARIO MARQUES DE VALDECILLA, Santander, Spain Introduction: Infectious diseases are important causes of morbidity and mortality in paediatric patients who undergo an allogeneic haemopoietic stem-cell transplantation (allo-HSCT). We analyze the infectious complications occurring during three different posttransplantation phases (days 0-30, 30-100, and 100-365 after allo-HSCT) and their effect on mortality in a high risk allo-HSCT pediatric cohort during the first year after allo-HSCT. Materials (or patients) and methods: Thirty one patients younger than 18 years underwent allo-HSCT in our center from 2004 to 2014. We retrospectively analyzed all documented
infections in 35 allo-HSCT(4 patients had a previous allo-HSCT) reviewing medical and microbiological records in the first year after allo-HSCT. No primary antibacterial or antifungal prophylaxis was employed. Acyclovir was used until immunosuppression was stopped and TMP-SMX until CD4 counts were greater than 200/mm3. Twenty patients were male. Twenty patients had a malignant disease. Median age at time of transplantation was 10 years (1-18). There were 15 mismatched donors (13 unrelated). The stem cell source was bone marrow (19), peripheral blood (12) and cord blood (4). Mieloablative conditioning regimen was used in 27 and timoglobuline was used in 16 allo-HSCT. Sixteen patients had acute graft versus host disease (aGVHD), but 8 had greater than grade 2 GVHD. Seven patients developed moderate-severe chronic GVHD during the study period. Results: One hundred forty-one infections were documented in 31 patients. Seventy-seven (55%) bacterial infections were registered, being more frequent in the first period (46 vs 26 vs 5 cases respectively). We found 46 Blood Stream Infections (BSI) which were the most frequent bacterial infection in first two periods (28 and 16 BSI respectively). Staphylococcus Epidermidis was the main pathogen of BSI (22 cases). Cumulative incidence for BSI in the 3 periods was 80%, 47% and 7% respectively. There were 7 cases of C. Difficile Infection (6 in first period). Forty-four viral Infections (31%) were documented, being equally prevalent in all 3 periods (15 vs 15 vs 14 cases) but with different etiologies at each period. Two patients developed Posttransplantation Lymphoproliferative Disease (PTLD) by Epstein Barr (EBV)(first and fourth month). Nineteen Fungal Infections (13%) were documented, being more frequent in the 2nd and 3rd period (1 vs 11 vs 7 cases). Candida Albicans was the major etiology (12 cases, all with oral candidiasis) and 7 invasive fungal infections (IFI) were detected. One patient (0,7%) was diagnosed of Cerebral Toxoplasmosis at 8th month. 365 days after allo-HSCT, 19 patients were alive. Infection was a direct cause of death in 2 patients with cord blood transplant (Early PTLD-EBV in Fanconi Anemia and IFI by Rhizopus sp in a patient with primary graft failure) during the first year after allo-HSCT. Seventeen patients are currently alive (54%) with a median follow up of 48 months (0-127). One death due to Pneumococcal sepsis on sixth year after allo-HSCT was registered in this period. Conclusion: Bacteria and viruses were the most prevalent infections documented in a high risk paediatric group of patients. However direct infection-related mortality was less than 10% with no bacterial-related mortality one year after allo-HSCT even though primary antibacterial or antifungal prophylaxis was not used. Disclosure of Interest: None declared. P262 In vivo B-cell depletion with Rituximab following transplantation with T-cell depleted haploidentical donor hematopoietic cells efficiently prevents EBV reactivation and EBV-PTLD without the need of ex vivo B-cell depletion of the garft C. Jepsen1,*, C. Pronk1, J. Toporski1, J. Dykes2, D. Turkiewicz1 1 Department of Pediatric Oncology and Hematology, 2Department of Immunology and Transfusion Medicin, University Hospital in Scania, Lund, Sweden Introduction: Haploidentical stem cell transplantation (haploSCT) is an established treatment for patients that require an allogeneic SCT but lack a HLA matched donor. Ex vivo T-cell depletion (TCD) efficiently prevents GvHD, but the consequential delayed immunologic recovery increases the risk of EBV reactivation. Herein we aim to investigate the efficacy of ex vivo B-cell depletion using one dose of Rituximab on day þ 1, in the context of TCD haploSCT, to preventing EBV reactivation and EBV-PTLD. Materials (or patients) and methods: We evaluated a total of 50 haploSCT performed in 42 patients, 29 males and 15 females, at the Dep of Paediatric Oncology and Haematology in Lund, Sweden between 2005 and 2014. Median age of
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patients was 7 years and 8 months (from 4 months to 18 years). Six children received more than one haploSCT due to graft failure or relapse. Twenty patients were diagnosed with a malignant disease (ALL 13, AML 4, MDS/JMML 2), 17 with solid tumours, (NBL 14, RMS 1, Ewing Sa 1, Wilms 1) and 8 with nonmalignant diseases (3 with SCID, 2 with MPS VI, 1 Fanconi Anemia, 1 Osteopetrosis and 1 SAA). In all cases, either donor or recipient or both were EBV serologically positive before haploSCT. Conditioning consisted of either Fludarabine or Clofarabine combined with Thiotepa and Melphane. One patient with SCIDXl did not receive any conditioning. For rejection prophylaxis either OKT3 (0.1 mg/kg until day þ 21) or ATG-Fresenius 10mg/kg/day (day -12 to -10) was used. To prevent GvHD, immunomagnetic TCD of either CD3 þ or TCRab þ T-cells was performed (CliniMACS, Miltenyi Biotec). One dose of Rituximab 375mg/m2 on day þ 1, without ex vivo CD19-depletion of the graft, was administered to all patients to prevent EBV associated complications. All patients were followed weekly with EBV PCR in plasma until recovered normal immunologic function and monitored clinically for the symptoms of EBV-PTLD. Results: Median follow-up was 428 days ranging from 9 days to 9 years. No case of EBV-PTLD was observed. Only 4 patients had at least one positive result of EBV-PCR resulting in cumulative incidence of EBV positivity of 0.08 (95% confidence interval 0.02 – 0.18). Time from haplosSCT to the first positive EBV-PCR result ranged from 88 to 159 days. The highest observed number of EBV copies ranged from 270 to 4600 copies/mL. In 3 patients, EBV-PCR positivity converted spontaneously within 2 weeks without intervention. One patient with a DLI-induced acute and chronic GvHD had repeated episodes of transient EBV positivity during 338 days but finally became negative. Three out of 4 patients were serologically EBV neg prior to haploSCT while the donors were positive. All long-term survivors recovered with normal B-cell function and do not require IVIG substitution. Conclusion: Prevention of EBV-PTLD after TCD haploSCT is achieved by either or both in vivo and/or in vitro B-cell depletion. Post-haploSCT in vivo B-cell depletion given one day after infusion of non-B cell depleted graft have an advantage of eliminating both residual autologous and donor B-cells. Our results strongly suggest that in vivo B-cell depletion with one dose of Rituximab is sufficient to prevent EBV reactivation and EBV-PTLD without the risk of long-term B-cell dysfunction. Disclosure of Interest: None declared. P263 Impact of donor-recipient ABO mismatch on allogeneic hematopoietic stem cell transplantation in pediatric thalassemic patients D. Atay1,*, F. Erbey1, A. Akcay1, G. Ozturk1 1 PEDI˙ATRI˙C HEMATOLOGY ONCOLOGY BONE MARROW TRANSPLANTATI˙ON UNIT, ACIBADEM UNIVERSITY MEDICINE FACULTY, Istanbul, Turkey Introduction: The ABO incompatibility between donor and recipient is not considered a barrier to successful allogeneic hematopoietic stem cell transplantation (HSCT), even if it can be associated with several immunohematologic complications. Nevertheless, conflicting data still exist about the influence of major, minor, bidirectional ABO incompatibility on graftversus-host disease (GVHD), venoocclusive disease (VOD) incidence, hematologic recovery and engraftment in pediatric thalassemic patients. Materials (or patients) and methods: From May 2011 to August 2014, 52 children with beta thalassemia major who underwent allogeneic HSCT and followed for a minimum of hundred days post-transplantation were enrolled this study. Results: The median age of patients at the time of transplant was 6 years (range ¼ 11 months-18 years). The distribution of
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Pesaro risk class I and II categories was 29 and 23 children. The median serum ferritin level was 1700 ng/mL. All patients received bone marrow (BM) stem cells from matched sibling (n ¼ 43), related (n ¼ 5) or unrelated (n ¼ 3) donors after myeloablative conditioning regimens, which include ATG in 14 patients. Except 6 patients (major:4, none:2), all patients achieved initial donor cell engraftment. Thalassemic reconstitution was observed in 6 of the transplant patients (major:1, minor:1, none:4). Post-transplant pure red cell aplasia occurred in two patients, both were major ABO mismatched patients. Among BM recipients, overall incidence of acute and chronic GVHD, VOD, neutrophil and platelet engraftment were similar in ABO-matched and ABO mismatched transplants (P40.05). However, especially in major and bidirectional ABO mismatched patients, a delayed erythroid recovery was recorded as compared to the group receiving an ABO compatible graft (p:0.02 and p:0.03). Median time to red cell transfusion independence was significantly longer in major ABO-incompatible BM recipients (median time, 95 days vs 32 days; p:0.001). This difference appears to be independent of the amount of cells infused during transplantation, with a median number of CD34 cells of 4.89x 106/kg in ABO-mismatched patients and 5.40x106/kg in control ABO-matched patients (P40.05). Conclusion: In our experience, major and bidirectional ABO incompatibility leads mainly to delayed RBC recovery after HSCT. Therefore, whenever feasible, major ABO-mismatched donors should be avoided in HSCT recipients, to prevent delayed erythroid recovery with prolonged RBC transfusion needs and impaired quality of life. Disclosure of Interest: None declared. P264 CD45RA depleted donor lymphocyte infusions for antiviral boost following ex-vivo T-cell depleted haploidentical hematopoietic stem cell transplantation D. Turkiewicz1,*, J. Dykes2, C. Pronk1, J. Toporski1 1 Department of Pediatric Oncology and Hematology, 2Department of Immunology and Transfusion Medicin, University Hospital in Scania, Lund, Sweden Introduction: Extensive ex-vivo T cell depletion is a very effective method of graft versus host disease prevention. It allows performing even a haploidentical stem cell transplantation (haploSCT) without any pharmacological post-transplantation immunosuppression. However, to be so effective T-cell depletion must be nearly total. This, in turn, leads to elimination of the early phase of T-cell recovery thus increasing the risk of viral infections. One possible way to manage the problem of T-cell gap and provide the patient with the donor antiviral immunity is by donor lymphocyte infusion. However, infusion of unselected lymphocytes poses a risk of severe graft versus host disease. Since the alloreactive T-cells are predominantly contained in the naı¨ve T-cell compartment, depletion of CD45RA positive cells from DLI decreases the frequency of GvHD initiating cells while preserving the pathogen specific memory lymphocytes. Infusion of CD45RA depleted donor lymphocytes might therefore allow for safe transfer of donor-derived immunity with a limited GvHD risk. Materials (or patients) and methods: Infusion of CD45RA depleted lymphocytes has been performed in 4 children after haploidentical SCT. The clinical data is summarized in a table below. All patients had an active viral infection at time of infusion, all were in complete remission of the underlying disease and had a profound T-cell lymphopenia. Two patients had clinical symptoms of viral diseases: HHV6 encephalitis (one patient) and concomitant active varicella, colitis and rotavirus infection (one patient). None of the children had any GvHD. Three patients received one infusion of 2,5x104/kg, one patient received two infusions: 2,5x104/kg and four weeks later 5x104/kg.
Results: UPN Disgnosis
1
2
3
4
Virus ifection
Clinical symptoms
CD45RA depleted DLI - cell dose
Outcome
Recovery VZV, CMV gan- varicella, colitis 2.5x104/ with loss of kg ciclovir resis- keratitisretinitis sight in one Day tant, Rotavirus eye no GvHD þ 80 CMV neg from day þ 73 HSCT from DLI VZV neg day þ 22 from DLI Rota neg day þ 22 from DLI ALL 2CR CMV - increasnone DLI#1: Full recovery ing number of 2.5x104/ no GvHD copies despite kg HAdV neg cidofovir treatday day þ 31 ment, HAdV þ 74 from DLI#2 from CMV neg HSCT day þ 84 DLI#2: from DLI#2 5x105/kg day þ 93 from HSCT Recovery ALL 3CR HHV6, HAdV encephalitis 2.5x104/ kg with residual day neurological þ 63 symptoms, from no GvHD HSCT HAdV neg day þ 26 from DLI 4 SAA HAdV - increasnone 2.5x10 / Full recovery kg ing copy numno GvHD day ber despite HAdV neg þ 68 cidofovir day þ 75 from treatment from DLI HSCT SCID IL7Ra
Conclusion: All patients survived, eliminated all viruses and none developed graft versus host disease. The depletion of CD45RA þ cells with CliniMACS system was reliable, effective and affordable. Since the depletion step does not alter the biological properties of the cells, the regulatory issues are simpler to comply. However, a clinical study is needed to establish the role of this method in decreasing morbidity and mortality of haploSCT. Disclosure of Interest: None declared. P265 Outcome following haploidentical haematopoietic stem cell transplantation using ex-vivo T-cell depletion and reduced intensity conditioning in children with acute leukemia D. Turkiewicz1,*, M. Ifversen2, J. Dykes3, C. Heilmann2, C. Pronk1, K. Mueller2, A. Fischer-Nielsen4, J. Toporski1 1 Department of Pediatric Oncology and Hematology, University Hospital in Scania, Lund, Sweden, 2Pediatric Clinic II, 4074, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 3Immunology and Transfusion Medicin, University Hospital in Scania, Lund, Sweden, 4Rigshospitalets Blodbank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Introduction: Haploidentical stem cell transplantation (HaploSCT) is an established treatment modality for children who require an allogeneic stem cell transplantation (alloSCT) but lack a matched (sibling or unrelated) donor. The reduced intensity conditioning permits performing alloSCT even in patients with a high risk of transplant related mortality (TRM). We present the results of TCD haploSCT in children with acute
leukemia performed between 2005 and 2014 in 2 Nordic HSCT centers in Lund and Copenhagen; a result of an established cooperation in the Oresund region. Materials (or patients) and methods: HaploSCT was performed in 23 children with acute leukemias, 15 with ALL and 8 with de novo AML. Four patients were transplanted with Z5% blasts in bone marrow and 19 were in complete remission (CR; CR1: 3, CR2: 9, CR42: 7). Eight patients (35%) had previously received an alloSCT, median time from the 1st alloSCT to haploSCT was 375 days (50 days-2.3 years). Conditioning regimen consisted of a combination of either clofarabine 200mg/m2 or fludarabine 125mg/m2 with 10mg/ kg Thiotepa and 120mg/m2 Melphalane. For rejection prophylaxis, either OKT3 (0.1mg/kg/day until day þ 21) or ATG-Fresenius (30mg/kg day -12 to -9) was used. GvHD prevention consisted of ex-vivo immunomagnetic depletion of either CD3 þ (n ¼ 9) or TCR ab þ (n ¼ 14) cells (CliniMACS, Miltenyi Biotech). Median number of transplanted CD34 þ cells/kg was 11.4x106. Median number of co-infused ab þ T-cells/kg was 2.7x104. One dose of Rituximab 375mg/m2 was given on day þ 1 to prevent EBV-PTLD. Sixteen patients received a short course of MMF (n ¼ 16) until day þ 28. Fourteen patients received low-dose donor lymphocyte infusions (DLI) either as a relapse prophylaxis or to clear viral infections. Results: 22/23 patients engrafted. One patient with primary graft failure was successfully regrafted with another haploidentical donor. Median time to ANC and PLT engraftment was 12 and 15 days, respectively. Cumulative incidence (CI) of primary grade I to II aGvHD was 0.21 (95% Confidence Interval 0.07-0.39). No primary grade III-IV aGvHD was observed. No cGvHD occurred in children who did not receive DLI. Post-DLI aGvHD was observed in 6/14 patients (43%), of which 5 had grade III/IV. Two patients developed extensive cGvHD following DLI. Thirteen patients are alive; 12 in CR, with median follow up of 2 years and 2 months (from 147 days to 9 years). Ten patients died: 6 of relapse (including 4 patients transplanted without remission) and 4 of TRM (2 post-DLI GvHD, 1 AdV infection and 1 secondary graft failure). For the whole cohort, 1 year EFS, CI of relapse and TRM were 0.5 (95% CI 0.32 – 0.76), 0.32 and 0.18 respectively. For patients transplanted in remission, 1-year EFS reached 0.6 (95%CI: 0.32 – 0.76), CI of relapse 0.17 and TRM 0.22. EFS for children with previous alloSCT was 0.47 (95% CI: 0.2 – 1) at 1 year. Conclusion: Our results show that TCD HaploSCT is a valid therapeutic alternative for children with high risk acute leukemia that achieve CR. Despite reduced conditioning, cumulative incidence of relapse is acceptable in this high risk population. The role of post-haploSCT DLI remains to be determined as it may cause life threatening GvHD despite the relatively low doses of lymphocytes infused. Disclosure of Interest: None declared. P266 Fertility Preservation in Female Pediatric HSCT Patients: A Multidisciplinary Approach for the French Speaking Part of Switzerland F. Bernard1,2,*, L. Crosazzo3, C. Girardin1, M. Yaron4, I. Andrieu-Vidal5, F. Phan-Hug3, D. Wunder6, M.-P. Primi6, J.-P. Simon7, V. Schwitzgebel1, I. Streuli4, Y. Chalandon8, M. Ansari1,2, M. Beck-Popovic3, F. Gumy-Pause1,2 1 Pediatrics, University Hospital of Geneva, 2CANSEARCH Research Center, Geneva, 3Pediatrics, University Hospital of Canton de Vaud, Lausanne, 4Gynecology-Obstetrics, 5Pediatric Surgery, University Hospital of Geneva, Geneva, 6Gynecology-Obstetrics, 7 Legal Affairs Unit, University Hospital of Canton de Vaud, Lausanne, 8Hematology, University Hospital of Geneva, Geneva, Switzerland Introduction: Conditioning regimens prior to hematopoietic cell transplantation (HSCT) are known to be highly gonadotoxic (especially total body irradiation and alkylating agents such as Busulfan (Bu), Cyclophosphamide (Cy) and Melphalan
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(Mel)). Unfortunately, impaired reproductive function and infertility are frequently seen in long term survivors. Ovarian failure rate post HSCT ranges from 72 to 100% and may negatively impact the survivor’s quality of life. Different fertility preservation options (FPO) are available for pubertal patients (sperm banking for males and cryopreservation of oocytes for females). Recently, ovarian tissue cryopreservation (OTC) has emerged as a new experimental option. This technique can be proposed to pubertal and prepubertal females. Theoretically, two different options are available once the patient is ready to conceive: ovarian auto-transplantation (experimental method, feasible today) or in vitro maturation of primordial follicles followed by in vitro fertilization (still in research, nowadays not feasible). Materials (or patients) and methods: In 2010, two multidisciplinary swiss pediatric teams in Lausanne (CHUV) and in Geneva (HUG) were formed under the auspices of the ‘‘Re´seau Romand de Cancer et Fertilite´’’. Each team consists of pediatricians specialized in hemato-oncology, gynecology, endocrinology, surgery, psychiatry, reproductive medicine, radiation oncology, adolescent medicine and genetics. Regular video-conference meetings are organized during which new cases are presented, with the objectives of determining 1) indications for fertility preservation and 2) the best FPO for the individual patient. Meetings also provide an opportunity to discuss guidelines, literature and difficult cases. Results: To date, ovarian fragments were collected and cryopreserved for 12 (7 autologous and 5 allogenic) HSCT prepubertal girls, with a median age of 5 years (range 6 months to 11 years). In all cases, an informed consent form was signed. Except one patient with hemophagocytic lymphohistiocytosis (HLH), all patients suffered from malignant diseases: metastatic neuroblastoma (n ¼ 6), acute lymphoblastic leukemia (n ¼ 4), and metastatic Ewing sarcoma (n ¼ 1). OTCs were realized before HSCT conditioning regimens (5 BuMel, 4 Bu-Cy, 1 Treosulfan-Mel, 1 CEM (Carboplatin-EtoposideMel) and 1 TBI-Etoposide). All patients except the one with HLH, underwent several courses of chemotherapy before OTC and conditioning phase, the number of courses was dictated by their disease. These chemotherapy courses are not known to have a sterilizing effect. Post surgery follow up was uneventful in all 12 patients. OTC was also proposed to 2 other HSCT patients (1 acute lymphoblastic leukemia and 1 juvenile myelomonocytic leukemia) but refused by the parents. Conclusion: Fertility preservation techniques are now also available for prepubertal females and continue to develop. The opportunity to provide accurate information and hope for future fertility in young patients is invaluable. In view of the paucity of cases, only unification of medical forces will enable us to offer for each of our patients the best possible care. Disclosure of Interest: None declared. P267 Low transplant-related mortality in children with acute leukemia given hla-haploidentical hematopoietic stem cell transplantation after removal of Alpha/Beta þ t cells and of CD19 þ b cells from the graft F. Locatelli1,*, A. Bertaina1, P. Merli1, B. Lucarelli1, L. P. Brescia1,
coupled with B-cell depletion accomplished through the use of an anti-CD19 monoclonal antibody to prevent the occurrence of Epstein-Barr Virus (EBV)-related post-transplant lymphoproliferative disorders (PTLD). Materials (or patients) and methods: Enrolled in the study were 70 patients (pts, 48 M, 22 F), with acute leukemia and a median age at HSCT of 11.3 yrs (range 0.6-17.9). Fifty-one pts had ALL and 19 AML. All children were transplanted in morphological complete remission (CR): 26 pts were transplanted in CR1, 41 in CR2 and 3 in more advanced CR. All pts transplanted in CR1 had either poor cytogenetic/molecular characteristics or high levels of minimal residual disease (MRD) at the end of induction therapy. The donor was either the mother (n ¼ 42) or the father (n ¼ 28); according to the model of KIR/KIR ligand disparity, 31 pts were transplanted from an NK-alloreactive donor. The median number of CD34 þ cells, NK cells, gd þ T cells, and ab þ T cells were 13.6, 34.7, 8.4, 0.08 and 0.04x106/kg, respectively. A myeloablative regimen, containing TBI in 52 cases, was given to all children, who also received rabbit ATG (12 mg/kg over 3 days, from -5 to -3). Rituximab (200 mg/m2) was administered on day -1 to further prevent EBV-related PTLD. Results: Primary engraftment occurred in 69/70 pts, the remaining child being successfully re-transplanted from the other parent. The median time to reach neutrophil and platelet recovery was 13 days (9-19) and 11 days (8-20). No child developed gut or liver acute GVHD. Twenty pts experienced skin-only grade I-II GVHD, this leading to a cumulative incidence (CI) of 28.6% (Standard Error, SE, 5.4). Only 3 pts developed skin limited chronic GVHD, the CI of this complication being 4.7% (SE 2.7). Three pts died for causes other than disease relapse (all in the first 100 days after HSCT), the CI of transplantation-related mortality (TRM) being 4.3% (SE 2.4). Fourteen pts relapsed, the CI of disease recurrence being 21.2% (SE 5.1). With a median follow-up of 27 months (range 6-48), the 2-year Kaplan Meier estimate of leukemiafree survival (LFS) was 74.5% (SE 5.4). The 2-year LFS probability of children with ALL and AML was 75.8% (SE 6.1) and 70% (SE 11.5), respectively. The 2-year LFS of pts who did or did not experience skin-only acute GVHD was 90% (SE 6.7) and 68.7% (SE 6.7, P ¼ 0.11), respectively, while that of children given or not TBI as part of the conditioning regimen was 82% (SE 5.5) and 51.5% (SE 12.7, P ¼ 0.009.) Conclusion: These data obtained in one of the largest single center cohorts of HLA haploidentical-HSCT in childhood indicate that a selective graft manipulation results into effective prevention of both acute and chronic GVHD, rapid recovery of neutrophil and platelet counts and low TRM. The 2-year LFS probability observed in these children favourably compares with that reported in studies where the donor was either an HLA-identical sibling or an unrelated donor of adult stem cells or cord blood cells. Disclosure of Interest: None declared.
D. Pagliara1, R. Pinto1, R. Masetti2, D. Pende3, S. Ceccarelli1, M. Falco4, A. Moretta5, L. Moretta4, G. Li Pira1
P268 Invasive Fungal Infection in Pediatric Hematopoietic Stem Cell Transplantation H. D. Rodriguez Vega1,*, C. Fuentes Socorro1, B. Torres Guerola1, J. M. Fernandez Navarro1 1 Pediatric Oncology, Hospital Universitari i Politecnic La Fe, Valencia, Spain
Introduction: We conducted a prospective, single center study (NCT01810120) in children with acute leukemia aimed at evaluating the safety and efficacy of HLA-haploidentical HSCT after a graft manipulation based on depletion of T lymphocytes carrying the ab chains of the T-cell receptor (TCR),
Introduction: Invasive fungal infections (IFI) are a major cause of mortality and morbidity among pediatric patients receiving hematopoietic stem cell transplantation (HSCT). Diagnosis and treatment of IFI are complex, so that it becomes necessary to know those features that allow to identify patients at high risk. Publications reporting data about IFI risk factors in pediatric population are scanty. Materials (or patients) and methods: We retrospectively analyzed medical records from children with oncohematological disorders who underwent HSCT in a reference Pediatric Oncology Service of a tertiary hospital between January 1991
1
BAMBINO GESU HOSPITAL, Rome, 2University of Bologna, Bologna, 3Istituto di Ricovero e Cura a Carattere Scientifico, Azienda Ospedaliera Universitaria San Martino , 4Istituto Giannina Gaslini, 5Dipartimento di Medicina Sperimentale and Centro di Eccellenza per la Ricerca Biomedica, Universita` di Genova, Genova, Italy
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and December 2013. We defined IFI cases based on current EORTC/MSG criteria. In our centre, we use fluconazol prophylaxis in autologous HSCT from the beginning of mucositis until neutropenia resolves. In allogeneic HSCT, we use voriconazol prophylaxis from the end of the conditioning to the immunosuppressants withdrawal. Since 2009, we twiceweekly do serum galactomanan leves in all HSCT patients during the more risky period. We initially described the incidence and epidemiological features of the different kinds of IFI in our centre. We also carried out an univariate analysis and subsequently a binary logistic regression in an attempt of identify risk factors for IFI. Results: The study population includes 403 pacients who underwent 445 HSCT. The median age was 7,26 years (±4,42). Acute Leukemia was the most frequent diagnosis (55,7% of HSCT). Autologous HSCT was the most common (66%). Sixteen percent of patients suffered from acute graft versus host disease (aGVHD) whereas 4,3% did it from the chronic form (cGVHD). We observed an overall incidence of IFI of 3,4%. Invasive Aspergillosis was the more frequent kind of IFI with 12 cases (incidence of 2,7%). Pulmonary Aspergillosis was the most frequent presentation form (75% of cases). Overall mortality was 44,9% among patients who underwent HSCT but 68% in the IFI group. Patients with a prior diagnosis of lymphoma showed the higher risk for IFI with an Odds Ratio (OR) 25 (2,18-306,98). Conclusion: In our centre, we found an incidence and mortality of IFI similar to that communicated in the literature. As in other centers, after using azole prophylaxis, number of Candida infections has decreased and Aspergillosis is the most frequent IFI. In the univariate analysis, prior diagnosis of lymphoma, allogeneic HSCT, and aGVHD were significantly related to the presence of IFI. After binary logistic regression only the diagnosis of lymphoma was risk factor for IFI with an Odds Ratio (OR) 25 (2,18-306,98). This could be explained by the presence of two cases of candidiasis among only 16 patients with lymphoma. Allogeneic HSCT, specially blood cord and haploidentical transplantation, showed trends of being a risk factor for IFI though it didn’t reach statistical significance. References: 1. Srinivasan A, Wang C, Srivastava DK, et al. Timeline, epidemiology, and risk factors for bacterial, fungal, and viral infections in children and adolescents after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 94–101. 2. Castagnola E, Faraci M, Moroni C, et al. Invasive mycoses in children receiving hemopoietic SCT. Bone Marrow Transplant 2008;41 Suppl 2:S107-11. Disclosure of Interest: None declared. P269 Allogeneic hematopoietic stem cell transplantation for pediatric T lineage acute lymphoblastic leukemia H. Sakaguchi1,*, A. Narita2, K. Narita1, S. Kataoka1, M. Hamada1, N. Murakami2, Y. Sekiya2, K. Suzuki2, N. Kawashima2, Y. Okuno2, S. Doisaki2, H. Muramatsu2, N. Yoshida1, N. Nishio2, A. Hama2, Y. Takahashi2, S. Kojima2, K. Kato1 1 Department of Hematology and Oncology, Children’s Medical Center, Japanese Red Cross Nagoya First Hospital, 2Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan Introduction: The historically unfavorable outcome of patients with T lineage acute lymphoblastic leukemia (T-ALL) has recently improved through the use of highly effective treatment protocols with risk classification. With appropriately intensive therapy, children with T-ALL have an outcome similar to that of children with B-precursor acute lymphoblastic leukemia. However, patients with T-ALL remain at higher risk for remission induction failure, early relapse, and isolated CNS relapse. These high-risk-patients indicate hematopoietic stem cell transplantation (HSCT). In this study, our purpose was to evaluate clinical importance of HSCT in treatment for pediatric T-ALL.
Materials (or patients) and methods: This retrospective study included 22 consecutive pediatric patients diagnosed with T-ALL who underwent HSCT at our institutes between Jan 1990 and Mar 2013. Both institute maintain a comprehensive and prospective database on all patients who undergo transplantation. We reviewed all available medical records for this study. The median age at diagnosis was 7.6 years old (range, 1.8-13.0); that of at transplantation was 8.3 years old (range, 3.3-16.2 years). The clinical status at HSCT was composed of first complete remission (1CR) for 9 patients (including 5 patients with delayed primary remission), second complete remission (2CR) for 7 patients, and advanced stage (AS) including third remission and non-remission for 6 patients. Conditioning regimens consisted of total body irradiation (TBI) based therapy (n ¼ 17), busulfan based myeloablative therapy (n ¼ 2), and reduced intensity therapy (n ¼ 3). Hematopoietic cells were donated from autologous bone marrow (n ¼ 1), human leukocyte antigen (HLA)-identical sibling or parent (n ¼ 11), HLA-haploidentical parent (n ¼ 3), volunteered unrelated donor (n ¼ 5), and unrelated cord blood (n ¼ 2). Overall survival (OS) was defined as the time from HSCT to death or last follow-up date. Disease free survival (DFS) was defined as the time from HSCT to relapse, death, or last follow-up date. Results: The median period of observation was 38 months (range, 4-195 months). Primary graft failures occurred in 1 patient (4%), who received cord blood graft and underwent second cord blood transplantation resulting primaly graft failure again, finally were rescued by HLA-haploidentical transplantation with sustained engraftment and complete remission. The probabilities of 3years OS/DFS were 65%/63% for total cohort, 100%/100% for patients at 1CR, 34%/43% for patients with 2CR, and 38%/38% for patients at AS, respectively. Among 13 patients at 2CR or AS, 6 patients survive with remission, including 3 patients who received HLAhaploidentical HSCT, 2 patients who received HLA-identical sibling HSCT with TBI-based myeloablative conditioning, and the other patient conditioned with nelarabine combined chemotherapy following cord blood transplantation. While, 7 patinets at 2CR or AS died during observation; cause of death consisted of progressive diaseas (n ¼ 4), respiratory distress (n ¼ 2), and hepatic veno-occlusive disease (n ¼ 1). Conclusion: Our findings suggested that HSCT yielded long term disease free survival to the patients at 1CR even with delayed primary remission, on the other hand, the prognosis of patients at 2CR or AS treated with conventional HSCT was poor; these patients could be rescued by more novel approach such as HLA-haploidentical HSCT or nelarabine combined conditioning. Disclosure of Interest: None declared. P270 Haploidentical hematopoietic stem cell transplantation using ex vivo T cell-depleted grafts in pediatric patients H. J. Im1,*, K. N. Koh1, J. K. Suh1, S. W. Lee1, E. S. Choi1, S. Jang2, J. J. Seo1 1 Pediatrics, 2Laboratory Medicine, Asan Medical Center, Seoul, Korea, Republic Of Introduction: Currently, haploidentical hematopoietic cell transplantation (HHCT) is considered a valid option for patients who have diseases curable with hematopoietic cell transplantation (HCT) but who lack a matched related or unrelated donor. The purpose of this study was to assess the efficacy and feasibility of transplantation using ex vivo T cell-depleted grafts from haploidentical family donors in children and adolescents with malignant or non-malignant diseases. Materials (or patients) and methods: Between July 2008 and November 2014, 57 pediatric patients underwent ex vivo T cell-depleted HHCT after reduced intensity conditioning (RIC). Twenty-eight received CD3-depleted stem cells (CD3-HHCT) and 29 received TCRab-depleted grafts (TCRab-HHCT). Among 57 patients, 22 had non-malignant disease (NM; 1 with Fanconi
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anemia, 19 with acquired SAA, 1 with CDA and 1 with WiskottAldrich syndrome), 31 had hematologic malignancy [6 with ALL (2 CR1, 2 CR2, 1 CR3, 1 non-CR), 16 with AML (6 CR1, 6 CR2, 4 non-CR), 1 with mixed lineage leukemia not in CR, 4 with MDS (2 RCMD and 2 RCC), 2 with JMML and 2 with NHL (1CR2, 1 CR3], and 4 had solid tumors [2 with rhabdomyosarcoma (1 CR2, 1 refractory), 1 with refractory neuroblastoma, and 1 with Ewing sarcoma in CR3]. Eight of 35 patients with malignant disease had refractory disease at the time of HHCT. RIC regimen consisted of fludarabine, cyclophosphamide and r-ATG. Low-dose total body irradiation was incorporated into the conditioning regimen in 45 patients. Results: Of a total of 57 patients, two patients failed to achieve primary engraftment, and five patients experienced graft rejection (GR) within 28 days post-transplant. All graft failure (GF) including GR occurred in CD3-HHCT. No patients with TCRab-HHCT experienced GF. The cumulative incidence (CI) of acute GVHD grade II-IV and III-IV were 31% and 14%, respectively. Five of 53 evaluable patients developed extensive chronic GVHD with a CI of 10%. Four patients from CD3-HHCT died of non-relapse causes [2 of CMV disease, 1 of encephalopathy, and 1 of autoimmune hemolytic anemia)], whereas none from TCRab-HHCT died of non-relapse cause. The non-relapse mortality (NRM) was 6.5% at 1 year and 9.2% at 2 years. Eleven patients relapsed and nine of them died of disease. Of 22 patients with NM, only one patient with CD3HHCT died, resulting in OS of 95% at 2 years. As for 35 patients with malignant disease, the estimated EFS at 1 year was 54%. Patients with refractory disease at the time of transplant had a poor outcome (EFS: 0% for 8 patients with refractory disease versus 75% for the remaining 27 patients, P ¼ 0.000). Conclusion: In this study, HHCT using ex vivo T-cell depleted grafts showed a favorable outcome with low NRM for pediatric patients with NM and malignant disease in CR at transplant. However, further study is warranted to improve the outcome for patients with refractory malignant disease. Disclosure of Interest: None declared.
P271 Zoledronic acid is able to potentiate cd T-cell antileukemic activity in patients receiving ab þ T cell and CD19 þ B lymphocytes depleted grafts from haploidentical donor I. Airoldi1,*, A. Bertaina2, A. Petretto3, B. Lucarelli2, A. Zorzoli3, P. Merli2, C. Lavarello3, G. Barbarito3, L. P. Brescia2, V. Bertaina2, E. Inglese3, G. M. Milano2, F. Locatelli2 1 Laboratory of Oncology, Istituto Giannina Gaslini, Genova, 2 IRCCS Bambino Gesu` Children’s Hospital, Rome, 3Istituto Giannina Gaslini, Genova, Italy Introduction: A new method of graft manipulation based on physical removal of ab þ T cells and CD19 þ B cells, leaving mature NK cells and gd T cells in the graft, has been recently employed for HLA-haploidentical HSCT. We demonstrated that gd T cells collected from transplanted patients (pts) are endowed with limited killing capacity of leukemia cells; we hypothesized that in vivo treatment with zoledronic acid (ZOL) may expand these lymphocytes and increase their ability to lyse primary ALL and AML blasts. Materials (or patients) and methods: Thirty-three pts with acute leukemias were enrolled in a phase I/II trial of allogeneic HSCT from an HLA-partially matched family donor after TCRab þ T cells/CD19 þ B lymphocyte negative depletion. Pts were treated with ZOL within 1 month after HSCT and then every 28 days for at least 2 times whenever possible (according to a protocol approved by the Ethical Committee). gd T cells from pts before and after ZOL treatment were studied till at least 7 months after HSCT by flow-cytometry, degranulation assay and high-resolution mass spectrometry. Results: ZOL treatment did not affect the relative proportion of Vd1 and Vd2 subsets (A), while it significantly decreased the central memory (CM) population in both Vd1 and Vd2 cells. ZOL also increased the terminally differentiated (TD) subset in the Vd2 cell subset. Both Vd1 and Vd2 cells from pts treated once with ZOL showed increased cytotoxicity against primary
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leukemia cells, as assessed by the degranulation assay (B). Such an ability was further increased in Vd2 cells, but not in Vd1 lymphocytes, in those patients that received more than one treatment (B). Proteomic analysis, evaluated through the use of high-resolution mass spectrometry (HRMS), performed on gd T cells purified from pts, before (n ¼ 5) and after (n ¼ 5) the first treatment with ZOL, revealed a significant change (6789 proteins identified, T-Test FDRo0.01 S040.5, 377 significant proteins) in the proteome signature (C). Noteworthy, gd T cells from treated pts revealed an up-regulation of proteins involved in activation processes and immune responses, paralleled by a down-regulation of proteins involved in the proliferation processes (D). The gd T proteotype and the associated activation pathways were further modulated upon repeated infusion of ZOL treatment. Conclusion: Analyses of gd T cells from patients given TCRab þ T cells/CD19 þ B lymphocyte depleted HLA-haploidentical HSCT showed that ZOL treatment is able to: i) promote an increase of TD Vd2 cells; ii) modulate pathways involved in activation/proliferation of gd T cells; iii) potentiate the cytotoxic killing of gd T cells. Altogether, our results provide a robust biological rationale for implementing clinical trials with ZOL infusions in the post-transplant setting aimed at reducing the risk of leukemia recurrence and of life-threatening infections. Disclosure of Interest: None declared. P272 Outcome after Hematopoietic Stem Cell Transplantation for Pediatric MDS J. A. Park1,*, Y. J. Lim2, K.-N. Koh3, J. J. Seo3, H. J. Im3 1 Pediatrics, Inje University Haeundae-Paik hospital, Busan, 2 Pediatrics, Chung-Nam University Hospital, Daejeon, 3Pediatrics, Asan Medical Center Children’s Hospital, Seoul, Korea, Republic Of Introduction: Myelodysplastic syndrome (MDS) in childhood is extremely rare and accounts for less than 5% of all hematopoietic malignancies. Allogeneic hematopoietic stem cell transplantation (HCT) is the only curative treatment option for MDS. However, due to its rarity, safety and efficacy of HCT for pediatric MDS have not been fully determined. We
investigated outcome of HCT and impact of WHO classification 2008 on outcome after HCT for pediatric MDS. Materials (or patients) and methods: We retrospectively analyzed transplant outcomes for 29 pediatric MDS patients, median age 10.9 years, undergoing HCT between 1997 and 2014. Three were secondary MDS, and 26 were primary MDS. By WHO 2008 classification, 19 had refractory cytopenia of childhood (RCC), 8 had refractory anemia with excess blasts (RAEB), and the remaining two had refractory anemia with excess blasts in transformation (RAEB-t). Donor was matchedrelated in 12, matched-unrelated in 6, mismatched-unrelated in 2, umbilical cord blood (UCB) in 2, and haploidenticalrelated in 7. The conditioning regimen was busulfan and cyclophosphamide based myeloablative (MA) in 18 and fludarabine based nonmyeloablative (NMA) in 11 patients. Results: Cumulative incidence of neutrophil engraftment by day 28 was 71%, but all patients with graft failure were rescued with second HCT. Cumulative incidence of acute GVHD by day 100, and chronic GVHD by 1 year were 43% and 21%, respectively. After median follow-up of 3.2 years, TRM and relapse were 15% and 24.3%. EFS and OS after HCT were 64% (54%474%, 95% CI) and 68% (59%478% 95% CI), respectively. WHO classification significantly affected EFS after HCT for pediatric MDS patients (P ¼ 0.0089; Fig. 1a). Shorter interval between diagnosis and HCT (o 6 months) was associated with more favorable outcome (P ¼ 0.017, Fig. 1b). No significant difference in EFS was seen in patients with pre-HCT complete remission or o5% blasts versus in patients with 45% blasts (P ¼ 0.39). EFS in patients who had received active treatment including chemotherapy and immunosuppressive treatment before HCT was lower, compared to patients who had not (P ¼ 0.075). EFS rates were similar between MA and NMA conditioning (P ¼ 0.26) and did not differed by stem cell donors (P ¼ 0.27). Conclusion: We confirmed that WHO classification 2008 for pediatric MDS was significantly associated with patients’ outcome after HCT. Additionally, our data suggested that pediatric patients with MDS had more favorable outcome if they were transplanted early in the course of their disease, and marrow blasts o5% at HCT did not improve EFS, but rather, active treatment before HCT showed a trend of decreasing EFS
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rates. This study needs to be confirmed in larger, prospective studies including more pediatric MDS patients. Disclosure of Interest: None declared. P273 Encouraging results of reduced-intensity allogeneic haematopoietic stem cell transplantation for paediatric patients with acute lymphoblastic leukemia in second complete remission K. Higuchi1,*, M. Sato1, O. Kondo1, A. Mayumi1, M. Shimizu1, A. Sawada1, M. Yasui1, M. Inoue1 1 Haematology/Oncology, Osaka Medical Center And Research Institute For Maternal And Child Health, Izumi, Osaka, Japan Introduction: We have been performing reduced-intensity stem cell transplantation (RIST) to avoid preconditioningrelated complications. However, the effectiveness of RIST in paediatric patients with acute lymphoblastic leukemia (ALL) remains to be clarified. Materials (or patients) and methods: We retrospectively reviewed 18 paediatric patients with ALL in second complete remission (CR2) who underwent first allogeneic haematopoietic stem cell transplantation (allo-SCT) between 2000 and 2013 in our institute. We compared the outcomes of RIST with those of myeloablative stem cell transplantation (MAST). Results: The median age at allo-SCT was 9 years (range, 2 to 18 years). There were B-lineage ALL only, and none of Philadelphia chromosome-positive ALL. Four patients received HLA-matched bone marrow (2 related; 2 unrelated), 9 HLAmismatched bone marrow (8 unrelated; 1 HLA haploidentical related), 4 cord blood, and 1 CD34 positive peripheral blood stem cells (HLA haploidentical related). In all patients, the 5year overall survival (5y-OS) rate and the 5-year event free survival (5y-EFS) rate were 65.0% and 55.0%, respectively. Seven patients underwent RIST and 11 patients underwent MAST. The median follow-up durations of RIST and MAST groups were 3.3 years (range, 0.9 to 8.2 years) and 2.1 years (range, 0.2 to 13.7 years), respectively. The 5y-OS rates in RIST and MAST groups were 85.7% and 53.0% (P ¼ 0.19), and the 5y-EFS rates were 71.4% and 45.5% (P ¼ 0.28), respectively. The 5-year cumulative transplant-related mortality (TRM) rates in RIST and MAST groups were 0% and 31.8% (P ¼ 0.16), and the 5-year cumulative relapse rates were 28.6% and 31.4% (P ¼ 0.81), respectively.
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Conclusion: In our series, the cumulative relapse rate in RIST group was similar with that in MAST group, and the cumulative TRM rate in RIST group was lower than that of MAST group. Therefore, both of the 5y-OS and the 5y-EFS rates in RIST group seem to be better than those in MAST group. The outcomes of RIST in our series do not seem to be poorer. Although further studies are needed because of the small size of patients’ population and short follow-up duration, RIST can be considered as the transplantation for paediatric patients with ALL in CR2. Disclosure of Interest: None declared. P274 Donor/recepient CMV serological status is a key prognostic factor for occurence of health- and lifethreatenig complications after haematopoietic stem cell transplantation in children J. Gozdzik1,2, M. Wozniak1,*, W. Czogala1, A. KrasowskaKwiecien1,3, A. Dluzniewska1, O. Wiecha1, S. Skoczen1, M. Sawa1 1 Transplantation Center, University Children’s Hospital, 2Immunology, Chair of Clinical Immunology and Transplantation, Jagiellonian University Medical College, 3Department of Transplantation, Chair of Clinical Immunology and Transplantation, Krakow, Poland Introduction: Cytomegalovirus (CMV) infection is still a reason of life-threatening complications in hematopoietic stem cell transplant (HSCT) recipients. Donor/recipient CMV serological status is an important risk factor. In Poland almost 90% of adult population is CMV-positive while many donors come from countries where CMV-positive population is in minority (o40%). The aim of the study was to assess the incidence, clinical course and effectiveness of treatment CMV infections after HSCT taking into account donor/recipient CMV serological status. Materials (or patients) and methods: Between 2005-2014 in our center 113 children underwent 126 HSCT (100 patients received one transplant, 12 – two transplants from the same donor and 1 patient received three transplants from 2 donors). The HSCT were performed for malignancy and non-malignant disease in 67 and 46 patients, respectively; 41 HSCT were from related donor and 72 from unrelated donor. The conditioning regimen was ablative (85 transplants), reduced toxicity (39), or non conditioned (2). The study group comprised the following recipient/donor pairs: CMV þ /CMV þ (52), CMV þ /CMV- (36), CMV-/CMV þ (15) and CMV-/CMV- (11). Congenital CMV infection were found in 3 children, and 1 was infected in early infancy. CMV DNA real-time PCR testing was performed
once weekly during immunosuppression. Positive evidence of CMV replication was an indication for ganciclovir (GCV) in a dose of 10 mg/kg (I-line treatment). In case of ineffectiveness foscarnet (FCV) was given in a dose of 120mg/kg (II-line). In case of further lack of effectiveness upon Z14-day therapy the third-line treatment was introduced (GCV and FCV, 120 mg/ kg). Due to resistance to GCV and FCV, cidofovir was administered (5-week regimen, 5 mg/kg, once a week [IV-line treatment]). The preemptive therapy was used until a negative result and continued with reduced dose for next 14 days. Results: After the first HSCT, 34 patients (30%) with CMV replication were identified, on average day þ 38 (0-224). 28 patients experienced a single episode lasting from 7 days to 4 months. In 5 patients recurrence of CMV reactivation was observed. First reactivation ocurred average on 14 day (7-30) and the second on 42 (30-180) day after HSCT; 1 patient (CMV þ /CMV-) experienced three episodes. In spite of treatment (I-IV line), he died of CMV pneumonia. There was 1 case of fatal hepatitis in CMV þ infant with congenital CMV transplanted from a CMV- alternative donor. Among the 12 patients undergoing second HSCT CMV replication was confirmed in 2 patients, both were CMV þ , with CMV- donors. After the first transplantation from the same donors no reactivation was observed. After the second HSCT, 1 patient had three reactivation episodes, with no symptomatic disease. The second patient with CMV reactivation developed retinitis, meningitis and CNS involving. In this case the treatment with two agents was long and successful. The patient, who underwent 3 HSCT (from different donors) experienced no CMV replication. Both the patient and the donors were CMV þ .
Conclusion: 1. Recepient/donor serological status CMV þ /CMVis an extremaly high risk factor of life-threatenig complications of CMV reactivation in children. 2. Even very intensive and longterm treatment (triple HSCT) in case of CMV þ recipient and donor is not a risk factor of CMV reactivation. Disclosure of Interest: None declared. P275 Allogeneic stem cell transplantation in pediatric myelodysplastic syndromes and juvenile myelomonocytic leukemia- a retrospective study of Polish national group M. Ussowicz1,*, J. Musial1, K. Drabko2, A. Zaucha-Praz˙mo2, A. Pieczonka3, J. Wachowiak3, J. Styczynski4, E. Gorczyn´ska1, K. Kalwak1, L. Gil5, A. Chybicka1 on behalf of Polish Pediatric Group for Hematopoietic Stem Cell Transplantation (PPGHSCT) 1 Department of Pediatric BMT, Hematology and Oncology, Wroclaw Medical University, Wroclaw, 2Department of Pediatric Hematology, Oncology and Transplantology, Medical University of Lublin, Lublin, 3Department and Clinic of Pediatrics Oncology, Hematology and Transplantation, Medical University in Poznan, Poznan, 4Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, 5 Department of Hematology, Poznan University of Medical Sciences, Poznan, Poland Introduction: Myelodysplastic syndromes (MDS) and Juvenile Myelomonocytic Leukemia (JMML) in childhood are rare malignancies treated with megachemotherapy and allogeneic stem cell transplantation (SCT). The Polish Pediatric Group for
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Hematopoietic Stem Cell Transplantation analysed the results of 96 children and adolescents transplanted in years 19972013. Materials (or patients) and methods: The group consisted of 36 girls and 60 boys (M/F ratio 0.375). Patients median age was 5.9 years (range 0.2-19 years), median interval from diagnosis to SCT was 6.43 months (range 1.5-103 months), the median follow up time after SCT was 21 months (range 0.1-209). Of 96 patients, 17 were transplanted with refractory cytopenia (RC), 40 with Refractory Anemia with Excess of Blasts (RAEB/RAEBT), 12 with Myelodysplasia Related Acute Myeloid Leukemia (MDR-AML), 5 with undefined MDS, and 22 with JMML. 53 patients were transplanted from matched unrelated donors (MUD), 27 from matched sibling donors (MSD), and 15 from haploidentical (haplo) donors. Graft material was BM in 43, PBSC in 50, and cord blood in 2 patients. Conditioning regimen was myeloablative (busulfan-based) in 76 patients or non-myeloablative in 20 (RIC, mostly treosulfan-based). Results: The neutrophil engraftement was achieved in 91,6% of patients, and platelet 4 20 K/uL in 82% patients. Acute GVHD was seen in 30% (29 patients), and chronic GVHD in 15% (12/77 patients surviving beyond day 90). Relapse incidence was 24% (16 patients) with median incidence of 6 months. The long term overall survival was 32% in JMML and 48% in MDS. In univariate analysis MDS subtype, graft material, conditioning intensity, incidence of aGVHD or cGVHD had not affected the SCT outcome either in MDS or in JMML. The OS was superior in patients transplanted earlier than 6 months from diagnosis (64% vs 32%, P ¼ 0.005). The choice of haploidentical donor was a factor of adverse prognosis in MDS patients (P ¼ 0.001). The OS was superior in patients transplanted after year 2005 (57% vs 30%, P ¼ 0.01). Conclusion: Early allogeneic SCT is advantageous in children with MDS or JMML. The outcome in MSD and MUD transplantations is comparable. All relapses were observed within 24 months from SCT, what warrants close follow up during this posttransplant period. Similarly to results observed in adults, progress in supportive care resulted in improved transplantation outcomes in children. Disclosure of Interest: None declared. P276 Treosulfan, melphalan and thiotepa megachemotherapy backbone is well tolerated in heavily pretreated children and facilitates reliable engraftment M. Ussowicz1,*, E. Wawrzyniak1, J. Weclawek-Tompol1, M. Mielcarek1, J. Musial1, J. Peregud-Pogorzelski2, K. Kalwak1 1 Department of Pediatric BMT, Hematology and Oncology, Wroclaw Medical University, Wroclaw, 2Department of Pediatrics, Hematology and Oncology, Medical University, Szczecin, Poland Introduction: The conditioning regimen is an important prerequisite in the stem cell transplantation settings which is responsible for both the anticancer effect and establishing of environment suitable for stem cell engraftment. Here we report the results of megatherapy containing the treosulfan, melphalan and thiotepa backbone (Treo-Mel-TT) in heavily pretreated children with solid tumours. Materials (or patients) and methods: The group consisted of 9 patients (pts) with disseminated, relapsing malignancies, with median age 5.7 years (range 3-14.8). Diagnoses were neuroblastoma (NBL, 7 patients), primary neuroectodermal tumour (PNET, 1 pt), and nephroblastoma (WT, 1 pt). 6 patients with NBL underwent busulphan-melphalan megachemotherapy with autologous SCT, and relapsed afterwards. The megatherapy backbone consisted of i.v. treosulfan at 10 g/ m2 daily from day -8 to -6 (total dose 30 g/m2), i.v. thiotepa at 5 mg/kg daily from day -4 to -3 (total dose 10 mg/kg), and i.v. melphalan at 70 mg/m2 daily from day -2 to -1 (total dose 140 g/m2). For patients undergoing the haploidentical transplantation, i.v. fludarabine was added at 40 mg/m2 daily from day -8 to -5 (total dose 160 mg/m2), and ATG in total dose of 30 mg/ kg, usually from day -12 to -9. After Treo-Mel-TT megatherapy,
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8 patients underwent haploidentical transplantation from the parent (with CD3/19 depletion – 4 pts, or with TCR alpha-beta depletion – 4 pts), and 1 patient – second autologous SCT. The patients toxicities during the transplant procedure were assessed and graded according to Common Terminology Criteria for Adverse Events (CTCAE), Version 4.0. Results: Neutrophil engraftment was achieved by day þ 12 (range 10-24), and in all patients transplanted from haploidentical donors stable full donor chimerism was achieved. The median hospitalisation length was 37 days (range 28-49). WHO grades for body weight loss were 1 (5 pts) or 2 (2 pts), for body weight gain 1 (3 pts) or 2 (4 pts), diarrhea lasted for 1 to 12 days with grade 1 (2 pts), 2 (2 pts) or 3 (3 pts), oral mucositis grade was 1 (1 pt) or 3 (6 pts). All 9 patients developed neutropenia grade 4, with lymphopenia grade 3 (2 pts) or 4 (7 pts). Kidney function was not impaired (9/9 pts with normal creatinine levels), only mild liver toxicities were observed (AlAT43 x upper limit – 3 pts, AspAT43 x upper limit – 1 pt, no increase in alkaline phospatase or billirubin levels). 4 patients remain alive and well in remission with median follow up of 19 months (range 6.6-32.7 months). Conclusion: Although the reported group is small and heterogenous, the strikingly dismal prognosis was obvious in all patients. Despite long and intensive multiple lines of pretransplant chemotherapy, the patients tolerated the megachemotherapy very well, and some of them achieved remission and are disease-free. The treosulfan-melphalanthiotepa conditioning is a protocol that can be safely used in patients with solid tumours due to good tolerability and sustained engraftment. Disclosure of Interest: None declared. P277 Late effects in children after allogenic stem cell transplantation (Allo-SCT) M. J. LLAMAS POYATO1,*, J. R. MOLINA HURTADO1, C. CHIC ACEVEDO1, E. GARCIA TORRES1, G. RODRIGUEZ GARCIA1, C. MARTIN CALVO1, A. RODRIGUEZ VILLA1, R. ROJAS CONTRERAS1, F. MARTINEZ GUIBELARDE1, C. HERRERA ARROYO1, P. GOMEZ GARCIA1 1 HEMATOLOGY, REINA SOFIA UNIVERSITY HOSPITAL, CORDOBA, Spain Introduction: Late effects (LE) after Allo-SCT can appear in long-term survivors. LE expresses themselves as structural or functional impairment of organs or tissues secondary to AlloSCT or related to a new tumoral growth. The aim of this study was to determine the incidence of LE that contribute to longterm morbidity after Allo-SCT in pediatric patients. Materials (or patients) and methods: We analyse retrospectively 126 consecutive pediatric patients (r18 age)
undergoing Allo-SCT in our institution from May 1981 to November 2011 with a minimum overall survival more than two years after transplant. Data were derived from review of clinical histories in the last year. Results: Median age at transplantation was 11 years (range: 118). Seventy-one patients (56.3%) were male and 55 (47.7%) female. The most common underlying disease was acute lymphoblastic leukemia (45.2%) while 51 (40.5%) patients were in complete remission at Allo-SCT. Bone marrow was the most common stem cell source (82.5%) and conditioning regimen was myeloablative in 97.6% of patients. Median follow-up was 88 months (range: 24-341) with a progression free survival of 80.2% and an overall survival of 84.1%. Major death cause was relapse in 14 patients. A total of 40 (31.7%) patients developed chronic graft versus host disease (cGVHD). The most common long-term LE were: skin lesions in 38 (30.2%) patients like scleroderma (n ¼ 12) and lichen planus (n ¼ 4). Ocular involvement in 19 patients (15.1%), highlighting keratoconjunctivitis sicca (n ¼ 13). Oropharyngeal injury in 26 patients (20.6%) highlighting the lichen planus (n ¼ 14). Chronic lung involvement in 16 patients (12.7%): 12 of them developed bronchiolitis obliterans and it was related to previously cGVHD (Po0.001) and prolonged use of high-dose steroids (Po0.001). Liver complications in 11 (8.7%) patients (mostly increased liver enzymes). Uro-vaginal complications in 2 patients. Skeletal complications in 12 (9.5%) patients: avascular necrosis in 8 of them. Bone damage was related to Total Body Irradiation in conditioning regimen (P ¼ 0.003). Two patients developed cardiac insufficiency. Endocrine-metabolic complications developed in 24 patients (19%): hypothyroidism (n ¼ 4), hypogonadism (n ¼ 11), hypercholesterolemia (n ¼ 5), diabetes mellitus (n ¼ 3) and hypertension (n ¼ 1). Seven patients (4.8%) developed secondary tumors (six of them were aconditioned with TBI): they were more common in the group with cGVHD (P ¼ 0.01) and those receiving long term high-dose steroids (P ¼ 0.016). Regarding the growth, 23 (18.3%) and 22 (17.5%) patients have a height and weight less than the 25th percentile for their age (last measurement in the clinical history),respectively. Multivariate analysis showed that the risk of developing any LE in Allo-SCT survivors was associated with female sex of receptor (P ¼ 0.008),cGVHD (P ¼ 0.008) and high-dose steroids (P ¼ 0.004). If we divide the patients into two groups according to the date of Allo-SCT (before or after 2000) there were not significant differences in OS (P ¼ 0.63) or PFS (P ¼ 0.45). In the first group (before 2000 LE were more common compared to the other group (45.9% vs 29.2%;P ¼ 0.045). Conclusion: LE secondary to Allo-SCT in pediatric patients affect a high percentage of them,but not impede a normal life. Early diagnosis of these LE and possible treatment should be ensured by monitoring patients maintained over time. The prolonged use of steroids and cGVHD are associated with the occurrence of LE. Disclosure of Interest: None declared.
administered four-times-daily during four days. However, this administration modality is nursing-time consuming, responsible for organizational difficulties and discomfort for the patient. Even if once daily schedule is used in the adult population, only few data regarding this administration in pediatric population exist. Our study aims to compare the two administration modalities of Bu: four-times-daily versus once daily in a pediatric cohort. Materials (or patients) and methods: We performed a prospective study in 20 patients at the Strasbourg University Hospital during august 2012 and june 2014. The cohort was divided in two groups: in the first one (A), Bu was administered four times daily (12 patients), and in the second (B), Bu was administered once daily (8 patients). The posology of the first dose of Bu was determined according to the child’s weight, then the following doses were adapted to the measured AUC. The target AUC was between 900 to 1500 mM.min in fourtimes-daily schedule and 3600 to 6000 mM.min in once daily schedule. The plasmatic Bu concentrations were measured by gas chromatograph mass spectrometer method. AUC and accumulated dose received by every patient were calculated and finally efficiency, toxicity, organizational consequences and patient comfort were evaluated Results: In total, 78 AUC were determined (47 in group A, 31 in group B). The plasmatic concentration of Bu, 30 minutes after the end of the drip were measured between 268 and 1836 ng/ ml (mean, 1054 ng/ml) and 1919 to 6726 ng/ml (mean, 3005 ng/ml) in group A and B, respectively. These data confirm the large interpersonal variability. In the group A, 10/12 patients
P278 Comparative pediatric study of two modalities of administration of intravenous Busulfan (Bu) : four-timesdaily versus once daily schedule M. A. Puel1,*, V. Kemmel2, A. Spiegel1, P. Lutz1, J. M. Lessinger2, C. Paillard1 1 pediatrie, CHU Hautepierre, 2Laboratoire de Biochimie et Biologie Mole´culaire, CHU Hautepierre, CHU Strasbourg, strasbourg, France Introduction: Busulfan (Bu) is well known to have a large inter and intra-patient variability, especially during childhood. Exposure of high Bu concentrations have toxicity consequences due to overexposure, while no engraftment is observed with too low concentrations, due to underexposure. This is why Therefore therapeutic drug monitoring by following of the area under the curve (AUC) is essential, in pediatric population. Intra-venous Bu is classically
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had an average of AUC in the therapeutic window and 8/8 patient in the group B. As shown in Figure 1, the amplitude of dose adjustment was greater in group A. So the initial dose calculated for the protocol B was more effective to obtain the therapeutic target. On clinic view, no failure of engraftment was noticed. 3 deaths were reported but none related to Bu use. The only one patient, who presented a veino-occlusive disease was out range Bu concentration and had a favorable evolution after the return of the Bu concentrations in therapeutic range. In term of organization, the once daily administration permits to reduce 5 hours a day the perfusion time. This extratime was also responsible of an increase in patient’s comfort. Conclusion: Our study underlines that both schedule of Bu administration permits therapeutic drug monitoring with an equivalent patients care. Moreover, patient’s comfort and the nursing time optimization seems to be improved by once daily administration in pediatric patient. Disclosure of Interest: None declared.
Acute leukaemia I P279 Azacitidine maintenance after allogeneic stem cell transplantation is feasible in patients with acute myeloid leukemia and myelodysplasia A. Antar1,*, M. Kharfan-Dabaja2, H. Abou Ghaddara1, R. Mahfouz3, A. Bazarbachi1 1 Internal Medicine/Hematology-Oncology, American University of Beirut, Beirut, Lebanon, 2Blood and Marrow Transplantation, Moffitt Cancer Center, Tampa, FL, United States, 3Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon Introduction: 5-Azacidine (5-AZA) is a DNA hypomethylating agent with proven clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A recent non-randomized study reported promising results with the use of lower doses of 5-AZA as maintenance therapy after hematopoietic stem cell transplantation (HSCT). It is important to note that 5-AZA has an immunomodulatory effect and might enhance the graft-versus-leukemia (GVL) effect. Here, we report the successful use of 5-AZA maintenance following allogeneic HSCT in patients with high risk AML and MDS. Materials (or patients) and methods: Nine patients (M ¼ 6, F ¼ 3; median age ¼ 49 (36-65) years ) with high-risk AML (n ¼ 6 including 2 abnormal karyotypes) or MDS (n ¼ 3
[P279]
Table 1.
including 1 abnormal karyotype) received 5-AZA as posttransplant maintenance at a dose of 32mg/m2 daily for 5 days every 4 weeks starting at a median time of 100 (30-210) days post-transplant. All patients were in complete remission at initiation of 5-AZA. A median of 12 cycles (1-18) were delivered. Patients’ characteristics and response to treatment are summarized in Table I. Results: After a median follow-up of 19 months post HSCT and 15 months after starting 5-AZA treatment, five patients with normal karyotype are still in CR. Conversely, all three patients with abnormal karyotype rapidly developed disease recurrence while they were receiving 5-AZA after a median of 3 months. Overall, the actuarial 1-year progression free and overall survival rates were 65% and 90%, respectively. 5AZA was generally well tolerated with only mild thrombocytopenia observed in 2 patients. No clinically evident graftversus-host disease exacerbation was observed. Stem cell source for all patients : peripheral blood; all patients received cyclosporine as GVHD prophylaxis; CR: complete remission; PR: partial remission; MRD: matched related donor; MUD: matched unrelated donor; FB4: 5 days fludarabine plus 4 days busulfan(130 mg/m2/day); FB3: 5 days fludarabine plus 3 days busulfan (130 mg/m2/day); FB2: 5 days fludarabine plus 2 days busulfan (130 mg/m2/ day); ATG: anti-thymoglobuline; DLI: donor lymphocyte infusion. Conclusion: These results suggest that Low-dose 5-AZA is an effective maintenance therapy post- allogeneic SCT in high-risk AML and MDS particularly when a normal diploid karyotype is present. The relative lack of efficacy in the presence of an abnormal karyotype is intriguing and questions whether these subjects might benefit from higher doses of 5-AZA or other novel therapies within the context of a well-designed clinical trial. Prospective clinical trials and longer follow-up are needed to confirm these observations. Disclosure of Interest: None declared. P280 Mutated regions of nucleophosmin 1 elicit CD8 þ T-cell responses in patients with acute myeloid leukemia A. Sun1,*, Z. Cheng1, G. Chen1, X. Yang1, D. Wu1 1 The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou, China Introduction: Stimulating lymphocytes from healthy donors by DCs loaded with NPM1mut peptides in vitro in order to induce specific cellular immune responses against the peptides. And detecting the NPM1mut peptide specific T-cell in peripheral blood of patients with NPM1mut positive acute myeloid leukemia (AML), to provide a theoretical basis for immune therapy of AML.
Patients characteristics and Outcomes After Azacitidine maintenance
Subject # Disease status at HSCT Donor type Conditioning Disease recurrence Salvage therapy if recurrence Progression free survival, months Status at last follow up Survival, months
1
2
3
4
5
6
7
8
9
CR2
CR3
CR1
CR1
Refractory
CR1
PR
PR
CR1
MRD FB2 þ ATG
MRD FB3 þ ATG
MRD FB3 þ ATG
MRD FB4 þ ATG
MRD FB4 þ ATG
MRD FB4 þ ATG
MRD FB3 þ ATG
MRD FB2 þ ATG
no
no
yes
no
MUD FB3 þ ATG þ TBI (4Gy) yes
no
no
no
yes
N/A
N/A
Chemotherapy followed by DLI
N/A
None
N/A
N/A
N/A
Chemotherapy followed by DLI
13 þ
24 þ
1
24 þ
3
19 þ
21 þ
18 þ
10
CR
CR
CR
CR
died
CR
CR
CR
CR
13 þ
24 þ
18 þ
24 þ
5
19 þ
21 þ
18 þ
34 þ
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Materials (or patients) and methods: 1. newly diagnosed patients of AML underwent gene mutations screening routinely. 2. peripheral blood mononuclear cells (PBMCs) were isolatedfrom healthy donors with HLA-A* 0201 or A*1101 and NPM1mut positive AML patients in complete remission. 3. Inducing differentiation of PBMCs into DCs 4. Generation of NPM1-specific cytotoxic T cells 5. ELISPOT analysis and intracellular staining Results: 1. In the Han Chinese population, the most common alleles of HLA-A loci were A* 1101, A*2402, and A*0201 allele. 2. Through prediction of the aforementioned software, HLAA*0201 restricted wild type NPM1 amino acid sequences (DLWQWRKSL), mutated NPM1-A/D amino acid sequences (AIQDLCLAV), and HLA-A*1101 restricted mutated NPM1-A/D amino acid sequences (AVEEVSLRK) were synthesized. There were no proper epitopes for either HLA-A*1101 restricted wild type NPM1 protein or HLA-A*2402 restricted wild type and mutated NPM1 protein. 3. Expression of surface antigens of DCs on day 7 were as follows: CD14(2.6%), CD80(43.4%), CD83(7.8%), CD86(99.9%), HLA-DR(67.1%), which was consistent with DCs phenotype. 4. About 10 days after DCs were co-incubated with their own PBMCs, the number of lymphocytes increased significantly, especially in the NPM1mut peptide pulsed group. On day 7 of co-incubation, ELISPOT analysis results of all samples were negative. However, 3 cases’ ELISPOT results were positive for the mutated peptide holes on day 14, in contrast, the results of the wild type peptide holes were negative. The NPM1mut peptide-specific T lymphocytes positive rate [ ¼ (average number of spots in mutated peptide holes-the average number of spots in negative control holes)/total lymphocytes per hole] was about 1/2500. Intracellular staining showed that in the aforementioned 3 cases, the proportion of CD8 þ IFN-g þ cells of the mutant peptide group was higher than either the wild type peptide group or negative control group, but there was no difference between the later two groups. 5. 6 peripheral blood samples of patients with NPM1mut þ AML were performed ELISPOT analysis, with only 1 case (16.7%) showing a approaching positive result. Conclusion: NPM1mut peptide pulsed DCs can stimulate their own PBMCs from healthy donors in vitro to produce mutated NPM1 specific T lymphocytes, and it is expected to be used as immune therapy of AML. The mutated NPM1 specific CTL in NPM1mut þ AML patients are almost undetectable that indicates immune system have been comprised because of chemotherapy and disease, and can not responses to antigens efficiently. Perhaps AML patients can accept CTL transfusion from their relatives. Our study provide an experimental basis for cellular immunotherapy of AML. Disclosure of Interest: None declared. P281 Effect of in vivo T-cell depletion with ATG on cytomegalovirus (CMV) induced antileukemic effect in patients with acute myeloid leukemia (AML) receiving grafts from unrelated donors A. Busca1,*, C. Dellacasa1, C. Frairia1, S. Aydin1, R. Passera2, M. Pini3, F. Zallio3, E. Audisio1, L. Giaccone1, E. Maffini1, C. Costa4, R. Cavallo4, S. Manetta1, B. Bruno1 1 Onco Hematology, 2AOU Citta’ della Salute e della Scienza, Turin, 3Hematology, AO Nazionale Alessandria, Alessandria, 4 Microbiology, AOU Citta’ della Salute e della Scienza, Turin, Italy Introduction: Several studies provided evidence of a consistent antileukemic effect induced by CMV replication in AML patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). It is conceivable that the ‘‘virus-versus leukemia’’ effect promoted by CMV reactivation requires a robust T- or NK-cell immune response to elicit a graft-versus leukemia effect. In vivo T-cell depletion is typically associated with a delayed T-cell reconstitution, and may potentially abrogate the protective effect of CMV infection. Nevertheless, the influence of antithymocyte globulin (ATG, Thymoglobulin)
on the antitumor effect of CMV replication after HSCT is rather unexplored. To address this issue, we retrospectively analyzed a cohort of 101 patients with AML who received grafts from an unrelated donor in two italian institutions between 2004 and 2014, after a conditioning regimen including ATG. Materials (or patients) and methods: Overall, 66 patients (65%) had early disease phase at the time of HSCT and 83 patients (82%) received myeloablative regimens. Prophylaxis of GVHD consisted of cyclosporine and short course MTX combined with ATG at a dose of 5-7.5 mg/Kg over two or three days. Real-time qPCR testing in whole blood for CMV reactivation was routinely monitored twice weekly. CMV viral load 42000 copies/mL was considered as CMV reactivation. Results: The cumulative incidence of CMV reactivation was 59% at 12 months. The 5-year cumulative incidence of relapse in patients with CMV reactivation was 29% compared with 37% for patients without CMV reactivation (P ¼ 0.279). The only factor associated with a reduced 5-year cumulative incidence of relapse was an advanced disease status at HSCT. Similarly, CMV reactivation was not associated with different OS or NRM rates (P ¼ 0.850 and P ¼ 0.297 respectively). In the multivariable model adverse cytogenetics (HR 2.42, 95% CI 1.02-5.72; P ¼ 0.044) and acute GVHD (HR 3.36, 95% CI 1.328.54; P ¼ 0.011) were independent risk factors for reducing OS, while the presence of chronic GVHD was associated with a better OS (HR 0.37, 95% CI 0.15-0.89; P ¼ 0.027). Conclusion: The results of present study showed that patients with AML receiving T-cell depleted HSCT using ATG did not benefit from CMV reactivation. Larger studies are mandatory to confirm our preliminary data. Disclosure of Interest: None declared. P282 Allogeneic hematopoietic cell transplantation in AML with normal karyotype and isolated NPM1 mutation: a retrospective analysis from the Acute Leukemia Working Party of EBMT A. Bazarbachi1,*, M. Labopin2, M. Kharfan-Dabaja3, R. Schwerdtfeger4, L. Volin5, J. H. Bourhis6, G. Socie´7, E. Deconinck8, T. GeddeDahl9, A. Rambaldi10, P. Jindra11, G. Schlimok12, D. Blaise13, P. Chevallier14, A. Nagler15, C. Schmid16, J. Esteve17, M. Mohty18 1 Internal Medicine, Bone marrow transplantation program, American University of Beirut, Beirut, Lebanon, 2Acute Leukemia Working Party of EBMT, Paris, France, 3Department of Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center/University of South Florida College of Medicine, Tampa, Florida, United States, 4Deutsche Klinik fu¨r Diagnostik KMT Zentrum, Wiesbaden, Germany, 5Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland, 6Division of Hematology, Department of Medical Oncology, Gustave Roussy, institut de cance´rologie, BMT Service, Villejuif, 7Dept. of Hematology – BMT, Hopital St. Louis, Paris, 8Service d’He´matologie, Hopital Jean Minjoz, Besancon, France, 9Rikshospitalet Department of Medicine, The National Hospital, Oslo, Norway, 10Azienda Ospedaliera Papa Giovanni XXIII Hematology and Bone Marrow Transplant Unit, Bergamo, Italy, 11Dept. of Hematology/Oncology, Charles University Hospital, Pilsen, Czech Republic, 12Klinikum Augsburg II Medizinische Klinik, Augsburg, Germany, 13Programme de Transplantation&Therapie Cellulaire, Centre de Recherche en Cance´rologie de Marseille, Institut Paoli Calmettes, Marseille, 14 CHU Nantes Dept. D’He´matologie, Nantes, France, 15Chaim Sheba Medical Center, Tel Aviv University, Tel-Hashomer, Israel, 16 SCT Unit, Klinikum Augsburg, Germany, 17Department of Hematology, Hospital Clinic, IDIBAPS, Barcelona, Spain, 18De´partement d’He´matologie, Hopital Saint Antoine, Paris, France Introduction: NPM1 mutation, in the absence of FLT3-ITD, confers a survival advantage and lower risk of disease relapse in AML with normal diploid karyotype. The prognostic significance of mutated NPM1 in the setting of allogeneic HCT has not been described. Materials (or patients) and methods: we evaluated outcomes of 156 AML patients (females ¼ 83, 53.2%), median age
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of 54 (19.5-71) years, with normal diploid karyotype and mutated NPM1, who underwent an allogeneic HCT between 2006 and 2012. Donor source was limited to matched-related or unrelated donors, and cell source consisted of bone marrow or mobilized peripheral blood stem cells. Patient-, donor-, and disease-characteristics are summarized in Table 1. Results: The median time from diagnosis to allogeneic HCT was 310 (89-3713) days and the median follow-up from allografting was 32 (2-86) months. Overall, the 2-year cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were 27% (20-35) and 13% (8-19) respectively, and the 2-year cumulative incidence of chronic GVHD was 37% (29-46). Finally, the 2-year leukemia-free survival (LFS) and the 2-year overall survival (OS) were 60% (51-68) and 70% (62-77), respectively. In univariate analysis, transplantation in CR1 was associated with lower 2-year CIR [CR1 ¼ 14% (7-23), CR2 ¼ 37% (23-51), advanced/active ¼ 48% (26-67), P ¼ 0.0009], superior 2year LFS [CR1 ¼ 75% (64-86), CR2 ¼ 51% (36-65), advanced/ active ¼ 30% (11-49), Po0.0001] and superior 2-year OS [CR1 ¼ 81% (72-91), CR2 ¼ 67% (54-80), advanced/active ¼ 39% (19-59), Po0.0001]. Patients allografted from unrelated donors have higher 100-day cumulative incidence of 4 grade 2 acute GVHD [MUD ¼ 28% (19-39) vs. MRD ¼ 12% (5-22), P ¼ 0.02]. Patients older than 54.3 years had higher 2-year cumulative incidence of NRM [20% (11-31) vs. 7% [2-14], P ¼ 0.03] and inferior 2-year OS [61% (49-72) vs. 78% (68-88), P ¼ 0.02]. In multivariable analysis, transplantation in CR1 resulted in lower 2-year CIR and superior 2-year LFS and OS. variables
Results N, (%) F ¼ 83 (53%) M ¼ 73 (47%) Yes ¼ 21 (14%) No ¼ 133 (86%) MRD ¼ 66 (42%) MUD ¼ 90 (58%) BM ¼ 30 (19%) PBSC ¼ 125 (80%) BM þ PBSC ¼ 1 (1%) CR1 ¼ 69 (44%) CR2 ¼ 64 (41%) Advanced/active ¼ 23 (15%) RIC ¼ 85 (54%) MAC ¼ 71 (46%) Yes ¼ 84 (54%) No ¼ 72 (46%) CSA þ MTX ¼ 77 (49%) CSA þ MMF ¼ 41 (27%) CSA alone ¼ 28 (18%) D þ /R þ ¼ 48 (31%) D þ /R- ¼ 14 (9%) D-/R þ ¼ 52 (34%) D-/R- ¼ 39 (26%)
Recipient gender Female donora`male recipient Donor source Cell source Disease status at allogeneic HCT Preparative regimen intensity ATG use GVHD prophylaxis Donor/recipient CMV serology
Abbreviations: F: female, M: male, MRD: matched-related donor, MUD: matched-unrelated donor, RIC: reduced-intensity conditioning, MAC: myeloablative conditioning, CSA:
[P283]
Table 1.
cyclosporine A, MTX: methotrexate, MMF: mycophenolate mofetil, D: allograft donor, R: allograft recipient Conclusion: AML disease status at allografting remains the most important predictor of post-allogeneic HCT outcomes despite expression of mutated NPM1. Survival outcomes are better when patients are transplanted in CR14CR24advanced/active. The impact of other molecular abnormalities in conjunction with NPM1 is yet to be established. Also, a future matched-controlled analysis comparing AML patients with mutated vs. wild-type NPM1 expression in the setting of allogeneic HCT is warranted. Disclosure of Interest: None declared. P283 Sorafenib Maintenance Appears Safe and Improves Clinical Outcomes in FLT3-ITD Acute Myeloid Leukemia after Allogeneic HCT A. Antar1, M. Kharfan-Dabaja2, R. Mahfouz3, A. Bazarbachi1,* 1 Internal Medicine/Hematology-Oncology, American University of Beirut , Beirut , Lebanon, 2Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, United States, 3Pathology and Laboratory Medicine, American University of Beirut , Beirut , Lebanon Introduction: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) gene is one of the most frequently observed genetic alterations in AML with an incidence of about 20–30%. FLT3-ITD is significantly associated with a poor outcome and it is recommended that patients harboring this mutation should undergo allogeneic hematopoietic cell transplantation (allo-HCT). Sorafenib is a tyrosine kinase inhibitor with activity against RAF, VEGF, and FLT3-ITD. It has been used off-label in FLT3-ITD AML. Here, we report the successful use of sorafenib following allo-HCT in patients with FLT3-ITD AML. Materials (or patients) and methods: Six male patients (median age ¼ 50(32-58) years) with normal karyotype FLT3ITD AML at diagnosis received sorafenib as post-transplant maintenance therapy (n ¼ 5) or as salvage therapy following a post allo-HCT relapse (n ¼ 1). Patient #6 was started on sorafenib in combination with chemotherapy for early relapse prior to transplant and was transplanted in CR2. The remaining 5 patients were transplanted in CR1 and received sorafenib post-transplant as maintenance therapy. Patients’ characteristics and treatment details are summarized in Table 1. Results: Sorafenib was well tolerated. Interestingly, skin GVHD grade II was observed in 5/6 patients and generally occurred within few days after initiation of sorafenib, but responded promptly to corticosteroids in all patients. No other manifestations of acute GVHD were observed out of the ordinary. All 6 patients are still alive and in complete remission with a median follow-up of 16 months (range, 10-29) since first induction, and a median follow-up of 12 months (range, 4-20) since initiation
Characteristics of patients and their treatment
Patient nb.
1
2
3
4
5
6
Molecular ( þ ) other than FLT3 ITD Induction
–
NPM1
–
–
NPM1, CEBPa
–
Daunorubicin þ Ara-C (3 þ 7) FB4 þ ATG 1Ctx,allo-sct,2Ctx
Idarubicin þ Ara-C FB3 þ ATG 2Ctx, allosct Post (71)
Daunorubicin þ Ara-C (3 þ 7) FB3 þ ATG 3Ctx,allo-sct
Daunorubicin þ Ara-C (3 þ 7) FB4 þ ATG 2Ctx, allo-sct
Daunorubicin þ Ara-C (3 þ 7) FB4 þ ATG 4Ctx,allo-sct
Daunorubicin þ AraC (3 þ 7) FB4 þ ATG 4Ctx
Post(35)
Post (40)
Post (30)
Pre(70)
Conditioning Therapy sequence before sorafenib Day on Sorafenib prepost HSCT Sorafenib dose Side effects (grade) Dose modification
Post (150) 400 mg BID Skin GVHD ( II) Liver toxicity 200 mg BID
400 mg BID None
400 mg BID
400 mg BID
400 mg BID
400 mg BID
Skin GVHD (II)
Skin GVHD (II)
Skin GVHD ( II)
No
No
No
No
Skin GVHD (II) Myocardial infarction 200 mg BID
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of sorafenib. Remarkably, all patients are in molecular remission. Stem cell source for all patients : peripheral blood ; All donors were matched related donor; all patients received cyclosporine as GVHD prophylaxis; FB4: 5 days fludarabine plus 4 days busulfan(130 mg/m2/day); FB3: 5 days fludarabine plus 3 days busulfan (130 mg/m2/day); ATG: anti-thymoglobuline; Ctx: chemotherapy. Conclusion: These results suggest that sorafenib appears to be an effective maintenance therapy post allo-HCT in FLT3-ITD AML with achievement of durable complete responses. This result may suggest an immuno-modulatory effect of sorafenib in the post transplant setting and warrant a broader clinical evaluation of the use of maintenance sorafenib in FLT3-ITD AML. Disclosure of Interest: None declared. P284 Value of pre-transplantation minimal residual disease in acute myelioid leukemia and myeloablative allogeneic hematopoietic cell transplantation M. Kwon1, A. C. Franco Villegas1,*, P. Balsalobre1, I. Bun˜o1, C. Martinez-Laperche 1, J. Anguita 1, A. Perez Corral 1, C. Pascual 1 , D. Serrano1, J. Gayoso1, J. L. Diez1 1 Hematology, University General Hospital Gregorio Maranon, Madrid, Spain Introduction: Relapse remains the main cause of treatment failure in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (SCT). The detection of minimal residual disease (MRD) in AML has improved in the past years with multiparametric flow cytometry (MFC) and molecular analysis (RT-PCR). However the prognostic impact of pre-transplantation MRD and the outcome after SCT is not well established. The aim of this study was to evaluate pre-transplantation MRD in patients in first complete remission (CR1) undergoing myeloablative allogeneic SCT. Materials (or patients) and methods: Consecutive adult patients with AML in CR1 after intensive chemotherapy with available MRD determination before transplant who underwent myeloablative SCT were retrospectively analyzed. MRD was studied by 4-color MFC on bone marrow aspirates, and quantitative RT-PCR (NPM1,WT1,MLL) on bone marrow and/or peripheral blood samples obtained within thirty days before transplant. Results: 46 patients consecutively transplanted in our institution between 1999 and January 2014 were included (Table 1).
Twenty-eight showed negative MRD pre-transplant whereas 18 showed positive values. Pre-transplant MRD negative patients included a higher proportion of high-risk cytogenetics LMA and HLA-identical siblings as stem cell source (Table 1). Within the MRD-positive group, 11/18 (61%) patients showed MRD-positive values by MFC: in 7 cases MRD was also detected by PCR, only 1 showed negative PCR and in the remaining 3 cases, PCR was not available. On the other hand, in 7/18 (38%) patients MRD was not detected by MFC, however, PCR detected MRD in all of the cases. With a median follow-up of the whole series of 35 months, 3-years estimates of overall survival were 66% (95% CI, 44.5-87) vs 41% (95% CI, 15-66.5) for MRD-negative and MRD-positive patients (P ¼ 0.12), respectively. Disease free survival was 68% (95% CI, 48-88) vs 36% (95% CI, 10-61) (P ¼ 0.06) whereas cumulative incidence of relapse was 23% (95% CI, 10-51) vs 31% (95% CI 14-67), respectively (P ¼ 0.54). In the MRD-negative group, cause of death was toxicity in 7% of the cases and relapse in 17%, while in the MRD-positive group, 22% of patients died due to toxicity and 27% due to relapse. Conclusion: Our data shows that the presence of MRD before allogeneic SCT in patients with AML in CR1 is associated with a significant worse DFS rate compared to patients with negative MRD, as well as a tendency towards a worse overall survival. The detection of MRD by MFC correlates with the detection by PCR in most of the cases. However, in a significant group of patients, MRD was detected only by PCR. This could be related to differences in sensitivity between both methods. Further studies including larger series are needed to confirm these observations. Disclosure of Interest: None declared. P285 Prognostic Impact of Pretransplantation Minimal Residual Disease detection for flow Cytometric, on Outcome of Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia. A Single Center Experience in Colombia over 14 Years A. A. Borda1,*, I. Cordoba1, V. Abello1, C. Rosales1, M. Rosales1, J. Figueroa1, E. Pedraza1, H. Esguerra1, M. Osuna2, A. Villegas1 1 Unidad de trasplante de me`dula o`sea, Clinica de Marly, 2 Hematologı`a, Hospital de San Jose, Bogota, Colombia Introduction: The accumulated evidence from studies of multiparameter flow cytometric MRD (MFC-MRD) assessment in AML leaves little doubt that this method of MRD detection can be used to risk stratify both younger and older patients at treatment time points. Persistence of disease or high levels of pretransplantation minimal residual disease (MRD) have been reported to predict disease relapse after Allogeneic bone marrow transplantation (BMT). The prognostic impact of MFCMRD is strong enough to have emerged despite study differences in the MFC assays and the limitations of now outdated restricted antibody panels. Aims: To determine the value of Minimal Residual Disease (MRD) assessed by Multiparameter Flow Cytometry (MFC) pretransplantation Allogeneic BMT, in predicting outcome in patients with acute myeloid leukemia (AML). Materials (or patients) and methods: We performed a retrospective analysis the predictive value of MRD assessment by MFC pre trasnplantation alogeneic in 119 patients (diagnosed AML treated between 2000 and 2014 submitted at our institution who had available MRD assessment). MRD by MFC on bone marrow specimens obtained approximately 30 days before transplantation. The detection threshold for defining pre transplantation positive MRD was 40.3% . Results: Of the 119 patients, 80 (67%) were in complete remission (CR1) , 31 (26%) CR2 and 4 CR2 8 (6%). Their median age was 38 years (Range, 10-64). Hyperleucocytosis in 39 (32%) and Cytogenetics was favorable risk in 32 (26%), intermediate risk in 39 (32.%), adverse risk in 35 (29%) and unknown in 13 (14%). There were a total of 44 deaths and 17 relapses; these contributed to the probability estimates for
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overall survival (OS) and disease free survival (DFS), stratified by MRD status and shown in figure 1. The median follow-up after BMT among survivors was 8.3 years (range, 6.9 to 9,6 years). The 7.5-years estimates of OS for MRD-positive and MRD-negative patients were 43.1% (range, 23,2% to 58,6%) and 68% (range 56% to 78.3%), respectively, and the 7,5 year estimates for DFS for MRD-positive and MRD-negative patients were 40.5% (range 21.4% to 52.6% ) and 56% (range 42.5% to 65.8%) respectively. After adjustment for various covariates, age, cytogenetics risk, hyperleucocytosis, secundary AML, the hazard ratios of MRD positive versus MRD negative were 2.06 (range 1.52 to 6.24; P ¼ 0,003) for overall mortality, 3.45 ( range 2.14 to 7.32; P ¼ 0.014) for DFS. Conclusion: That detection of MRD pre transplantation define a population of patients with AML who are at higher risk for adverse outcome, even after adjusting for other factors that influence post-BMT outcome. Disclosure of Interest: None declared. P286 Mild chronic GvHD may alleviate the poor prognosis associated with FLT3-internal tandem duplication in adult patients with AML following alloHSCT with myeloablative conditioning in first complete remission B. Nasilowska-Adamska1,*, A. Czyz2, K. Halaburda1, A. Tomaszewska3, M. Markiewicz4, P. Rzepecki5, B. Piatkowska-Jakubas6, M. Paluszewska7, R. Szydlo8, I. Solarska3, K. Borg1, S. KyrczKrzemien4, M. Komarnicki2, A. Skotnicki6, W. W. Jedrzejczak7, K. Warzocha1 1 Institute of Hematology and Transfusion Madicine, Warsaw, 2 K.Marcinkowski University of Medical Science, Poznan, 3Institute of Hematology and Transfusion Medicine, Warsaw, 4Silesian Medical Institute, Katowice, 5Military Medical Institute, Warsaw, 6 Collegium Medicum of Jagiellonian University , Krakow, 7Warsaw Medical University, Warsaw, Poland, 8Hammersmith Hospital, London, United Kingdom Introduction: Internal tandem duplication (ITD) of FLT3 is the most common mutation in AML with the highest frequency in patients with normal karyotype. The aim of this study was to estimate the significance of FLT3-ITD at diagnosis on the outcome of alloHSCT. Materials (or patients) and methods: A total of 140 patients at median age of 38 years (range 18- 68) with the analysis of FLT3-ITD performed at diagnosis who underwent first alloHSCT after myeloablative conditioning were included in this retrospective study. All patients were transplanted in first complete remission (CR) of AML between the years of 2006-2013 in six Polish centers. Results: FLT3-ITD was detected in 42/140 (30%) patients. Compared with FLT3-ITD-negative patients, FLT3-ITD positive patients had higher median white blood cells count (WBC) at
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diagnosis (71x109/l vs 28x109/l, Po0,001). Other characteristics were similar in the FLT3-ITD positive and negative groups of patients. At three years, relapse incidence (RI) was significantly higher in AML patients with intermediate or normal karyotype and WBC count 425 x109/L at diagnosis [12,7% (95%CI 5-29%) vs. 51.6% (95%CI 35-68%), Po0,001] or FLT3-ITD positive [20.4% (95%CI 11-34%) vs.52.9% (95%CI 33-72%), P ¼ 0,002] in univariate analysis. In multivariate analysis, we found that these two variables also significantly influenced RI following alloHSCT in AML patients with normal or intermediate karyotype [WBC425 x109/L (HR 4,2; 95%CI, 1,6-10,8, P ¼ 0,003), FLT3-ITD (HR 2,2; 95%CI, 0,99-5,0, P ¼ 0,053)]. Three-year leukemia free survival (LFS) following alloHSCT did not significantly differ between FLT3-ITD positive and negative groups, although, we found significant difference in LFS between patients FLT3-ITD positive and negative and normal or intermediate karyotype, respectively 44,7% (95% CI 26,3-64,7%) and 67,6% (95% CI 54,5-78,4%), P ¼ 0,06. Threeyear overall survival (OS) did not significantly differ between FLT3-ITD positive and negative groups, even in the group of patients with normal and intermediate karyotype. We also found that in patients who experienced chronic GvHD (cGvHD), the RI was significantly lower than in patients not affected with cGvHD [14,2% (95% CI 6-30) vs. 37,8% (95%CI 2750), P ¼ 0,041]. It is interesting, that patients with mild cGvHD had significantly lower RI when compared to patients with moderate or severe grade of cGvHD or those who did not experienced cGvHD, respectively 4,8 % (95%CI 1-25%) vs. 36,0% (95%CI 16-62%) vs. 27,8% (95%CI 18-41%), P ¼ 0,032. We did not observe any differences in LFS in accordance to overall incidence of cGvHD, but we found that mild cGvHD may positively influence LFS when comparing to the group of patients with no cGvHD or moderate/severe grade of cGvHD [respectively, 88.4% (95%CI 65-97%) vs. 67.5% (95%CI 55-78%) vs. 61.7% (95%CI 35-83%), P ¼ 0,06]. Conclusion: We observed that FLT3-ITD was harboring a poor prognosis in AML patients who underwent alloHSCT with myeloablative conditioning in first CR, mainly in those with intermediate or normal cytogenetic profile at diagnosis and significantly increased risk of relapse. Therefore, patients with high molecular risk AML may not be curable even following alloHSCT, unless they develop symptoms of mild cGvHD and concomitant GvL which may decrease RI and improves LFS. Disclosure of Interest: None declared. P287 Early Cumulative Incidence of Relapse in 80 Acute Myeloid Leukemia patients after chemotherapy and Transplant post-consolidation treatment: prognostic role of postinduction WT1 D. Capelli1,*, I. Attolico2, F. Saraceni1, G. Mancini1, S. Mancini1, A. R. Scortechini1, M. Montanari1, I. Scortechini1, N. Viola3, S. P. Pascale2, M. Chiarucci1, P. Leoni1, A. Olivieri1 1 Ospedali Riuniti di Ancona, Clinica di Ematologia, Torrette di Ancona, 2Divisione di Ematologia, Ospedale San Carlo, Potenza, 3 Ospedali Riuniti di Ancona, Servizio di Immunologia, Torrette di Ancona, Italy Introduction: Post consolidation minimal residual disease (MRD) evaluation represents a strong prognostic factor for AML outcome, while the significance of post induction MRD is still uncertain. Materials (or patients) and methods: We evaluated post induction and post consolidation bone marrow MRD in 80 AML patients aged in median 57 years (range: 17-80) with 16 months median follow-up (range 2-55). We analysed abnormal leukemia immunophenotype by multiparameter flow cytometry (MPFC) and WT1 by RT-PCR as described by Buccisano et al and Cilloni et al. The ELN cytogenetic molecular risk was determined in 62 patients (24 favorable, 24 INT-1, 14 unfavorable). WT1 was þ ve in 63/75 patients (85%) at diagnosis (median 1,056; range:259-232,056), in 10/59
P288 Comparison of The Results of Acute Leukemia Patients Who Had Undergone Haploidentical and HLA-I˙dentical Allogeneic Stem Cell Transplantation: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, C. Bal3, I. Dora1, B. Kosan1, E. Er1, B. Sarıtas1, N. Baskan1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, 3 Biostatistics Department, Osmangazi University, Kocaeli, Turkey
(16,9%) post induction (median 21; range:0,6-134.633) and in 8/50 (16%) post consolidation (median 18,6; range:0,8-45.338). MPFC MRD was þ ve in 27/56 (48.2%) patients after induction and in 16/44 (36.4%) after consolidation. Results: We analysed overall, 3, 12 month Cumulative Incidence of Relapse (CIR) by MRD status adjusted by ELN risk, gender, secondary disease, hyperleukocytosis, age and kind of post consolidation treatment. 68 patients achieved CR, 27 patients relapsed in a median of 6.6 months (1-34 months) with a 45% CIR: 12 patients at 3 months (13%), 23 at 12 months (33%). Post consolidation treatment consisted of chemotherapy in 34, Autologous and Allogeneic Tansplant in 32 patients, performed after 1 induction (37 high dose Cytarabine based, 43 standard dose Cytarabine based) and 1 high dose Cytarabine consolidation course. We observed 40%, 24.3% and 25.5% 12 month CIR after chemotherapy, Autologous and Allogeneic Transplant respectively. We also observed a 12 month CIR of 14.6% in favorable, compared to 23.3% in NPM-FLT3-, 57% in FLT3 þ and 61.6% in unfavorable ELN risk patients respectively. Patients with WT1 þ ve post induction and consolidation had a 12 month CIR of 90% and 62.5% respectively. Patients with MPFC þ ve post induction and consolidation had a 12 month CIR of 51.5% and 22.7% respectively. Multivariate analysis identified post induction WT1 þ ve status and not MPFC MRD status as the main predictor of 12 month CIR. Patients with WT1 þ ve after induction had a 14.3 RR to relapse at 12 months (Po0.0001) in comparison with WT1 -ve patients (Figure 1). Hyperleukocytosis (WBC count 450,000/mcl at diagnosis) and age460 yrs also significantly predicted 12 month CIR with a RR of 7.15 (P ¼ 0.004) and 12.3 (P ¼ 0.001) respectively. Conclusion: In conclusion post induction WT1 positivity confirmed its prognostic significance in our series, indipendently from ELN risk and post consolidation treatment, targeting a subset of early relapsing patients with an extremely poor outcome. References: Buccisano F, Maurillo L, Del Principe MI et al. Prognostic and therapeutic implications of minimal residual disease detection in acute myeloid leukemia. Blood. 2012; 119: 332–341. Cilloni D, Renneville A, Hermitte F et al. Real-time quantitative polymerase chain reaction detection of minimal residual disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study. J Clin Oncol. 2009; 27: 5195–5201. Disclosure of Interest: None declared.
Introduction: HLA-identical hematopoietic stem cell transplantation (HSCT) is now considered as an established therapeutic modality in several hematological malignancies. However, only 30-50% of the patients have a HLA-identical donor. In the present study, we report and compare the results of acute leukemia patients who were in complete remission at the time of transplantation and who had undergone HLAidentical or haploidentical hematopoietic stem cell transplantation. Materials (or patients) and methods: We analyzed retrospectively, the results of acute leukemia patients who were in complete remission at the time of transplantation and who had undergone allogeneic stem transplantation between July 2010 and December 2013. No T cell depletion were performed. Characteristics of the patients are shown in Table 1. Results: Characteristics of the patients in the 2 groups were similar (Table 1). Documented viral infection rate was higher in the haploidentical HSCT group in comparison to HLA-identical HSCT group (80% vs 50% respectively, P ¼ 0.003). Median hospitalization duration was 35 days in haploidentical HSCT group and 28 days in the HLA-identical HSCT group (Po0,001). Relapse and mortality rates (First 100 day, 1 year, 2 years) were similar in the 2 groups. Disease free survival and overall survival of haploidentical HSCT group and HLA-identical HSCT group did not differ significantly (P ¼ 0.46, P ¼ 0.42, respectively). Clinical results are shown in Table 2. Conclusion: According to our results, disease free survival and overall survival are similar in haploidentical and HLA-identical HSCT. However, in haploidentical HSCT time to neutrophil and thrombocyte engratment and hospitalization durations are longer, viral infection rates and GVHD rates are increased when compared to HLA-identical HSCT. More immunosuppressive drug usage is obligatory in haploidentical HSCT. Increased rate of viral infections and GVHD and longer hospitalization durations leads to increased costs in haploidentical HSCT and emphasizes the importance of experience in disease management. Disclosure of Interest: None declared. P289 Prospective study on 220 neutropenic episodes in 109 AML patients. Role of bed-side ultrasound (BUS) in neutropenic enteroclitis (NEC): early diagnosis, incidence and survival with different chemotherapy regimen E. Benedetti1,*, R. Giubbolini2, R. Morganti1, I. Bertaggia1, B. Bruno3, P. Lippolis4, F. Caracciolo5, E. Orciuolo1, G. Buda1, F. Cerri6, D. Caramella6, M. Petrini2 1 Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, 2Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Hematology Unit Ospedale S. Chiara Pisa, Pisa, 3U.O Ematologia Trapianti di midollo, University of Torino, Torino, 4UO Chirurgia D’Urgenza , Azienda Ospedaliero-Universitaria Pisana, 5U.O Ematologia Trapianti di midollo, Hematology Unit Ospedale S. Chiara Pisa, 6 Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, U.O. Radiologia Universitaria Pisa, Pisa, Italy Introduction: Neutropenic enterocolitis (NEC) is a life threatening complication of leukemic patients (pts) treated with chemotherapy (CHT) with mortality rate up to 50%. It is
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[P288]
characterized by abdominal pain (AP), fever (F) and diarrhoea (D). Ultrasound (US) was used to evaluate bowel-wall thickening (BWT), and 44 mm is considered diagnostic of NEC. Early diagnosis is crucial to start conservative medical management (CMM) which appears the optimal strategy for most cases. Objective: evaluate prospectively if US can detect early signs of NEC and guide a prompt treatment (CMM or surgical) in order to reduce mortality and to evaluate the impact of different CHT regimens on mucosal damage and NEC occurrence. Materials (or patients) and methods: In the last 7 years all AML pts admitted in our center undergoing CHT were enrolled (n ¼ 109). Abdominal US was performed, baseline before treatment, and as only one symptom (or a combination) appeared within 12h from onset: F and/or D and/or AP in CHTrelated neutropenic pts. Results: N ¼ 220 chemotherapy-related neutropenic episodes (NE) occurred in 109 pts. N ¼ 17 NEC were diagnosed (7.7 %). N ¼ 2 pts had 2 episodes of NEC and both survived. CHT regimens received (number of cycles/NEC episodes) was: 3 þ 7 (Idarubicin þ ARAC) N ¼ 62/10, Idarubicin (AML-M3) N ¼ 9/1, 3 þ 3 þ 5 (Idarubicin,VP-16,ARAC) N ¼ 48/3; 2 þ 5 (Idarubicin þ ARAC) N ¼ 24/1; Clofarabine (40mg) N ¼ 5/0; Clofarabine (20mg) 14/0; Clofarabine þ ARA-C (Cofa 20mg) N ¼ 18/1; FLANG N ¼ 13/1; HD-ARA-C (3gr/mq for 3 consecutive days) N ¼ 26/0. Overall 3 pts died out of 17 NEC episodes (17.6%). All pts were promptly treated as soon as BUS was diagnostic of NEC. Median time to response from beginning of CMM was 24h and the first sign of was a decrease in AP followed by F, D and BWT. Amelioration of symptoms occurred with pts still neutropenic. Symptoms at Dx were:F þ AP þ D N ¼ 9, F þ D N ¼ 1, F þ AP N ¼ 1, Dia þ AP N ¼ 4,D N ¼ 0, F N ¼ 0, AP N ¼ 2.
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Fever at Dx was absent in N ¼ 6 episodes (35%). Statistically (St) CHT regimens mostly associated with NEC were: 3 þ 7 odds ratio (OD) 6.00 (Po0.0001), 3 þ 3 þ 5 OD ¼ 1.75 (Po0.0001). There was not a St significant association of the following CHT regimens with NEC occurrence: Clofarabine (20 or 40mg) N ¼ 0 NEC, 2 þ 5 (P ¼ 0.125), Idarubicin (AML-M3) (P ¼ 0.111), Clofa 20mg þ ARAC (P ¼ 0.167), HD-ARAC N ¼ 0 NEC, FLANG (P ¼ 0.143). The association of ARAC to Clofarabine vs Clofarabine (20 or 40mg) alone was not St significant in NEC occurrence (P ¼ 0.9). The likelihood of NEC Dx in a discriminant model (Bayes theoreme) for pts with BWT and AP ¼ 100%, AP þ D ¼ 100%, AP þ D þ F ¼ 100%, AP þ F ¼ 100%. Conclusion: BUS allowed to detect early signs of NEC and to start prompt treatment in this life threatening complication, with a 76% survival rate. With BUS pts do not live the isolation room. Early diagnosis and intervention allowed to reduce mortality. Images of BUS and CT were superimposable. Fever is not a condition sine qua non for NEC diagnosis. Different chemotherapy regimens do have a different impact on mucosal damage. A prompt BUS in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical) eventually reducing mortality. Disclosure of Interest: None declared.
P290 BID, BAK AND BCL-XL expression in childhood acute lymphoblastic leukemia bone marrow M. Panagouli1, G. Martimianaki1, M. Pesmatzoglou1, E. Stiakaki1,* 1 Dept of Pediatric Hematology-Oncology, University Hospital of Heraklion, University of Crete, Heraklion, Greece Introduction: The apoptosis which may be involved in the pathogenesis of acute lymphoblastic leukemia (ALL) is known to be regulated by the receptor-mediated (extrinsic) and the mitochondria-mediated (intrinsic) pathway. The latter is controlled by the family of BCL-2 proteins, which are critical regulators of the apoptotic response with pro-apoptotic (BAX, BAK) and anti-apoptotic (BCL-2, BCL-XL, MCL-1) members. BAX and BAK play a pivotal role to the pro-apoptotic activity of BCL-2 family regulated by anti-apoptotic members of BCL-2 and BCL-XL while BID is considered one of the main activators of apoptosis. Aim: The quantitative determination of the expression levels of anti-apoptotic BCL-XL, and proapoptotic molecules BAK and BID in bone marrow (BM) of children with acute lymphoblastic leukemia at diagnosis and after remission achievement and of children with Solid Tumours (ST) without bone marrow involvement. Materials (or patients) and methods: Twenty eight children (15 boys, median age 7,68±1,06 years) with ALL (23 B lineage ALL, 5T ALL), 25 children at diagnosis (ALLd), 23 after remission was achieved (ALLr), and 38 with Solid Tumors at diagnosis without bone marrow involvement, were studied. Total RNA was isolated from bone marrow mononuclear cells. The mRNA levels corresponding to BCL-XL, BAK, and BID were quantified by real-time polymerase chain reaction (PCR) analysis using GAPDH as normaliser. All reactions were performed in duplicates. For quantification of the genes a standard curve was made by serial dilutions of a reference line cDNA (Tanoue). Results: The expression levels of anti-apoptotic BCL-XL were determined significant higher in ALL blasts at diagnosis compared with remission (ALLdvsALLr:3,16vs0,97, P ¼ 0,00001), as well as with ST(ALLdvsST:3,16vs0,64, P ¼ 0,000000013). The expression levels of BID were determined low at diagnosis of ALL and high enough in remission with statistically significant difference (ALLdvsALLr:4,57vs18,07, P ¼ 0,000001), result which is in accordance with its apoptotic role. High levels were also observed in ST without BM involvement (ALLdvsST:4,57vs10,329, P ¼ 0,003). Concerning the expression of apoptotic BAK gene, the levels were found statistically significant higher at ALLd compared with ALL in remission (ALLdvsALLr:4,73vs1,84, P ¼ 0,001). Similar high levels were determined in Solid Tumors with statistically significant differences compared with ALLr (STvsALLr:5.44 vs 1,844, P ¼ 0,011). Conclusion: The over-expression of BCL-XL at ALL diagnosis seems to be suppressed by the induction treatment and remission achievement. The expression of BID is amplified in remission as a pro-apoptotic molecule stimulating apoptosis. The role of BAK levels at diagnosis and remission in respect to its pro-apoptotic function warrants further investigation. References: 1. Sawata S, Suyama E and Taira K. A system based on specific protein–RNA interactions for analysisof target protein–protein interactions in vitro: successful selection of membrane-bound Bak–Bcl-xL proteins in vitro.Protein Eng Des & Sel 2004; 17: 501-508. 2. Addeo R, Caraglia M, Baldi A, D’Angelo V. Prognostic Role of bcl-xL and p53 in Childhood Acute Lymphoblastic Leukemia (ALL). Cancer Biology & Therapy 2005; 4: 32-38. 3. Du H, Wolf J, Schafer B, Moldoveanu T, et al. BH3 Domains other than Bim and Bid can Directly ActivateBax/Bak. Journal of Biol Chem 2011; 286: 491–501. Disclosure of Interest: None declared.
P291 Allogeneic haematopoietic stem cell transplantation in patients older than 65 years with high risk acute myeloid leukemia: results in 46 consecutive patients E. Sala1,*, M. G. Carrabba2, C. Messina1, F. Pavesi1, E. Xue1, B. Gentner1, L. Vago1, M. Morelli1, A. Assanelli1, S. Marktel1, M. T. Lupo-Stanghellini1, C. Corti1, J. Peccatori1, F. Ciceri1, M. Bernardi1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 2Unit of Hematology and Bone Marrow Transplantation, San Raffaele Scientific Institute, Milan, Italy Introduction: Allogeneic haematopoietic stem cell transplantation (allo-HSCT) from related or unrelated donors is the only curative option for patients (pts) with high risk acute myeloid leukemia (AML). Most of these pts are older than 60 years. Elderly pts have been excluded from allo-HSCT option until recently. The reduction of therapy-related complications and the introduction of reduced-intensity conditioning regimens has increased the upper age limit. Encouraging results of alloHSCT in pts up to the age of 70 years have been reported. Materials (or patients) and methods: We conducted a restrospective analysis of 46 consecutive pts, 65 yrs of age or older, with high risk AML, who received allo-HSCT at our institute between 2004 and 2014. Twenty pts out of 46 had a diagnosis of de novo AML (adverse or intermediate-2 risk profile according to ELN stratification was evaluated in 14/20, while 6 had a chemorefractory disease), 23/46 had AML secondary to previous Myelodisplastic or Myeloproliferative disorder and finally 3 pts had a diagnosis of therapy-related AML. Data were reviewed from the local database and updated at last follow up for pts still alive with a minimum follow up of 6 months. Results: Between August 2004 and July 2014, 46 pts, median age 66 years (range 65-78) underwent allo-HSCT. At transplant 28 pts were in complete remission (CR), 18 were in persistence of disease (PD). The stem cells source was in 8 (17%) cases a matched related donor (MRD), 14 (30%) a matched unrelated donor (MUD), 23 (50%) a haploidentical donor (HAPLO), in 1 case (3%) cord blood (CB). All pts transplanted from an adult donor received mobilized peripheral blood stem cells. Conditioning regimen was a reduced toxicity combination Treosulfan-based in most of the pts (36 out of 46, 78%). Graft versus Host Disease (GvHD) prophylaxys was cyclosporine þ methotrexate in 19 cases, rapamycine þ mofetil mycophenolate in 24 cases, ex vivo T-cell depletion in 3 cases. Median follow-up after transplant was 328 days (range 13-2050). Early deaths after transplant (before 30 days) were 4 (8.6%), 1 for disease progression and 3 for infection during aplasia. Engraftment rate was 97% (41/42 pts). Severe acute GvHD incidence (grade III-IV) during follow-up was 9% (4 out of 42 evaluable pts with at least 100 days of follow up), while severe chronic GvHD was detected in 3 pts out of 42 (7%). At last follow-up 17/46 pts (37%) are alive and 16 in CR, 29 pts (63%) were dead: 12 died because of disease relapse/progression, 16 for causes related to transplant toxicities (TRM), 1 patient was lost at follow up. The overall survival (OS) from diagnosis was 80% at 1 year and 38% at 3 years, while the OS from allo-HSCT was 55% at 1 year and 36% at 3 years, EFS at 1 year from alloHSCT was 45%. OS 1 year after allo-HSCT was 69% for pts who were in CR at transplant and 33% for pts who were in PD (p 0.0036). TRM at 1 year was 32%. Conclusion: Allo-HSCT from both matched related and alternative donors is feasible and potentially curative in pts older than 65. Disclosure of Interest: None declared.
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P292 Determination of copy number variation (CNV) helps in assessing the risk factor of response to HSCT and standard chemotherapy in AML patients E. Jaskula1,2,*, M. Mordak-Domagala1, M. Sedzimirska1, A. Sobczynska-Konefal1, J. Lange1, N. Surgut1, D. Owczarek1, A. Siemienska1, A. Lange1,2 1 Lower Silesian Center for Cellular Transplantation with National Bone Marrow Donor Registry, 2L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland, Wroclaw, Poland Introduction: For the purpose of this study, copy number variations were determined with the use of Agilent Technologies microarray plates (ShurePrint G3 Cancer CGH þ SNP 4x180 K) which were read after the completion of hybridization in the Roche instrument (NimbleGen MS200 Microarray Scanner) with use of Nexus Copy Number Software (BioDiscovery). Gains and losses were summarized together, giving the number of CNV in tested samples. Materials (or patients) and methods: Baseline characteristics of the cohort – transplanted (non-transplanted): F/M: 13/14 (29/33), median age: 42 (60), karyotype – favorable/other: 2/25 (9/43), Flt3positive/negative: 9/18 (18/34), HSCT – myeloablative/RIC: 22/5 non-transplanted – fit: standard intention to treat/unfit – low dose AraC - 42/10. Results: Overall survival was significantly better in the transplant group as compared to the standard chemotherapy group: 0.45 vs. 0.09, Po0.04 at 2-year cut-off point. Survival of patients assessed 18 months post-HSCT was better in the group with CNVs not exceeding 20 as compared to those with higher values (9/6 vs. 4/8, n.s.) irrespective of the presence of karyotype abnormalities. Survival of non-transplanted patients was significantly affected by the presence of CNVs above 20 (Po0.039, log rank test) (Figure 1). The adverse effect of CNVs above 20 was seen irrespective of the presence of FLT3 mutation in both transplanted and nontransplanted groups. Conclusion: Higher numbers of CNV in AML patients may assist both the decision-making process as to the transplantation, and – if transplantation is performed – advance planning
of the post-transplant treatment modalities knowing the CNVassociated risk factor of relapse. Supported by INNOMED/I/1/NCBR/2014 CellsTherapy NCBiR grant. Disclosure of Interest: None declared. P293 Is allogeneic stem-cell transplantation an option for patients with acute leukemia and myelodiplastic syndrome transplanted with active disease? A center experience E. Pe´rez-Lo´pez1,*, O. Lo´pez-Godino1, M. Cabrero1, J. Labrador2, F. Sa´nchez-Guijo1, L. Va´zquez1, B. Vidriales1, L. Lo´pez-Corral1, D. Caballero1 1 Hospital Universitario de Salamanca, Salamanca, Spain, 2 Hematology department, Hospital Universitario de Salamanca, Salamanca, Spain Introduction: The prognosis of patients with refractory/ relapsed acute leukemia (rAL) or myelodisplastic syndrome (MDS) is very poor. Although patients with refractory disease are not good candidates for allogeneic stem cell transplantation, this could be the only curative treatment for these patients. Unfortunately, reports of long-term follow up in this population and doubt exist about if this strategy should be offer to these patients The purpose of the present study is to evaluate the outcomes and efficacy of salvaged HSCT for rAL and MSD in our centre. Materials (or patients) and methods: We examined retrospectively 74 patients with rAL (myeloid & lymphoblastic n ¼ 36/10) and MDS (n ¼ 28) who underwent allogeneic transplantation at our centre since 1999 to 2013 with active disease at transplant. Feasibility and efficacy of HCT was analyzed in terms of TRM, event free survival (EFS) and overall survival (OS). Results: The main characteristics of the patients and transplant were: median age 55 years (14-69), status disease at transplant: refractory 37 (50%), relapse 17 (23%), untreated 8 (10%) and aplastic 12 (30%) with a median blasts of 10% (572) in those with burdern leukaemia. Median time from diagnosis to transplant was 7 months (1-160).
[P292]
Figure 1.
Overall survival of non-transplanted AML patients on chemotherapy.
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Conditioning regimen was myeloablative in 22 (30%) and reduced intensity (RIC) in 52 (70%) 8 patients (11%) have received a prior transplant, 4 of them an allogeneic transplant. The source was peripheral blood in all but 5 (3 bone marrow and 2 cord blood). The donor was: related in 45 (60%), unrelated in 23 (32%) and 6 haploidentical (8%). Graft-versushost disease (GVHD) prophylaxis was calcineurin inhibitorsregimens ( þ MMF or þ methotrexate) in 38 (52%) and tacrolimus and rapamicine in 25 (34%), and other inmunosupression in 10 (14%). All the patients but one engrafted. The incidence of acute-GVHD (aGVHD) was 61% (14% grade III-IV) with median onset of 26 days (8-130); in evaluable patients, chronic-GVHD (cGVHD) was 45% (24% severe) with a median onset of 163 days (84-451). Regarding response to transplant at day þ 100: 43 patients (58%) achieve complete remission (CR), 13 (17%), progression disease and 18 (24%) were death previously, mostly from relapse (n ¼ 12). From those patients without CR at þ 100, 11/ 13 death because relapsed disease. With a median follow up of 10 months, the estimated OS and EFS at 1 year was 50% and 38%, respectively. In the univariate analysis, achieve CR at þ 100 and development of cGVHD (yes/no and grade) were associated with better OS (Po0.05); these variables remained in the multivariate analysis. The same factors were associated with better EFS in the univariate and multivariante analysis. Status disease at transplant and leukaemia burden has not significance in both OS and EFS. TRM was 17% (10% at day þ 100). The main cause of death was progression disease (n ¼ 29 40%). From those patients alive who achieve CR at þ 100, 31% of them (23/74) remained in CR. Conclusion: Our results indicate that patients with rAL or MDS can benefit from HSCT even with high leukemia burden. We have shown that HSCT can induce long-term disease remission and possibly cure in those who developed cGVHD especially who achieve CR at þ 100. Disclosure of Interest: None declared. P294 Maintenance treatment with the association of deferasirox and Vitamin D improves survival in AML after allogeneic hematopoietic stem cell transplantation E. Paubelle1,*, A. Brignier2, F. Lemonier1, A. Marc¸ais1, D. Sibon1, R. Delarue1, O. Hermine1, F. Suarez1 1 Hematology, Hoˆpital Necker, 2Cellular Therapy, Hoˆpital Saint-Louis, Paris, France Introduction: Currently, the most efficient curative treatment for patients with Acute Myeloid Leukemia (AML) is allogeneic hematopoietic stem cell transplantation (HSCT). However, many patients relapse because of the presence of quiescent leukemia initiating cells (LIC). It has been shown that deferasirox(DFX) is able to induce AML cells apoptosis in vitro through inhibition of the NF-kB pathway. In addition, our group has shown that iron chelators are able to promote in vitro monocyte differentiation in both normal hematopoietic progenitors and primary AML cells and that iron chelators act synergistically with vitamin D (VD) to promote cell differentiation. Morerover, both iron chelators and VD analogues have a favorable safety profile and are often used in this patient population. We report here the outcome of patients who receivedDFX-VD combination therapy as maintenance treatment after allogeneic HSCT for AML. Materials (or patients) and methods: From June 2007 to June 2014, 33 patients diagnosed with AML and receiving allogeneic HSCT in Necker Hospital were included in the study.DFX-VD maintenance was left to the discretion of the referring physician. Overall, 10 patients received DFX-VD. The dose of DFX was adapted on ferritin and creatinine levels. DFX was initiated at 20–30 mg/kg per day and VD at 100,000 units orally weekly. Overall survival (OS) was defined as the time from the first day of induction to death from any cause and
was censored at the date of last visit. Survival was estimated by the Kaplan-Meier method and was comparisons were performedwith the log-rank test. Multivariate analysis was performed using Cox proportional hazard model. Statistical significance was defined as p-value o0.05. Results: Median age was 54 and sex ratio 1.4. According to molecular and cytogenetic prognosis group, 2 patients (6%) had favorable risk, 26 (79%) intermediate and 11 (33%) unfavorable risk. Risk data were not available for 4 (12%) patients. At diagnosis, medium serum ferritin was of 603ng/mL and medium transferrin saturation of 52%. Baseline characteristics did not differ between the 10 patients who received DFX-VD maintenance and the 23 patients who did not. Medium time between HSCT and the beginningof DFX-VD was of 121 days with a median duration of 19 months without significant adverse event.Incidence of graft versus host disease was similar. Relapse rate was 43.5 and 10%, P ¼ 0.06 for control and DFX-VD patients respectively, and treatment related mortality of 13 and 20%, P ¼ 0.62. DFX-VD maintenance treatment improved significantly survival as median OS had not been reached versus 20.1 months in no maintenance group, P ¼ 0.04. In multivariate analysis, only maintenance treatment with DFX-VD had a favorable impact with aHazardRatio of 0.2 (0.1 – 0.9), P ¼ 0.03. Conclusion: Based on this small observational study, DFX-VD maintenance treatment may improve overall survival after allogeneic HSCT in AML patients by preventing relapse.As shown for As2O3 and retinoids, VD might affect LICs. Therefore, further studies should evaluate the impact of DFX-VD on LIC. These encouraging results from this retrospective study need to be validatedin a large randomized prospective multicenter study. Disclosure of Interest: None declared. P295 Chemotherapy followed by donor stem cell infusion provides a better survival than chemotherapy alone for patients with acute leukemia relapsed after allogeneic hematopoietic stem cell transplantation Y. Zhao1, Y. Luo1, Y. Tan1, J. Shi1, W. Zheng1, H. Huang1,* 1 Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Introduction: Relapse remains a major obstacle to the success of allogeneic hematopoietic stem cell transplantation (HSCT)in acute leukemia, yet little is known about the relevant prognostic factors after relapse, and the best treatment strategy has not been demonstrated. Thus, this retrospective study was designed to compare the treatment efficacy of chemotherapy alone and chemotherapy followed by donor stem cell infusion (DSI) in patients with relapsed leukemia after HSCT, and also to investigate the prognostic factors in these patients. Materials (or patients) and methods: Forty-nine patients who relapsed after HSCT at the First Affiliated Hospital of Zhejiang University between January 2006 and August 2013 , were nonrandomly assigned to treatment with chemotherapy alone (n ¼ 29) or chemotherapy followed by DSI group (n ¼ 20) according to the patient’s own choice. Results: The 1-year postrelapse overall survival (OS) rate and diseasefree survival (DFS) for entire cohort was 16.5% and 14.3%, respectively. In patients receiving chemotherapy followed by DSI, complete remission rate was significantly higher (40.0% vs. 13.8%, P ¼ 0.036), and 1-year overall survival (OS) and disease-free survival (DFS) was significantly improved (1-year OS: 32.3% vs. 10.3%, P ¼ 0.005; 1-year DFS: 25.8% vs. 6.9%, P ¼ 0.005), compared with patients receiving chemotherapy alone. For the patients with acute myeloid leukemia (AML), chemotherapy followed DSI was superior to chemotherapy alone, with 1-year OS and PFS rates of 56.3% vs. 11.1% (P ¼ 0.026) and 56.3% vs. 11.1% (P ¼ 0.038), respectively, however, for the patients with acute lymphoid leukemia
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(ALL), the benefit of DSI was not obvious (1-year OS: 10% vs 10%, P ¼ 0.175;1-year DFS: 10% vs 5%, P ¼ 0.182). There was no significant differences in treatment related mortality (TRM) between the two intervention groups (34% vs 33.7%, P ¼ 0.712). Risk factors for mortality after relapse included shorter time to relapse, disease type (ALL), higher marrow blast cells at relapse, intervention group (chemotherapy alone), and no chronic graft-versus-host disease (cGVHD) occurring after intervention, but only cGVHD occurring after intervention was an independent predictor after multivariate analysis (RR ¼ 0.425, P ¼ 0.043). The 1-year OS and DFS was significantly improved in the patients harboring cGVHD after intervention compared with no cGVHD patients (1-year OS: 41.9% vs 6.3%, P ¼ 0.00; 1-year DFS: 41.9% vs 0%, P ¼ 0.00). Conclusion: The study suggest that chemotherapy followed by DSI had a stronger anti-leukemic effect and provided a better survival in relapsed leukemia after HSCT, especially for those AML. Furthermore, cGVHD occurring after intervention was associated with a longer survival for relapsed patients. Disclosure of Interest: None declared. P296 Clinical outcomes of CLAG/CLAGM or fludarabine based chemotherapy following allogeneic SCT in relapsed/ refractory acute myeloid leukemia patients H. Park1,*, Y. Koh1, J.-H. Youk1, I. Kim1, S. Park1, J.-O. Lee2, S.-M. Bang2, S.-S. Yoon1 1 Seoul national university hospital, Seoul, 2Seoul National University Bundang Hospital, Seongnam, Korea, Republic Of Introduction: Substantial number of acute myeloid leukemia (AML) patients does not respond to induction chemotherapy (refractory) or relapse after achieving complete remission (CR). In relapsed/refractory AML, therapeutic options are still undefined and unsatisfactory. We intended to reveal the efficacy of new agents followed by allogeneic stem cell transplantation (ASCT) in relapsed/refractory AML patients. New agents included cladribine (CLAG/CLAGM) and fudarabine (FLAG/FLAGI/mFLAI). Materials (or patients) and methods: Relapsed/refractory AML patients who were treated with cladribine or fludarabine based chemotherapy at two institutions (Seoul National University Hospital and Seoul National University Bundang Hospital) were retrospectively reviewed. We analyzed receive of ASCT, relapse free survival (RFS), and overall survival (OS). Results: A total of 118 patients (Male : Female ¼ 71:47, median age ¼ 53 years) were analyzed; 66 patients were treated with CLAG/CLAGM and 52 patients were treated with FLAG/FLAGI/ m-FLAI. Twenty-nine patients underwent ASCT after reinduction chemotherapy; The 19 patients after CLAG/CLAGM and 10 patients after FLAG/FLAGI/mFLAI. The median OS of patients who received CLAG/CLAGM was 13.5 months; 39.1 months for ASCT group and 12.9 months for non-transplant group (P ¼ 0.238). For patients who achieved complete remission (CR) after CLAG/CLAGM, significantly better OS was observed in patients who underwent ASCT post-CLAG/CLAM chemotherapy compared to those who did not (median OS not reached vs. 12.3 months, P ¼ 0.036). In fludarabine-based chemotherapy group, the median OS was 15.1 months; 30.0 months for ASCT group and 14.7 months for non-transplant group (P ¼ 0.089). For patients who achieved CR after fludarabine-based chemotherapy, better OS was observed in patients who underwent ASCT after chemotherapy (median OS 30.0 months vs 14.7 months. P ¼ 0.046). As for RFS (CR patients ¼ 57, nonCR patients ¼ 36, not evaluated patients ¼ 25), the probability of 2yr RFS was 61.9%, the median RFS was not reached in CLAG/CLAGM group. 2Yr RFS rate was 77.8% for ASCT group, and was 55.6% for non-transplant group (P ¼ 0.144). Similarly, in FLAG/FLAGI/ mFLAI group, no significant difference in 2yr RFS rate according to subsequent ASCT was observed (62.5% vs 14.7%, P ¼ 0.184). The probability of 2yr RFS was 14.7 % in non-transplant group, was 62.5% in ASCT group.
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In cladribine-based chemotherapy groups, CR rate was 61.5%, which was not significantly different with fludarabine-based group (61.0%) (P ¼ 0.562). Also, the median OS of ASCT group after CR was not significantly different between two groups (median OS 31.0 months (cladribine group) vs 30.0 months (fludarabine group). P ¼ 0.911). Conclusion: For patients who receive reinduction chemotherapy using cladribine or fludarabine, consolidation ASCT is definitely necessary to improve survival outcome. Disclosure of Interest: None declared. P297 Allogeneic transplantation for acute lymphoblastic leukemia (ALL) in adults 2008-2013: results from Denmark I. Schjoedt1,*, D. Hovgaard1, L. Vindeløv1, L. Friis1, N. Smedegaard Andersen1, S. Lykke Petersen1, H. Sengeløv1 1 Hematology, RIGSHOSPITALET , Copenhagen, Denmark Introduction: The study is a retrospective single-center analysis of survival from allogeneic transplantation for B-ALL or T-ALL from Rigshospitalet University Hospital, Copenhagen over a six-year period. Materials (or patients) and methods: Data from all ALLpatients transplanted at Rigshospitalet University Hospital, the only Danish ALL- transplant center during the period 20082013, were retrospectively evaluated. Patients with an adult donor and myeloablative conditioning received Etoposide 60 mg/kg x 1 and total body irradiation (TBI) 12 Grey. If TBI was not to be given, patients received Etoposide 60 mg/kg x 1 and Busulphane 3.2 mg/kg x 5 days instead. If the donor source was UCB, conditioning consisted of Fludarabine 25 mg/m2 x 3 days, and Cyclophosphamide 60 mg/kg x 2 days plus TBI 12 Gy. Non-myeloablative conditioning consisted of Fludarabine 30 mg/m2 x 3 days plus TBI 2 Gy. No CNS-irradiation was given as part of the conditioning regimen. Rejection- and gvh profylaxis was MTX and Cyclosporine for the myeloablative conditioning or Tacrolimus and Mycophenolate Mofetil for the non-myeloablative conditioning. Results: In total 63 patients, 47 (75%) with B-ALL and 16 (25%) with T-ALL were transplanted during this period. Of these 41 (66%): 31 B and 10 T were transplanted in 1.CR, whereas 22 (33%): 16 B and 6 T were in a 2. or later remission. The male:female ratio was 2:1. The median age was 20 and range 18-63. Most patients (59 (94%)) had a myeloablative conditioning, as the non-myeloablative conditioning (4 (6%)) for ALL was not conducted at Copenhagen until 2013. The median observation time was 878 days (81 – 2200 days). Overall survival was 72% for all 63, with a plateau reached after approximately 800 days. 16 patients died, 5 from relapse (8%), 7 from gvhd (11%), 3 from infection (5%) and 1 (2%) from a non-hematologic cancer. The stem cell source had no significant impact on overall survival. 39 were transplanted with bone marrow, 20 with peripheral blood stem cells, and 4 with double umbilical cords.
There was no significant difference in survival according to donor source, 51 (81%) had a matched unrelated (MUD) donor and 12 (19%) a sibling donor. Conclusion: This retrospective single-center study demonstrates excellent survival for patients with either B-ALL or T-ALL, treated in Denmark over the six-year period from 20082013. Compared to other published data, overall survival is high and a true plateau is reached after approximately two years. The intensive pre-transplant nordic (Nopho) treatmentregimen for younger patients, the short delay from treatment to transplantation and the timely well-structured CMV-survey are important contributing factors, however several other factors may influence these positive results. Disclosure of Interest: None declared. P298 Second Haploidentical Hematopoietic Stem Cell Transplantation with Treosulfan-based Conditioning Regimen for Acute Leukemia in Relapse J. Peccatori1,*, L. Crucitti1, R. Greco1, M. T. Lupo Stanghellini1, L. Vago2, N. Cieri3, F. Giglio1, M. Morelli1, A. Assanelli1, M. Marcatti1, M. Carrabba1, S. Marktel1, T. Perini1, F. Lorentino1, S. Girlanda1, C. Corti1, M. Bernardi1, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, 2Unit of Molecular and Functional Immunogenetics, 3Experimental Hematology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: In the last years, HSCT using haploidentical T-replete source has become more and more popular, thanks to acceptable rates of GvHD and NRM. Acute leukemias (AL) relapsing after allo-HSCT have a dismal prognosis. Strategies to prevent post-transplant disease recurrence are limited. We evaluated the outcome of haploidentical transplant performed as second transplant (HSCT2) for patients with AL, relapsing after a first allo-HSCT (HSCT1). Materials (or patients) and methods: We analyzed 42 patients (median age 48 years) who underwent to HSCT2 from a haploidentical donor, different from the previous one, between 2007 and 2013 in our Institute. Patients suffered of myeloid AL (n ¼ 31), lymphoblastic AL (n ¼ 9) or biphenotypic AL (n ¼ 2) relapsing after HSCT1 (matched related: 21; unrelated: 17; mismatched related: 4; cord blood: 4). Thirtyseven patients (88%) received re-induction: 13 of them achieved complete remission (CR), 16 did not respond and 8 patients were transplanted in aplasia. In 5 cases HSCT2 was performed without re-induction therapy, in active disease (AD). Median HCT-Co-morbidity Index was 2 (range 0-6). All patients received treosulfan (14 g/m2 for 3 days) and fludarabine (30 mg/m2 for 5 days) as part of preparative regimen for HSCT2; conditioning was intensified with total body irradiation (4 Gray) in 18 (43%) or with melphalan (70 mg/m2 for 2 days) in 6 patients (14%). GvHD prophylaxis consisted of mycophenolate mofetile and sirolimus combined with pre-transplant ATG-Fresenius (n ¼ 36) or with posttransplant cyclophosphamide (n ¼ 6). Unmanipulated PBSCs were used as graft source in all cases. Results: Three patients (7%) died during aplasia, all the others achieved neutrophil engraftment (n ¼ 39, 93%), with no primary graft rejection. Complete remission at day þ 30 was reached in 37 out of 39 evaluated cases (95%). Estimated OS and LFS rates at 1 year from HSCT2 were 37% ± 8% and 35% ± 7%, respectively. One-year RI was 32% ± 7% and NRM to 31% ± 7% (7 patients died for infectious, 5 for GvHD and 1 for CNS toxicity). Sixteen patients (38%) suffered of acute GvHD grade II-IV and 20 (47%) of chronic GvHD. Disease status at HSCT2 (AD vs CR) had no impact on OS (HR:1.26, 95% CI 0.582.76, P ¼ 0.564) and relapse (HR:1.35, CI 95% 0.46-3.76, P ¼ 0.61). Conversely, time of remission from HSCT146 months provided the better outcome in terms of estimated OS at 1 year (50% ± 10%) compared to patients with shorter duration of remission (18% ± 9%) (HR:0.49, 95% CI 0.24-1.02, P ¼ 0.05) and reduced risk of relapse (HR:0.28, 95% CI 0.11-
0.75, P ¼ 0.011). After a median follow-up time of 22 months (range 6-68), 11 patients (26%) were alive with ongoing leukemia remission. Conclusion: These results demonstrate the feasibility of second allo-HSCT from a haploidentical donor as treatment for AL relapse, showing rates of OS, LFS and NRM comparable to the reported in literature for HSCT2 from matched related or unrelated graft. Interestingly, disease status at HSCT2 did not influence the outcome of transplant. While, in accordance with previous reports, lasting time of remission after HSCT1 is the major factor involved in predicting outcome of a second transplant due to an increased relapse incidence, reflecting diseases biologically different. If confirmed in a larger cohort, these data could be useful in decision making after HSCT1 failure for disease recurrence. Disclosure of Interest: None declared. P299 The double-negative NPM1/FLT3-ITD mutation shows long term outcomes after allogeneic transplant similar to those of the ‘favourable’ risk group according to the ELN risk classification, distinct from other intermediate-I risk in normal-karyotype AML J.-S. Ahn1,*, H.-J. Kim1, Y.-K. Kim1, S.-H. Jung1, D.-H. Yang1, J.-J. Lee1, N. Y. Kim2, S. H. Choi2, M. D. Minden3, C. W. Jung4, J.-H. Jang4, H. J. Kim5, J. H. Moon6, S. K. Sohn6, J.-H. Won7, S.-H. Kim8, D. D. H. Kim3 1 Department of Hematology-Oncology, 2Genomic Research Center for Hematopoietic Diseases, Chonnam National University Hwasun Hospital, Jeollanam-do, Korea, Republic Of, 3Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada, 4Division of Hematology-Oncology, Samsung Medical Center, 5Department of Hematology, The Catholic University of Korea, Seoul, 6 Department of Hematology-Oncology, Kyungpook National University Hospital, Daegu, 7Department of Hematology-Oncology, Soon Chun Hyang University Hospital, Seoul, 8Department of Hematology-Oncology, Dong-A University College of Medicine, Busan, Korea, Republic Of Introduction: The prognostic significance of the European LeukemiaNet (ELN) molecular risk classification was examined in patients with normal-karyotype acute myeloid leukaemia (NK-AML) after allogeneic haematopoietic cell transplantation (HCT). Materials (or patients) and methods: In total, 121 patients received allogeneic HCT for NK-AML and were evaluated for ‘‘fms-related tyrosine kinase 3-internal tandem duplication’’ (FLT3-ITD), nucleophosmin 1 (NPM1), and ‘‘CCAAT/enhancer binding protein a’’ (CEBPA) mutations in diagnostic samples, and analysed in terms of the long-term outcomes following HCT. Results: The prevalences of FLT3-ITDpos, NPM1mut, and CEBPAmut were 32.2%, 43.8%, and 29.2%, respectively. The 5year OS in the favourable (n ¼ 60) versus the intermediate-1 group (n ¼ 61), according to the original ELN risk, was 69.0% versus 48.1% (P ¼ 0.038). Among the intermediate-1 group, the 5-year OS was 57.6% in NPM1wild/FLT3-ITDneg (n ¼ 25), 45.8% in NPM1mut/FLT3-ITDpos (n ¼ 24), and 28.1% in NPM1wild/FLT3ITDpos (n ¼ 12) patients. Thus, we reclassified the doublenegative NPM1wild/FLT3-ITDneg group to the favourable from the intermediate-1 risk group. With this revised ELN risk classification, the 5-year OS was 65.2% in the favourable (n ¼ 85) and 40.4% in the intermediate-1 groups (n ¼ 36; P ¼ 0.004). Thus, reclassification of the NPM1wild/FLT3-ITDneg double-negative group into the favourable risk group improved the prognostic stratification power of the ELN risk classification significantly (Po0.001). Conclusion: A revised ELN risk classification, reclassifying double-negative patients into the favourable risk group, can improve risk stratification in NK-AML patients following allogeneic HCT. Disclosure of Interest: None declared.
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P300 Myeloablative Haploidentical Stem Cell Transplantation (MAC-HAPLO) With Post-Transplant Cyclophosphamide (PTCy) As Gvhd Prophylaxis In High Risk Leukemias/ Myelosdisplastic Syndromes (MDS) J. Gayoso1,*, P. Balsalobre1, M. Kwon1, A. Sampol2, P. Montesinos3, D. Serrano1, P. Herrera4, M. J. Pascual5, A. Bermu´dez6, C. Castilla-Llorente7, L. Bento2, D. Champ1, A. Pe´rez-Corral1, E. Buces1, J. L. Dı´ez-Martı´n1 on behalf of Grupo Espan˜ol de Trasplante Hemopoye´tico (GETH) 1 Hematology, HGU Gregorio Maran˜o´n, Madrid, 2Hematology, Hosp. Son Espasses, Palma Mallorca, 3Hematology, Hosp. La Fe´, Valencia, 4Hematology, Hosp. Ramo´n y Cajal, Madrid, 5Hematology, CHU Ma´laga, Ma´laga, 6Hematology, Hosp. Marque´s de Valdecilla, Santander, 7Hematology, Hosp. Morales Messeguer, Murcia, Spain Introduction: Allogeneic transplantation is the only curative option for patients with high risk leukemias or MDS. Only one third of them have an HLA identical sibling donor and around 60-70% will find an unrelated donor, that´s why haploidentical stem cell transplantation (HAPLO-HSCT) offers a therapeutic option to most of these patients. Myeloablative conditioning (MAC) used to obtains better disease control than reduced intensity conditioning regimens (RIC), but with higher toxicity, rendering long term similar results Materials (or patients) and methods: We retrospectively evaluated the results in patients diagnosed with high risk leukemias or MDS of our MAC-HAPLO regimens (Fludarabine 30 mg/m2 x5 days (-6 to -2), Cyclophosphamide14,5 mg/kg x2 days (-6 to -5), IV Busulfan 3,2 mg/kg x 3 days (BUX3) on days 4 to -2, or Fludarabine 40 mg/m2 x4 days (-6 to -2) and IV Busulfan 3,2 mg/kg x 4 days (BUX4)) with GVHD prophylaxis based on PTCy (50 mg/kg on days þ 3 and þ 4) and a calcineurin inhibitor plus mycophenolate from day þ 5, performed in GETH centers. Results: From Feb-2011, 24 MAC-HAPLO have been done in 7 centers. Median age was 37 years (15-65), 58% were males and all were in advanced disease phase or presented high risk features (AML 16/ALL 4/MDS 2/ CML-BC 1/ CMML 1). Previous HSCT had been employed in 21% (autologous in 1, allogeneic in 4), and in 79% the HAPLO-HSCT was their first transplant. Disease status at HAPLO-HSCT was morphologic CR in 83%, but with persistent disease (morphologic or MRD þ by flow or molecular markers) in 67%. BM was the graft source in 3 patients (12.5%) and PBSC in 21 (87.5%), non T-cell depleted in all cases. The haploidentical donor was the patient´s mother (21%), father (12.5%), siblings (41.5%) or offspring (25%). MAC regimen was BUX3 in 8 (33%) and BUX4 in 16 patients (67%). Median infused CD34 þ cells were 4,93x10e6/kg (3,20-8,43). Median neutrophils engraftment was reached at day þ 16 (1329) and platelets 420K at day þ 27 (11-131). Complete chimerism was obtained at a median of 22 days (13-44) in 21 evaluated patients. Cumulative incidence (CI) of non-relapse mortality (NRM) was 21.5% at 1 year. CI of grade II-IV acute GVHD was 45.5% at day þ 100, and grade III-IV was 9%. CI of chronic GVHD at 1 year was 35%, being extensive in 8% . No differences in acute or chronic GvHD CI were seen when comparing BUX3 against BUX4. After a median follow-up of 15 months (3-31), estimated 18-months event-free survival (EFS) and overall survival (OS) were 63% and 75% respectively. CI of relapse or progression was 19.5%. No significant differences in NRM, EFS, OS and relapse incidence were detected between BUX3 and BUX4. The effect of CR prior to MAC-HAPLO has not been apropiately assesed due to the limited number of events in our series Conclusion: IV Busulfan based MAC-HAPLO with PTCy in the treatment of high risk leukemias and MDS offers good disease control with manageable toxicity, with either BUX3 or BUX4. Disclosure of Interest: None declared.
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P301 Safety and efficiency of bortezomib to improve GVHD control in TCR-alpha/beta depleted transplantation in children with acute myeloid leukemia L. Shelikhova1,*, M. Maschan1, D. Balashov1, J. Skvortsova1, E. Boyakova1, V. Kalinina2, D. Shasheleva1, M. Persiantseva1, G. Novichkova1, A. Maschan1 1 Hematopoietic stem cell transplantation, 2Molecular Biology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: Relapse of malignancy and graft-versus-host disease (GvHD) remains the most important cause of failure after allogeneic transplantation for acute leukemia in children. Negative depletion of a/b( þ ) T cells and CD19 þ B lymphocytes is a new technology of graft manipulation, which improved GVHD control and immune reconstitution in our pilot trial. Bortezomib, a proteasome inhibitor capable of direct antitumor effects, has shown activity in GVHD prevention and therapy. In vitro data suggest that bortezomib sensitizes tumor cells to the cytotoxicity of NK cells. In this pilot trial we evaluated the safety and effects of bortezomib added to our backbone of intensive chemotherapy-based conditioning and TCR-alpha/beta depletion in haploidentical and matched unrelated transplantation (MUD). Materials (or patients) and methods: A total of 19 pediatric patients with acute myeloid leukemia (AML), 7 female, 12 male, median age 10,6 years, underwent allo HSCT between January 2014 and august 2014. Eight patients were transplanted from haploidentical donors and 11 from MUD. Patients were divided in 2 groups according to remission status: complete remission (CR) - 13 pts, active disease (AD) - 6 pts. All patients with AD were transplanted from haploidentical donor. Preparative regimen included Fludarabine 150 mg/m2, Treosulfan 42 g/m2, Melphalan 140 mg/m2, ATG (rabbit, Thymoglobuline) 5 mg/kg and rituximab 100mg/m2. Bortezomib 1,3mg/m2 was used at days -5,-2, þ 2, þ 5. PBSC were depleted of TCR-alpha/beta and CD19 þ B-cell according to manufacturer’s recommendations. The median dose of infused CD34 þ cells was 7,2x106/kg (range 4,2-17), TCR a/b -13,8 x103/kg (range 0,6-68). No additional post-transplant immune suppression was used in 18 pts, 1 pt received additional tacrolimus till day þ 30. Results: Primary engraftment was achieved in 16 of 19 pts, the median time to neutrophil and platelet recovery was 13 days. Two pts transplanted in AD had active disease on day þ 30, 1 pt in active disease had primary graft failure with no evidence of leukemia and was retransplanted from MUD. None of the patients died of transplant-related cause, pTRM - 0%. No patient had significant acute organ toxicity (renal, cardiac, hepatic, bladder, CNS). Febrile neutropenia was registered in 5 (26%), mucositis grade II - in 5 pts (26%), severe infection, requiring inotropic support, developed in one case (5%). Bortezomib-induced peripheral neuropathy (BINP) grade 1 was observed in 3 pts (15%), dose reduction was required in 1 pt (5%). Cumulative incidence of aGvHD grade II was 5,9% (95% CI: 039), no case of grade III-IV aGvHD was observed. Posttransplant steroids were administered to 1 (6%) pt. With a median follow-up of 5,8 months (3-9) 15 of 19 pts are alive (3 transplanted in AD, 12 in CR, P ¼ 0,029), 12 disease-free. Disease status at HSCT significantly influenced the relapse rate: cumulative incidence of relapse was 27 % in CR patients and 83% in AD (Grey test P ¼ 0,001). Conclusion: We demonstrate that bortezomib can be safely administered to children with AML in the context of treosulfan/melphalan chemotherapy with minimal neurologic and low visceral toxicity. We suggest that peritransplant administration of bortezomib might further improve GVHD control provided by depletion of TCR-alpha/beta lymphocytes from the graft and allow for immune-suppression-free posttransplant environment. Disclosure of Interest: None declared.
P302 TCRab þ /CD19 þ -depletion in hematopoietic stem cells transplantation from matched unrelated and haploidentical donors following treosulfan-based conditioning in pediatric acute lymphoblastic leukemia patients L. Shelikhova1,*, M. Maschan1, D. Balashov 1, J. Skvortsova1, V. Kalinina2, D. Shasheleva1, G. Novichkova1, A. Maschan1, M. Ilushina1 1 Hematopoietic stem cell transplantation, 2Molecular Biology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: Graft-versus-host disease (GvHD) and GVHDassociated morbidity and mortality are major obstacles to the success of transplantation from unrelated and haploidentical donors. Negative depletion of a/b( þ ) T cells and CD19 þ B lymphocytes is a new technology of graft manipulation with a potential to improve GvHD control and immune reconstitution. Materials (or patients) and methods: A total of 34 pediatric patients with acute lymphoblastic leukemia (T-ALL- 16, B-ALL18, 13 female, 19 male, median age 8,6 years) underwent allogeneic HSCT between May 2012 and december 2013. Thirteen - from haploidentical donor and 21 from matched unrelated donor. Preparative regimen included Fludarabine 150 mg/m2, Treosulfan 42 g/m2, Melphalan 140 mg/m2 and ATG (horse, ATGAM) at 25 mg/kg/day, d -8,-9 (11 pts) or d -5,-4 (18 pts), or (rabbit, Thymoglobuline) at 2,5mg/kg/day, d -5,-4 (5pts). PBSC were depleted of TCR-alpha/beta and CD19 þ B-cell on CliniMACS Plus, Miltenyi Biotec, according to the manufacturer’s recommendations. The median dose of infused CD34 þ cells was 9,1x 106/kg (range3,9-18), TCRa/b 17 x103/ kg (range 0,7-190). Seven pts recieved no GvHD prophylaxis after HCT, tacrolimus (tacro) and short methotrexate (Mtx) were used for 24 pts, tacro - 2 pts, Mtx - 1 pts. Patients were divided in 4 groups according to remission status: CR1 (11pts), CR2 (16 pts), CR42 (6pts.), active disease (AD) (1pt.) and according to CNS involvemen: CNS þ (10 pts), CNS - (24pts). Results: Primary engraftment was achieved in 33 of 34 pts, the median time to neutrophil and platelet recovery was 13 and 14 days, respectively. Early mortality was low with a 100-day pTRM - 2,9% (95% CI: 0,2-20), 2-year pTRM – 14,9% (95%CI: 836). The 2 early deaths included bacterial sepsis and viral infections, three late: bacterial sepsis in 2 pts. and rhinocerebral mucormycosis in 1pt, all with extensive chronic GVHD. Cumulative incidence of acute GvHD grade Z II was 29% (95% CI: 17-49), grade III–IV 9 % (95%CI: 3-26). No correlation between graft composition and aGVHD was noted. Cumulative incidence of relapse at 2 year is 48,6% (95%CI: 34-69). Two year pEFS is 36,5% (95%CI: 19-53), 2-year pOS - 39% (95%CI: 17-61). There was no significant difference in survival and relapse rate
according leukemia subtype and donor type. Patient’s CNS status had a trend to influence significantly the relapse rate: pRFS for CNS (-) 59% (95%CI: 37-80), for CNS ( þ ) 34% (95%CI: 13-65), P ¼ 0,054. Conclusion: We confirm that depletion of TCR-alpha/beta and CD19 lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in ALL patients. All major outcomes were equivalent between transplantation from unrelated and haploidentical donor. The anti-leukemic activity of this regimen was obviously not optimal. We suggest that TCRalpha/beta depletion in ALL should be further explored in combination with TBI-based conditioning regimen and/or novel targeted immunotherapy approaches. Disclosure of Interest: None declared. P303 Exon 23 DNA methyltransferase 3A gene mutations in patients with acute myeloid leukemia receiving hematopoietic stem cell transplantation after non myeloablative conditioning L. B. Bonifacio1,*, L. Schmalbrock1, M. Bill1, M. Jentzsch1, K. Schubert1, K. Wildenberger1, L. Kloss1, H. Weidner1, T. Gaber1, W. Po¨nisch1, V. Vucinic1, G.-N. Franke1, T. Lange1,2, M. Cross1, G. Behre1, D. Niederwieser1, S. Schwind1 1 Department of Hematology, Medical Oncology and Hemostaseology, University of Leipzig, Leipzig, 2Hematology, Asklepios Hospital , Weissenfels, Germany Introduction: Some studies revealed a negative impact of mutations (mut) in the DNA methyltransferase 3A (DNMT3A) gene on outcome in acute myeloid leukemia (AML) subgroups. However, the prognostic impact of DNMT3A mut in patients (pts) receiving hematopoietic stem cell transplantation after non myeloablative conditioning (NMA-HCT) is unknown. Here, we determined the prognostic significance of exon 23 DNMT3A mut in NMA-HCT treated AML pts. Materials (or patients) and methods: We analyzed the coding region of exon 23 of the DNMT3A gene by Sanger sequencing of genomic DNA from pre-treatment samples of 104 AML pts (median age 64.5 years (y), range 47-75y). 67 (64%) pts had de novo AML. A normal karyotype (NK) was found in 50 (48%) pts. All pts underwent NMA-HCT (Fludarabine 30mg/m2 from day -4 to -2 followed by 2 Gy total body irradiation at day 0). 17 (16%) pts had a matched related, 57 (55%) pts a matched unrelated & 30 (29%) a mismatched unrelated donor. All pts were also characterized for NPM1, CEBPA, IDH1 & IDH2 mut & the presence of FLT3-ITD. Pts were classified according to the European LeukemiaNet (ELN) cytogenetic risk groups: Favorable n ¼ 20 (19%), Intermediate-I n ¼ 32 (31%), Intermediate-II n ¼ 23 (22%) & Adverse n ¼ 28 (27%). Median follow-up was 2.8y. The prognostic
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impact on Overall Survival (OS) & Event-Free Survival (EFS) & associations with clinical parameters & molecular aberrations were evaluated. Results: In our pts set, 17 (16.3%) pts had an exon 23 DNMT3A mut at codon R882 (n ¼ 16) or P904 (n ¼ 1). The presence of exon 23 DNMT3A mut was significantly associated with older age (P ¼ .04) & de novo AML (P ¼ .11) by trend. Remarkably, 14 out of the 17 exon 23 DNMT3A mut pts had at least one mutation in the NPM1, CEBPA, IDH1 or IDH2 gene or a FLT3-ITD. Exon 23 DNMT3A mut were significantly associated with the presence of NPM1 mut (P ¼ .01), especially in pts with NK (Po.001). Exon 23 DNMT3A mut significantly associated with the occurrence of IDH1 mut (P ¼ .01) in the whole pts set. We did not find any differences in exon 23 DNMT3A mutation frequency in the four ELN genetic groups. Furthermore, there was no impact of exon 23 DNMT3A mut on OS (P ¼ .71; Figure A) or EFS (P ¼ .48; Figure B) in our pts set. Conclusion: In conclusion, there was no significant impact of exon 23 DNMT3A mut status on outcome of AML pts treated with NMA-HCT. This study suggests that NMA-HCT might abrogate the negative impact of DNMT3A mut in some AML subgroups. Disclosure of Interest: None declared.
Aplastic anaemia P304 Donor lymphocyte infusions (DLI) for mixed chimerism in patients with severe aplastic anemia A. Bacigalupo1,*, A. Ghiso1, A. M. Raiola1, F. Gualandi1, A. Dominietto1, R. Varaldo1, S. Bregante1, C. Di Grazia1, T. Lamparelli1, S. Luchetti1, S. Geroldi1, E. Tedone1, F. Galaverna1, M. T. van Lint1 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: Patients with acquired severe aplastic anemia (SAA) are often found to have mixed chimerism (MC ) both in unfractionated bone marrow cells (BM) and in CD3 þ selected peripheral blood cells (CD3 þ PB). MC can be transient, permanent or progressive (Lawler et al BJH 2009). Progressive MC predicts graft rejection. The role of donor lymphocyte infusions (DLI) in treating MC in SAA patients is unclear and poorly reported. Materials (or patients) and methods: In this study we analyze the outcome of 14 SAA patients receiving DLI for the treatment of mixed chimerism. Patients.The median age was 28 years (23-53). The donor was an HLA identical sibling (n ¼ 11) or an alternative donor (n ¼ 3). The median interval between transplant and the first DLI was 161 days (51-2263). Only 4 patients were given DLI beyond 1 year from BMT: all four had a sudden fall in platelet counts, together with MC. Five patients have severe thrombocytopenia; one patient had severe neutropenia, four patients were anemic. DLI were started at 10^6/kg CD3 þ cells for SIB, and 10^5/kg for alternative donors. DLI were always given together with cyclosporin A (CyA). All paients had mixed chimerism in both BM and PBCD3 þ cells: the median donor chimerism at the time of DLI was 89% for BM ( 21-99%) and 36% for PBCD3 þ (14- 92%). Results: Patients received a median of 4 DLI (range 1-10). MC chimerism was converted to complete donor chimerism (100%) in unfractionated BM cells in 9/14 patients (64%) ; in PBCD3 þ cells it became complete donor in 7/14 (50%). The median BM donor chimerism went from 89% (average 79%), to a median of 100% (average 95%). The median PBCD3 þ donor chimerism went from 36% (average 44) to 63% (average
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79%). Four patients who were cytopenic, recovered their counts. Acute GvHD grade I occurred in 3 patients (21%) and grade III in 1 patient (7%). Two patients died (14%) : one of GvHD and one of graft failure, despite full donor chimerism and repeated infusions of donor marrow cells. Twelve patients are alive and well at a median interval of 2155 days (215-6306) from transplant. The 10 year actuarial survival of this group of patients is 77%. Three patients are off CyA (21%), 9 patients are still on CyA (64%). At last follow up 11/14 patients have normal peripheral blood counts. Conclusion: DLI , administered together with CyA , because of mixed chimerism with or without cytopenia , can induce complete donor chimerism in over 50% of the patients. A proportion of these can be taken off immunosuppression. Several patients remain permanent MC especially in PBCD3 þ cells. The risk of lethal GvHD, in the current series 7%, should be taken in to account when considering DLI for the treatment of mixed chimerism. Disclosure of Interest: None declared. P305 Bone marrow transplantation (BMT) for acquired severe aplastic anemia A. Bacigalupo1,*, A. Ghiso1, A. M. Raiola1, F. Gualandi1, A. Dominietto1, R. Varaldo1, S. Bregante1, C. Di Grazia1, T. Lamparelli1, S. Luchetti1, S. Geroldi1, E. Tedone1, M. T. van Lint1 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: The outcome of transplants for SAA has improved over the past decades. This study was done to assess changes in BMT protocols, possibly relevant for patients with acquired severe aplastic anemia (SAA) in the periods 1991-2000, 2000-2009, 2010-2014 Materials (or patients) and methods: 103 SAA patients were grafted betweeen 1991 and 2014. Median age was 29 years (962). The donor was an identical sibling (63%), a mismatched relative (10%), a matched unrelated donor (UD) (25%), a cord blood unit (2%). The number of patients in the 3 periods was 40, 41, and 22. The proportion of alternative donor was 10%, 49%, 64%. The conditioning regimen was cyclophosphamide 200 (CY200), FLU CY low dose (FC-LD), FLU CY high dose and TBI 200 rads. GvHD prophylaxis was Cyclosporina and methotrextae for all patients. UD grafts also received antithymocyrte globulin 3m,75 mg/kg x2 (Thymoglobulin , Sanofi). The source of stem cells was bone marrow (n ¼ 99), peripheral blood (n ¼ 2) and cord blood (n ¼ 2). Results: More patients in the first period received CY200, more patients in the last period received a combination of fludarabine CY (FC) or FC with TBI200, due to a change in donor type, with over 60% of donors other than SIBS (Table 1). Table 1. 19901999
20002009
20102014
N¼ Median age yy range Alternative donors CY 200 GvHD II-IV
40 23 16-60 10%
41 29 9-53 49%
22 28 17-62 64%
66% 20%
22% 12%
11% 22%
Causes of Death Rejection Infections GvHD MOF Alive
2.5% 10% 10% 10% 60%
5% 15% 2% 2.5% 68%
9% 0% 0% 0% 91%
P
0.6 o0.0001 o0.0001
o0.0001 o0.03
Patients surviving in the 3 periods were as follows 60%, 68%, 91%. Causes of death stratified for transplant period, showed no reduction in death due to rejection, but a reduction of infections (from 10% to 0%), of GvHD (from 10% to 0%) and multiorgan failure (from 10% to 0%). The actuarial 4 year survival for the 3 groups is 60%, 67%, 90%. The infectious cause of death in the first period were Pseudomonas (n ¼ 2), Serratia (n ¼ 1) Aspergillus (n ¼ 1); in the second period the etiology was Aspergillus (n ¼ 1), Pseudomonas þ EBV (n ¼ 1), fusariosis (n ¼ 1), EBV alone (n ¼ 1), bacterial sepsis (n ¼ 2). Conclusion: Despite a significantly greater proportion of alternative donors in recent years, the outcome of transplants for aquired SAA has significantly improved: the reason for improvement is not entirely clear: there has been a reduction of infections, possibly due to better diagnosis - early treatment, including mabthera prophylaxis of EBV for unrelated transplants, but also to the widespread use of new antifungal agents. Disclosure of Interest: None declared. P306 Allogeneic hematopoietic stem cell transplantation in severe aplastic anemia – low incidence of graft versus host disease and excellent results with graft failure rescue F. Moita1,*, B. Velosa Ferreira1, M. J. Gutierrez1, G. Teixeira1, I. Sousa Ferreira1, N. Miranda1, M. Abecasis1 1 Bone Marrow Transplant Unit, Instituto Portugueˆs de Oncologia de Lisboa, Francisco Gentil, Lisbon, Portugal Introduction: Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains the only curative therapy for patients with aplastic anemia (AA). It is, however, only recommended as first line treatment in young patients with a matched sibling donor (MSD), while alloHSCT from alternative donors is only indicated after failure of immunosuppressive therapy (IST). Materials (or patients) and methods: A retrospective analysis of all patients with the diagnosis of severe acquired AA that underwent alloHSCT in our institution, between 1987 e 2014, was conducted. Results: Fifty alloHSCT were performed, in a total of 40 patients. The median age at transplantation was 15,8 years (range 3,3 - 55,8 years). Thirty-four cases received alloHSCT from a MSD and 16 cases from a matched unrelated donor (MUD) (13 with a 9/10 HLA match; 3 with a 10/10 HLA match). Bone marrow was the chosen stem cell source in 42 cases. T-cell depletion with ATG or alemtuzumab was used in association with the conditioning regimen in 70% of cases. The median time between diagnosis and alloHSCT was 7 months (3 for MSD and 11,5 months for MUD). Fifteen patients received IST prior to alloHSCT. Extensive (grade 3/4) acute graft versus host disease (GVHD) was documented in only one patient. Chronic GVHD occurred in 34,2% patients, with an incidence of 32,1% in MSD and 40% in MUD. Severe chronic GVHD was only documented in 2 patients (7%), both transplanted with a MSD. Graft failure was documented in 11 alloHSCT (8 patients), at a median time of 5 months after alloHSCT, with no significant difference between donor source (20,6% in MSD and 26,7% in MUD). The incidence of graft failure was superior in patients that did not receive T-cell depletion in the conditioning regimen (33,3% versus 17,1% for T-cell depleted alloHSCT). Seven of these 8 patients proceeded to a subsequent alloHSCT. All of the re-transplanted patients are alive, with a median follow-up of 11 years after the last alloHSCT. Four patients died, all due to infectious complications, at a median time of 4,5 months after alloHSCT. The median followup for living patients is 12 years (minimum 0,5; maximum 26,6 years). The median overall survival was not reached and the overall survival at 12 years is 88,4% (CI 95%: 78,2%4100%). Conclusion: In this small series, alloHSCT was associated with excellent long term outcomes, with no significant differences between MSD and MUD in terms of extensive GVHD and graft
failure. The low mortality and morbidity make it a very appealing procedure for this disease, particularly when compared with the outcome of IST. In conclusion, our data support the use of alloHSCT as first line treatment in young patients with severe AA, even in the absence of a MSD. Disclosure of Interest: None declared. P307 Outcome of allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome or leukemia evolving from aplastic anemia I. Golubovskaya1,*, E. Morozova1, Y. Rudnickaya1, T. Rudakova1, V. Ovechkina1, A. Rats1, A. Kulagin1, B. Afanasyev1 1 First Pavlov State Medical University of St. Petersburg, Raisa Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, St Petersburg, Russian Federation Introduction: Long-term survivors of acquired aplastic anemia (AA) have an increased risk of transforming into myelodysplastic syndrome or acute myeloid leukemia (MDS/AML) after immunosuppressive therapy. Allogeneic stem cell transplantation (HSCT) is optimal option to improve prognosis in patients with secondary MDS/AML evolving from aplastic anemia. The number of patients transplanted to date is limited and impact of post-transplant therapy has not been extensively studied. Overall survival (OS) in this extremely unfavorable cohort of patients is significantly lower than in patients with de novo MDS/AML. Reduced intensity conditioning regimen (RIC) and myeloablative conditioning regimen (MAC) result in similar OS in MDS/AML (Kro¨ger et al., ASH 2014). The aim of our study was to analyze the effectiveness of HSCT in secondary MDS/ AML after AA. Materials (or patients) and methods: We analyzed 14 patients with MDS/AML transformation after treatment of acquired aplastic anemia who underwent allo-HSCT between September 2007 and January 2014 from matched related (n ¼ 5), matched unrelated (n ¼ 7) or haploidentical donor (n ¼ 2). Primary diagnoses were severe AA (n ¼ 6) and non-severe AA (n ¼ 8). The median time between primary diagnoses and transformation to MDS/AML was 33 months (range 7-204). Monosomy 7 was detected in 8 (57%) patients. The median age at HSCT was 19 years (range 10-37) and the median time from diagnosis MDS/AML to HSCT was 6 months (range 1-18). Eight patients failed to respond to previous chemotherapy or hypomethylating therapy (refractory disease), 9 patients were heavily transfused and the median number of RBC and PLT transfusions was 40 and 47 units, respectively. The conditioning regimen was fludarabine-containing RIC in 12 (86%) and MAC in 2 (14%) cases. The median dose of Infused CD34 þ cell was 3,3#106/kg (range 1-9). Results: Sustained engraftment was achieved in 8 of 14 patients (57%). One patient died of acute GVHD Grade IV. Two patients with refractory MDS (RAEB-2, CMML) after MAC relapsed at day þ 60 and þ 350 and received donor lymphocyte infusion (DLI) in combination with 5-azacytidine as further therapy. Despite of second HSCT both patients died of disease progression. Six patients who failed to engraft received second HSCT from the same (n ¼ 2) or alternative donor (n ¼ 4). All of these patients had refractory disease and were heavily transfused (median number of RBC and PLT transfusions was 45 and 44 units vs 15 and 20 units in engraftment group, respectively). They died of infectious complications due to primary graft failure (n ¼ 5) or acute GVHD Grade IV (n ¼ 1). Five patients (36%) are alive with the median follow-up of 30 months (range 11-88). Four of them received 2 cycles of hypomethylating agents as a prophylaxis of relapse after HSCT. All survivors are in complete remission with signs of limited chronic GVHD. Conclusion: Primary graft failure and disease relapse remain a significant problems that have an impact on the outcome of HSCT in MDS/AML evolving from AA. Higher incidence of graft failure after HSCT may be associated with lack of immunoablation in fludarabine-containing RIC in heavily transfused patients.
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Further optimization of conditioning regimens can be a facility for improvement of HSCT results. Our limited experience suggests that post-transplant hypomethylating therapy may be a promising option for the better disease control. Disclosure of Interest: None declared. P308 Ex-vivo T-cell depleted peripheral blood stem cell transplantation from HLA-matched sibling donors for the treatment of severe aplastic anemia I. Cano-Ferri1,*, F. Moscardo1, J. Sanz1, M. A. Dasi1, P. Solves1, I. Lorenzo1, L. Senent1, P. Montesinos1, F. Lopez-Chulia1, A. Lancharro1, J. Martinez1, N. Carpio1, R. Rodriguez-Veiga1, M. A. Sanz1, G. Sanz1 1 HOSPITAL UNIVERSITARIO LA FE, Valencia, Spain Introduction: Bone marrow is the preferred stem cell source for allogeneic transplantation (allo-SCT) in acquired severe aplastic anemia (SAA) due to a lower risk of graft-versus-host disease (GVHD). However, it has not been compared with exvivo T-cell depleted peripheral blood (TCD-PB). Materials (or patients) and methods: Between July 1996 and March 2014, 22 consecutive patients with SAA underwent alloSCT with TCD-PB grafts. Graft manipulation consisted of a CD34 positive selection followed by a T-cell add-back to achieve 3x105/kg. All patients received Cyclophosphamide 200 mg/kg y anti-thymocite globulin (ATG) for conditioning regimen and cyclosporine A (CSA) combined with long-course prednisone for GVHD prophylaxis. Results: Median age was 35 years (range, 3-63) and 10 (45%) were male. Prior immune suppressive therapy with ATG and CSA was administered in 8 patients (36%). Median CD34 þ cells and CD3 þ cells were 5.1x106/kg and 3.6x105/kg, respectively. Female donor to male recipient combination was present in 4 cases (18%). Sixteen donor/recipient pairs (73%) tested positive for cytomegalovirus (CMV) serostatus. Two patients initiated allo-SCT with uncontrolled infection and died on days þ 1 and þ 2. All remaining patients engrafted at a median time of 12 days (range, 10-14). Cumulative incidence (CI) of neutrophil recovery was 91%. An additional patient died on day þ 21 due to cerebral hemorrhage. All the remaining patients had platelet recovery at a median time of 18 days (range, 8-53). CI of platelet recovery was 86%. Two patients developed grade 1 acute GVHD while no patients showed acute GVHD grade Z2. Two patients had limited and 1 patient had extensive chronic GVHD. Only 1 patient remains on immunosuppressive therapy. CMV reactivation was observed in 4 patients but none of them developed organ disease. After a median follow-up of 90 months (range, 8-184), no relapses were observed and all patients maintained stable blood counts. 8-year CI of non-relapse mortality was 14%. 8-year disease-free and overall survivals were 86%. Conclusion: Allo-SCT from HLA-matched sibling donors using ex-vivo T-cell depletion achieved excellent short and longterm outcomes with low GVHD and good quality of life in patients with SAA. This procedure should be compared with bone marrow allo-SCT. Disclosure of Interest: None declared. P309 Allogeneic Hematopoietic Stem Cell Transplantation in Adult Patients with Paroxysmal Nocturnal Hemoglobinuria: Single Centre Experience M. Merter1,*, U. Sahin1, E. Atilla1, S. Civriz Bozdag1, S. Kocak Toprak1, M. Kurt Yu¨ksel1, K. Dalva1, P. Topcuoglu1, O. Arslan1, M. Ozcan1, T. Demirer1, O. Ilhan1, H. Akan1, M. Beksac1, N. Konuk1, G. Gurman1 1 Hematology Department, Ankara University Medicine Faculty, Ankara, Turkey Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hemopoietic stem cell disorder which is
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characterized by complement-mediated hemolytic anemia, thrombophilia and bone marrow failure. Despite the introduction of eculizumab, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still the only curative treatment. Materials (or patients) and methods: In this retrospective study we assessed 9 PNH patients who received HSCT in our transplantation unit between 2005 and 2014. Median age of patients was 29 (range 21-40). Three of patients were male and 6 of patients were female. Results: Four patients had a history of treatment with eculizumab before transplantation. There were hemolytic component in 8 patients, venous thrombosis in 3 patients and bone marrow failure in 1 patient. Eight patients were transfusion dependent. All donors were HLA-identical (8 siblings and 1 unrelated). Median age of donors was 24(1746). Median time from diagnosis to transplantation was 11(2180) months. Seven patients received a reduced intensity conditioning (RIC) consisting of fludarabin, cyclophosphamide and ATG (Flu-Cy-ATG), one patient received a myeloablative conditioning consisting of cyclophosphamide and ATG (CyATG) and one patient received a myeloablative conditioning consisting of busulfan, cyclophosphamide and ATG(Bu-CyATG). We used peripheral blood stem cells in 3 patients and bone marrow in 6 patients as stem cell source. Two patients who had a history of thrombosis, died due to sepsis during the aplasia period of the transplantation. Transplant-related mortality for our patients was 22%. There was primary engraftment failure in one patient (11%). The transfusion need disappeared in this patient after the transplantation. As of December 1 2014, 7 patients are alive. Six patients have been complete hematologic recovery with no evidence of PNH following a median follow-up of 69 months (18-109 months). No thromboembolic event has been observed in early and long-term follow-up in these six patients. Besides, we did not see any TE event in the one patient with primary engraftment failure. Conclusion: Our findings confirm that hematopoietic stem cell transplantation is a definite curative treatment for PNH and Flu-Cy-ATG is a feasible regimen for transplantation. In patients who have a history of thromboembolism, other treatment strategies may be more appropriate rather than transplantation. Disclosure of Interest: None declared. P310 Outcome of allogeneic hematopoietic stem cell transplantation in pediatric patients with acquired severe aplastic anemia: a Turkish multicenter study U¨. Koc¸ak1,*, A. Ku¨pesiz2, S. Aksoylar3, V. Hazar4, M. Karaku¨kc¸u¨5, ¨ zbek8, T. ˙Ileri9, M. Elli10, S. Kansoy3, G. Karasu6, V. Uygun7, N. Y. O ¨ ztu¨rk11, E. U¨nal5, Z. Kaya1, S. Go¨zmen3, M. Ertem9, S. Çakı G. O ¨ zyu¨rek13, A. Akc¸ay 11, T. Fıs¸gın13, E. U¨nal9, Kılıc¸12, B. Tunc¸8, E. O 14 O. Gu¨rsel , D. Atay15, M. A. Yes¸ilipek6 on behalf of Turkish Pediatric Bone Marrow Transplantation Study Group (TPBMT-SG) 1 Pediatric Hematology and BMT Unit, Gazi University School of Medicine, Ankara, 2Pediatric Hematology/Oncology and BMT Unit, Akdeniz University School of Medicine, Antalya, 3Pediatric Oncology and BMT Unit, Ege University School of Medicine, ˙Izmir, 4 Pediatric Hematology/Oncology and BMT Unit, Medipol University School of Medicine, ˙Istanbul, 5Pediatric Hematology/ Oncology and BMT Unit, Erciyes University School of Medicine, Kayseri, 6Pediatric BMT Unit, Bahcesehir University School of Medicine Medical Park Goztepe Hospital, ˙Istanbul, 7Pediatric Hematology/Oncology and BMT Unit, Bahcesehir University School of Medicine Medical Park Antalya Hospital, Antalya, 8 Pediatric Hematology/Oncology and BMT Unit, Ankara Child Health and Diseases Hematology and Oncology Training and Research Hospital , 9Pediatric Hematology and BMT Unit, Ankara University School of Medicine, Ankara, 10Pediatric Oncology and BMT Unit, Ondokuz Mayıs University School of Medicine, Samsun, 11 Pediatric BMT Unit, Acıbadem University Atakent Hospital, 12 Pediatric BMT Unit, Go¨ztepe Medical Park Hospital, ˙Istanbul, 13 Pediatric BMT Unit, Bahcesehir University School of Medicine
Medical Park Samsun Hospital, Samsun, 14Pediatric Hematology and BMT Unit, Gulhane Military Medical Academy, Ankara, 15 Pediatric BMT Unit, Medical Park Bahcelievler Hospital, ˙Istanbul, Turkey Introduction: Acquired severe aplastic anemia (SAA) during childhood is a rare bone marrow failure which is classically treated with allogeneic hematopoietic stem cell transplantation (HSCT) from a sibling donor if available. Although the second option currently is immunosuppressive therapy (IST), with a merely alternative donor (AD) search when IST fails, both the high rate of failure and long term complications are undesirable particularly in the pediatric population that have a long life expectancy. The outcome of HSCTs performed in a country between Asia and Europe has not been previously investigated. Materials (or patients) and methods: The outcome of 121 children with acquired SAA that were transplanted in 12 pediatric centers in Turkey between 1999-2014 was retrospectively analyzed. Results: There were 121 patients [48 (39.7%) male, 73 (60.3%) female] with a median age of 11 years (1-20 years). The donor was HLA matched related (MRD) in 86 (71%) (71 matched sibling, 14 related family donor), alternative (AD) in 35 (29%) (23 matched unrelated, 5 mismatched unrelated, 7 mismatched family donors) patients. Graft source was bone marrow in 62 (51.2%), peripheral stem cells in 51 (42.1%), cord blood in 4 (3.3%) and both bone marrow and peripheral stem cells in 4 (3.3%) patients. The median time to neutrophil recovery was 16 days (8-34 days) in MRD, 15 days (8-45 days) in AD transplants. Graft failure was observed in 14 patients (11.6%) (6 MRD, 8 AD). No secondary malignancy was observed in any of the patients. The median time to transplant from the diagnosis was 5.1 months (0-98.6 months) in MRD, 21.2 months (8-119 months) in AD transplants. Graft versus host disease (GVHD) prophylaxis was with cyclosporine alone or combined in all patients. AD transplants were complicated with more grade II/IV acute GVHD (aGVHD) (25% vs 4.7%) than MRD transplants. There was no grade III/IV aGVHD in MRD transplants and only 1.2% limited and 1.2% extensive chronic GVHD (cGVHD) whereas there were 11.1% III/IV aGVHD, 2.8 % limited and 5.6% extensive cGVHD in AD transplants. Five year event free survival was 84.7% in MRD, 61.1% in AD transplants (Figure 1). Five year overall survival in patients transplanted from a MRD was 92.9 % with a median follow up 4.1 years (0.3-15 years) whereas it was 69.4 % in AD with a median follow up 3.7 years (1.3-14 years) (Figure 2). Graft failure and aGVHD was associated with poor survival in all transplants (Po0.001). Conclusion: This is the first report of pediatric patients with acquired SAA from Turkey presenting that outcome of upfront MRD transplants are comparable to that of the developed
countries. The relatively lower outcome of patients that were transplanted from ADs on the other hand might be due to the occurrence of more aGVHD or the longer time elapsed from diagnosis to transplant. We believe that improving supportive care and earlier availability of unrelated donors would lead to better results in AD transplants as well. Disclosure of Interest: None declared. P311 Hematopoietic Stem Cell Transplantation for Aplastic Anaemia : Outcomes from Argentine Group of Haematopoietic Stem cell transplant (GATMO) V. Milovic1,*, A. Basquiera2, J. Garcı´a2, S. Formisano2, R. Ferna´ndez2, G. Jaimovich2, G. Kuminsky2, J. Milone2, M. Rivas2, S. Yantorno2, J. Jarchum2, M. Rizzi2, J. Martı´nez Rolo´n2, C. Foncuberta2, R. Burgos2, G. Balladares2, S. Saba2, G. Drelichman2, J. Real2, L. Feldman2, J. E. Roberti1 1 Hospital Aleman, 2GATMO, Buenos Aires, Argentina Introduction: Acquired aplastic anaemia (AAA) is a serious and rare disease characterised by peripheral blood pancytopenia, bone marrow aplasia or hypoplasia. For patients with severe and very severe AAA younger than 40 years of age, hematopoietic stem cell transplantation (HSCT) is the treatment of choice. The use of ATG/Cyclosphosphamide (Cy) conditioning regimen has been associated with a 5-year survival rate of 60-90%. Since 2002, rATG is the only available ATG in the Argentine market. We describe the outcomes of HSC transplantation (tx) in pts with AAA, who received rATG/ Cyclophosphamide (Cy) as conditioning regimen Materials (or patients) and methods: Retrospective cohort study. We analysed data from 84 transplants performed in 82 pts between 1998-2014 in 11 Argentine centres. Patients: Mean age at transplant was 19.264±10.09 years; 46(55%) pts were o20 years old, and 36(43%) were 420 years old. Median interval from diagnosis to transplant was 141(21-4429) days. Nineteen patients (25%) had received a previous treatment, 3(3.4%) were second transplants; 74(88%) patients received HSC from related donors; the source of HSC was: bone marrow in 56(68%), and peripheral blood in 21(25.6%) patients. Mean infused cell dose was 4.3±3x106/kg. Results: Median time to neutrophil count41000/mm3 was 20(9-42) days, and to platelet count 420000/mm3 was 23(11108) days. Four (4.8%) patients had early rejection and 4(4.8%) had late graft rejection, with a median time to rejection of 371(137-745) days in the late rejection group. Seven (8.3%) patients developed cGvH, with no significant difference according to SC source. With a median follow-up of 823(14429) days, 23(28%) patients died; the main cause of death was infection in 15(71%) cases. The overall survival at 100, 365 and 4429 days was 89%(CI80-94), 79%(CI68-86) and 67%(CI54-
[P310]
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77), respectively, without statistically significant difference between age groups. Conclusion: Most patients with AAA were able to receive SCT as the first line therapy, with a low early graft rejection rate. Overall survival and cGvH rate are similar to those reported internationally. Disclosure of Interest: None declared. P312 Immunosuppressive treatment combined with cord blood infusion for patients with severe aplastic anemia Y. Cai1,*, C. Wang2, J. Yang2, J. Jiang2 1 hematolgy, 2hematology, Shanghai General Hospital, Shanghai, China Introduction: Treatment of acquired severe aplastic anemia (SAA) consists of immunosuppressive therapy and allogeneic stem cell transplantation. Patients usually receive immunosuppressive therapy (IST) as first-line treatment. The median time to transfusion independence was 96 days. Patients experienced prolonged neutropenia, a high rate of infection. The response of IST was 70-80%. Cord blood transplantation (CBT) has become a promising therapeutic option for patients with nonmalignant diseases.Unrelated donor CBT has not yet been recommended for SAA patients. Doctor SUN et al reported a single-center experience in treating 18 patients with SAA who received unrelated CBT. Their conditioning regimen composed of cyclophosphamide, rabbit antithymocyte globulin and fludarabine. All patients were given a combination of cyclosporine (CsA) and mycophenolate mofetil for GVHD prophylaxis. Cord Blood failed to engraft in all cases, but may facilitate autologous recovery and improve survival with low risk of transplantrelated mortality. Materials (or patients) and methods: We report a singlecenter experience in treating 12 patients with SAA who received immunosuppressive treatment combined with unrelated cord blood infusion and Cord Blood may facilitate neutrophil recovery. Between May 2010 and January 2014, 12 patients (3 male and 9 female) were enrolled in a study. The median age at diagnosis was 18 (5-55) years. Seven patients were newly diagnosed and 5 patients received cyclosporine±testosterone /undecanoate before. All patients were treated with fludarabine at 30mg/m2/day for 5 days without CTX , 6 cases received Thymoglobuline at 2-3 mg/kg/day for 5 days.And another 6 cases received ATGFresenius at 5-6 mg/kg/day for 5 days.All patients were treated with cyclosporine. Cord unit selection was based on the best HLA compatibility, with at least four of six loci matching. The median nucleated cell was 2.8 (1.9-14.2) 107 cells/kg. And CD34 positive cell was 0.9 (0.4-3.6) 105 cells/kg. Unrelated cord blood was infused on d0. Results: The median time to ANCZ0.5 109 /L was 20 (11 to 66)days .It may be favorable to control infection. And the median time to plateletsZ20 109/L was 96 (12 days-7 months)days. Only 1 patient acquired unrelated cord stem cell engraftment. The remaining 11 patients experienced graft rejection and autologous recovery. One patient achieved 95-98.5% donor cells continuously. One case showed 95-98.5% donor cells in 1 month and donor cells declined to 0% in 3M. Four cases showed11.6-68.9% donor cells on day 14 and declined to 0% on day 28.The remaining 6 cases showed no donor cells at all test points. Eleven patients have survived for 11 – 48 months (median follow-up was 14 months). CR and PR was 7 and 5 cases, respectively. Three cases relapsed in 4 months, 6 months and 24 months, respectively. Among them, 2 cases relapsed after termination of immunosuppressant. The median follow-up was 14 months. One patient relapsed at 2 years for termination of immunosuppressant and died of
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infection in 3 years. 1-year and 2-year survival rates were 100%. 3-year survival rate was 91.7%. Conclusion: Compared to traditional immunosuppressive treatment, IST plus unrelated CBT achieved faster hematopoiesis recovery and reduced death caused by infection.Compared to stem cell transplantation, IST plus unrelated CBT might facilitate autologous recovery, minimize transplantrelated mortality and have no GVHD. Disclosure of Interest: None declared. P313 Determination of effective blood concentrations of cyclosporine in the treatment of severe aplastic anemia in children M. Philippe1, E. He´nin2, Y. Bertrand3,*, D. Plantaz4, S. Goutelle5, N. Bleyzac1 1 Pharmacy Department, Pediatric Hematology and Oncology Unit, IHOP, 2Departement of Clinical Pharmacology & Clinical Trials, Hospices Civils de Lyon, 3Transplantation Unit, Pediatric Hematology and Oncology Unit, IHOP, Lyon, France, Lyon, 4 Pediatric Hematology and Oncology Unit, CHU Grenoble, Grenoble, 5Pharmacy Department, Groupement hospitalier de ge´riatrie, Lyon, France Introduction: Optimal immunosuppressive therapy in acquired severe aplastic anemia (SAA) remains to be refined, especially cyclosporine (CsA) use. Current recommendations state that CsA trough blood concentrations (TBC) should be maintained between 200 and 400 ng/mL despite the lack of supporting data. This study aimed at quantifying relationships between exposure to CsA and hematological response, and at determining the optimal CsA TBC target. Materials (or patients) and methods: Twenty-three pediatric patients with SAA treated with CsA were retrospectively analyzed. Nonlinear mixed effects modelling approaches were used to develop a Pharmacokinetic-Pharmacodynamic model. The pharmacokinetic model described the relationships between CsA doses and TBC. The pharmacodynamic model allowed to estimate boundaries for optimal CsA effects, neutrophils being used as biomarker of response. A time-toevent model linked effective concentration to time-totherapeutic success. Simulations were subsequently performed using the model to evaluate the chance of therapeutic success according to various CsA TBC target, and thus estimate the optimal CsA exposure. Time-to-therapeutic success was externally predicted in two additional patients not included in the model building. Results: Twenty-three pediatric patients with SAA were included. The median (min-max) age and weight were: 8.5 (8-15) years, and 34 (9.8-79.3) kg, respectively. Fifteen out of 23 patients responded to the IST (65.2%) with a median time of response of 69 days (min 19- max 182). CsA TBC were adequately described by a two-compartment model with firstorder absorption, a lag-time and a linear elimination. The optimal target range of CsA TBC was estimated between 87 and 120 ng/mL (relative standard erroro5%). Model-based simulations showed that CsA TBC should be maintained around 100 ng/mL to expect a maximal response rate; higher TBC would be associated with lower response rate. Using the model, time-to-therapeutic success could adequately be predicted for two new patients. Conclusion: This original modelling approach was successful in describing the relationship between CsA TBC and the neutrophil response in patients with SAA. While further research in a larger population is necessary to confirm our findings, this work suggest that a CsA TBC target of 100 ng/mL, much lower than that currently recommended, would be associated with a better response rate in children with SAA. Disclosure of Interest: None declared.
Lymphoma
Table 1. Treatment-relative mortality (TRM) and 2-years Overall Survival (OS) of children with pediatric lymphoma who underwent allogeneic stem cell transplantation.
P314 Hematopoietic stem cell transplantation for refractory or recurrent Non-Hodgkin Lymphoma in children and adolescent. A retrospective study from the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) A. Spiegel1,*, N. Rauss2, Y. Bertrand2, J.-H. Dalle3, G. Michel4, F. Rialland5, P. Schneider6, C. Jubert7, J. Kanold8, L. Brugie`res9, P. Lutz1, C. Paillard1 1 Service d’onco-he´matologie pe´diatrique, Centre Hospitalier Universitaire de Strasbourg, STRASBOURG, 2IHOP, Centre Hospitalier Universitaire de Lyon, LYON, 3Service d’he´matologie pe´diatrique et de greffe de moelle osseuse Robert Debre´, APHP, PARIS, 4Service d’he´matologie pe´diatrique et de greffe de moelle osseuse, Centre Hospitalier de Marseille, MARSEILLE, 5 Centre Hospitalier Universitaire de Nantes, NANTES, 6Centre Hospitalier Universitaire de Rouen, ROUEN, 7Centre Hospitalier Universitaire de Bordeaux, BORDEAUX, 8Centre Hospitalier Universitaire de Clermont-Ferrand, CLERMONT-FERRAND, 9Institut Gustave Roussy, AP-HP, PARIS, France Introduction: Despite excellent outcome with current riskadapted combined modality first-line treatment for children with non Hodgkin lymphoma (NHL), treatment of patients failing first-line therapy or relapsing remains a major challenge. Allogeneic HSCT have become part of salvage therapy strategies for these patients. The potential benefit of a graft-versus-lymphoma effect has been counterbalanced by high transplant related mortality in this population. The purpose of the current study is to describe outcomes in a large cohort of pediatric patients with lymphoma who received an allo-HSCT in recent years. Materials (or patients) and methods: Clinical data were prospectively collected using ProMISe (Project Manager Internet Server), an internet-based data registry system shared by all centers of the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC). Between 2000 and 2014, this French retrospective multicenter study involved 98 consecutive patients under age of 18 years who received a first allo-HSCT for NHL. Their clinical features as well as their therapies and evolution are described and compared to results of literature. Results: Sex ratio is about 1:2. Median age of diagnosis is 8,2 years. There are three major subgroups of lymphomas: Anaplastic large-cell lymphomas (ALCL) with T or null phenotype (35 cases), Lymphoblastic lymphomas (LBL) (18 cases), Mature B-cell lymphomas including Burkitt lymphoma (BL) (11 cases) and diffuse large B cell lymphoma (DLBCL)(15 cases). Data are incomplete for 19 children. Median age of HSCT is 9.8 years old and median time of allogeneic stem cell transplantation to diagnosis is 417 days. Complete remission (CR) before HSCT is reached in 55/79 cases mainly CR2 (37/55 cases). Myeloablative conditioning regimen is described in 71/ 79 cases. Total Body Irradiation (TBI) with myeolablative dose is used in 71% of patients. The use of bone marrow is most frequent (41/79 cases). Sibling donor is found in 1/3 cases. Engraftment is obtained in 87% of patients and median time of neutrophils recovery is 21 days. Acute graft versus host disease is observed in 45/79 cases mainly stage I (17/45 cases) and stage II (15/45 cases). Chronic graft versus host disease is noticed in 12/79 cases. 31/79 patients are death, mainly due to disease progression or relapse (16/31cases). For all patients, toxicity related mortality is 17.7% (14/79 cases). 2-year overall survival is 60.8%. Conclusion: The present data and previous studies clearly demonstrate that allogeneic HSCT may be curative for selected patients with NHL. Several questions remain to be determined, among them the optimal time point for transplantation and choice of the best conditioning regimen.
TRM All patients ALCL LBL BL&DLBCL
17.7% 20% 27.8% 7.7%
(14/79) (7/35) (5/18) (2/26)
2-years OS 60.8% 71.4% 33.3% 69.2%
(48/79) (25/35) (6/18) (18/26)
Some patients may be selected for RIC regimens that are associated with lower TRM. Disclosure of Interest: None declared. P315 Autologous stem cell transplantation for children and adolescents with relapsed and refractory Hodgkin’s lymphoma A. Hedayati Asl1,* on behalf of Mehrvar A, Zangooei R, Faranoush M, Dinarooni P, Olad E, Jenabzadeh M, Athari G, Fallah V, Haghighi M 1 Pediatric Hematology-Oncology, MAHAK, Tehran, Iran, Islamic Republic Of Introduction: Despite the generally excellent prognosis of children and adolescents with Hodgkin’s lymphoma, approximately 20% of patients relapse. High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a recognized treatment option for patients with relapsed Hodgkin’s lymphoma. This study evaluates the results and outcome of non-cryopreserved autologous stem cell transplant of 23patients with Hodgkin lymphoma. Materials (or patients) and methods: Twenty-three patients age 5 to 25 years (median 14.5 years, M/F ¼ 17/6), with relapsed, refractory or poor prognosis HD, underwent ASCT in our hospital (from 2012-2014). Status at transplant was: second complete remission (CR2): n ¼ 13; further CR (CR 42): n ¼ 8, partial remission (PR): n ¼ 2. Twenty-three patients received chemotherapy- based conditioning regimens: cyclophosphamide, carmustine and etoposide (CBV): 6, CCNU, etoposide, cytarabine and melphalan (CEAM): 17, Peripheral blood (PB) was the source of progenitor cells in 23 patients. All patients engrafted. Results: The median mononuclear cell dose was 5.4 108/ kg. The median time to reach absolute neutrophil count 4 0.5 109/L was 11 days, and the median time to platelet count 4 20 109 was 13 days. Grade 2 and grade 3 mucositis was seen in 60% our patients. Transplant-related mortality at 100 days not occurred. Only two patient relapsed 15 and 18 months after transplant (mean 16.5 m.). With a median followup of 19.5 months (5-29 months) after transplant the eventfree survival were 85%. All patients remain alive. No significant different between CBV group vs. CEAM group in engraftment day. Conclusion: High-dose therapy with stem cell rescue can lead to durable remissions in children and adolescents with advanced HD. Future investigations should focus on strategies designed to decrease relapse after auto-transplantation, particularly in patients at high risk for relapse. Our analysis suggests that these regimens (CEAM, CBV) are feasible in pediatric patients with acceptable engraftment and toxicity. Disclosure of Interest: None declared.
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P316 Application of LYSA risk factors in patients with relapse/ refractory Hodgkin Lymphoma undergoing Autologous Stem Cells Transplantation: a retrospective single center study from Argentina A. Zenatti1, A. L. Basquiera1,2,*, P. Garcı´a1, G. Sturich1, L. Guanchiale1, J. C. Damonte1, G. Caeiro1, P. Abichain1, C. Hollman1, A. Berretta1, J. J. Garcı´a1,2 1 Hematology and Oncology, Hospital Privado Centro Me´dico de Co´rdoba, 2Instituto Universitario de Ciencias Biome´dicas de Co´rdoba (IUCBC), Co´rdoba, Argentina Introduction: Patients with relapse/refractory (R/R) Hodgkin Lymphoma can achieve long-term remission with high dose therapy followed by autologous hematopoietic stem cell transplantation (ASCT). However, a number of patients will relapse after ASCT and for this group of patients a further treatment after ASCT may be necessary. Thus, identification of these patients before ASCT is the paramount importance. An expert panel from the Lymphoma Study Association (LYSA) defined a risk stratification for patients with R/R Hodking Lymhoma. This classification took into account three prognostic factors considering the setting of the relapse previous ASCT: primary refractory disease, early relapse (less than 12 months after end of treatment) and stage III/IV at relapse. Patients were separated into three risk groups: standard, intermediate and high risk of relapse after ASCT. Materials (or patients) and methods: Our objective was to apply the LYSA classification in all patients with R/R Hodgkin lymphoma underwent ASCT in the Hospital Privado de Co´rdoba, Argentina. We reviewed clinical data of 97 patients who received an ASCT between 1991 and 2013. 91 out of 97 patients were included in the analysis (64 men; median age at ASCT 25 years, range 5-62). Results: Distribution of risk factors was as follow: 31 patients were refractory, 30 had early relapse and 49 patients relapsed as advance stage. According to LYSA recommendations 15/91 patients were standard risk, 45/91 were intermediate risk and 31/91 were high risk. Conditioning regimen was CVB (n ¼ 39; 43%) and BEAM (n ¼ 52; 57%). Median follow-up of the surviving patients was 22.4 months after transplantation. Cumulative incidence of non-relapse mortality was 4% at 6 months. Relapse occurred in 38 patients after ASCT in a median time of 5.1 months. We found a statistically difference between the groups in terms of five-year overall survival (OS) (93% vs. 67% vs. 55%; P ¼ 0.035) and cumulative incidence of relapse (13% vs. 47% vs. 53%; P ¼ 0.022) for standard, intermediate and high risk respectively. Conclusion: Our series contribute to demonstrate that risk factors at relapse can identify patients with adverse outcome after ASCT. For these patients additional treatment strategies may be considered. Disclosure of Interest: None declared. P317 The role of pre-transplant positron emission tomography (PET), in patients with Hodgkin disease undergoing a haplo transplants with post-transplant cyclophosphamide C. Marani1, A. M. Raiola1, A. Dominietto1, R. Varaldo1, F. Gualandi1, C. Di Grazia1, T. Lamparelli1, S. Bregante1, M. T. van Lint1, F. Galaverna1, A. Ghiso1, A. Bacigalupo1,* 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: Patients with Hodgkings disease (HD) relapsing after an autologous stem cell transplants, or after second/third line therapy, represent a group of patients with a very dismal outcomes. The Baltimore/Seattle groups have reported encouraging results in these high risk HD patients, using a non myeloablative (NMA) conditioning regimen followed by unmanipulated haploidentical bone marrow transplantation (haplo-BMT) and high dose post-transplant cyclophosphamide (PT-CY) (Lutznik 2008). Recently, we have confirmed these data
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(Raiola 2014). However, relapse after allogenic BMT continued to be major problem. Materials (or patients) and methods: We hypothesized that, in HD patients treated with the Baltimore protocol, positron emission tomography (PET) status before a haplo-BMT would be predictive of relapse. Patients with a negative PET scan or with a reduction in the number of hypermetabolic areas after salvage chemotherapy were defined as ‘‘chemosensitive’’; patients with an unmodified or enhanced PET signal after salvage chemotherapy were considered ‘‘chemorefractory’’. Patients. 32 patients with relapsed HD received a haplo-BMT between September 11th 2009 and March 4th 2014. The donor was a haploidentical relative in all cases. All patients had relapsed after an autologous graft and underwent a PET scan before transplant. The median age was 31years(18-59). For patients classified as ‘‘chemosensitive’’ (n ¼ 21) the PET scan was negative in 12 and was borderline positive in 9. For patients classified as ‘‘chemorefractory’’ (n ¼ 11) the PET scan was strongly positive. Results: All patients engrafted with 1 exception, a patient who showed autologous recovery. The median day for 500 neutrophils was 18 days (14-26) for chemosensitive and 18 days (13-25) for chemorefractory patients. Acute GvHD grade IIIV was diagnosed in 24% and 18% of chemosensitive/ chemorefractory patients (P ¼ 0.1). Moderate severe chronic GvHD was diagnosed respectively in 19% and 18% (P ¼ 0.9). Transplant related mortality (TRM) was 0% for chemosensitive and 9% for chemorefractory patients. With a median follow-up of 2 years for surviving patients , 16 have relapsed (50%): the relapse rate was 29% in chemosensitive vs 91% in chemorefractory patients (P ¼ 0.0008). The 2-year progression free survival is 63% for chemosensitive patients and 0% for chemorefractory patients (P ¼ 0.0001). The overall survival is respectively 86% and 71% (P ¼ 0.1) Conclusion: This study proves that a pre-transplant ‘‘chemosentive’’ PET-based status is essential to achieve long term remissions in patients with HD undergoing a haplo BMT with PT-CY. Disclosure of Interest: None declared. P318 Efficacy of upfront autologous stem cell transplantation and safety of high dose ranimustine regimen (MEAM) in the treatment of diffuse large B cell lymphoma A. Nakaya1,*, S. Fujita1, A. Satake1, Y. Azuma1, Y. Fujita1, M. Hotta1, H. Yoshimura1, K. Ishii1, T. Ito1, S. Nomura1 1 First Department of Internal Medicine, Division of Hematology and Oncology, Kansai Medical University, Osaka, Japan Introduction: In patients with diffuse large cell lymphoma (DLBCL) classified as high-intermediate risk or high risk on the international prognosis index (IPI), the efficacy of high-dose chemotherapy (HDT) together with autologous stem cell transplantation (ASCT) during the first remission remains controversial in the rituximab era. In our institute, patients with DLBCL in both the high-intermediate and high IPI groups initially underwent R-CHOP followed by HDT/ASCT in an upfront setting. Various high-dose conditioning regimens have been developed for treatment prior to ASCT. However to date an optimal conditioning regimen with the lowest toxicity and highest therapeutic efficacy has not been established. Conditioning HDC for ASCT is widely performed using the BEAM (BCNU[carmustine]/VP-16/ARA-C[cytarabine]/L-PAM[melphalan]) regimen. However, one of the components of the BCNU regimen has not been approved by the Japanese National Health Insurance scheme and has been replaced with the alkylating agent ranimustine, MCNU; this revised regimen has been termed the MEAM regimen (MCNU/VP-16/ARA-C/L-PAM). The MEAM regimen is one of the most frequently used conditioning regimens for lymphoid malignancies in Japan. However, there is very limited information regarding its safety profile.
Materials (or patients) and methods: To investigate if upfront HDT/ASCT could improve the prognosis of high risk DLBCL patients, we retrospectively analyzed 46 patients who had all been treated with HDT/ASCT in both upfront and salvage settings. Furthermore, we examine the safety and feasibility of the MEAM regimen. Results: Forty six DLBCL patients underwent ASCT following treatment with the MEAM regimen between 2006 and 2013 at Kansai Medical University Hospital. Twenty four patients received upfront HDT/ASCT and twenty two patients received salvage therapy. Median patient age was 58.4 years in the upfront setting and 54.1 years in the salvage group. In the upfront setting, with the exception of one patient, 23 patients had a high-intermediate or high risk IPI. All patients in the upfront setting were diagnosed as having DLBCL, whereas only three patients in the salvage group were diagnosed as having DLBCL that had transformed from follicular lymphoma. Three-year overall survival (OS) and progressionfree survival (PFS) in the upfront setting were 95.0 %(P ¼ 0.00249) and 83.3 %(P ¼ 0.11), respectively. In the salvage group, 3-year-OS and PFS were 53.6% and 65.6%, respectively. The major toxicities were anorexia, nausea, diarrhea, and febrile neutropenia (FN). In the salvage group, two patients died from regimen-related toxicities. One patient became fatal because of FN and the other died of interstitial pneumonia. Therapy related mortality was 4.3%. Vascular sarcoma and squamous cell carcinoma in skin were observed as secondary malignancy. Conclusion: Upfront HDT/ASCT in patients with high-risk DLBCL is feasible and can improve therapeutic outcome; and the MEAM regimen could be a tolerable, effective and curative conditioning regimen regarding ASCT. Disclosure of Interest: None declared. P319 Outcome of Hodgkin’s Lymphoma Patients Relapsing after Allogeneic Hematopoietic Stem Cell Transplantation B. Sarina1,*, A. Vai1, S. Garcia2, A. Granata2, R. Crocchiolo1, S. Bramanti1, D. Coso2, R. Bouabdallah2, F. Broussais2, L. Giordano1, L. Morabito1, A. Santoro1, D. Blaise2, L. Castagna1 1 Humanitas Cancer Center, Rozzano (Mi), Italy, 2Institut Paoli Calmettes, Marseille, France Introduction: Relapse in Hodgkin’s lymphoma (HL) patients (pts) who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still high and no standardized treatment is currently available. Materials (or patients) and methods: We retrospectively analyzed 99 consecutive HL pts who underwent allo-HSCT at two European transplant centers between 1999 and 2013. Treatment choice at relapse after allo-HSCT was made by treating physician, mainly based on previous therapies, relapse characteristics and clinical condition. The aim of this study was to describe the outcome of HL pts relapsing after allo-HSCT. Results: The median pts’ age was 30 (range: 16-62). 77 (78%) pts were in complete (CR) or partial response status before allo-HSCT. More than 90% of the pts received a previous autologous HSCT. 47 (47%) pts received allo-HSCT from an HLA-identical sibling donor, 14 (14%) pts from an unrelated donor, 36 (36%) from a haploidentical donor and 2 (2%) from cord blood units. 35 out of 99 (35%) pts relapsed or progressed after allo-HSCT. The characteristics and therapies of the relapsed pts are shown in Table 1. The median time to relapse or progression after allo-HSCT was 4 months (0-18). 4 (11%) pts were candidate to supportive therapy, because of poor performance status. 6 (17%) pts underwent haploidentical transplantation following relapse and their data were censored at the time of transplant. After a median follow-up (FU) of 29 (6-101) months from relapse, overall survival (OS) was 34% and progression-free survival (PFS) was 26%. At the last follow-up among the 15 pts who received DLI (DLI group), 7 pts (47%) were alive and 6 pts in CR (40%), meanwhile among the 15 pts who did not receive DLI (no-DLI group), 5
Table 1. Characteristics and therapies of patients relapsing/ progressing after allo-HSCT n ¼ 35 Patients treated Median time to relapse (months) Acute graft-versus-host disease Before relapse After DLI
30 (86%) 4 (0-18) 14 (40%) 11 2
Number of therapy lines after relapse 1 2 42
3 (9%) 13 (37%) 14 (40%)
Treatment at relapse DLI DLI þ RT DLI þ CT DLI þ Brentuximab Brentuximab ± CT CT RT
15 (43%) 2 8 5 2 (6%) 10 (29%) 3 (9%)
(33%) pts were alive and 2 (13%) pts in CR (P ¼ 0.11 and P ¼ 0.03, respectively). Conclusion: The prognosis in relapsed HL pts is still poor. However, DLI group seem to compare favourably with no-DLI group. On the basis of these results, new therapeutical approaches, including prophylactic or pre-emptive strategies, are warranted. Disclosure of Interest: None declared. P320 Comparison of HLA Identical and Haploidentical Hematopoietic Stem Cell Transplantation in Non Hodgkin Lymphoma patients: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, I. Dora1, B. Kosan1, E. Er1, N. Baskan1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, Kocaeli, Turkey Introduction: Allogeneic hematopoietic stem cell transplantation (AHSCT) is an established therapeutic modality in NonHodgkin lymphoma (NHL) patients relapsing after autologous stem cell transplantation. In the present study, we compare the results of Non-Hodgkin lymphoma patients who had undergone haploidentical or HLA identical allogeneic hematopoietic stem cell transplantation in our center. Materials (or patients) and methods: We analyzed the results of 14 NHL patients who had undergone haploidentical hematopoietic stem cell transplantation and 17 NHL patients who had undergone HLA identical hematopoietic stem cell transplantation between July 2010 to December 2013. 50% response to previous chemotherapy were considered as the eligibility criteria for transplantation. While,71,4% of the patients had active disease prior to transplantation in haploidentical AHSCT group, 70,6% of the patients in the HLA identical AHSCT group had active disease. No T cell depletion were performed. Cyclophosphamide, Cyclosporine and Mycophenolate mofetil were used for GVHD prophylaxis. Posacanazole and acyclovir were started at the day of transplantation and piperacillin-tazobactam were started when neutropenia ensues prophylactically. Characteristics of the patients are shown in Table 1.
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Results: Characteristics of the patients are shown in Table 1 and clinical results are shown in Table 2. There were no statistically significant difference between overall survival and disease survival of haploidentical AHSCT patients and HLA identical AHSCT patients (P ¼ 0,66, P ¼ 0,95, respectively). Kaplan-meier overall survival and disease free survival curves of the haploidentical AHSCT and HLA identical AHSCT groups are shown in Figure 1. Conclusion: For patients without HLA-identical or unrelated donor and relapsing after autologous hematopoietic stem cell transplantation, haploidentical hematopoietic stem cell transplantation could be an effective mode of treatment. Disclosure of Interest: None declared.
P321 Comparison of total body ultrasonography with total body computerized tomography (CT) and 18-Fluorodeoxyglucose positron emission tomography (PET/CT) to detect recurrence of disease in patients with Hodgkin and non-Hodgkin lymphoma E. Benedetti1,*, F. Martini1, R. Morganti1, F. Caracciolo1, N. Cecconi1, G. Cervetti1, D. Caramella2, L. Rigacci3, M. Petrini4 1 Depart. of Oncology, Hematology Unit Ospedale S. Chiara Pisa, 2 Depart. of Oncology, U.O. Radiologia Universitaria Pisa, Pisa, 3 Universita’ di Firenze, U.O Ematologia Firenze , Firenze, 4Depart. of Oncology, U.O. Ematologia Pisa, Pisa, Italy Introduction: Patients with lymphoma may relapse with asymptomatic relapse in approximately 30% of cases. Total body CT (TB-CT) scan is the NCCN recommended imaging technique with a biannual basis frequency for the first 5 years. PET is strongly recommended for 18 fluorodeoxyglucose (18FDG) avid lymphoma such as Hodgkin lymphoma (HD). Nevertheless radiation exposure during follow up is becoming a concern. The development of new generations ultrasound sonographer may help in the follow up of lymphoma patients, with less radiation exposure and less expenses.
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Materials (or patients) and methods: In our Center we retrospectively evaluated our Lymphoma data-base and in the last 4 years, we selected 150 lymphoma patients (pts) who experienced relapse and received chemotherapy. Out of the 150 pts, 50 relapsed pts were identified who were investigated with both Ultrasound (US) and TB-CT (N ¼ 43) or PET(N ¼ 7). The examinations were performed within 2 weeks from each other. US was performed double blinded, always before CT or PET. US was accompanied by a chest X ray (CXR) in most cases during the routine follow up. Pts were assessed on a 3 monthly bases. Total body US assessed: superficial nodes (S): laterocervical, supra and sub clavicular, axillary, and abdominal (A) and, if possible, anterior mediastinal (M) lymph nodes (Ly) stations. As a control group N ¼ 50 randomly chosen complete remission (CR) pts out of the 150 pts were analyzed. Results: Histologies of the 50 pts were as follows: HD N ¼ 10, Follicular (Fol) NHL N ¼ 9, Marginal (MZL) N ¼ 3, MALT N ¼ 3, Mantle cell (MCL) N ¼ 11; not specified N ¼ 2, DLBCL N ¼ 9, Burkitt N ¼ 1; T-NH N ¼ 2. In 32 cases there was a concordance between US and CT or PET/CT regarding S and A Ly. One case CT scan revealed M relapse in respect to US. In 9 cases of contemporaneous M þ A þ S involvement CT was superior to detect only M involvement. In 3 cases CT or PET/CT revealed lung and or bone involvement with concordance with US in S and A ly. Out of 3 cases of negative US 2 positive cases of CT scan regarded M involvement and 1 A Ly. Nevertheless, overall, there was not a statistically (ST) significant difference in detection of lymphoma relapse between US and CT or PET/ CT (P ¼ 0.186).Pts have been stratified according to BMI (BMI4 ¼ 25 and o25): BMI does not influence concordance in relapse detection between US and CT or PET/CT (P ¼ 0.8). There was not a ST significant difference in relapse detection according to Histologies: HD vs Follicular NHL (P ¼ 0.650), HD vs MCL (P ¼ 0.183), Fol vs MCL (P ¼ 0.617),Fol vs DLBCL (P ¼ 0.9). In the CR control group: N ¼ 44/50 there was concordance US vs CT or PET. In N ¼ 3 pts PET was a false positive. In N ¼ 1pts/50 US was positive vs neg CT and the pts relapsed 3 months later. N ¼ 2 pts US revealed increased and dishomogeneous spleen vs normal CT and the pts experienced relapse 2.8 months later. In CR pts US specificity is 100%. Conclusion: In our retrospective study TB-US was helpful to detect lymphoma relapses without a St significant difference in respect to CT and PET/CT. US allows a close follow up, it is radiation-free, easily performed also in renal insufficiency pts in whom CT contrast media may be contraindicated, and is less expensive. Further prospective studies are warranted in order to assess its usefulness in lymphoma patients. Disclosure of Interest: None declared. P322 A Potential opportunity for improving outcomes of allogeneic hematopoietic stem cell transplantation in relapsed / refractory classic Hodgkin lymphoma E. Borzenkova1,*, A. Alyanskiy 1, M. Popova1, I. Osipov1, E. Kondakova1, M. Ivanova1, E. Babenko1, O. Slesarchuk1, A. Kozlov1, S. Bondarenko1, N. Mikhaylova 1, B. Afanasyev1 1 Raisa Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation First Pavlov State Medical University of St. Petersburg, St.-Petersburg, Russian Federation Introduction: The relapsed/refractory classical Hodgkin Lymphoma (r/r-cHL) pts comprise about 30% of overall cHL patient’s population. High-dose chemotherapy followed by autologous stem cell transplantation induces long-term response in 50% of pts. Brentuximab-vedotin (BV) is an antiCD30 drug-antibody conjugate, which allowed achieving good response in majority of r/r-cHL pts. In pts, who had relapse/PD after auto-HSCT, allo-HSCT could be a therapeutic option. The outcome of allo-HSCT in pts with active disease is poor. Most of the pts have relapse or PD of cHL early allo-HSCT before "graft versus cHL" effect appears.
The aim of this study was to estimate efficacy and safety of BV administration before and after allo-HSCT in pts with r/r-cHL. Materials (or patients) and methods: From Jan 2002 to Nov 2014, 37 pts with r/r-cHL have undergone RIC allo-HSCT: Fludarabine/Melphalan or Busulfane (n ¼ 22), Fludarabine/ Bendamustine (n ¼ 13) or Fludarabine/2GyTBI (n ¼ 2). GVHD prophylaxis included calcineurin inhibitors (ICN) and MTX or MMF (from MRD), and ATG (from MUD) or cyclophosphamide, ICN and MMF (from MUD or MMUD). Primary objective was to estimate ORR to BV before alloHSCT and for relapse/PD therapy in the early period after alloHSCT. Secondary objective was to estimate TRM, OS and PFS at 100 days after allo-HSCT and at 180 days after relapse/PD onset. BV was administrated at a dose 1.8 mg/m2 (IV) every 3 weeks. Results: BV as a )bridge* to allo-HSCT, was used in 10 pts with progression of cHL with median number of cycles 6 (1-9). The control group consisted of 27 pts who received CT before alloHSCT with median number of cycles 2 (1-4). Relapse or PD after allo-SCT was observed in 14 pts. Three pts didn’t receive any therapy. BV after allo-HSCT was administrated in 5 pts: BV þ DLI (n ¼ 3), BV þ CT (n ¼ 1), BV alone (n ¼ 1). Control group included 5 pts with CT þ DLI administration after alloHSCT and 1 pts with DLI alone. The ORR of BV administration before allo-HSCT was 60% (including 30% who achieved PET – negative state) vs 38% in CT group. ORR of BV administration after allo-HSCT for relapse therapy ( þ /- DLI/CT) was 60% (3 of 5 pts achieved PET-negative status) vs 16% in CT group. PFS at 100 days after allo-HSCT was 60% in BV group and 48.1% in CT group (P ¼ 0,2). OS at 100 days after allo-HSCT was 100% in BV group vs 66,7% in CT group (P ¼ 0,048). TRM at 100 days after allo-HSCT was 0% in both groups. PFS2 at 180 days after relapse or PD was 60% in BV group and 30% in control group. OS at 180 days after relapse or PD was 80% and 16,7%, respectively. We did not observe toxicity grade 3-4 after BV administration. The pts treated with BV before and after allo-HSCT had the highest rate of overall survival at 2 years after allo-HSCT – 83,3%, followed by pts who received BV only before allo-HSCT – 75%. Two-years OS in CT group was 44,0%. Conclusion: BV has high clinical efficacy in patients with r/rcHL, both before and after allo-HSCT. Use of BV as a )bridge* to allo-HSCT may help achieve optimal disease status, which improves the results of allo-HSCT. BV could be safely administrated for relapse or PD treatment after allo-HSCT and was also associated with better PFS and OS. Prospective studies on BV with allo-HSCT are needed. Disclosure of Interest: None declared. P323 Excellent outcome of reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation in patients with Se´zary Syndrome F. Onida1,*, G. Saporiti2, D. Vincenti2, C. Annaloro2, E. Tagliaferri2, B. Motta2, S. Alberti Violetti3, E. Berti3, A. Cortelezzi1 1 Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico - University of Milan, 2Hematology, 3Dermatology unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Introduction: Patients with Se´zary Syndrome (SS) have a dismal life-expectation, with reported median survival ranging from 24 to 48 months and only 25 % of patients surviving beyond 5 years from diagnosis. Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only curative treatment option for patients with SS, with reduced-intensity (RIC) or non-myeloablative (NMA) regimens as the generally preferred conditioning strategies due to significantly lower morbidity and mortality in comparison to myeloablative conditioning. Materials (or patients) and methods: In our BMT Center, since April 2004 to June 2014 eight patients with refractory SS patients (5 males and 3 females) underwent allo-HSCT after a
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pentostatin/TBI200 NMA or a fludarabine/melphalan RIC regimen. Graft-versus-Host Disease (GvHD) prophylaxis included CsA and MMF ( þ ATG in MUD transplants). Kaplan-Meier survival cumulative probability estimates were calculated from the time of transplant. Results: Median age at the time of transplant was 61 years (range 43-66). Median time from diagnosis to transplantation was 34 months (range 13-252) and median number of previous treatment lines was 6 (range 2-8). Donors were HLA-identical sibling in 2 patients and HLA-matched unrelated in 6 (fully matched in 3 and with at least 1 allelic mismatch in 3). Source of stem cells was peripheral blood in all patients. Full donor chimerism was obtained within 3 months in all patients but one, who eventually experienced a graft failure and autologous hematopoietic reconstitution. Acute GvHD occurred in 3 patients (2 grade I-II, 1 grade III), whereas chronic GvHD was observed in 2 patients, of limited stage in both. Overall, clinical CR was obtained and maintained in 7 out of 8 patients. With a median follow-up of 25 months (range 5-128), all the 8 patients are currently alive with an estimated DFS at 5 years of 87% (Fig.1). Worth mentioning, a complete blood and marrow disease clearance was achieved also in the patient who did not show allogeneic engraftment, possibly as a result of the immunological mechanisms also responsible for the graft failure. Conclusion: In conclusion, although on a limited number of patients, our experience confirm the value of RIC/NMA alloHSCT as a therapeutic strategy for high-risk patients with refractory Se´zary Syndrome, showing an excellent disease-free survival rate and supporting the existence of a clinically relevant graft-versus-lymphoma effect in inducing and maintaining remission. Disclosure of Interest: None declared. P324 Autologous Stem Cell Transplantation in Refractory Hodgkin Lymphoma G. D. Petrova1,*, K. Melkova1, T. Chernyavskaya1, N. Gorbunova1, V. Kostrykina1 1 stem cell transplantation, Federal State Scientific Institution "Russian Cancer Research Center named N.N.Blokhin", Moscow, Russian Federation Introduction: Today the role of single or double Autologous Stem Cell Transplantation (ASCT) in patients (pts) with refractory Hodgkin Lymphoma (HL) is not clearly understood. We evaluated the feasibility and efficacy of single and double ASCT in pts with refractory to induction chemotherapy HL. Materials (or patients) and methods: Between 2007 and 2014 62 pts (34 female) aged 16-52 (median 28) with biopsy
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proved HL who never achieved complete remission (CR) were enrolled in study. All pts received salvage therapy with stem cell mobilization, after which 39/62 (63%) patients achieved disease control (DC) (5CR, 15PR, 19SD) and 23/62 (37%) pts had PD. Stem cell collection was effective in all 62 pts and 26/ 62(42%) pts had stem cell harvest adequate for 2 ASCT. Of the 62 pts 9 (15%) did not receive ASCT. Results: First ASCT was administered to 53 pts (27 female), 10 pts received 2 ASCT. Median follow-up was 3 years (range 1-7). At the time of first transplantation 79% pts had more than minimal disease (Z2 cm), 53% pts had extranodal involvement and 21% pts had B-symptoms. The time interval between diagnosis setting and 1ASCT was 5-124 mo (median 19). Number of previous treatment cycles was 6-27 (median 11), lines - 1-7 (median 3). Induction radiotherapy was performed in 55% pts. Disease status at 1ASCT: 3CR, 15 PR, 17 SD, 18 PD. After 1ASCT DC was achieved in 68% pts (13CR, 8PR, 15SD), 32% pts (17) with PD were not considered to be off the study. Equal number of patients (9 in each group) with PD at 1ASCT had DC (1CR, 1PR, 7SD), and PD. In a period of 3-9 mo (median 6) 10 pts received 2ASCT. At time of 2ASCT equal number of patients - 5/10 (50%) had chemoresistant (2SD, 3PD) and chemosensitive disease (5PR). Results of 2ASCT in pts with PD were very poor: two patients died due to treatmentrelated toxicity and in one case there was further PD. Median overall survival (OS) in a group of 17/53 (32%) pts with PD after 1ASCT was only 1,5 years and didn’t differ in groups with 2ASCT and alternative treatment (P ¼ 0,8). After 2ASCT among 8 alive pts DC was achieved in 88% (5CR, 1PR, 1SD), 1PD; estimated 5-year OS and progression free survival (PFS) were 50% and 38% respectively. Estimated 5-year OS and PFS for all 53 pts were 42% and 26% respectively. Estimated 5-year OS for 23/53(43%) pts with PR/SD after 1ASCT OS was 100% in a group of 6 pts with 2ASCT and 12% in a group of 17 pts without 2ASCT (P ¼ 0,006). Estimated 5-year OS for 13/53 (24%) pts with CR after 1ASCT was 100%.Univariate analysis identified that pts had a superior outcome if they had interval between diagnosis setting and 1ASCT less than 5 years, had chemosensitive disease and less than 14 cycles or 5 lines of previous therapy at time of 1ASCT and achieved CR/PR after 1ASCT. Conclusion: ASCT is an effective treatment option for pts who never achieved CR. Double ASCT proved to be highly effective - according to an intention to treat analysis the number of patients with CR increased from 25% after the 1ASCT therapy to 34% after the 2ASCT. Second ASCT is a treatment of choice for pts with PR/SD after 1ASCT. Second ASCT doesn’t improve outcome of pts with PD after 1ASCT. Alternative variants of treatment are needed for this patient group. Disclosure of Interest: None declared.
P325 Analysis of clinical outcomes and prognostic factors in 191 patients with diffuse large B cell lymphoma who underwent autologous stem cell transplantation at any time H.-J. Shin1,*, D. H. Yoon2, H. S. Lee3, S. Y. Oh4, D. H. Yang5, H. J. Kang6, S. Y. Chong7, Y. Park8, Y. R. Do9, S.-N. Lim10, J.-C. Jo11, W. S. Lee12, M.-K. Song1, J. S. Chung1 1 Pusan National University Hospital, Busan, 2Asan Medical Center, Seoul, 3Kosin University College of Medicine, 4Dong-A University College of Medicine, Busan, 5Chonnam National University Hwasun Hospital, Hwasun, 6Korea Cancer Center Hospital, Seoul, 7Bundang Cha Hospital, Bundang, 8Korea University College of Medicine, Seoul, 9Dongsan Medical Center, Daegu, 10Haeundae Baek Hospital, 11Ulsan University Hospital, 12 Busan Paik Hospital, Busan, Korea, Republic Of Introduction: The exact timing of autologous stem cell transplantation (ASCT) for patients with diffuse large B cell lymphoma (DLBCL), especially with high-intermediate and high IPI, has been still conflicted. Recent randomized trials revealed upfront ASCT improved progression-free survival (PFS), however, the advantage in PFS does not translate in overall survival (OS) benefit. Materials (or patients) and methods: We retrospectively collected and reviewed the clinical data of 191 patients with DLBCL who underwent ASCT at any time. Results: Median age was 50 years (range, 15-65 years) and male to female ratio was 1.16:1. The median follow up duration was 39 months (range, 5-210 months). The estimated 4-year OS rate was 64.0% ± 3.8%. In analysis with disease status at time of ASCT, patients with first complete remission (CR) had excellent OS (4-year OS rate, 79.9% ± 5.8%) (Po0.001). Patients with first partial remission (PR) and second CR (4-year OS rate, 60.3% ± 9.2% vs. 73.8% ± 6.9%) showed similar OS rates. The estimated 4-year OS rate was only 52.9% ± 8.2% in patients with second PR. In the subgroup analysis, for low/low-intermediate risk age-adjusted (AA) IPI patients, first CR/PR revealed favorable OS (estimated 4-year OS rates of 2 groups were more than 90%) while 4-year OS rate of second CR was superior compared to second PR (4-year OS rate, 83% ± 9.0% vs. 44.1% ± 10.9%) (Po0.001). It reflected in patients with low/low-intermediated AA-IPI, it is important to make CR prior to ASCT after relapse. For high-intermediate/high risk AAIPI patients, 4-year OS rate for patients with first CR was 77.0% ± 7.4% compared to those with first PR who had 48.5% ± 11.0% of 4-year OS rates (P ¼ 0.002). Therefore, for patients with high-intermediate/high AA-IPI, achievement of CR before
upfront ASCT is very important to prolong survival. For upfront ASCT, survival graphs showed well-validated as each AA-IPI risk group (Po0.001) while OS was not different between each AA-IPI risk group for salvage ASCT after relapse. This means AA-IPI at diagnosis can predict survival outcome only in patients who underwent upfront ASCT not salvage ASCT. OS was similar between BuCy-based and classic BEAM/BEAC conditioning regimens. Notably, BuCy-based conditioning regimens showed improved OS compared to BEAM/BEAC conditioning regimens in patients who were exposed to rituximab prior to ASCT (4-year OS rate of upfront ASCT, 78.1% ± 6.2% vs. 66.2% ± 14.0%; 4-year OS rate of salvage ASCT, 72.7% ± 9.0% vs. 50.0% ± 17.7%). It shows BuCy containing conditioning regimen may be appropriate compared to classical regimens in R-CHOP era compared to CHOP chemotherapy. Age older than 48.5 years, disease status at time of ASCT, and best response for ASCT revealed independent prognostic factors for DLBCL patients who underwent ASCT at any time. Bulky mass and bone marrow involvement did not impact on survival. Conclusion: In current study, it is suggested that achievement of first CR followed by upfront ASCT is the most important strategy to prolong OS in high risk (high-intermediate/high IPI) patients with DLBCL who is going to undergo ASCT. Disclosure of Interest: None declared. P326 Autologous-stem cell transplantation in Hodgkin’s Lymphoma: the Tuscany experience L. Iovino1, I. Donnini2,*, L. Rigacci2, E. Orciuolo1, B. Puccini2, S. Kovalchuk2, L. Rigacci2, F. Mazziotta1, B. Puccini2, A. Fabbri3, E. Cencini3, M. Bocchia4, M. Petrini1, A. Bosi2 1 Hematology, University of Pisa, Pisa, 2Hematology, University of Florence, Florence, 3Hematology, University of Siena, 4Hematology, University of Florence, Siena, Italy Introduction: Hodgkin Lymphoma (HL) is a very chemosensitive disease. Near a 35% of patients results refractory after a first-line regimen (primary refractory), or relapse after having achieved complete remission. In this cases, high-dose therapy and autologous stem cell transplantation (ASCT) are recommended as salvage treatment. The objective of the this study was to investigate the results of ASCT for HL during recent years and to identify variables predictive for outcome. Materials (or patients) and methods: For this retrospective study, patients aged between 18 and 70 who were treated with a first ASCT between 2009 and 2013 in three Tuscan
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Centers (Firenze, Pisa, Siena) were eligible. Baseline patient’s, disease’s, and transplant’s data were collected from MED-A forms. Centers with potentially eligible patients were contacted to provide additional treatment and follow-up information including a written histopathology report. Statistical univariate analysis used log rank’s test to assess the impact of baseline characteristics on survival endpoints. Results: 32 patients with HL were eligible for this analysis. Fifty-three percent were male, median age was 35 years (range 19-50). Twenty-eight percent of the patients presented with advanced Ann-Arbor stage at diagnosis, and 37% had B symptoms. Twenty-eight percent of the patients received more than two treatment lines prior to transplant. Remission status at the moment of ASCT was complete remission (CR) in 44% of the patients, partial remission (PR) in 41%, and refractory disease in 15%. Second line therapy was a high-dose of ARA-C with a platinum-based drug (oxaliplatinum and cisplatinum) in 47% of patients, IGEV in 47% of patients, 6% received another second line scheme. Response after ASCT was CR in 81% of patients, 9% achieved a PR, 6% of patients did not achieved a response (stable disease or progression). One patients died in first 30 days for transplant-related mortality (TRM). At the last follow-up, 84% of patients were alive. Two of them were alive but relapsed after the achievement of CR. With a median follow-up of 42 months, the 5-years overall survival (OS) and progression-free survival (PFS) for the whole series were 82% and 80%, respectively. The trend of survival curves for OS and PFS are notably identical, as confirmation of the impact of relapse on the OS (Figure 1). Five-year OS and PFS was better in patients who achieved a CR before ASCT, but not statistically significant, whereas the response after ASCT affected distinctly both OS than PFS (Po0,0001). The type of second line (DHA-platinum, IGEV or others) did not affect OS and PS, as the number of previous lines, the Ann-Arbor stage and the presence of B symptoms. Conclusion: In conclusion, this study confirms the feasibility of ASCT in HL and his benefit as salvage therapy, with a role in prevention of relapse. High-dose therapy is effective when the bulk of disease is notably reduced, independently of number and type of previous lines of therapies. New drugs (i.e. Brentuximab-vedotin) can improve the rate of CR sparing the toxicity of a heavy pre-treatments with conventional drugs. Disclosure of Interest: None declared. P327 The influence of rituximab, high dose therapy followed by autologous stem cell transplantation and age in patients with primary CNS-lymphoma M. Madle1, I. Kra¨mer1,*, N. Lehners1, M.-A. Schwarzbich1, P. Wuchter1, G. Egerer1, A. D. Ho1, M. Witzens-Harig1 1 Department of Hematology , University of Heidelberg, Heidelberg, Germany Introduction: For patients with diffuse large B-cell lymphoma without the involvement of the CNS, the addition of rituximab to standard chemotherapy has significantly improved survival. Thus far, there have been no prospective randomized trials evaluating the impact of rituximab as part of the primary treatment of primary CNS-lymphoma (PCNSL). Materials (or patients) and methods: In this single-center, retrospective analysis, a total of 79 PCNSL-patients treated in our institution between 2000 and 2011 were included. Beside first-line chemotherapy with or without rituximab, we evaluated the impact of age (r/4 60 years), autologous stem cell transplantation (ASCT þ /-) and other factors upon overall survival (OS) and progression-free survival (PFS). Results: In patients treated with rituximab (n ¼ 27), 3-year OS was 77.8% (95%4CI: 62-93%). In contrast, in patients treated without rituximab (n ¼ 52), 3-year OS was only 39.9% (CI: 2753%, Figure 2). The difference in OS was significant in the univariate (P ¼ 0.002) as well as the multivariate analysis (P ¼ 0.049, Hazard ratio (HR) ¼ 0.248). In the rituximab group, 80.8% were free of progression at the date of analysis (median
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not reached), whereas median PFS in the group without rituximab was only 17 months (CI: 8-26), (P ¼ 0.001). Patients r 60 years of age (n ¼ 28) had a 3-year OS of 78.2% (CI: 6394%) and a median PFS of 75 months, in patients 4 60 years (n ¼ 51) 3-year OS was 38.7% (CI: 25-52%) and median PFS was 39 months (CI: 6-72). Patients who received high dose therapy and autologous stem cell transplantation (ASCT) had a 3-year OS of 85.2% (CI: 72-99%) and 65.1% were alive up to the time of analysis (range 9-131 months). Without ASCT median OS was only 16 months (CI: 11-21) and 3-year OS was 35.2% (CI: 22-48%). Age and ASCT were significantly associated with better OS in univariate (P ¼ 0.002 and P ¼ 0.001) as well in multivariate analysis (P ¼ 0.004, HR ¼ 0.023 and P ¼ 0.001, HR ¼ 0.014). Conclusion: Rituximab treatment, ASCT and age are independent prognostic factors for overall survival in the first-line treatment of PCNSL. Disclosure of Interest: M. Madle: None declared, I. Kra¨mer Funding from: Gilead Sciences GmbH Germany, N. Lehners: None declared, M.-A. Schwarzbich: None declared, P. Wuchter Employee of: Sanofi and ETICHO, G. Egerer: None declared, A. D. Ho: None declared, M. Witzens-Harig: None declared. P328 Factors predicting early immune recovery after autologous transplantation in patients with non-Hodgkin lymphoma J. Valtola1,*, V. Varmavuo1, A. Ropponen2, H. Kuitunen3, O. Kuittinen3, L. Keskinen4, E.-R. Savolainen5, R. Silvennoinen1, T. Kuittinen1, T. Nousiainen1, J. Pelkonen2, P. Ma¨ntymaa6, E. Jantunen1 1 Department of Medicine, Kuopio University Hospital, 2Department of Clinical Microbiology, University of Eastern Finland, Kuopio, 3Department of Medicine, Oulu University Hospital, Oulu, 4 Department of Oncology, Tampere University Hospital, Tampere, 5Laboratory of Northern Finland, Oulu, 6Laboratory of Eastern Finland, Kuopio, Finland Introduction: Rapid lymphocyte recovery on day 15 (ALC15) after auto-SCT has been associated with improved outcome in patients with non-Hodgkin lymphoma (NHL). The chosen mobilization method affects the graft composition and earlier evidence indicates that various graft constituents may alter the course of immune recovery and outcome post-transplant. Limited prospective data is available assessing the possible factors predicting early immune recovery after auto-SCT. Materials (or patients) and methods: Seventy-one patients with NHL were included into this prospective study. There were 34 males and 37 females with a median age of 62 years. The most common histology was diffuse large B-cell lymphoma (DLBCL) (44%) and 79% of the patients were in first complete or partial remission at the time of the auto-SCT. All patients were mobilized with chemotherapy plus G-CSF. In addition, plerixafor was given for 24 patients. Cryopreserved graft samples were analyzed with flow cytometry for CD34 þ cells, T and B lymphocytes and NK cells. Complete blood counts were evaluated at þ 15 days and 1 month posttransplant and a flow cytometry of lymphocyte subsets (T, NK, B) was performed one month after the graft infusion. Associations of various factors (age, gender, histology, disease status, use of plerixafor or rituximab, graft CD34 þ counts and lymphocyte subsets) to various cut-off levels of ALC15, ALC30 and lymphocyte subsets at 1 month post-transplant were evaluated. Results: Histology of DLBCL (P ¼ 0.006) and the use of plerixafor (P ¼ 0.016) were associated with ALC15 Z0.5 x 109 /L. Also the total number of infused T lymphocytes, the amount of CD3 þ CD4 þ and NK cells in the graft correlated with ALC15 Z0.5 x 109 /L. ALC30 Z1.0 x 109/L was associated with prior rituximab use and the number of CD3 þ (P ¼ 0.024) and CD3 þ CD4 þ cells (P ¼ 0.018) in the graft. Blood CD3 þ CD4 þ level of Z0.3 x 109/L (lower normal range) at one month was associated with the number of CD3 þ CD4 þ cells in the graft (P ¼ 0.022). The NK cell level of Z0.2 x 109 /L in the
blood at one month was associated with male gender (P ¼ 0.005), the use of plerixafor (P ¼ o0.001) and the amounts of CD3 þ (P ¼ o0.001), CD3 þ CD4 þ (P ¼ 0.001), CD3 þ CD8 þ (P ¼ 0.002) and NK cells (P ¼ o0.001) in the infused grafts. Conclusion: The number of CD34 þ cells infused does not affect the early immune recovery. The amount of various lymphocyte subsets in the grafts seems to be of greater importance. Patients receiving plerixafor after chemomobilization appear to have more rapid immune recovery especially if defined with ALC15 Z 0.5x 109/L or NK cells Z 0.2 x 109/L at 1 month post-transplant. The potential effects of early immune recovery to post-transplant infectious complications and longterm outcome will be evaluated in the ongoing GOA study (Graft and Outcome in Autologous stem cell transplantation). Disclosure of Interest: J. Valtola: None declared, V. Varmavuo: None declared, A. Ropponen: None declared, H. Kuitunen: None declared, O. Kuittinen Employee of: Received lecture fees from Roche and Mundipharma, L. Keskinen Employee of: Received lecture fees from GlaxoSmithKline, E.-R. Savolainen: None declared, R. Silvennoinen Employee of: Received lecture fees from Sanofi, T. Kuittinen: None declared, T. Nousiainen: None declared, J. Pelkonen: None declared, P. Ma¨ntymaa: None declared, E. Jantunen Employee of: Received lecture fees from Genzyme and Sanofi, Conflict with: Participated in EU Leadership meeting organized by Genzyme as well as Medical Advisory Board meeting organized by Genzyme. P329 The Efficacy of CD34 cell mobilization regimens in patients with lymphoma and myeloma at a single institution J. Romejko-Jarosinska1,*, P. Lidia1, L. Targonski1, M. Szymanski1, E. Mroz Zycinska1, Z. Pojda1, J. Walewski1 1 Maria Sklodowska Curie Memorial Cancer Centre, Warsaw, Poland Introduction: The outcome of autologous hematopoietic cell transplantation (auto-HCT) is essentially related to CD34 cell dose with the generally accepted minimum of 2x10^6 per kg of patient body weight. Patients who are unable to reach this threshold are usually deprived of this potentially curative procedure. We retrospectively evaluated efficacy of a number of mobilization procedures at our institution and analyzed factors predicting the poor harvest Materials (or patients) and methods: We reviewed reports of the 220 mobilization procedures in a total of 202 adult patients with Hodgkin lymphoma (48), DLBCL (51), mantle cell lymphoma (37), myeloma (43), and other lymphoma subtypes (21), who were planned for auto-HCT between January 2012 and November 2014. Median (range) age was 51 (19-69), and 47 patients were 60 y/o or more. The following mobilization regimens were used: G-CSF þ high dose cyclophosphamide (n ¼ 97), G-CSF þ high or intermediate dose cytarabine (n ¼ 96), G-CSF þ ICE (n ¼ 19), G-CSF alone (n ¼ 5). In addition, plerixafor was used in 21 patients. 90 patients were mobilized after first-line treatment (41%). Effective mobilization was defined as a count of 2.0x10^6/kg CD34 cells. Results: Overall, 69% of patients had a peak of 410/ml CD 34 cells in blood and 29% had 430/ml. Sufficient number of CD 34 cells were collected in 76% of patients, including 39% in first apheresis. Majority of CD34 cells were collected in the first (43%) and in the second (39%) day of apheresis. There was no difference in successful mobilization between myeloma and lymphoma patients. The use of platinum based regimen in previous treatment, number of days on G-CSF more than 10, and CD34 cell count less than 5/ml on the first day of collection correlated to poor mobilization in univariate analysis. There was no difference in apheresis outcome between chemotherapy regimens used for mobilization. 18 of 48 poor mobilizers had a second mobilization. Eleven of them collected sufficient CD34 cell count. Only 11 of 21 patients who received plerixafor reached a threshold CD 34 cell harvest.
Conclusion: More than 24% of patients needed a second mobilization cycle. Number of G-CSF administration days, maximum peripheral CD34 cell count, and platinum treatment were predictive for poor harvest. Chemotherapy regimen selection was non-predictive for the harvest Disclosure of Interest: None declared. P330 Chemosensitivity and not histology determines outcome of allogeneic stem cell transplantation in patients with advanced-phase lymphoma J. Apostolidis1,*, F. Panitsas1, S. Gigantes1, I. Baltadakis1, K. Kaisari1, V. Pardalis1, I. Markou1, M. Gonianaki1, E. Xenou1, K. Xirokosta1, T. Loidoris1, M. Bouzani1, T. Karmiris1, D. Karakasis1, N. Harhalakis1 1 Department of Hematology, BMT Unit, Evangelismos Hospital, Athens, Greece Introduction: Patients with relapsed/refractory (R/R) Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL) have poor prognosis. Allogeneic stem cell transplantation (allo-SCT) is a potentially curative strategy, albeit at the cost of significant mortality. However, its application has been facilitated over the last 15 years with the use of reduced-intensity conditioning (RIC) regimens. The aim of this study is to evaluate the outcomes of allo-SCT in a recent cohort of patients with advanced phase HL/NHL from a single institution. Materials (or patients) and methods: Between 01/2001 and 10/2013, 73 consecutive patients, aged 21-65 (median: 41) years, received allo-SCT at our center. Diagnoses were aggressive NHL in 17 patients (23%), indolent NHL in 34 (47%) and HL in 22 (30%). Patients had previously received a median of 3 lines of chemotherapy (range: 1-8), while 30 (41%) had relapsed after autologous transplant (ASCT). At transplant, 18 (25%) were in complete remission (CR) and 35 (48%) had chemosensitive disease (1st/2nd partial remission, Z 3rd CR), whereas the disease was chemorefractory in 20 (27%). Donors were HLA identical siblings in 36 (49%) cases, unrelated in 35 (48%) and umbilical cord blood in 2 (3%). Reduced intensity conditioning (RIC) was used in 60 (82%) cases, mainly a Fludarabine/Melphalan regimen. Results: With a median follow-up of 38 months (range: 4-118), 3-year overall survival (OS), 3-year progression-free survival (PFS) and 3-year relapse rate (RR) were 47% [95% CI: 34-58%], 41% [95% CI: 29-52%], and 32% [95% CI: 21-43%], respectively. The 3-year OS for patients with aggressive NHL, indolent NHL and HL were 37%, 52%, and 46%, respectively. Non-relapse mortality (NRM) was 22% at 1 year. The incidence of acute graft-versus-host disease (GVHD) was 46% (grade II-IV: 32%), and of chronic GVHD 66% (extensive: 49%). In multivariate analysis, age, donor type, and histology did not have any prognostic impact on outcome. On the other hand, disease status at transplant (chemorefractory versus CR/chemosensitive disease) was the only significant factor influencing OS [HR: 1.77 (95% CI: 0.9-3.3), P ¼ 0.07] and disease-free survival [HR: 1.89 (95% CI: 1.0-3.5) P ¼ 0.04]. Conclusion: Allo-SCT may offer prolonged PFS in about 40% of patients with advanced-phase HL/NHL with an acceptable risk of NRM in the era of RIC regimens. Referral of patients prior to the development of chemoresistance is critical for the successful outcome of allo-SCT. Moreover, novel agents (brentuximab vedotin, ibrutinib, PD-1 inhibitors) may serve as a bridge to overcome chemoresistance and improve outcome in this patient population. Disclosure of Interest: None declared.
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P331 Simultaneous inhibition of CXCR4 and VLA-4 exhibits combinatorial effect in overcoming stroma-mediated chemotherapy resistance in mantle cell lymphoma cells K.-S. Eom1,2,*, Y.-R. Kim3, S. Lee1 1 Hematology, Catholic Blood & Marrow Transplantation Center, Seoul St. Mary’s Hospital, The Catholic University of Korea, 2 Cancer Research Institute in the Catholic University of Korea, 3 Cancer Research Institute in teh Catholic Univeristy of Korea, Seoul, Korea, Republic Of Introduction: Mantle cell lymphoma is a rare, aggressive and distinct subtype of non-Hodgkin’s lymphoma. Despite substantial efforts to improve clinical outcomes, DFS remains low due to MRD. Cells constuting MRD have capacity to find refuge in protective microenvironments and the selective pressure of chemotherapy promotes the outgrowth of MRD. CXCR4/ CXCL12 axis has been considered as a major contributor to environment-mediated drug resistance (EMDR). Cell adhesion mediated by VLA-4 confers protection against chemotherapy in various cancers. The interactions between CXCR4 and VLA-4 constitute a 2-way pathway where both receptors work cooperatively. Therefore, it is expected that the combined
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blockade of both CXCR4 and VLA-4 is more effective in overcoming EMDR than soley blocking the respective receptor. Here, the combined use of specific Abs for both receptors was tested to see the effect on EMDR. Materials (or patients) and methods: MCL cell line Jeko-1 and the murine stromal cell line M2-10B4 were used. Chemotaxis and migration inhibition assay were performed using Transwell inserts. Purified NA/LE mouse anti-human CD184, FITC anti-human CD49d and FITC mouse anti-human CD45. For migration inhibition experiments, anti-CXCR4 Ab and anti-integrin alpha 4 Ab azide free for VLA-4 were purchased. Results: Strong expression of both CXCR4 and VLA-4 were observed. Chemotaxis was significantly inhibited by antiCXCR4 Ab. Pseudo-emperipolesis of Jeko-1 pre-treated with anti-VLA4 antibody was markedly reduced. Combined use of both antibodies (anti-CXCR4 and anti-VLA-4 antibody) reduces migration of MCL cells in a combinatorial manner. Blocking antibodies could reverse the protective effect of MSC on MCL cells. These effect was most prominent when Abs were used simultaneously, demonstrating a combinatorial effect on inducing apoptosis (Figure 1). Both antibodies resulted in decreased phosphorylation of downstream signaling proteins
(ERK1/2, AKT, NF-kB). The reduction in phosphorylation of the proteins was most prominent when the cells were treated simultaneously with both Abs. Combined use of the Abs induces a combinatorial increase in apoptosis in the upper chamber of Transwell insert, while the migrated cells in the lower chamber, and in turn, adhered to MSCs or underwent pseudoemperipolesis demonstrated marked decrease in apoptosis. Conclusion: The combined blockade of CXCR4 and VLA-4 resulted in combinatorial effects in reducing migration and inducing apoptosis of MCL cells. These findings are associated with reduced phosphorylation of downstream signaling proteins. The result of our study may provide a basis for future development of therapeutic tools targeting both CXCR4 and VLA-4 such as bispecific antibody to improve treatment outcome of MCL. Disclosure of Interest: None declared. P332 Radioimmunotherapy and BEAM chemotherapy versus BEAM as the conditioning regimen for autologous stem cell transplantation (ASCT) in relapsed follicular lymphoma (FL): a retrospective matched-control study of the LWP-EBMT L. Bento1,*, A. Boumendil1, H. Finel1, P. Chevallier1, S. Amorim1, H. Monjanel1, D. Blaise1, J.-O. Bay1, E. Nicolas-Virelizier1, G. McQuaker1, G. Rossi1, R. Johnson1, A. Huynh1, N. Fegueux1, A. Rambaldi1, G. Salles1, C. Craddock1, K. Orchard1, D. Pohlreich1, H. Tilly1, G. Bandini1, X. Poire´1, F. Guilhot1, A. Haenel1, C. Crawley1, B. Metzner1, J. Gribben1, N. H. Russell1, G. Damaj1, K. Thomson1, P. Dreger1, S. Montoto1 on behalf of EBMT Lymphoma Working Party, Paris, France 1 EBMT, Paris, France Introduction: Relapse after transplant remains the most common cause of treatment failure in patients receiving an autologous stem cell transplant (ASCT) for recurrent follicular lymphoma (FL). We hypothesized that the incorporation of radioimmunotherapy (RIT) in the conditioning regimen of ASCT might reduce the relapse rate and improve the outcome in patients with FL. Materials (or patients) and methods: Between 2004 and 2012, 2359 patients (57% male, median age at ASCT: 56– range:19-77) who had an ASCT for relapsed FL with BEAM conditioning regimen with or without RIT (Ibritumomabtiuxetan: Zevalins) were reported to the EBMT and included in this study. Z-BEAM (n ¼ 207), R-BEAM (n ¼ 179) and BEAM
(n ¼ 1973) were analysed excluding addition of other drugs. We conducted a matched-cohort analysis of BEAM versus Zevalins-BEAM (Z-BEAM) including 282 and 154 patients, respectively. R-BEAM cases were not considered for this analysis. The variables used to match the pairs were age, time from diagnosis to transplant, gender, disease status at ASCT, performance status (PS) and year of transplant. Overall survival (OS) and event-free survival (EFS) were determined using the Kaplan-Meier method, and curves were compared by log-rank test.In the matched sample, outcome in BEAM vs Z-BEAM were compared using a Cox model stratified on matched pairs. Results: The matched-cohort analysis of BEAM versus Z-BEAM was described. The majority of the patients had a good PS at transplant (KarnofskyZ80% in 96% and 94% of patients receiving BEAM and Z-BEAM, respectively) and were transplanted in good response (at least sensitive relapse in 96% of patients receiving BEAM and in 94% of the Z-BEAM cohort). After a median follow-up of 18.7 months (range: 1-85.7), 25% of patients in the BEAM group relapsed, in comparison with 34% in the Z-BEAM cohort. Forty (14%) and 20 (13%) patients died in the BEAM and Z-BEAM cohorts, respectively. There were no significant differences in non-relapse mortality (HR: 0.5 [95%CI: 0.13-1.93]; P ¼ 0.3), nor on cumulative incidence of relapse (HR ¼ 1.55 [0.96-2.51]; P ¼ 0.07). Similarly, no significant differences between BEAM and Z- BEAM cohorts were found in OS (HR ¼ 0.77 [CI95%: 0.41-1.46]; P ¼ 0.43) or EFS (HR ¼ 1.34 [0.86-2.08]; P ¼ 0.2). Conclusion: Based on the matching factors used here, this study failed to show a benefit of adding RIT to BEAM in ASCT for relapsed FL. However, the limitations of registry analyses have to be taken into account, and prospective trials are needed for definite information. Disclosure of Interest: L. Bento Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Boumendil Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, H. Finel Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, P. Chevallier Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, S. Amorim Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, H. Monjanel Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, D. Blaise Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, J.-O. Bay Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, E. Nicolas-Virelizier Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany,
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G. McQuaker Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Rossi Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, R. Johnson Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Huynh Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, N. Fegueux Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Rambaldi Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Salles Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, C. Craddock Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, K. Orchard Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, D. Pohlreich Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, H. Tilly Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Bandini Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, X. Poire´ Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, F. Guilhot Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Haenel Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, C. Crawley Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, B. Metzner Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, J. Gribben Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, N. H. Russell Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Damaj Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, K. Thomson Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, P. Dreger Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, S. Montoto Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany. P333 Autologous-stem cell transplantation in diffuse-large B cell lymphoma: the Tuscany experience L. Iovino1,*, I. Donnini2, L. Rigacci2, B. Puccini2, S. Kovalchuk2, E. Orciuolo1, F. Mazziotta1, A. Fabbri3, E. Cencini3, M. Bocchia3, M. Petrini1, A. Bosi2 1 Hematology, University of Pisa, Pisa, 2Hematology, University of Florence, Florence, 3Hematology, University of Siena, Siena, Italy Introduction: Patients with diffuse-large B cell lymphoma (DLBCL, NOS) who doesn’t achieve a complete remission (CR) after a first-line chemotherapy regimen, young high-risk
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patients, and patients who relapsed after CR, are candidate to high-dose therapy and autologous stem cell transplantation (ASCT). The objective of the present study was to investigate the results of ASCT for DLBCL during recent years in Tuscany network. Materials (or patients) and methods: For this retrospective study, patients aged between 18 and 70 who were treated with a first ASCT between 2009 and 2014 in three Tuscan Institutions (Firenze, Pisa, Siena) with complete follow-up data were eligible. Baseline patient’s, disease’s, and transplant’s data were collected from MED-A forms. Statistical univariate analysis used log rank’s test to assess the impact of baseline characteristics on survival endpoints. Results: thirty patients with DLBCL were eligible for this analysis. 57% were male, median age was 41 years (range 2161). Forty-seven percent of the patients presented an advanced Ann-Arbor stage at diagnosis, and 27% had B symptoms. 27% of the patients received 3 or more treatment lines prior to transplant. Remission status at ASCT was CR in 43% of the patients, PR in 50%, and refractory disease in 7%. Second line therapy was a high-dose of ARA-C with a platinum-based drug in 66% of patients. Response after ASCT was CR in 22 patients (73%). Two patient achieved a PR, and two patients did not response. Three patients (10%) died in first three months for transplant-related mortality (TRM), and were not included in survival analyses. At the last follow-up, 23 patients (77%) were alive. Two of them (7%) relapsed. With a mean follow-up of 81 months, the 5-years overall survival (OS) and progression-free survival (PFS) for the whole series were 75% and 68%, respectively. Five-year OS and PFS was significant better in patients who achieved a CR after ASCT (p 0,005) (Figure 1). The type of second line did not impact on OS and PFS. Univariate analysis showed a significant trend (p 0,026) in PFS in favor of patients who achieved at least a PR pre-ASCT, whereas no differences were observed in OS for this variable. Other variables did not show a significant prognostic value. Conclusion: this study confirms the role of ASCT in DLBCL, and his benefit as salvage therapy, with a role in prevention of relapse and an high percentage of conversion in CR since PR. High-dose therapy is effective when the disease still chemosensitive. Disclosure of Interest: None declared. P334 Allogeneic stem cell transplantation for relapsed or refractory Hodgkin lymphoma: a better therapeutical perspective M. M. Greco1,*, A. M. carella1, E. Merla1, N. Cascavilla1 1 Hematopoietic Stem Cell Transplantation Unit, IRCSS Casa Sollievo Della Sofferenza, San Giovanni Rotondo (FG), Italy Introduction: The current standard of care of relapsed or resistant Hodgkin lymphoma is a salvage chemotherapy
consisting of a conclusive myeloablative chemotherapic regimen with autologous stem cells transplantation (Auto-SCT), if indicated followed by radiotherapy. Patients with relapsed or refractory disease after autologous bone marrow transplantation have a poor survival and the management of the disease is challenging. In the last decade, several factors have refined the role of allogeneic transplantation such as reduced intensity conditioning (RIC) transplantation, donor lymphocytes infusion, biologically based treatments, and monoclonal antibody drug-coniugate anti-CD30 Bretuximab Vedotin. Materials (or patients) and methods: Between 2007 and 2014 14 patients whose disease progressed or relapsed after Auto-SCT underwent an allogeneic RIC stem cell transplantation (RIC Allo-SCT). Four patients were male and ten female, median age was 29 years and age range was 25-40 years. Disease status at SCT was complete remission (CR) (n ¼ 5; 35%), partial remission (n ¼ 6; 45%) and progression disease (PD) (n ¼ 3; 20%). The median time from Auto-SCT to Allo-SCT was 6 months (range 2-8). The conditioning regimen was FluMel (n ¼ 1), BU-Flu (n ¼ 4) and Thio-BU-Flu (n ¼ 9). The donor was an HLA-identical sibling (n ¼ 4), a matched unrelated donor (MUD) (n ¼ 4) or a familiar aploidentical (n ¼ 6). Graft versus host disease (GVHD) profilaxis consisted of Cyclosporin (CSA) and Methotrexate (MTX) short-course in simbling transplants, CSA þ MTX and rabbit ATG in MUD, Cyclofosphamide 50 mg/kg day þ 3, þ 4, associated to CSA þ Mycophenolate by day þ 5. The Bretuximab Vedotin was utilized in 5 patients from 2013. Results: All patients engrafted and the median of neutrophils recovery was 14 days. Acute GWHD Z2 was documented in 6/ 14 patients (43%). 7/14 patients dead, the cause of death was infectious disease in 2 patients, respectively located in lung and CNS. 2 patients affected by chronic GVHD dead because of congestive heart failure and obliterans bronchiolytis and 3 patients dead because of the progression of the disease. The last 3 patients were primary refractory with Bulky disease. 7 patients (50%) are alive in CR. Conclusion: We conclude that Bretuximab Vedotin can induce CR or high citoreduction promoting a favorable outcome of transplantation. The early and late TRM is similar to that one detected in other hematological malignancies. The Bulky and the primary refractory disease represent a biological barrier of the tumour and its microenvironment to allogeneic activity of transplantation. Disclosure of Interest: None declared. P335 Differences in the outcome of Hodgkin’s lymphoma treated with high dose chemotherapy and autologous stem cell transplantation M. R. Todorovic1,*, B. Balint2, D. Stanisavljevic3, B. Andjelic 4, J. Bila1, D. Antic4, D. Vujuic5, N. Kraguljac Kurtovic4, D. Sefer4, J. Jeleicic4, B. Mihaljevic1 1 Medical faculty University of Belgrade, Clinic for hematology CCS, 2Institute for transfusiology and Hemobiologyof MMA, 3 Institute for statistics, Medical faculty University of Belgrade, 4 Clinic for hematology CCS, 5Medical faculty University of Belgrade, Belgrade, Serbia
regimen in first ASCT was BEAM. Second ASCT performed with CBV regimen in five patients relapsed after first transplantation. Results: Data on 80 patients who received ASCT between Jan 2006 and Dec 2013 were reviewed. Median time from first treatment to ASCT was 2.6 years (0.8-8.4). Demography at ASCT was 62.5% stage III and IV, median age 29.8 (18-47), median prior treatment regimens 3 (2-7), median Hasenclever index 3 (0-6); Initially, 43 patients had responding disease [20 (25%) complete remission (CR), 23 (28.7%) partial remission (PR)], and 37(46.3%) patients had stable disease/progressive disease. Post-first ASCT, an overall response rate (CR þ PR) was 76.2% with CR (38.7%) and PR (37.5%). Mean time to second ASCT was 18.6 (3-39) months. Post-second ASCT all 5 patients upgraded their response, with two consecutive CR after brentuximab vedotin, one CR after radiotherapy, and two after chemotherapy. Median overall survival (OS)/progressionfree survival (PFS) from first ASCT was 105 months/51months. Nineteen patients died due to progressive disease at the end of follow up period. Patients with initial CR 4 12 months had double longer OS, than patients with CRo12 months (110 months vs. 60 monts). Positive impact on OS had initial Hasenclever indexo3, CR after first line therapy, attaining CR at D þ 100 after ASCT, while number of CD34 þ peripheral blood stem cells 4 5.5x10e9/L had positive impact on PFS after ASCT. Multivariate Cox regression defined that independent prognostic factor for OS was favorable therapeutic outcome at D þ 100 (P ¼ 0.001). Conclusion: Although, the results of ASCT vary significantly depending on a number of prognostic factors, it is the standard of care for patients with relapsed HL and leads to a better outcome improving overall response rate for 22.5%. Disclosure of Interest: None declared. P336 Outcome of autologous stem-cell transplantation in Hodgkin and non hodgkin lymphoma patients N. Soyer1,*, C. Atmaca Mutlu2, F. Vural1, M. Tombuloglu1, M. Tobu1, F. Sahin1, G. Saydam1 1 Hematology, 2Internal Medicine, Ege University Medical Faculty, IZMIR, Turkey Introduction: Autologous hemopoetic stem cell transplantation (aHSCT) can offer potential long-term remission or cure in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Materials (or patients) and methods: One hundred eight NHL and 56 HL patients treated with autologous stem-cell transplantation in Ege University Hospital from January 2008 through December 2014 for relapsed or refractory HL and NHL were analyzed retrospectively.
Introduction: Hodgkin’s lymphoma (HL) is highly curable disease to conventional chemotherapy. Even though, some patients are primary refractory or relapsed after first-line chemotherapy. For them autologous stem cell transplantation (ASCT) is the standard of care. The results of ASCT vary significantly depending on a number of prognostic factors. The purpose of this study was to assess prognostic factors and outcome of patients with relapsed/refractory HL who received high-dose chemotherapy and ASCT. Materials (or patients) and methods: Patients with relapsed or refractory HL who were initially treated with ABVD therapy, were analyzed. In the course of relapse or refractory disease, all patients received DHAP as first salvage, six patients received ICE as second salvage therapy, while conditioning
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Results: Data of the 164 lymphoma patients treated with aHSCT were evaluated retrospectively. Median age of HL and NHL was 40 years (22-75 years) and 53 years (19-76 years) respectively. Forty one (%38) patients were female, 67 (62%) were male from all of 108 NHL patients. 20 (35%) were female, 36 (65%) were male from all of 56 HL patients. Transplantation related mortality was 0.8% (13 of 164 patients;1 HL, 12 NHL). Complete remission and partial remission was obtained in 37 (67%) and 9 (16%) of HL; 52 (48%) and 25(23%) of NHL. Refractory disease was in 9 (16%) in HL and 19 (17%) in NHL. After a median follow-up of 13.5 months (range, 3- 110 months), 55 of 108 (50.9%) patients with NHL were alive. The estimated 5 year OS was % 50 for NHL patients. After a median follow-up of 62 months (range, 12180 months), 46 of 56 (82.1%) patients with HL were still alive. The 5-year overall survival (OS) were 86% for HL patients. Progression free survival of HL and NHL patients was shown Figure 1. Conclusion: Autologous stem-cell transplantation remains to be the standard treatment of relapsed or refractory HL and NHL patients. Prognosis of relapsed/refractory HL and NHL patients ineligible for autologous stem-cell transplantation and treated with several lines of standard chemotherapy ± radiotherapy is poor. Improving the treatment of patients at high risk of failure at diagnosis, evaluation of new chemotherapeutic agents, further development of immunotherapy and radioimmunoconjugates require further study. Disclosure of Interest: None declared. P337 Results of 10 years high dose chemotherapy with Tutshka protocol followed by autolougous stem cell transplantation in diffuse large B cell lymphoma R.-M. Hamladji1,*, N. Ait Amer1, M. Benakli1, R. Ahmed Nacer1, A. Talbi1, F. Harieche1, F. Mehdid1, R. Belhadj1 1 Hematology-Bone Marrow Transplantation Department. Pierre and Marie Curie Center, Algiers, Algeria Introduction: Despite numerous studies on the long term outcome of patients (pts) with diffuse large B cell lymphoma (DLBCL) treated with high dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) choosing the best effective HDT protocol is not yet known. This study reports the results of 10 years Tutshka protol applied to pts with DLBCL. Materials (or patients) and methods: Between January 2004 and December 2013, 94 DLBCL pts including 28 mediastinal large cell lymphoma pts (MLCL) underwent, before ASCT, HDT with Tutshka protocol that combines oral Busulfan (16 mg/Kg) and intra-venous cyclophosphamide (120 mg/Kg), intra venous Etoposide (30 mg/Kg) have been added since 2012. Median age is 37 years ( 17 to 65) and sex ratio (M/F) 1.2. For the 28 pts with MLCL median age is 31 years ( 17 to 56), sex ratio: 0.6 and their pre transplant disease status as follow: 14 pts in complete remission (CR) and 14 pts in partial remission (PR) after 2 to 3 lines of treatment. For the others 66 DLBCL pts the median age is 45 years (17 to 65), sex ratio:2.7 and their pre transplant disease status: 44 pts in CR within 10 pts after 2 to 3 lines of treatment and 22 pts in PR. All pts receive, after thawing, mobilized peripheral blood stem cells obtained by G-CSF mobilization (15mg/Kg/d, 5 days) alone and froze in liquid nitrogen. The median rate CD34 þ cells infused is 3.14 x 106/Kg (0,87-17,36). The median follow-up at 10/30/ 2014 is 48 months (4-120). Results: The median time to graft (PNC 4 0.5 x 109/l) was 12 days (10- 60). Five early deaths were observed including 4 infection (TRM: 4%) and one in disease progression at 3 months. After 3 months of 89 evaluable pts (27 MLCL and 62 others DLBCL) relapse was observed in 28/89 pts (22.5%) and it was earlier relapse in a period of 24 months in 15 pts (75%).
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Deaths were among 23/94 pts (24.5%): MLCL 10/28 (36%) and others DLBCL 13/66 (20%) with one very late death after second relapse at 101 months. Persistent CR was achieved in 66/89 pts (74%) including 16/27 (59%) MLCL and 50/66 (75.5%) others DLBCL. The overall survival (OS) and event free survival (EFS) at 10 years are respectively 66% and 59.5%. In terms of OS and EFS there is no difference between MLCL (60% and 57.5% respectively) and others DLBCL (66% and 72% respectively). Conclusion: Our results show that Tutshka protocol is well tolerated and is an alternative in DLBCL including MLCL. Disclosure of Interest: None declared. P338 Hodgkin lymphoma with early relapse (3-12 months) behaving clinically similar to primary refractory Hodgkin lymphoma (primary progressive: partial response: relapse within three months). Single institution analysis of 201 patients S. Akhtar1,*, M. S. Rauf1, T. A. Elhassan1, I. Maghfoor1 1 Oncology Center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia Introduction: The outcome of high dose chemotherapy (HDC) and auto-SCT in primary refractory Hodgkin lymphoma (PR-HL) is not as encouraging as in relapsed HL. Patient relapsing between 3-12 months (relapse 3-12) also have worse outcome. We compared HDC auto-SCT in PR-HL against relapse 3-12 group. Materials (or patients) and methods: From 1996 to 2013, patients with HL who received HDC auto-SCT for PR-HL and for relapse 3-12 were identified. PR-HL is defined as partial response (PR), no response (NR), stable disease (SD), progressive disease (PD), relapsing within 3 months (relapse 0-3) of finishing the planned treatment. Competing risk analysis method was used to estimate disease specific death (DSdeath) and disease specific event (DS-event) from the day 0 of auto-SCT. Results: Male 100 (50%), female 101 (50 %), Median age at diagnosis was 22.2 years (12-61 years) and at ASCT was 24 years (13.8-62 years). Initial chemotherapy was ABVD in 168 (84%), MOPP/COPP alternating with ABV or ABVD in 17 (8%), median chemotherapy cycles were 6. 58 patients (29%) also had radiation therapy (XRT) after initial chemotherapy. Response to chemo þ XRT was PR in 78 (39%), PD in 48 (24%), relapse 0-3 in 25 (12%) and relapse 3-12 in 50 (25%). ESHAP as first line salvage in 178 (89%); median cycles given were 3. Post salvage / prior to HDC auto-SCT disease status was PR in 126 (63%), CR in 61 (30%) and stable disease/no response in 14 (7%). BEAM was used as HDC. Median follow-up is 64 months from auto-SCT. Post auto-SCT overall response in 140 patients (70%); CR in 123 (61 %), PR in 17 (8.5 %), NR / SD in 1 (0.5%) and PD in 50 (25%) patients, unknown in 9 (4.5%). 66 patients had XRT post auto-SCT. Type of first event was no event in 94 (47%), persistent disease in 18 (9%), progressive disease in 50 (25%), relapsed disease in 28 (14%), treatment related mortality in 7 (3.5%) and died of other cause 4 (2%). At last follow-up in December 2014, 102 patients (51%) are alive with no disease, 14 (7%) alive with disease, 71 (35%) died of disease and 14 (7%) died of treatment related mortality or other causes. For whole group estimated cumulative risk of DS-death (36%) and DS-event (47%) is shown in Table and Figure. Factors Whole group Progressive disease Partial response Relapse 0-3 months Relapse 3-12 months
Total N DS-Death % P-value DS-Event % P-value 201 48 78 25 50
36 40 36 34 32
x 0.4
47 52 51 44 36
x 0.1
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Conclusion: In patients with all types of PR-HL and those with relapse 3-12, ESHAP þ BEAM combination results in high CR rate. Despite this high CR rate, one third of patients died of disease post auto-SCT. There is no statistically significant difference in various PR-HL types and relapse 3-12 group. Relapse 3-12 has high incidence of disease specific deaths and events. This cohort is behaving more like a PR-HL and would be more appropriate to be referred as refractory HL in future studies for better management and outcome. Disclosure of Interest: None declared. P339 Outcome of HDC auto-SCT in adolescent and young adults (AYA) patients with Hodgkin lymphoma: Primary refractory vs early relapse (3-12 months) vs relapse after 12 months: A single institution result of 216 patients S. Akhtar1,*, M. S. Rauf1, T. A. Elhassan1, I. Maghfoor1 1 Oncology Center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia Introduction: The outcome of high dose chemotherapy (HDC) and auto-SCT in adolescent and young adults (AYA) is not commonly reported separately. Outcome in primary refractory Hodgkin lymphoma (PR-HL) and relapsing between 3-12 months (relapse 3-12) is not as encouraging as in relapsed HL after 12 months (relapse 412). We are reporting HDC auto-SCT results in PR-HL vs relapse 3-12 vs relapse 412 in AYA. Materials (or patients) and methods: From 1996 to 2013, all adolescent (14-21 years) and young adults (421 to 30 years) (at the time of auto-SCT) patients with HL and HDC auto-SCT were identified. PR-HL is defined as partial response (PR), no response (NR), stable disease (SD), progressive disease (PD), relapsing within 3 months (relapse 0-3) of finishing the planned treatment. Competing risk analysis method was used to estimate disease specific death (DS-death) and disease specific event (DS-event) from the day 0. Results: Male 107 (49.5%), female 109 (50.5 %), Median age at diagnosis 19 years (5-29) and at ASCT 22.4 years (13.8-30). Adolescent 94 (43.5%), young adults 122 (56.5%). Initial chemotherapy was ABVD in 176 (81%), MOPP/COPP alternating with ABV or ABVD in 20 (9%), median chemotherapy cycles were 6. Sixty-eight patients (31%) also had XRT after initial chemotherapy. Reason for HDC auto-SCT: refractory disease in 129 (60%), relapse 3-12 in 33 (15%) and relapse 412 in 54
(25%). ESHAP was used as first line salvage in 181 patients (84%) and in another 20 (9%) as 2nd line salvage; median 3 cycles (3 in 63% and 4 in 14%). Post salvage / prior to HDC auto-SCT disease status was PR in 132 (61%), CR in 73 (34%) and stable disease/no response in 11 (5%). BEAM was used as HDC. After auto-SCT, overall response in 157 patients (73%) (CR in 140 (65 %), PR in 17 (8%)), NR/SD in 2 (1%) and PD in 48 (22%) patients, unknown in 9 (4%). 71 (33%) patients had XRT after auto-SCT. After auto-SCT in 26 (12%) patients had relapsed disease, treatment related mortality in 5 (2%) and died of other cause 2 (1%). Median post auto-SCT follow-up is 63 months. Currently 123 patients (57%) are in CR, 14 (7%) alive with disease, 70 (32%) died of disease. For whole group estimated risk of DS-death (33%) and DS-event (45%) and according to PR-HL vs relapse 3-12 vs relapse 412 is shown in Table and Figure. Factors
Total N
DS-Death %
Whole group Adolescent (14-21 yrs) Young adults (421-30 yrs) Primary refractory Relapse 3-12 months Relapse after 12 months
216 94 122
33 32 34
119 37 60
43 32 12
Pvalue 0.7 0.0001
DS-Event % 45 49 41 57 35 27
P-value
0.4 0.00003
Conclusion: In this relatively high risk group of AYA, ESHAP þ BEAM combination resulted in high CR rate. Even PR-HL and relapse 3-12 have encouraging survival. There is no difference in the outcome of adolescent vs young adults. Relapse 3-12 has high incidence of disease specific deaths and events. This cohort is behaving more like a PR-HL and would be more appropriate to be referred as refractory HL in future studies for better management and outcome. Disclosure of Interest: None declared.
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P340 High dose chemotherapy and auto-SCT for nodular lymphocyte-predominant Hodgkin lymphoma: Single institution results of sixteen patients treated with and without rituximab containing salvage chemotherapy S. Akhtar1,*, M. S. Rauf1, T. A. M. Elhassan1, I. Maghfoor1 1 Oncology Center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia Introduction: Nodular lymphocyte-predominant Hodgkin lymphoma (NLP-HL) is a distinct subtype of Hodgkin lymphoma with unique clinical and histological features and indolent behavior with frequent recurrences. We are reporting our experience of NLP-HL patients who underwent High dose chemotherapy (HDC) and auto-SCT for refractory or relapsed disease. Materials (or patients) and methods: From 1996 to 2013, 290 patients with relapsed / refractory HL received HDC autoSCT and 16 were identified as NLP-HL. Refractory disease is defined as partial response (PR), no response (NR), stable disease (SD), progressive disease (PD) or relapsing within 3 months (relapse 0-3) of finishing the planned treatment. Kaplan-Meir method was used for survival analysis from day 0. Results: Sixteen patients underwent HDC auto-SCT for NLP-HL. At the time of auto-SCT, 13 (81%) had NLP-HL and 3 (19%) had transformed diffuse large B cell lymphoma (DLBCL) (1 had
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DLBCL and 2 had T cell histiocytic rich B cell lymphoma). Male 14 (88%), female 2 (12 %), Median age at diagnosis 22 years (10-54 yrs) and at auto-SCT 27 years (15-55 yrs). At initial presentation patients characteristics were stages I:II:III:IV, 2:6:4:4 respectively. Primary treatment was ABVD in 13 (81%) and 3 (19%) received 1 of each MOPP, R-CHOP and radiation alone. 2 patients also had radiation therapy (XRT) after chemo. 12 (75%) had tissue confirmation for disease prior to salvage chemo. Reason for HDC auto-SCT was refractory NLP-HL in 10 patients (PD in 4, PR in 4, and relapse within 3 months in 2) and relapsed disease in 6. Prior to salvage chemo, stages I:II:III:IV were 2:4:5:5 respectively. Bulky disease in 2 (12%), spleen involvement in 8 (50%) and extranodal-site involvement in 5 (31%), mediastinal involvement in 6 (38%) patients and positive bone marrow in 2 (12%). ESHAP was used in 15/ 16 patients: as first line salvage in 10 (63%), 2nd and 3rd line 3 (19%); median number of cycles given were 3. 5 patients also had rituximab with ESHAP. Response to salvage was PR in 7 (44%), CR in 9 (56%). All patients had BEAM as HDC. Median follow-up is 65 months from auto-SCT. Post auto-SCT overall response was observed in 14 patients (88%); CR / CRunconfirmed in 13 (81%) and PR in 1 (6%). 2 (13%) patients had PD patients. 6 (38%) patients had XRT post auto-SCT; for consolidation post CR in 4 patients and 1 each for eradication of residual and progressive disease. 11 patients who received ESHAP only, 4 of them had post auto-SCT disease specific events (persistent disease in 1, relapsed in 1 and PD in 2
patients) as compared to no event was observed in 5 patients who received rituximab þ ESHAP. Currently 15 (94%) patients are alive, 14 with no evidence of disease and only 1 with disease (on observation for slowly progressive disease for 7 years). Five year event free survival is 75% and overall survival is 94%. Conclusion: In patients with NLP-HL, even in refractory disease, attainment of long term disease free survival is possible after HDC auto-SCT. Except one, all patients are alive. Post auto-SCT relapse or progression can still be managed with chemo / chemo þ immunotherapy. Although limited number of patients received rituximab, results are very encouraging. Integration of rituximab in salvage setting should be explored further in this unique set of patient population. Disclosure of Interest: None declared. P341 In vivo purging may not be required in the era of universal use of rituximab containing chemo-immunotherapy in patients with follicular and mantle cell lymphoma: A single center experience A. Singh1, C. Divine1, O. Aljitawi1, S. Abhyankar1, J. McGuirk1, S. Ganguly1,* 1 Hematology/Oncology, UNIVERSITY OF KANSAS MEDICAL CENTER, Westwood, United States Introduction: Although, rituximab containing chemotherapy regimens have improved response rates in B-cell lymphomas, a significant percentage of patients will eventually relapse. Autologous stem cell transplantation (Auto SCT) may provide long term remission in some of these patients. Tumor contamination of harvested stem cell graft has been postulated as a reason for relapse after Auto SCT. Single agent rituximab has been used for in-vivo purging of CD20 þ tumor cells with positive results. In this study, we aimed to evaluate whether use of rituximab based in-vivo purging is still beneficial in the era of universal use of rituximab containing chemotherapy in patients with follicular (FL) or mantle cell lymphoma (MCL). Materials (or patients) and methods: Twenty consecutive patients with a diagnosis of relapsed CD20 þ FL or transplant eligible MCL were included in this study. All patients received rituximab containing chemotherapy. Restaging was done after completion of chemotherapy and those with chemo-sensitive disease (n ¼ 14) were considered for Auto SCT. Polymerase Chain Reaction (PCR) analysis for bcl2 and bcl1 were done on apheresis products from patients with FL and MCL respectively. In-vivo purging with single agent rituximab was planned if any stem cell product were to be found PCR positive prior to Auto SCT. Results: A total of 19 patients were eligible for analysis. Patient characteristics are shown in the accompanying table. The bcl-2/bcl-1 status of the graft was available in 14 patients (FL; n ¼ 10 and MCL; n ¼ 4). The stem cell products in all 14 patients tested negative by PCR for both bcl2 (FL) and bcl1(MCL). None of these patients required additional rituximab treatment for in-vivo purging. Eleven patients (FL; n ¼ 7 and MCL; n ¼ 4) underwent Auto SCT. Three Patients with FL opted for ‘Harvest and Hold’ approach and delayed transplantation. Median (range) PFS and OS of patients with FL undergoing Auto SCT were 2 yrs. (9 mo-5 y) and 3 yrs. (2-5 y) and those with MCL were 3 yrs. (2-4y) and 4 yrs. (2-5 y) respectively (median duration of follow up-3yrs.). Conclusion: In this small cohort of patients with FL and MCL treated by R-chemotherapy, 100% of the harvested stem cell products were tumor free by PCR analysis. Our findings suggest that with universal use of rituximab containing chemo-immunotherapy in patients with FL and MCL, additional in-vivo purging may not be required. We may also conclude that persistence of residual/refractory disease after
auto SCT rather than tumor contamination of the graft was the most likely cause of relapse in this cohort. Disclosure of Interest: None declared. P342 Adequacy of peripheral blood stem cell mobilizatıon in patients with relapsed B-cell non-Hodgkin’s lymphoma (NHL) treated wıth bendamustine: a pilot project and a proof of concept study A. Iliff1, C. Divine1, F. Diaz2, O. Aljitawi1, S. Abhyankar1, J. McGuirk1, S. Ganguly1,* 1 Hematology/Oncology, 2Biostatistics, UNIVERSITY OF KANSAS MEDICAL CENTER, Westwood, United States Introduction: Although rituximab containing chemo-immunotherapy regimens have improved response rates in B-cell non Hodgkin lymphomas (B-NHL), a significant percentage of patients will eventually relapse. Autologous stem cell transplantation (auto SCT) may provide long term remission in some of these patients. A pre-requisite for successful transplantation is the ability to collect adequate number of stem cells. Use of alkylating agents has been historically thought to be stem cell toxic. Repeated or prolonged exposure to such chemotherapy may lead to poor mobilization of peripheral blood stem cells, adversely affecting outcome after auto SCT. Bendamustine, an alkylating agent, with or without rituximab have been used in the treatment of various lymphomas in both upfront and salvage settings. Materials (or patients) and methods: The primary objective of this study was to determine if use of bendamustine in combination with rituximab (BR) in patients with relapsed B-NHL, leads to adequate response and collection of stem cells prior to proceeding with high dose chemotherapy and auto SCT. Patients with relapsed or refractory CD20 þ B-NHL, eligible for high dose chemotherapy and auto SCT were enrolled for this study. Enrolled patients received salvage regimen with BR for two to three cycles, depending on the disease response. In evaluable, chemo-sensitive patients, G-CSF was used for stem cell mobilization. A minimum of 2 million CD34 þ cells per Kg of body weight was accepted as adequate collection. All eligible patients underwent high dose chemotherapy followed by auto SCT. Results: Three patients with relapsed diffuse large B cell lymphoma (DLBCL) and 15 patients with relapsed follicular lymphoma (FL) were enrolled and participated in the study. None of the 3 patients with DLBCL responded to BR salvage. Twelve out of 15 patients (83%) with FL had response (9 CR and 3 PR). Ten patients (All with Follicular Lymphoma) underwent stem cell harvest followed by auto SCT. Of the 10 patients, who proceeded onto auto SCT, we were able to collect 42 million CD34 þ stem cells per Kg of body weight in all patients. Only 2 patients had to use plerixafor according to institutional guidelines. All 10 patients engrafted (median neutrophil recovery-11 days; range 10-16 days and median platelet recovery-15.5 days; range 0-21 days). BR was well tolerated with most common AE being GI toxicity followed by neutropenia. No patient died from salvage BR related toxicity. With a median duration of follow up of 2 years, 100% and 42% overall and disease free survival were noted at 3 years post transplantation, respectively. Conclusion: This study shows that combined chemotherapy with BR had a high efficacy in the treatment of relapsed or progressive FL with 12 out of 15 patients (83%) demonstrating response to therapy. Additionally we were able to harvest sufficient stem cells for all patients that proceeded onto auto SCT. In future, we propose to use Bendamustine based salvage chemotherapy followed by Bendamustine containing high dose chemotherapy and auto SCT in the management of relapsed/refractory FL. In this small
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study, we did not see any response to BR salvage in patients with relapsed DLBCL. Disclosure of Interest: A. Iliff: None declared, C. Divine: None declared, F. Diaz: None declared, O. Aljitawi: None declared, S. Abhyankar: None declared, J. McGuirk: None declared, S. Ganguly Funding from: Pharmaceutical Funded Project. P343 Clinical outcome of a consecutive series of lymphoma patients fulfilling indication to allogenic stem cell transplantation S. Bramanti1,*, R. Crocchiolo2, B. Sarina1, L. Morabito1, A. Vai1, C. Carlo Stella1, A. Santoro1, L. Castagna1 1 IST. CLINICO HUMANITAS, rozzano, Italy, 2ISTITUTO CLINICO HUMANITAS, IST. CLINICO HUMANITAS, rozzano, Italy Introduction: Allogeneic stem cell transplantation (allo-SCT) is a curative salvage options for relapse/refractory lymphoma patients with a dismal prognosis. Materials (or patients) and methods: From 2009 to 2013, we searched for all lymphoma in our center, with an indication to proceed to allo-SCT. Indications to allo-SCT were: relapse after high dose chemotherapy and refractoriness to salvage chemotherapy or to more CT lines. The aim of this monocentric retrospective study was to analyze the clinical outcome of an unselected consecutive lymphoma patients for whom an allogeneic transplant was planned. Results: 120 patients were retrieved and 117 were evaluable. 79 patients (67%) underwent to allo-SCT and 41 (35%) did not. The median follow up was 42 months (range 4-85). Patient characteristics were reported in Table 1. Main causes to inelegibility to allo-SCT were: disease progression (56%) and patient refusal (35%). In most of the patients (65%) the donor was haploidentical. The 4 years OS (Figure 1) of the allo group and no allo group were 46% and 20% respectively (P ¼ 0.0001). 4-year NRM in the allo group was 29%. Overall 52% (41) of the patients in allo group is still alive in complete remission. Only 1 patient is alive with active disease. In the no allo group, 9(23%) patients are alive, 5 (13%) in complete remission, 4 with active disease.
Table 1.
Patient characteristics
Allo N 78 (%)
No allo N 39 (%)
P
LNH HD
40 (51%) 38 (49%)
24 (61%) 15 (38%)
0.30
Indication for allo Relapse after auto Relapsed/Refractory High risk Relapse after allo
43 31 3 1
12 26 1 1
0.001
(55%) (39%) (4%) (1%)
(31%) (66%) (2%) (2%)
Donor availability at the time of indication no donor / 11 (28%) Haplo 51 (65%) 18 (46%) HLA id sib 18 (23%) 7 (18%) HLA id MUD 7 (9%) 3 (8%) mmMUD 2 (2%) / Unrelated donor 32 (41%) 7 (18%) search activation Median time from 23 18 diagnosis to indication months months to allo-SCT (4-147 ) (2-234)
0.01 0.12
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Conclusion: Even though allo-SCT is an effective approach in poor prognosis lymphomas, it is still difficult to fully appreciate the real impact on survival because of the lack of prospective randomized study. Furthermore, in published studies, it was reported only on the outcome of patients receiving allotransplanted, and few informations were available of patients potentially candidate to transplant. Similarly to donor vs no donor analysis, this retrospective study suggests that receiving allo-SCT conferred survival advantage in refractory lymphoma patients. Disclosure of Interest: None declared. P344 Prognostic factors and long term outcome after autologous hematopoietic stem cell transplantation in refractory or recurrent Hodgkin’s lymphoma of children and adolescents: a multicenter survey of Turkish Pediatric BMT Study Group V. Kesik1,*, E. Atas1, M. Karakukcu2, S. Aksoylar3, F. Erbey4, N. Tacyildiz5, A. Kupesiz6, H. Oniz7, E. Unal2, S. Kansoy3, G. Ozturk4, M. Elli8, Z. Kaya9, E. Unal5, V. Hazar6, S. Yilmaz Bengoa10, G. Karasu11, D. Atay4, A. Dagdemir8, H. Oren10, U¨. Kocak9, A. Yesilipek11 1 Department of Pediatric Oncology, Gulhane Military Medical Academy, Ankara, 2Department of Pediatric Hematology&Oncology and BMT Unit, Erciyes University Faculty of Medicine, Kayseri, 3 Department of Pediatric Oncology and BMT Unit, Ege University Faculty of Medicine, Izmir, 4Pediatric BMT Unit, Medical Park Bahc¸elievler Hospital, Istanbul, 5Department of Pediatric Oncology and BMT Unit, Ankara University Faculty of Medicine, Ankara, 6Department of Pediatric Hematology&Oncology and BMT Unit, Akdeniz University Faculty of Medicine, Antalya, 7 Department of Pediatric Oncology and BMT Unit, Tepecik Education and Research Hospital, Izmir, 8Department of Pediatric Oncology and BMT Unit, On Dokuz Mayıs University Faculty of Medicine, Samsun, 9Department of Pediatric Hematology and BMT Unit, Gazi University Faculty of Medicine, Ankara, 10Department of Pediatric Hematology and BMT Unit, Dokuz Eylu¨l University Faculty of Medicine, Izmir, 11Pediatric BMT Unit, Medical Park Go¨ztepe Hospital, Istanbul, Turkey Introduction: We examined the effect of some factors and age-adjusted international prognostic index (AAIPI) on relapse and survival of the children with Hodgkin’s lymphoma (HL) undergone autologous stem cell transplantation (ASCT) as a multicenter study in Turkey. Materials (or patients) and methods: Sixty-one children from 10 centers in Turkey who underwent autologous ASCT because of refractory or recurrent HL were retrospectively
P345 Autologous/Allogeneic Stem Cell Transplantation following High-dose Sequential Chemotherapy in Patients with Stage III-IV Burkitt and Lymphoblastic Lymphoma : Long term survival data from LLBL2001 protocol B. Gungor1, C. Unsal1, I. Barista2, M. Adam1, Y. Koc1,* 1 SCT Unit, Medical Park, Antalya, 2Oncology, Hacettepe University, Ankara, Turkey
analyzed. The evaluated possible prognostic factors were age, relapse number, stage, Hodgkin type, bulky tumor, spleen, extranodal bone and bone marrow involvement, B symptoms, involvement, hemoglobin, WBC, lymphocyte, monocyte, MPV, ferritin, albumin, LDH, sedimentation at diagnosis, pretransplant Karnofsky, PET positivity, number of chemotherapy regimens, radiotherapy, postransplant day 15 and 100 lymphocyte, neutrophil /lymphocyte ratio, MPV, and day 100 PET positivity. Risk factors affecting relapse free survival (RFS) were evaluated using stratified Cox regression. Results: At the time of study, 44 of 61 patients were alive, 15 patients were expired, and 2 patients were lost. Nine patients due to progressive disease, 2 patients due to infection, 4 patients due to other reasons except relapse were dead. Nonrelapse mortality rate was 40%. Median relapse time was 11 months (range, 1-105). Three-year overall survival (OS)/RFS were 77.3% and 68.5% with a median follow-up of 27 months (range, 1-114 months) for all patients, respectively. Subtype of HL (P ¼ 0.042, HR ¼ 1.6), WBC 410000/mm3 at postHSCT day100 (P ¼ 0.009, HR ¼ 5.4) no RT before HSCT (P ¼ 0.019, HR ¼ 3.1), no remission at HSCT (P ¼ 0.012, HR ¼ 3.2), refractory disease (P ¼ 0.003, HR ¼ 4.1), type of relapse (P ¼ 0.003, HR ¼ 0.17), PET-CT positivity at post-HSCT day 100 (P ¼ 0.009, HR ¼ 4.4), serum albumin under 2.5 mg/dl (P ¼ 0.035, HR ¼ 13.1) were predictive of relapse free survival (RFS). The factors effecting survival were represented on Table 1. When AAIPI was adapted to children according to LDH 500 IU/L instead of 250 IU/L, we called this new term as CAAIPI. Threeyear DFS rates were 100% for Score 0, 61.3 % for Score 1, 52.7 % for Score 2 (P ¼ 0.26) and OS rates were 80.0% for Score 0 (exitus due to infection), 93.3% for Score 1, 77.0% for Score 2 (P ¼ 0.50) according to CAAIPI. Conclusion: Although ASCT increase the survival rate, survival is worse among patients that relapse after HSCT, with no remission at the time of HSCT, and bone marrow positivity at diagnosis. Therefore, we suggest more aggressive therapies for children with bone marrow involvement at diagnosis and early ASCT at the first remission. We also suggest different treatment approaches for the latter. The ones with high IPI or AAIPI score can also be treated in this manner. We suggest that CAAIPI score that use LDH level as 500 IU/L is more useful in childhood. In addition, the characteristics that showed up significance in univariate but not in multivariate analysis appears to have an influence as well and might show a stronger correlation in larger trials. Disclosure of Interest: None declared.
Introduction: In patients with advanced stage highly-agressive NHL, a satisfying response rate and long-term survival cannot be achieved by conventional standard lymphoma chemotherapy regimens. Despite reports of high-dose chemotherapy regimens coupled with or without stem cell transplantation (SCT), medical literature lacks a standard treatment protocol in the management of patients with stage III-IV Burkitt (BL) and Lymphoblastic Lymphoma (LL). In this study, we report disease-free (DFS) and long-term overall survival (OS) of patients treated with LLBL2001 treatment protocol consisting of high-dose chemotherapy and autologous or allogeneic SCT in the past 12 years. Materials (or patients) and methods: Thirty newly diagnosed patients with LL (n ¼ 18) and BL (n ¼ 12) were treated with the LLBL2001 protocol consisting of induction (CTX þ DOX þ VCR þ PRED), consolidation with high-dose chemotherapy (2 cycles of HIDAC þ HDMTX, 3g/m2 each) and pegylated L-asparaginase, prophylactic intrathecal MTX þ Ara-C, pre-transplant conditioning with IIVP and HD-VP16 chemotherapy (2 g/m2), followed by autologous SCT with HD-MTZ (60 mg/m2) and HD-MEL (180 mg/ m2) conditioning. Patients with B-cell phenotype received invivo purging with rituximab prior to Auto-SCT. Protocol did not include prophylactic cranial radiotherapy. In cases with relapse or CNS involvement during administration of the LLBL2001 protocol, patients proceeded with allogeneic SCT after CR was achieved. Median age of the patient group was 30 (17-48) and 86% had bulky disease Z 6 cm. Forty-seven percent of patients had bone marrow involvement at diagnosis. Results: Following induction phase, 87% patient achieved CR, and transplantation could not be performed in 4 patients due to refractory lymphoma or social reasons. Twenty-six patients completed the treatment protocol (23 Auto-SCT, 3 Allo-SCT). Eight patients died due to relapsed/refractory disease (n ¼ 5), sepsis (n ¼ 2), and cGVHD (n ¼ 1). Significant complications observed in the patient cohort are as follows; neutropenic fever (25), CNS recurrence (4), sepsis (3), VZV reactivation (3), acute GVHD (3), chronic GVHD (3), aseptic femoral head necrosis (3), CMV reactivation (2), tumor lysis (2), BK virus cystitis (1), fungal pneumoniae (1), HSV keratitis (1), IVC thrombosis (1). When analysed by intent-to-treat principle, 12 year DFS and OS were 81% (88% in BL, 78% in LL, P ¼ 0.56) and 73% (88% in BL, 67% in LL, P ¼ 0.32), respectively. No relapse was observed beyond 3 years.
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Conclusion: LLBL2001 protocol is capable of inducing high long-term disease-free and overall survival rates and provides a curative option for patients with advanced stage highlyaggressive bulky NHL. Disclosure of Interest: None declared. P346 Allogeneic hematopoietic stem cell transplantation in patients with T cell lymphoma is prefer to chemotherapy Y. Luo1,*, Y. Wu2, Y. Tan2, H. Huang2 1 Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, 2Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Introduction: To compare the efficiency and safety of allogeneic hematopoietic stem cell transplantation versus chemotherapy in patients with T cell lymphoma. To analyze the clinical features and prognostic factors of these patients. Materials (or patients) and methods: 59 patients who were diagnosed as T cell lymphoma by pathology between Jan 1st 2007 to Dec 31 2012 in the First Affiliated Hospital of Zhejiang University School of Medicine were enrolled and analyzed retrospectively. All of the cases were determined according to WHO 2008 classification and classified by Ann Arbor staging system. These patients were all younger than 60 years old at clinical stage III/IV period and have completed two or more courses chemotherapy. 19 patients finally received allogeneic stem cell transplantation and 40 patients still received chemotherapy, followed up by telephone to Dec 31 2013. We evaluate the PFS and OS between two groups. At the same time, some clinical factors such as the level of serum lactate dehydrogenase (LDH) , B symptoms, and international prognostic index (IPI) were collected to analyze the relation between these parameters and survival. Results: After a median follow-up of 21 months, one-year overall survival(OS) of SCT and CTx were 94.4% and 65.0% (Po0.05). Three-year OS of SCT and CTx were 57.2% and 39.1% (Po0.05). Five-year OS of SCT and CTx were 45.8% and 23.1% (Po0.05). One-year progression-free survival (PFS) of SCT and CTx were 88.5% and 47.5% (Po0.001). Three-year PFS of SCT and CTx were 51.5% and 22.5% (Po0.001). Five-year PFS of SCT and CTx were 41.2% and 7.5% (Po0.001). The treatment related mortality (TRM) rate has no statistical significance between the two groups. 100-day TRM of SCT and CTx were 2.5% and 10.5% (P ¼ 0.190). One-year TRM of SCT and CTx were 17.5% and 15.8% (P ¼ 0.870). Three-year TRM of SCT and CTx were 27.5% and 21.1% (P ¼ 0.595). Five-year TRM of SCT and CTx were 30.0% and 26.3% (P ¼ 0.770). The level of serum LDH, extranodal violations, Ann Arbor stage and international prognostic index (IPI) were statistically relevant in predicting the prognosis of the overall survival (OS). Multivariate analysis showed that only treatment is an independent prognostic factor. Conclusion: Allogeneic hematopoietic stem cell transplantation is safe and effective for T cell lymphoma patients and can result in long-term disease control. Disclosure of Interest: None declared.
Multiple myeloma P347 The impact of autologous stem cell transplantation (ASCT) on survival of patients with multiple myeloma (MM) aged under 65 years in compliance with risk stratification on the modified scales mSMART 1.0 and mSMART 2.0 A. Garifullin1,*, I. Martynkevich1, S. Voloshin1, L. Martynenko1, A. Kuvshinov1, L. Stelmashenko1, E. Kleina1, G. Salogub2, E. Karyagina3, A. Schmidt1, A. Kuzyaeva1, K. Abdulkadyrov1 1 Russian Research Institute of Hematology and Transfusiology, 2 Saint-Petersburg Pavlov State Medical University, 3City Hospital N15, Saint-Petersburg, Russian Federation Introduction: Seventy percent of patients with MM have chromosomal aberrations. Part of them have prognostic value. That allows to stratify patients in separate groups of risk and select optimal strategy of treatment. Autologous stem cells transplantation is one of the effective methods for patients aged under 65 years with MM. Materials (or patients) and methods: We retrospectively analyzed 121 patientsr65 years (median age 56 years, range 26 - 65; male/female – 1:1.24). The incidences of genetic abnormalities were determined in all cases. Cytogenetic studies were performed on bone marrow samples using standard GTD-method. Metaphase FISH analyses were performed according to the manufacturer’s protocol using DNA probes: LSI 13(RB1)13q14, IGH/CCND1, IGH/FGFR3, LSI TP53 (17q13.1) (Abbott). Stratification of patients was carried in groups of risk according to the modified molecular classification mSMART 1.0 and mSMART 2.0. Patients with complex karyotype (3 and more chromosomal aberrations) were additionally entered in high risk group of both systems. Results: In group of patients after ASCT (single and tandem) (n ¼ 45) 5-years OS was 92% vs 86% (P ¼ .14) in group without ASCT (n ¼ 76). 5-years OS (mSMARTmod 1.0) in standard risk groups (mSMARTmod 1.0 and mSMARTmod 2.0) of patients with ASCT (n ¼ 34) and without ASCT (n ¼ 65) was 87% and 83%, respectively (P ¼ .68). In high risk group (mSMARTmod 1.0) 5years OS of patients with ASCT (n ¼ 11) was 100%, without ASCT (n ¼ 11) – 55% (P ¼ .04). Results 5-years OS (mSMARTmod 2.0) in intermediate risk group in patients with ASCT (n ¼ 9) was 100%, without ASCT (n ¼ 7) – 64% (P ¼ .12). In high risk group (mSMARTmod 2.0) was reached only 2-years OS of patients with and without ASCT which made 100% and 50%, respectively (P ¼ .36). The median of OS wasn’t reached in all groups of patients. Conclusion: ASCT improves treatment outcome in young MM high risk patients. Disclosure of Interest: None declared. P348 Outcome improvement in newly diagnosed Multiple Myeloma patients elegibile for Autologous Stem Cell Transplant: a single centre retrospective analysis A. Natale1,*, S. Pulini1, L. Torti1, A. M. Morelli1, P. Ranalli1, S. Angelini2, A. Spadano1, P. Di Bartolomeo1 1 Clinical Hematology, Department of Hematology, Transfusion Medicine and Biotechnology, ‘‘Spirito Santo’’ Civic Hospital, Pescara, 2Hematology, Mazzoni Hospital, Ascoli Piceno, Italy Introduction: Overall survival (OS) in Multiple Myeloma (MM) patients (pts) significantly improved during the past decade after the introduction of new drugs (proteasome inhibitors and immunomodulators). Materials (or patients) and methods: We retrospectively analyzed the outcome of 165 newly diagnosed MM pts transplanted in our institution. Various induction regimens were administered during different time periods: conventional chemotherapy-based (VAD 27 pts, 17% of total; TT2-like 37,
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22%; EDAP 1, 1%), thalidomide-based (TD 15, 9%), bortezomibbased (VTD 62, 37%; VD/VCD 11, 7%; PAD 3, 2%), lenalidomide-based (RD 9, 5%). We analyzed data of the 4 more representative regimens (VAD, TT2-like, TD and VTD) considering the number of the pts treated. Results: From November 1999 to December 2013 165 MM pts received at least one autologous stem cell transplant (ASCT) after induction. Median age at start therapy was 58 years (range 36-71) and median follow up time 52 months (range 7-179). 66% of the pts achieved at least very good partial response (VGPR) after induction therapy: sCR (stringent Complete Response) was achieved in 1%, CR in 26%, VGPR in 39%. After VTD induction regimen pts obtained the overall best quality of response (86% of them at least in VGPR) and the highest percentage of sCR (2%). 22 pts (13%) received a salvage therapy before ASCT: 13 pts experienced PD (Progressive Disease), 9 pts were considered in suboptimal or unsatisfactory response (4 of them were in Stable Disease, SD and 5 in Partial Remission, PR). After induction therapy 67 pts received a single ASCT, 98 pts double ASCT; 11 patients received tandem autologous and allogeneic SCT. ASCT improved quality of response in all patients, irrespective of the induction regimen received. None of the pts examined underwent PD after ASCT. After ASCT 87% of the pts were at least in VGPR: sCR was achieved in 5%, CR in 48%, VGPR in 34%. Also after transplant VTD induction confirmed to be the regimen with the best quality of response (93% were at least in VGPR) and the highest percentage of sCR (11%). MM relapsed after ASCT in 86 pts, median time from ASCT to relapse was 24 months (range 4-113). At last follow-up 113 (69%) pts were alive and 52 (31%) pts died, the main cause of death was PD (85%). The median OS was 9 years and 11 months, after 15 years the probability of survival was 35%. Considering the different induction regimen the best probability of survival (77% after 7 years and 6 month from start therapy) was achieved after VTD (median OS not reached, more than 7.5 years), followed by VAD (probability of survival 35% at 14 years and 11 months from start therapy, median OS 11 years), TT2-like (probability of survival 45% at 14 years and 8 months, median OS 9 years and 11 months) and TD (probability of survival 38% at 7 years and 8 months, median OS 6 years and 11 months). Conclusion: The introduction of novel agents improved outcome of MM pts transplanted in our center, according with published data. The improvement of quality of response after induction therapy results in better long term control of the disease. Moreover the avaibility of new drugs in relapse post transplant for patient treated with first line chemotherapy regimen improved OS also in this setting. ‘‘Does the dogma of multiple myeloma is incurable still hold?’’, Dr. Bart Barlogie recently asked (Blood, 2014). We are moving forward. Disclosure of Interest: None declared. P349 Polyclonal activation of the immunoglobulin determined by free-light assay after lenalidomide-treatment ın patients wıth multiple myeloma prolongs time to treatment failure A. L. Kluger1,*, G. Scho¨n2, A. Gerritzen3, U.-M. von Pein1, F. Ayuk1, C. Wolschke1, N. Kro¨ger1 1 Department of Stem Cell Transplantation, 2Institute of Medical Biometry, University Medical Center Hamburg, Hamburg, 3 Medical Laboratory Bremen, Bremen, Germany Introduction: Lenalidomide is an immunomodulatory drug, which induced T-cell and NK-cell activation and is approved for treatment of patients with multiple myeloma. Here we investigated the effect of lenalidomide on polyclonal activation of immunoglobuline detected by free-light assay and on its impact on outcome in patients with multiple myeloma.
Materials (or patients) and methods: In this study, we included 61 patients with multiple myeloma (male ¼ 42, female ¼ 18) with a median age of 61 years (range 39-77 years), who received lenalidomide at a median dose of 15 mg either as maintenance (n ¼ 28) or as salvage therapy (n ¼ 33) from 2006 to 2013 in our institution. The median duration of lenalidomide therapy was 14 months (range 2-69 months). 29.51% (n ¼ 18) received lenalidomide as single agent and 70% (n ¼ 43) in combination with Dexamethasone. All patients were inspected for the presence of a polyclonal activation on the basis of free-light chain serum levels. "Polyclonal activation" was defined as an increase of both free-light chain serum levels out of normal range (range k: 3.3mg/l-19,4mg/l; l: 5.7mg/l-26.3mg/l). Results: A total of 23 patients (37,70%) had shown a polyclonal activation during lenalidomide-treatment as described above. Polyclonal activation was seen in 11/23 patients with partial response, in 9/23 with complete response and in 2/23 with stable disease. One response-level was not specified. Comparing the time to treatment failure of the polyclonal activation population (n ¼ 23) and the non-polyclonal activation population (n ¼ 38), the patients with polyclonal activation had a significant higher median time to treatment failure (41.03 vs.16.9 months, P ¼ 0.049). The significance was meanly seen in patients with relapsed myeloma (58.0 vs. 12.94 months, P ¼ 0.043) while in maintenance the difference (41.03 vs. 26.0 months) was not significant (P ¼ 0.40). Conclusion: Our study suggests that a polyclonal activation measured by free-light chain assay after lenalidomide therapy resulted in a prolonged time to treatment failure, especially for the relapse therapy population. Disclosure of Interest: None declared. P350 Albumin is the main predıctor of survival after Autologous Stem Cell Transplantation in Multiple Myeloma patients E. Birtas Atesoglu1,*, A. Hacihanefioglu1, Z. Gulbas2 1 Department of Hematology, Kocaeli University, 2Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, Kocaeli, Turkey Introduction: Multiple myeloma(MM) remains a rarely curable disease. However, studies have shown benefit for overall survival (OS) and progression free survival (PFS) by the use of high-dose chemotherapy with subsequent autologous stem cell transplantation (ASCT). The aim of the present study was to retrospectively analyze the impact on outcomes of various factors especially albumin levels at the time of ASCT in MM patients. Materials (or patients) and methods: We analyzed retrospectively 137 multiple myeloma(MM) patients who had undergone autologous stem cell transplantation from August 2010 to January 2013. Detailed information concerning patients presentation at diagnosis as well as the disease status and other variables of known prognostic importance were evaluated before the initiation of conditioning chemotherapy for AHSCT. Results: Median follow-up time is 22 (1-46) months. When we analyze, the impact of gender, b2microglobulin levels (o3,5 vs Z3,5), ISS stage, LDH (normal limit vs Z upper normal limit), ferritin (o1000 vs Z1000), hemoglobin level (o12 vs Z12), age (r65 vs 465), remission status before transplantation, tandem vs single transplantation on overall survival (OS) and progression free survival (PFS), none of them had an impact on OS or PFS (P40,05). Only patients with albumin levels 43,5 had significantly better OS and PFS in comparison to patients with albumin levels o3,5 (P ¼ 0,02, P ¼ 0,04, respectively) (Figure 1). Conclusion: We conclude that albumin level measured before ASCT is a useful predictor for both OS and PFS in MM patients. Disclosure of Interest: None declared.
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P352 Minimal residual disease by multiparameter flow cytometry in Mieloma patients undergoing an up-front tandem ASCT: a real life single-institution study I. Cordone1, F. Marchesi2,*, S. Masi1, V. Summa1, M. L. Dessanti2, S. Gumenyuk2, R. Merola1, G. Cigliana1, G. Orlandi1, F. Palombi2, F. Pisani2, A. Romano2, A. Spadea2, L. Conti1, M. C. Petti2, A. Mengarelli2 1 Clinical Pathology Unit, 2Hematology and Stem Cell Transplantation Unit, Regina Elena National Cancer Insitute, Rome, Italy Introduction: Multiparameter Flow Cytometry (MFC) is a powerful technique in both diagnosis and Minimal Residual Disease (MRD) detection in multiple myeloma (MM) patients. We performed a prospective single center study on real-life MM patients who underwent up-front tandem autologous stem cell transplantation (ASCT) utilizing an innovative MFC MRD monitoring. The aims of the study were 1) to establish whether the 2nd ASCT allows a better quality of response in terms of MRD assessment; 2) to evaluate patients’ clinical outcome after 2nd ASCT according to MRD status and to postASCT treatment. Materials (or patients) and methods: From January 2006 to January 2012, 50 consecutive MM patients, median age 56 years (41-68) treated with a program containing an up-front tandem ASCT (Melphalan 200 mg/m2) entered the study. As induction, 23 patients received 3 courses of VAD chemotherapy and 27 received 3 courses of novel agents (Bortezomibbased n ¼ 21; immunomodulators only n ¼ 6). After the 2nd ASCT 28 patients underwent a consolidation/maintenance treatment based on Bortezomib (n ¼ 14), immunomodulators only (n ¼ 12) or others (n ¼ 2). Plasma-cells (PC) MFC characterization was performed on bone marrow (BM) samples at diagnosis utilizing the antibody combinations (Fitc/PE/PerCP/PE-Cy7/APC/APC-CY7): I)CD28/CD138/CD45/ CD38/CD33/CD20; II)CD38/CD138/CD45/CD56/CD117/CD19. The PC phenotypic aberrancies detected in the surface characterization were used as patient-specific disease markers (PSDM) to document intra-cytoplasmic immunoglobulin (cy-Ig) light chains restriction: III)cy-Ig Lambda/cy-Ig Kappa/CD19/ CD38/anti-PSDM/CD45 at diagnosis, after induction and at day þ 100 approximately after 1st and 2nd ASCT. Patients were
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considered to be in immunophenotypic complete response (iCR) when pathological PC were undetected at the MFC sensitivity limit of 10-4 cells, allowing a maximum sensitivity of detection of 0.001% in all cases. Treatment response by MFC criteria and progression-free survival (PFS) according to MRD status after 2nd ASCT were evaluated. Results: A statistically significant decrease of the mean (±SD) percentage of residual pathologic PC (2.6% [±6.3] vs 0.2% [±0.3]; P ¼ 0.01) and a significant increase of the proportion of patients in iCR (13 [26%] vs 29 [58%]; P ¼ 0.001) were observed comparing induction and 1st ASCT but not comparing 1st and 2st ASCT (0.2% [±0.3] vs 0.1% [±0.8]; P ¼ 0.764 and 29 [58%] vs 36 [72%]; P ¼ 0.236). The 4 years-PFS (median follow-up from diagnosis) of patients in iCR after the 2nd ASCT was significantly better than that of MFC-MRD positive patients (72% vs 23%; P ¼ 0.002). In 36 patients in iCR and 14 patients MFC-MRD positive after 2nd ASCT, no significant differences were observed in terms of PFS between those receiving (19 vs 9) or not (17 vs 5) a consolidation/maintenance treatment (4yPFS: 73% vs 57% [P ¼ 0.336] and 47% vs 20% [P ¼ 0.703], respectively). Conclusion: The cy-Ig light restriction on PSDM is a powerful tool of clinical relevance in MM MRD monitoring. In MM patients undergoing an up-front tandem ASCT, the 2nd ASCT doesn’t seem to add significant advantages in terms of quality of response assessed by MFC compared with the first one. With the caution due to the low number of patients, our data seem also to suggest that a consolidation/maintenance treatment in patients in iCR after 2nd ASCT may not be translated into significantly better PFS. Disclosure of Interest: None declared. P353 23 year Iranian experience in HSCT in multiple myeloma H. Kamranzadehfumani1,*, K. Alimoghadam1, M. Jahani1, M. Vaezi1, B. Bahar2, A. Mousavi1, A. Ghavamzadeh1 1 HORCSCT, 2Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic Of Introduction: Aim: Autologos HSCT is standard of care for eligible myeloma patients and allogenic HSCT may be the only curable treatment. We began this procedure in 1991 and later we performed allogenic HSCT in some cases. Materials (or patients) and methods: Methods: This retrospective study is a report of 557 patients who underwent HSCT in our center. The conditioning regimen was melphalan for autologos and melphalan þ fludarabine for allogenic
HSCT.MTX þ Cyclosporine A were used as GVHD prophylaxis. All cases of allogenic transplantation, were performed from match sibling donor. Results: Results: 557 patients ( 348 male,209 female) from 1991 to September 2014 resived HSCT. Mean age at transplant is 51 years (range 24-72). 489 autologos and 68 allogenic HSCT performed. Median follow up time is 27.7 months in autologos and 27.2 months in allogenic group. 97 patients (17.4%) died (1.9%in allogenic, 15.5% in autologos). The major cause of death in two group was relapse. 168 relapses ocured (31.3% of autologos, 10.2% of allogenic). Mean time to relapse is 24.7 months. In allogenic group 36% had acute and 31.34% had chronic GVHD. Most of them were limited. 2 patients died from GVHD(acute and chronic) and one patient die from CMV infection and one from DVT. Conclusion: Conclusion: We found relapse rate is lower in allogenic HSCT than autologos (P ¼ 0.01), but there is no difference in overall mortality rate. Acute or chronic GVHD had no relationship with relapse rate and it may be due to short time of follow up and few patients number in allogenic group. we found no relationship with sex, mortality and relapse. Disclosure of Interest: None declared. P354 The impact of pre-transplant remission status on posttransplant disease outcomes in patients with multiple myeloma T. Toptas1, I. Kaygusuz–Atagunduz2,*, F. Pepedil1, A. Eser1, O. Kara1, A. Sezgin1, T. Ozgumus1, C. Adiguzel1, T. Firatli–Tuglular1 1 Marmara University Hospital, Istanbul, Turkey, 2Department of Hematology, Marmara University Hospital, Istanbul, Turkey Introduction: We aimed to investigate the impact of pretransplant response depth on myeloma outcomes. Materials (or patients) and methods: We retrospectively analyzed the data of a total of 56 patients with multiple myeloma who underwent autologous stem cell transplantation (SCT) between 2000 and 2010. Response status was defined according to IMWG criteria. Post-hoc power analysis revealed that study show a hazard ratio (HR) of 0.5 with a power of 72% and an overall two-sided type I error of 5%, with use of a two-sided log-rank test. Results: Time-to-transplantation, median follow-up, and treatment related mortality were 12 (6-36) months, 32 months, and 3.6%, respectively. For all cohort, median PFS, TTP, TNT, and OS were 25, 32, 47, and 84 months respectively. 5- and 10-year OS were 60.9%, and 26.6%. The proportion of patients who achieved Z very good partial response (VGPR) before transplantation was 41.9%. Post-transplant ratio of patients with ZVGPR estimated to be 75.8%. There were no significant differences between patients with a pre-transplant response ZVGPR and partial response (PR) in terms of PFS (median 33 vs 24 months, P ¼ 0.3), TTP (not reached vs 25 months, P ¼ 0.2), TNT (not reached vs 33 months, P ¼ 0.09), and OS (not reached vs 71 months, P ¼ 0.8). Conclusion: Patients with multiple myeloma who achieve PR or ZVGPR before transplantation have similar disease outcomes. Since only 30% of patients will have a ZVGPR with novel agents, at least PR is a reasonable pre-transplant treatment goal. Disclosure of Interest: None declared. P355 bortezomib targets side population of human myeloma cells enriched clonogenic tumor initiation subpopulations J.-H. Won1,*, D. S. Hong2, K. H. Kim1, J. Yun2, H. J. Kim3, S. Kim2, H. J. Kim2, S.-C. Lee3, S. B. Bae3, N. S. Lee1, K.-T. Lee3, S. K. Park2 1 Soonchunhyang University Hospital, Seoul, 2Soonchunhyang University Hospital, Bucheon, 3Soonchunhyang University Hospital, Cheonan, Korea, Republic Of Introduction: Cytotoxic chemotherapy based treatment of multiple myeloma (MM) is not curative and the disease eventually recurs. This is in part because although currently
available antiMM strategies have been effective in targeting the bulk of tumor cells, they do not target the tumor initiating subpopulation or cancer stem cells. Materials (or patients) and methods: In our study, we performed flow cytometry based Hoechst 33342 staining to evaluate the existence of side population (SP) cells and investigated the prevalence and biologic function of SP cells in multiple myeloma cell lines. Results: Frequency of SP cells was heterogenous: most cell lines (ARH77, IM9, and MM.1R) had a less than 1% of SP cells but RPMI8226 had around 10% of SP cells. Sorted SP cells showed a capacity of higher proliferation abilities and clonogenecity than main population (MP) in RPMI8226 myeloma cell line and also observed the ability of SP cells to regenerate original population. We detected higher activity of ABCG2 in SP cells which associated with high rates of proliferation. But we could not found the difference of expression of specific surface markers between SP and MP, such as expression of CD138, CD34, CD38, CD19, CD20, and CD27. We compared relative sensitivity of SP and MP to clinical anti-myeloma agents (bortezomib, dexamethasone, doxorubicin, and melphalan), bortezomib only exhibit substantial activity to SP cells. Conclusion: Our studies demonstrate SP fraction of myeloma cells contained clonogenic tumor initiating potential and provide new mechanisms of action for bortezomib to target the SP cells. Further studies to define the myeloma initiating cell markers leave in abeyance. Disclosure of Interest: None declared. P356 Additional therapy before autologous transplantation improves the quality of response but not the outcome of Multiple Myeloma patients: a single centre experience L. Torti1,*, S. Pulini1, A. Natale1, A. M. Morelli1, R. Spadano1, S. Angelini2, A. Spadano1, P. Di Bartolomeo1 1 Clinical Hematology, Department of Hematology, Transfusion Medicine and Biotechnology, ‘‘Spirito Santo’’ Civic Hospital, Pescara, 2Hematology, Mazzoni Hospital, Ascoli Piceno, Italy Introduction: Newly diagnosed Multiple Myeloma (MM) patients (pts) who are eligible for high dose chemotherapy (CHT) and autologous stem cell transplant (ASCT) receive induction therapy before ASCT. Results from randomized clinical trials incorporating novel agents suggest that the quality of response after induction correlates with a longer control of MM. However, data published in the literature show that ASCT may improve the quality of response even in pts refractory to induction. It is not clear if pts with suboptimal response really benefit from additional treatment before ASCT. Materials (or patients) and methods: We retrospectively analyzed 165 newly diagnosed MM pts transplanted in our institution from November 1999 to December 2013. After different induction regimen (CHT-based, thalidomide-based,
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bortezomib-based or lenalidomide-based) 143 pts underwent ASCT; 22 pts (13%) received additional therapy before ASCT. Results: Among the 22 pts analyzed median age at start therapy was 58 years (range 36-66 years) and median follow up time 47 months (range 18-144 months). As first line therapy 6 pts received conventional CHT, 7 bortezomib basedregimen, 4 thalidomide-based and 5 lenalidomide-based. After induction 13 pts were in PD (Progressive Disease), 9 pts were considered in suboptimal/unsatisfactory response (4 in Stable Disease, SD and 5 in Partial Response, PR). They therefore underwent additional therapy: 15 pts were treated according bortezomib-based regimen, 4 lenalidomide-based, 3 conventional CHT. 18% of the pts achieved Complete Response (CR), 45% Very Good Partial Response (VGPR), 32% PR and 5% SD. All of the 22 pts received at least one ASCT, 11 pts double ASCT, 5 tandem autologous and allogenic SCT. ASCT improved quality of response in all patients and none of them underwent PD after transplant. After ASCT 23% patients were in CR, 41% in VGPR and 36% in PR. 15 pts experienced at least one MM relapse with a median time from ASCT of 16 months (range 4-51). At last follow up 10 pts were alive and 12 were dead, 11 for PD and 1 patient for complications related to Allogenic BMT (acute GvHD and mycotic pneumonia). The median Overall Survival (OS) for pts who received addition therapy before ASCT was 4 years and 4 months and the probability of survival was 23% at 12 years, significantly inferior respect to pts that did not receive additional therapy (median OS 11 years, 38% at 14 years and 11 months), p-value ¼ 0.0009. Conclusion: There is no agreement if MM patients with suboptimal response to induction may benefit from additional alternative therapy with the intent to maximize pretransplant response and therefore the control of MM. In our retrospective analysis pts that received additional therapy before ASCT improved depth of response but this result did not translate in improvement of OS. Our study has some limitations (retrospective study, number of pts, heterogeneous therapy administered, lack of biological data) but it addresses a significant clinical issue. In the era of the new drugs we aim to achieve the best response before ASCT, but if the quality of the response does not correlate with outcome do we need to postpone ASCT considering that high-dose melphalan remains one of the most active treatment in MM? We hope that prospective, randomized clinical trials will clarify the importance of depth or response before and after ASCT. Disclosure of Interest: None declared. P357 Tandem autologous plus reduced-intensity allogeneic stem cell transplantation without standard immunosuppression in young patients with relapsed and refractory multiple myeloma O. Pokrovskaya1,*, L. Mendeleeva1, E. Makunina1, L. Kuzmina1, M. Firsova1, M. Nareyko1, E. Urnova1, I. Galtseva1, T. Sorokina1, E. Parovichnikova1, V. Savchenko1 1 Bone Marrow Transplant Department, National Research Center for Hematology, Moscow, Russian Federation Introduction: Multiple myeloma (MM) remains an incurable disease and almost all patients (pts) will relapse or develop refractory disease even after autologous stem cell transplantation (autoSCT). Allogenic bone marrow transplantation (alloBMT) carries the benefit of graft versus myeloma effect but also high risk of treatment related mortality. Combining benefits of cytoreductive-therapy with adoptive immunotherapy forms the basis of a tandem auto-allo SCT treatment strategy which is very actual, especially in young pts with MM. Materials (or patients) and methods: Since April 2012 to December 2014 four young pts (three men and one woman, 26-40 year old) with advanced multiple myeloma underwent tandem autoSCT and alloBMT in the department of Bone Marrow Transplantation. 3 of them had myeloma G and one revealed secretion of immunoglobulin light chains only. At the
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time of diagnosis all pts had negative prognostic factors, such as unfavourable cytogenetics, extramedullary disease, high LDH level, ISS stage III. All patients received first-line induction chemotherapy with bortezomib þ dexamethasone þ /cyclophosphamide þ /- adriamicin. 3 pts progressed during induction and received 2 to 4 lines of therapy containing lenalidomide, bendamustine and cisplatin. One patients underwent irradiation of extramedullary lesion. The mobilization regimen was cyclophosphamide (4 g/m2) followed by daily administration of G-CSF. AutoSCT entailed conditioning with melphalan 200 mg/m2. Conditioning regimen before alloBMT was composed of fludarabine, busulfan and horse anti-thymocyte immunoglobuline. There was no standard acute graft-versus-host disease (GVHD) prophylaxis. For this purpose cyclophosphamide at dose 50 mg/kg at day þ 3, þ 4 was used. Also all pts received mesenchymal stem cells for acute GVHD prophylaxis at the day of WBC recovery 41*10^9/l. Response was defined according to IMWG criteria. Results: After induction only 1 pt achieved partial response, 1 had minimal response and 2 pts had refractory disease. No increase of antimyeloma effect was seen after autoSCT. The median time between auto-SCT and allo-BMT was 5 months (2-5). Neutrophil engraftment was achieved at a median day þ 17. Late acute GVHD had occurred in 1 case. This pt responded to initial treatment with prednisone 1 mg/kg. Three pts achieved complete response (CR) in 2-4 months after alloBMT, (with stringent CR in one case) and 1 pt remained in very good partial response after 4 months after alloBMT. All patients are alive. The median time of follow-up after alloBMT was 7 months (4-18,5). Conclusion: Despite high risk of treatment related mortality, even performed in advanced stage of MM, tandem autoSCT plus alloBMT without standard acute GVHD prophylaxis may result in high CR rate and long-term overall and progressionfree survival. Disclosure of Interest: None declared. P358 The efficacy and tolerability of pomalidomide treatment for multiple myeloma in patients after allogeneic hematopoietic stem cell transplantation S. Janjetovic1,*, F. Ayuk1, M. Christopeit1, C. Wolschke1, U.-M. von Pein1, T. Stuebig1, N. Kroeger1 1 University Medical Center Hamburg-Eppendorf, Hamburg, Germany Introduction: Disease outcome in patients suffering from multiple myeloma (MM) has dramatically improved with the introduction of novel agents, including immunomodulators and proteasome inhibitors. Increased rates of complete remission translated into increased overall survival. Pomalidomide is one of a new immunomodulatory agent approved for the treatment of advanced MM patients. The goal of our study was to determine the tolerability and efficacy of pomalidomide in the therapy of MM patients after allogeneic hematopoietic stem cell transplantation (HSCT). Materials (or patients) and methods: Our study included nine patients, who received pomalidomide therapy (2 to 4 mg) in combination with (n ¼ 7) or without (n ¼ 2) low dose dexamethasone (median dose 20 mg) as salvage (n ¼ 8) or due to persistence of disease (n ¼ 1) after allogeneic HSCT. Results: Pomalidomide therapy started after a median of 13 months (range 4 to 19 months) after allogeneic HSCT. Overall, pomalidomine therapy was well tolerated. In three patients infectious complications like pneumonia and gastroenteritis were observed leading to a temporary discontinuation of the drug. Four patients developed neutropenia (grade I) while one patient developed thrombocytopenia (grade II), which led to a dose reduction of pomalidomide. One patient developed a mild cutaneous Graft-versus-Host-Disease (grade I). Patients, who received 4 mg of pomalidomide, complained more about weakness, when compared to those who received 2 mg. Three out of nine patients showed a very good partial response, four
a partial response, and two stable disease during the therapy. The median duration of response was 8 months (range 4 to 13 months). Conclusion: Although our study group was small, we observed that the therapy with pomalidomide was effective and generally well tolerated after allogeneic HSCT. However, we observed hematologic toxicities and infectious complications requiring temporary discontinuation of the treatment. The incidence of Graft-versus-Host-Disease was not increased. Therefore, we conclude that pomalidomide can be applied safely as salvage treatment in MM patients after allogeneic HSCT. Disclosure of Interest: None declared. P359 Prognostic impact of STAT3 expression in patients with multiple myeloma treated with front-line autologous stem cell transplantation S.-H. Jung1,*, H.-W. Choi2, M.-G. Shin2, D.-H. Yang1, J.-S. Ahn1, Y.-K. Kim1, H.-J. Kim1, J.-J. Lee1 1 Department of Hematology-Oncology, 2Department of Laboratory medicine, Chonnam National University Hwasun Hospital, Hwasun, Korea, Republic Of Introduction: The signal transduced and activator of transcription 3 (STAT3) is considered as prognostic marker in various type of cancer. In this study, we investigated the prognostic significance of STAT3 expression in patients with multiple myeloma (MM) that treated with front-line high-dose therapy and autologous stem cell transplantation (HDT/ASCT). Materials (or patients) and methods: Sixty-nine patients with MM treated with front-line HDT/ASCT were enrolled between June 2005 and March 2013. Dual immunochemical staining (IHC) for phosphotyrosine-STAT3 (PY-STAT3) and CD138 on paraffin-embedded bone marrow sections at diagnosis was performed to evaluate tumor cell-specific PYSTAT3 expression. Results: PY-STAT3 was detected in 7 patients (10.1%) at diagnosis, and there were no significant differences of baseline clinical characteristics according to PY-STAT3 expression. After completion of induction chemotherapy, HDT/ASCT was performed at median 6.3 months. With a median follow-up of 37.7 months, the median progression-free survival (PFS) and
overall survival (OS) were 24.6 months and 76.0 months, respectively. Patients with PY-STAT3 expression had significantly shorter PFS compared to patients without PY-STAT3 expression (10.8 vs. 26.8 months, P ¼ 0.012). In addition, patients with PY-STAT3 expression had poor OS compared to patients without PY-STAT3 expression (57.1 vs. 76.0 months, P ¼ 0.018). Conclusion: This data suggest that PY-STAT3 expression using on dual IHC method is associated with poor survival outcomes in patients with MM treated with upfront HDT/ASCT. Disclosure of Interest: None declared. P360 Successful Stem Cell Mobilization and Autologous Stem Cell Transplantation after Pretreatment consisting of Bendamustine, Prednisone and Bortezomib (BPV) in 35 Patients with newly diagnosed/untreated Multiple Myeloma W. Poenisch1,*, M. Ploetze1, B. Holzvogt1, M. Andrea1, T. Schliwa1, M. Bourgeois1, S. Heyn1, G. N. Franke1, M. Jentzsch1, S. Leiblein1, R. Krahl1, S. Schwind1, V. Vucinic1, H. K. Al-Ali1, D. W. Niederwieser1 on behalf of OSHO (East German Study Group of Hematology and Oncology) 1 Department of Hematology and Oncology, University of Leipzig, Leipzig, Germany Introduction: Bendamustine is a bifunctional alkylating agent with low toxicity that produces both single- and double-strand breaks in DNA, and shows only partial cross resistance with other alkylating drugs. Treatment of patients with newly diagnosed multiple myeloma (MM) using bendamustine and prednisone in comparison to melphalan and prednisone results in superior complete response rate and prolonged time to treatment failure (Poenisch et al, Res Clin Oncol 132: 205-212;2006). So far, however, reliable information on stem cell toxicity and mobilization of stem cells for autologous stem cell transplantation (SCT) after induction treatment with a combination of bendamustine, prednisone and bortezomib (BPV) is missing. Materials (or patients) and methods: A retrospective analysis of peripheral blood stem cell mobilization and autologous SCT was performed in 35 patients with MM who had received at least one cycle of a BPV induction therapy consisting of
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bendamustine 60mg/m2 on days 1 and 2, bortezomib 1.3mg/ m2 on days 1, 4, 8 and 11, and prednisone 100mg on days 1, 2, 4, 8 and 11 between October 2008 and May 2014. The mobilization regimen consisted of cyclophosphamide 4 g/m2 and G-CSF (2x5mg/kg). Apheresis was started as soon as peripheral blood CD34 þ counts exceeded 20x106/L with a harvest target of 8x106 CD34 þ /kg. The minimal accepted target was 2x106 CD34 þ /kg. The pre-transplantation conditioning therapy consisted of melphalan 200mg/m2. Results: A median number of two (range 1–5) BPV treatment cycles were given to the patients. The majority of the patients (n ¼ 31, 89%) responded with 2 sCR, 5 nCR, 11 VGPR, and 13 PR. Three patients had MR, and 1 SD. Stem cell mobilization and harvest was successful in all patients. In 19 of 35 patients (54%) a single apharesis was sufficient to reach the target. The median number of aphareses was one (range 1–4) and the median CD34 þ cell-count/kg was 13.5 (range 3.2–33.1) x106. All patients received an autologous SCT. Engraftment was successful in 34 of 35 patients. The median time to leukocytes count 4lx109/L was reached after 11 days and the time to untransfused platelet count of 450x109/L was 13 days. 34 patients (97%) responded after the autologous SCT with 11 sCR, 2 CR, 7 nCR, 7 VGPR, and 7 PR. The progression free survival at 18 months was 87% and overall survival was 92%. Conclusion: The stem cell mobilization and autologous SCT is feasible in MM patients who have received BPV induction therapy. Disclosure of Interest: None declared.
Myelodysplastic syndromes P361 Prospective analysis of prognostic pre-transplant factors in MDS primarily treated by allogeneic hematopoietic stem cell transplantation: a study on behalf of the MDS subcommittee of the Chronic Malignancies Working Party of the EBMT E. M. Cremers1,*, T. de Witte1, A. van Biezen2, E. Kno¨dler2, J. Finke3, D. Beelen4, G. Socie´5, A. Nagler6, G. Kobbe7, L. Volin8, T. Gedde-Dahl9, J. Sierra10, Y. Beguin11, G. Sucak12, P. Ljungman13, A. Anagnostopoulos14, M. Robin5, N. Kro¨ger15 on behalf of Chronic Malignancies Working Party 1 Hematology, Radboud University Medical Center, Nijmegen, 2 Leiden University Medical Center, Leiden, Netherlands, 3University of Freiburg, Freiburg, 4University Hospital, Essen, Germany, 5 Hopital St. Louis, Paris, France, 6Chaim Sheba Medical Center, Tel-Hashomer, Israel, 7Heinrich Heine Universita¨t, Du¨sseldorf, Germany, 8HUCH Comprehensive Cancer Center, Helsinki, Finland, 9Rikshospitalet, Oslo, Norway, 10Hospital Santa Creu i Sant Pau, Barcelona, Spain, 11University of Liege, Liege, Belgium, 12 Gazi Universitesi Tip Faku¨ltesi Hastanesi, Ankara, Turkey, 13 Karolinska University Hospital, Stockholm, Sweden, 14George Papanicolaou General Hospital, Thessaloniki, Greece, 15University Hospital Eppendorf, Hamburg, Germany Introduction: Allogeneic HSCT is the most potent curative modality in MDS. Major cause of death after HSCT is NRM. NRM has decreased after introduction of reduced intensity regimens (RIC) and has allowed HSCT in more advanced age groups. In this large prospective analysis, performed by the Chronic Myeloid Malignancies Party (CMWP) of the EBMT, prognostic pre-transplant factors in MDS have been analyzed with the focus on the impact of red blood cell transfusions (RBCT) on outcome. Materials (or patients) and methods: All 225 patients in this study received upfront allogeneic HSCT after bone marrow ablative or RIC regimens, between January 2009 and January 2014. Data were collected at diagnosis, at transplantation and 6 weeks, 100 days, 1 year and 2 years after SCT. We analyzed
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the impact of WHO classification, number of transfusions, iron parameters, comorbidities, (non-)infectious complications on the occurrence of non-relapse mortality and overall survival. Results: 48% of the patients showed unilineage or multilineage MDS with o5% marrow blasts, 62% had 45% marrow blasts. According to IPSS 48% had lower risk MDS and 52% had higher risk MDS, at HSCT. 61 Patients have received RBCT before HSCT. 45% of patients received HSCT after standard intensity conditioning regimens and 55% after RIC regimens. Median interval between diagnosis and SCT was 10 months (range 1-128 months). Analyzed pre-transplant factors were: transplant and donor characteristics, comorbidities (present in 41% of the patients), median number of RBCT in 139 patients treated with RBCT before HSCT: 13 units (range: 1-146), ferritin levels (median 715 ng/ml; range 10-9033), median transferrin levels: 201 mg/dL (range 2-1946), unbound serum iron (median 116ug/dL; range 0-463). Twenty-five patients have been treated iron chelation prior to HSCT, and 25 patients at 2 years after SCT. 6 Patients received chelation both prior to and after HSCT. Nine patients underwent phlebotomies after HSCT. Two years OS was 64%, NRM 22% and RI 25%; 14% of patients died after relapse or progression of disease. Within this upfront allografted MDS cohort WHO-classification and cytogenetics had only minimal impact on treatment outcome (data not shown). Age had a significant impact on OS (HR 2.22; P ¼ 0.008), but not on NRM or RI. This might be explained by the influence of aging and increased comorbidities at higher age. Patients transplanted with peripheral blood as stem cell source had a significantly lower risk of death, OS (HR 0.46; P ¼ 0.049) and NRM (HR 0.32; P ¼ 0.017), but impact on RI was absent. We found a significant impact of transfusion burden (o20 RBCT versus 420 RBCT) on NRM (HR 1.89; Po0.05), without a significant effect on OS (HR 1.48 P ¼ 0.116). Ferritin levels (41000 ng/ml) had nonsignificant (P ¼ 0.06) negative impact on survival after SCT. Conclusion: This large observational study is the first prospective study in HSCT for MDS with detailed information of several important prognostic factors including disease modalities, transfusional data and iron parameters. Diseasespecific modalities had only minor impact on treatment outcome. However, age and transfusion burden predicted outcome after allogeneic HSCT. The impact of iron chelation before and after HSCT will be analyzed in mi ¼ ore detail in subsequent analyses. Disclosure of Interest: None declared. P362 Characterization and MicroRNA Expression Profile Analysis of Mesenchymal Stem Cells Derived From Myelodysplastic Syndrome and Acute myeloid leukemia (AML) Patients and Comparison with Healthy Controls H. Ozdogan1,*, B. Gur Dedeoglu1, A. Atalay1, S. Kose2, Z. A. Yegin3, F. Avcu4, D. Uckan Cetinkaya2, O. Ilhan5 1 Biotechnology Institute, Ankara University, 2Stem Cell Research and Application Center, Hacettepe University, 3Hematology, Gazi University, 4Hematology, Gulhane Military Medical Academy, 5 Hematology, Ankara University, Ankara, Turkey Introduction: Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) represent the heterogenous groups of clonal diseases that affect the stem cells in bone marrow. Their pathogenesis is complex and likely depends on interplay between aberrant hematopoietic cells and hematopoietic microenvironment (HM). The key component of the HM is mesenchymal stem cells (MSC). Only a few studies characterized abnormalities in the HM in MDS and AML. Recent findings suggest that a specific deletion of Dicer1 in MSC-derived osteoprogenitors triggers several features of MDS and AML in a murine model. Dicer1 is an RNase III endonuclease essential for microRNA (miRNA) biogenesis and RNA processing. miRNAs are 18 to 25 nucleotide non-coding RNAs regulating gene expression. In recent years, several studies have reported that the miRNAs play important roles in the regulation of
expression of the cytokine molecules as well as in the regulation of the differentiation of MSC and hematopoietic stem cells (HSC). These data suggest that there may be functional roles of miRNAs in the regulation of HM and miRNA deregulation in MSC may play a role in the pathology of MDS and AML. The aim of the present study is to analyze miRNA expression in MSC obtained from bone marrow (BM) of MDS and AML patients. Materials (or patients) and methods: In this study miRNA expression profiles of MSCs from BM samples of MDS (n:3 RAEB and RA)/AML patients (n:3 M1, M4) and healty donors (n:2) were evaluated. BM aspirates were collected from MDS and AML patients and healty donors. MSCs from MDS and AML were expanded in vitro and characterized. RNA isolation was performed from MSC cultures at passage 3. miRNA expression profiles were identified by miRNA microarray analysis (Agilent). miRNA microarray data analysis was performed by BRB Array Tools. Statistical methods such as ANOVA and t-test were used for the determination of miRNAs differentially expressed in MDS and AML patients compared to that of healty donors. The targets of differentially expressed miRNAs were identified by using bioinformatics tools and pathway enrichment analysis was performed with these target genes. Results: Three sets of differentially expressed miRNAs were obtained at the end of microarray experiments. To find out the miRNAs of MSC specific to AML and MDS, the two former group was compared to heathy donors and 287 miRNAs were found to be differentially expressed in MDS while it was 87 for AML patients compared to healthy donors. Additionally the comparison of MDS and AML resulted 34 differentially expressd miRNAs (Po0,05 and fold change 42). The pathway enrichment analysis was conducted with the target genes of top 3 miRNAs from each group. The target genes were found to be enriched in the pathways, which are important in the regulation of HM and cancer development and progression; MAPK, chemokine signaling pathway, cytokine-cytokine receptor interaction, hedgehog signaling pathway, wnt signaling pathway, VEGF signaling pathway, notch signaling pathway and TGF-beta signaling pathway. Conclusion: With the preliminary findings obtained from this study it could be suggested that miRNA exspression changes in MSCs might be involved in MDS and AML pathogenesis and miRNAs might be new players in the regulation of HM by targeting the genes taking part in pathways important in HM. Disclosure of Interest: H. Ozdogan Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest , Personal Interest: There is no any conflict of interest , Conflict with: There is no any conflict of interest, B. Gur Dedeoglu Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, A. Atalay Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, S. Kose Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, Z. A. Yegin Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, F. Avcu Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, D. Uckan Cetinkaya Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, O. Ilhan Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest
P363 Superior long-term outcomes of allogeneic hematopoietic cell transplantation when transplanted in response compared to relapsed or refractory after hypomethylating agents for patients with lower risk myelodysplastic syndrome J. H. Moon1,*, S. K. Sohn1, J. S. Ahn2, H. J. Kim2, Y.-R. Do3, M. K. Song4, H. J. Shin4, W. S. Lee5, S. M. Lee5, Y. D. Joo6, H. Kim7, H. S. Lee8, Y. S. Kim8, Y. Y. Cho9, S. H. Bae9 1 Hematology/Oncology, KYUNGPOOK NATIONAL UNIVERSITY HOSPITAL, Deagu, 2Hematology/Oncology, Chonnam National University Hwasun Hospital, Hwasun , 3Hematology/Oncology, Keimyung University Dongsan Medical Center, Deagu, 4Hematology/Oncology, Busan National University Hospital, 5Hematology/ Oncology, Inje University Busan Baik Hospital, 6Hematology/ Oncology, Inje University Haewoondae Baik Hospital, Busan, 7 Hematology/Oncology, Ulsan University Hospital, Ulsan, 8Hematology/Oncology, Kosin University Gosper Hospital, Busan, 9 Hematology/Oncology, Daegu Catholic University Medical Center, Deagu, Korea, Republic Of Introduction: Allogeneic hematopoieic cell transplantation (Allo-HCT) is generally recommended for lower-risk myelodysplastic syndrome (LR-MDS) patients with relapsed or refractory to HMA treatment. It is still under debate when LR-MDS patients should be transplanted in response or at the time of loss of response. This study retrospectively evaluated the outcomes of allo-HCT who transplanted in response or relapsed/refractory after HMA treatment. Materials (or patients) and methods: The data of 307 patients diagnosed with LR-MDS from Oct 1992 to Jul 2013 were retrospectively evaluated. To address the time dependence of allo-HCT, the methods of Simon and Makuch and the Mantel-Byar test was used. Results: Among 307 patients with LR-MDS, 200 patients were treated with HMA. Sixty-three (31.5%) patients showed response (CR/PR/HI) to HMA and 119 (59.5%) were relapsed/ refractory (SD/PD). Prior to Allo-HCT, 7 patients were in response and 26 patients in relapsed/refractory. Median time to allo-HCT were 2.8 years in responders and 1.9 years for patients with relapsed/refractory. Among HMA responders, the 5-year overall survival (OS) rate were 83.3% with allo-HCT (n ¼ 7) and 34.2% with continued HMA treatment (n ¼ 53) (P ¼ 0.07). Among patients with relapsed/refractory, the 5-year OS rate were 47.0% with allo-HCT (n ¼ 26) and 29.4% with BSC after HMA treatment (n ¼ 93) (P ¼ 0.45). There showed a trend in the benefits of OS for patients treated with allo-HCT in response compared to allo-HCT in SD/PD (P ¼ 0.17). In the multivariate analysis, ECOG-PS 2-3 (HR 3.030, P ¼ 0.006), IPSS blast Z0.5% (HR 2.003, P ¼ 0.018), poor cytogenetic risk group
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(HR 12.362, Po0.001), and non-response to HMA (HR 2.487, P ¼ 0.001) were unfavorable factors for OS among LR-MDS patients. Conclusion: Allo-HCT in response to HMA showed superior OS compared to allo-HCT in relapsed/refractory after HMA for patient with LR-MDS. To elucidate best time points of allo-HCT for LR-MDS patients, a large scale study will be needed. Disclosure of Interest: None declared. P364 The outcome of allogeneic stem cell transplantation in myelodysplastic syndrome is related to disease burden at transplant: a multicenter study of the Polish Adult Leukemia Group L. A. Gil1,*, J. Dwilewicz-Trojaczek2, M. Komarnicki1, S. Kyrcz-Krzemien3, M. Markiewicz3, M. Dzierzak-Mietla3, P. Zielinska3, J. Styczynski4, M. Borowczyk1, A. Nowicki1, M. Baranska1, M. Bieniaszewska5, A. Hellmann5, G. Basak6, W. W. Jedrzejczak6, K. Madry6, A. Lange7, K. Halaburda8, B. Jakubas9, P. Mensah-Glanowska9, P. Rzepecki10, A. Wierzbowska11, M. Ussowicz12, J. Dybko12, T. Wrobel12, T. Czerw13, S. Giebel13 1 Department of Hematology, Poznan University of Medical Sciences, Poznan, 2Department of Hematology, Oncology and Internal Diseases, Medical University of Warsaw, Warszawa, 3 Depatment of Hematology and BMT, Medical University of Silesia, Katowice, 4Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, 5Department of Hematology, Medical University of Gdansk, Gdansk, 6Department of Hematology, Oncology and Internal Diseases, Medical University of Warsaw, Warszawa, 7 Department of Hematology, Lower Silesian Center for Cellular Transplantation, Wroclaw, 8Department of Hematology, Institute of Hematology and Transfusion Medicine, Warszawa, 9Department of Hematology, Collegium Medicum, Jagiellonian University, Krakow, 10Department of Hematology, Military Institute of Medicine, Warszawa, 11Department of Hematology, Medical University of Lodz, Lodz, 12Department of Hematology, Wroclaw Medical University, Wroclaw, 13Department of Hematology and BMT, Cancer Center and Institute of Oncology, Gliwice, Poland Introduction: Allogeneic stem cell transplantation (alloSCT) is the only curative strategy for patients (pts) with myelodysplastic syndrome (MDS), however numerous controversies exist regarding timing of transplant, disease burden at transplant, the need for pretransplant therapy, conditioning intensity and stem cell source. Therefore, the Polish Adult Leukemia Group (PALG) analyzed the outcomes of MDS pts transplanted between 2000 and 2013 with respect to pretransplant factors. Materials (or patients) and methods: Two hundred and sixteen pts with median age of 47 years (range: 18-69) were retrospectively analyzed. The study cohort included pts with intermediate-2/high risk according to IPSS (101), unfavorable cytogenetics (70), secAML (34) and therapy related disease (tMDS) (26). Seventy four (34%) pts were older than 50. Before transplantation 90 pts had o5% bone marrow blasts, while 43 (20%) pts underwent alloSCT with blasts 410%. Ninety three pts were reported to be dependent on blood transfusion. Ferritin level was available for 48 pts, with elevated level 41000 ng/mL found in 28. In 68 (31%) pts pretransplant intensive therapy was applied. For conditioning, RIC protocols based on fludarabine were used in 113 (53%) pts and 103 pts were treated with MAC, mainly based on busulfan (66) or treosulfan (34). Stem cells from related (113) or unrelated (103) donors were grafted in median dose 4.6 (0.5-12.5)x106 of CD34 þ cells/kg. PB stem cells were transplanted in 168 (78%) pts. Twenty five pts received allograft from HLA mismatched donors. Results: Engraftment occurred in 206 (95%) pts and 6 pts died before þ 21 day after alloSCT. Infectious complications were observed in 189 (87%) pts during neutropenia (FUO 120, bacterial 45, fungal 17, bacterial and fungal 7). Late infections
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occurred in 106 pts, and were related mainly to CMV infection. Toxic complications (CTCAE grade 3-4) developed in 42 (19%) pts. Acute GVHD grade 2-4 occurred in 85 (36%) pts, while chronic extensive GVHD in 32 (15%) pts. Forty two pts experienced disease relapse after treatment. Over a median follow-up of 43 months (range: 15-70) 117 (54%) pts were alive. In 29 cases deaths were related to relapse, 20 pts died due to GVHD, 36 due to infection and 13 because of organ toxicity. Probability of 3-years overall survival (OS), disease free survival (DFS), relapse incidence (RI) and treatment related mortality (TRM) were 0.396±0.051, 0.384±0.05, 0.28±0.042 and 0.421±0.056, respectively. Among factors studied in univariate analysis those associated with lower OS were: tMDS (P ¼ 0.04; HR 2.2), bone marrow blasts 4 10% before alloSCT (P ¼ 0.04, HR 1.7) and PB as a source of stem cells (P ¼ 0.05; HR 1.7). In multivariate analysis the only factor associated with lower OS was bone marrow blasts 410% (P ¼ 0.023, HR 1.7). In subgroup analysis ferritin level 41000 ng/mL was significantly related to lower OS (P ¼ 0.001; HR 4.6). There were no statistically significant differences in OS, DFS, RI and TRM with respect to age, IPSS risk group, cytogenetics, dependence on transfusion, pretransplant therapy, intensity and type of conditioning, donor choice and HLA compatibility. Conclusion: Allogeneic SCT is an option for MDS patients in different age groups. Disease status at time of transplant is the most important determinant for the outcome. High rate of TRM remains a concern. Disclosure of Interest: None declared. P365 Next generation sequencing – based delineation of mutational spectrum in 40 patients with MDS uniformly treated with sequential FLAMSA-RIC-conditioning and allogeneic hematopoietic stem cell transplantation M. Christopeit1,*, A. Badbaran1, T. Zabelina1, N. Kro¨ger1 1 Department of Stem Cell Transplantation, University Hospital Eppendorf, Hamburg, Germany Introduction: Prognosis of patients with advanced MDS is poor. Allogeneic stem cell transplantation is a curative treatment. Current prognostic schemes take, amongst other parameters, cytogenetics into account. A classification that relies on the mutational status of recurrently mutated genes in MDS has not been developed. To correct for potential bias, correlation of mutational status with outcome best is performed in uniformly treated groups of patients. Materials (or patients) and methods: To this end, the mutational status of 40 patients suffering MDS was evaluated and correlated with overall survival (OS). Conditioning consisted of sequential chemotherapy (FLAMSA scheme) and busulfan-based reduced-intensity conditioning. Patients suffered RAEB-I (n ¼ 6), RAEB-II (n ¼ 18), or other forms of MDS (n ¼ 16). Median age was 62 years (range 39-73). Patients were transplanted from a matched related donor (n ¼ 4) or an unrelated donor (n ¼ 36). Results: DNA was isolated following standard procedures. Semiconductor based Next Generation Amplicon Sequencing (NGS) was carried out using an Ion Torrent Personal Genome Machine (PGMTM, Thermofischer). A mutational panel was constructed and consisted of ASXL1 (exon 12), BRAF COSMIC mutation (COSM476), CBL (exon 8, 9), CEBPA (all coding exons), DNMT3A (all coding exons), FLT3 (codons 676, 830850), GATA2 (all coding exons), IDH1 (exon 4), IDH2 (exon 4), JAK2 (exon 14), KIT (exons 8, 10, 11, 17), KRAS (exons 2, 3, 4), NPM1 (exon 12), NRAS (exons 2, 3, 4), PTPN11 (exons 3, 7, 8, 13), RUNX1 (exons 3-8), TET2 (all coding exons), TP53 (all coding exons), WT1 (exons 7, 9). One run was finished within two days. Mutations were found in 38/40 patients (95%, median number of mutations 2, range 0-6, exactly 1 mutation in 15 patients, 2 mutations in 13 patients, 3 mutations in 4 patients, 4 mutations in 3 patients, 5 mutations in 2 patients, 6 mutations in 1 patient). Mutated genes were ASXL1 (6 pts; missense, ms, 2, frameshift, fs, 3, ms þ fs 1), BRAF (ms 1),
CEBPA (8 pts; ms 5, fs 2, silent, sil, 1), DNMT3A (8 pts; ms 6, fs 2), FLT3 (ms 2), GATA2 (12 pts; ms 10, fs 2), IDH2 (ms 4), KIT (ms 1), KRAS (4 pts; ms 1, sil 1), NPM1 (fs 2), NRAS (ms 5), RUNX1 (12 pts; ms 2, fs 6, stop codon 4), TET2 (13 pts; ms 3, fs 2, stop codon 1, 2xms 1, ms þ sil 1, ms þ stop 1, 2xms þ stop 1, 2xms þ sil 1, 2xms þ stop 1, 2xstop 1), TP53 (5 pts; ms 4, 2xms 1). No significant correlation between the number of mutations and OS was found (P ¼ 0.672, log rank test). In multivariate analysis including all mutations discovered as covariates, FLT3 mutations were a significant covariate for OS (Po0.001) with an adverse prognostic impact. Conclusion: In conclusion, semiconductor based Next Generation Sequencing analysis of amplicon mutations in patients with MDS before allogeneic HSCT might prove a feasible means to a timely risk stratification. Further follow up analysis should cover the mutational status of patients missing complete hematologic remission in order to unravel treatable remaining malignant clones and the prognostic power of minimal residual disease. Unbiased analysis of mutations by whole genome sequencing might lead to further marker mutations and mechanistic insights. MC and AB contributed equally to this work. Disclosure of Interest: None declared. P366 Use of azacytidine as a bridge to allogeneic stem cell transplantation in MDS R. R. Pol1,*, K. Raj2, I. Lazana3, N. Russell1, P. Ferguson4, C. Craddock4, P. Mehta5, S. Killick6, E. Das-gupta1 1 Nottingham University hospital, U.K., Nottingham, 2Kings College hospital, Haematology, 3Kings College hospital, Kings College hospital, London, 4Queen Elizabeth Hospital Birmingham, Birmingham, 5University Hospitals Bristol NHS Foundation Trust, Bristol, 6Royal Bournemouth General Hospital, Bourmouth, United Kingdom Introduction: Allogeneic stem cell transplantation (alloSCT) is the only potential curative therapeutic approach in myelodysplastic syndrome (MDS). AML induction chemotherapies are associated with toxicities which could prohibit proceeding to alloSCT. Hence we investigated the impact of prior-totransplantation azacytidine (AZA) on patient outcome for MDS. Materials (or patients) and methods: We included 58 consecutive patients from 5 centres who underwent alloSCT for MDS between November 2006 and July 2014. They were male-37, female-21. The median age was 57 (range 24-75). WHO diagnosis were RCMD-3, RAEB1-19, RAEB2 -22, CMML-6, Fibrotic MDS-3, MDS/MPN – 2 and AML – 3. At diagnosis of MDS, 46/52 (86.5%) had intermediate-2 or high risk MDS as per IPSS and 7/52 (13.4%) had IPSS-1. Median number of days from diagnosis to HSCT was 414 (range 120-2920). Median Aza cycles to best response was 4 (range 1-9). Single agent azacytidine was used as a bridge to alloSCT in 46/58 patients. Additional chemotherapy was required in the remaining 12/58
patients post azacytidine but prior to HSCT to achieve remission (AML-8, SD-2, PR-1, CR-1). Additional treatment was FLAG-2, Flag-Ida -2, DA-3, DA þ Myelotarg-4, Clofarabine 1). Conditioning was myeloablative in 4 and RIC in 54. Graft was T replete in 5 (FLAG - 3, Flu-Cy-TBI-2) and T cell deplete in 50 (ATG/Alemtuzumab). The donor source was from siblings – 15, Cord-3, Haploidentical -2, and MUD-38. Results: Response to azacytidine before alloSCT was CR-30 (51.7%), PR-10 (17.2%), SD-10 (17.2%), and failure-8 (13.7%). Additional chemotherapy achieved CR in 11 and SD in 1. All patients who received azacytidine only (46) and additional chemotherapy (12) successfully received HSCT. After a median follow-up of 405 days (range 38-1882), the median survival was 480 days (1 year 3 months). OS was 64.7% at 1 year, 38% at 2 years and 34.7% at 3 years. Relapse at 1 year was seen in 16/58 patients (27.5%). Nonrelapse mortality was seen in 9/58 (15.5%) at 1 year. Mortality due to disease relapse/progression was seen in 9/58 (15.5%). DFS was 56% at 1 year. The median time to neutrophil engraftment (4 0.5) was 12 days (range 623) and median time to platelets engraftment (4 50) was 14 days (range 1-41). 56 patients were evaluable for aGVHD and this was seen in 34(60.7%). Grade I and II in 22(39.2%), Grade III and IV in 12(21.4%). Out of the 54 evaluable patients, 23 (42.5%) developed cGvHD (extensive in 11 (20.3%) and limited in 12 (22.2%). Conclusion: Single agent Aza is of value in stabilizing, achieving remission and allowing time for patients to reach transplantation. This is with less toxicity than conventional intensive chemotherapy and is useful as a bridge to alloSCT without affecting transplant outcomes. Disclosure of Interest: None declared. P367 Allogeneic stem cell transplantation for MDS patients more than 70 years of age. A report of MDS subcommittee of CMWP of EBMT S. Heidenreich1,*, T. Zabelina1, A. van Biezen2, J. Finke3, U. Platzbecker4, D. Niederwieser5, H. Einsele6, W. Bethge7, R. Schwerdtfeger8, D. Beelen9, J. Tischer10, A. Nagler11, P. Zache´e12, C. Scheid13, T. de Witte14, M. Robin15, N. Kro¨ger1 1 University Hospital Eppendorf, Hamburg, Germany, 2EBMT Data Office, Leiden, Netherlands, 3University of Freiburg, Freiburg, 4 University Hospital, Dresden, 5University Hospital, Leipzig, 6 University Hospital, Wu¨rzburg, 7University of Tu¨bingen, Tu¨bingen, 8Deutsche Klinik fu¨r Diagnostik, Wiesbaden, 9University Hospital, Essen, 10Klinikum Grosshadern, Munich, Germany, 11 Chaim Sheba Medical Center, Tel-Hashomer, Israel, 12ZNA, Antwerp, Belgium, 13University of Cologne, Cologne, Germany, 14 Radboud University Medical Centre, Nijmegen, Netherlands, 15 Hopital St. Louis, Paris, France Introduction: Myelodysplastic syndromes (MDS) are diagnosed at median age of 75 years. Allogeneic stem cell transplantation (HSCT) is the only curative treatment option, but with an increasing age, the number of comorbidities is escalating. Treatment guidelines suggest HSCT for intermediate-II and high risk constellations up to the age of 65 years, and reduced intensity conditioning (RIC) regimens are commonly used up to 70 years of age. However, increasing life expectancy, availability of RIC regimens and good Karnofsky performance status (KPS) of MDS patients more than 70 years of age, has led to an increased use of HSCT. We performed a retrospective analysis to investigate results after HSCT for those patients and influence of KPS on outcome. Materials (or patients) and methods: We analyzed data of 345 patients in the EMBT database older than 70 years with MDS (n ¼ 238) and secondary acute leukemia (sAL, n ¼ 107) which have undergone HSCT from related (n ¼ 89) and unrelated (n ¼ 256) donors between 2000 and 2014. Median follow up was 13 months (4-37 months). Median age at transplantation was 72 years (70-79 years) with male:female ratio of approximately 2:1. KPS was defined in 295 cases.
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Presence of acute graft-versus-host disease (aGVHD) (n ¼ 129) was documented for 320 patients. Univariate (Kaplan-Meier) and multivariate analysis (Cox regression test, competing risks - Fine & Gray test) were used to calculate overall survival (OS), transplantation related mortality (TRM), Relapse rates (REL) in relation to RIC (n ¼ 261) versus MAC (n ¼ 84) regimes, related or unrelated donor, KPS 90-100% versus r80% and presence of CMV antibodies. Due to missing data, FAB or WHO subclassification, IPSS scores, cytogenetics and information about HLA-match of the graft, as well as occurrence of chronic GVHD could not further be considered. Results: The number of HSCT for MDS patients of 70 years or more has increased over time. While 2000-2004 only 19 patients received transplantation, the following 3-year periods included 28 (2005-2007), 97 (2008-2010) and 200 (2011-2013) patients, respectively. 3-year estimated OS and REL were 34% (28-40%) and 44% (34-54%), respectively. TRM after 1 year was 40% (34-46%). KPS of 90-100% had a significant benefit for OS (P ¼ 0.037) and TRM (P ¼ 0.129) but not for REL. These results were confirmed in the multivariate analysis. Use of RIC regimens was superior in OS (P ¼ 0.037) and TRM (P ¼ 0.016) but did not result in higher REL. Patients with related donors had more REL (P ¼ 0.0008) without influence on OS. The presence of CMV antibodies in the patient improved OS (P ¼ 0.015). No significant influence on OS was seen for diagnosis at transplantation (MDS/sAL), complete remission before transplantation, use of TBI, in vivo t-cell depletion and use of bone marrow versus peripheral blood stem cells. Conclusion: HSCT for MDS patients 70-years of age or older is a possible choice of treatment with a 3-year OS of 34%. Good performance, determined by KPS, increases the chance of survival as well as the use of RIC regimens, which is not compromised by higher relapse rates. According to these data, MAC regimens are associated with high risk for TRM. Relevance of IPSS, cytogenetics, MDS subclassification and HLA-match are unclear due to a lack of data and should be considered in subsequent investigations. Disclosure of Interest: None declared. P368 Abstract Withdrawn
Solid tumours P369 Peripheral blood stem cell autografts for the treatment of children neuroblastoma A. Hedayati Asl1,* on behalf of Tashvighi M, Zangooei R, Mehrvar A, Iravani N, Astaraki S, Odameh Sh,Fallah V 1 Pediatric Stem cell transplantation, MAHAK, Tehran, Iran, Islamic Republic Of Introduction: The long-term survival probability for children with high-risk neuroblastoma was less than 15 percent. Better results have been achieved using an aggressive multimodality approach that includes chemotherapy, surgical resection, high-dose chemotherapy with hematopoietic stem-cell rescue, and radiation therapy. The prognosis of high-risk neuroblastoma after conventional chemotherapy is generally poor. The use of high-dose chemotherapy with autologous hematopoietic stem cell rescue in consolidation has resulted in improvements in survival, although further advances are still needed. Materials (or patients) and methods: seventeen patients age 5 to 20 years (median 4 years, M/F ¼ 13/4), with high risk, relapsed or metastatic underwent ASCT in our hospital (from 2012-2014). 15 of the 17 patients underwent a first HDCT/ASCR
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and 2 patients underwent a second HDCT/ASCR. 7 of the 17 patients who were evaluated for N-myc amplification were positive. Status at transplant was: second complete remission (CR2): n ¼ 11; CR3: n ¼ 3, (CR 43) n ¼ 3. All patients received chemotherapy- based conditioning regimens: MEC regimen consisting of carboplatin for 3 days, VP-16 for 3 days and melphalan in 2 days was the primary cytoreductive regimen. Peripheral blood (PB) was the source of progenitor cells in 17 patients. All patients engrafted. Results: The median mononuclear cell dose was 4.8 108/ kg. The median time to absolute neutrophil count 4 0.5 109/L was 11 days, and the median time to platelet count 4 20 109 was 14 days. One patient out of 17 (5.8%) experienced transplant-related mortality. Three patients relapsed after transplant (mean 8m.). With a median followup of 11 months (3-20 months) after transplant the event-free survival (95% confidence interval) was 0.64. 14 patients remain alive. Conclusion: Longer follow-up is required to evaluate fully efficacy and long term survival of our patients. However, further studies will be needed to decrease the toxic death rate in the transplantation. Disclosure of Interest: None declared. P370 Potential use of cytokine activated patients’ NK cells in metastatic colorectal cancer refractory to conventional therapies D. Montagna1,*, F. Ferulli2, S. Brugnatelli2, I. Turin2, M. Tanzi2, S. Delfanti2, C. Klersy2, D. Lisini3, E. Todisco4, D. Pende5, R. Maccario2, P. Pedrazzoli2 1 Fondazione IRCCS Pol. San Matteo, Universita` di Pavia, 2 Fondazione IRCCS Pol San Matteo, Pavia, 3Fond. IRCCS Istituto Neurologico C. Besta, Milano, 4Humanitas Cancer Center, Rozzano (MI), 5AOU San Martino-IST Genoa, Genova, Italy Introduction: The failure of conventional treatment to significantly improve outcomes in mCRC has prompted the development of immune-based therapies, including natural killer (NK) cells-based strategies. We investigate the capacity of patients-derived NK cells, either resting or after cytokine activation, to lyse autologous metastatic colorectal cancer (mCRC) cells which have been analyzed for expression of HLA Class I and of ligands for adhesion and triggering NK receptors involved in their recognition and killing. We also evaluated whether KRAS mutated mCRC cells were susceptible to antiEGFR-induced ADCC mediated by NK cells. Materials (or patients) and methods: After obtaining signed informed consent, 25 mCRC patients have been enrolled. mCRC cells, from primary or metastatic tissue, were expanded in vitro and analyzed to confirm their neoplastic origin. Ligand expression was evaluated by cytofluorimetric analysis and gene expression on mCRC cells. Purified NK cells were activated overnight (ON) and for five days with IL-2 or IL-15 and were analyzed for expression of triggering and inhibitory receptors and for their ability to kill autologous mCRC cells alone or previously coated with antiEGFR mAbs. Results: mCRC were successfully expanded from 21 of 25 samples. Experiments from 10 patients showed a substantial inability of patients resting NK cells to lyse mCRC cells. ON activation resulted in greatly increased NK cytotoxic potential (IL-2: mean 28%; range:10-68; and IL-15: mean 38%; range 1881 at E:T ratio of 40:1). Five days of NK cell activation were able to further enhance their lytic capability and to reduce the variability of the results obtained in ON condition(IL-2: mean 43%; range: 33-56; and IL-15: mean 58%; range 36-87 at E:T ratio of 40:1). Experiments performed in 6 patients (3 wild type and 3 KRAS mutated) demonstrated that the incubation of mCRC cells with anti-EGFR mAbs increase their susceptibility to NK-mediated lysis irrespective of KRAS status of tumor cells (mCRC alone: IL-2: mean 22%; range:17-39; and IL-15: mean 41%; range 18-87; mCRC EGFR-coated: IL-2: mean
32%; range: 22-48; and IL-15: mean 53%; range 44-84 at E:T ratio of 20:1). NKp30 and NKp46 were variably expressed by patient NK cells and could be up-regulated after cytokine activation, while mCRC expressed different levels of most NK ligands. Conclusion: The demonstration that mCRC cells can be efficiently lysed after in vitro NK cells activation support the ongoing design of immunotherapy with autologous NK cells for poor prognosis mCRC patients. References: Disclosure of Interest: None declared. P371 Pediatric Solid Tumors: an in vitro model to improve the efficacy of haploidentical stem cell transplantation M. Comini1, F. Bolda1,*, A. Beghin1, C. D’Ippolito2, D. Alberti3, L. Ruggeri4, L. Bercich5, G. Carella6, F. Porta2, A. Caruso7, A. Lanfranchi1 1 Stem Cell Lab, Children’s Hospital, 2Pediatric Oncohaematology and BMT Unit, Children’s Hospital, 3Pediatric Surgery Unit, Children’s Hospital, Spedali Civili di Brescia, Brescia, 4Division of Hematology and Clinical Immunology, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, 5 Department of Pathology I, University of Brescia, 6Reumatology and Clinical Immunology Lab, Spedali Civili di Brescia, 7Section of Microbiology, University of Brescia, Brescia, Italy Introduction: Pediatric solid tumors are often characterized by a poor prognosis. Some of these, such as Rhabdomyosarcoma (RMS), Neuroblastoma(NB), Ewing Sarcoma(ES), are defined high-risk and disease free survival is less than 20%. In these patients a standard approach with chemotherapy and/or radiotherapy is not sufficient to allow a good prognosis. This fact highlights the need for new therapeutic strategies as the haploidentical hematopoietic stem cell transplantation (HSCT) that could represent a therapeutic immunological approach thanks to GvL and GvT. The aim of the study is to design an in vitro model to select the best donor to use in haploidentical HSCT in a solid tumor setting. Materials (or patients) and methods: We enrolled 29 patients with age between 0 and 16 years (mean: 5.4, median: 4). There were 8 RMS, 12 NB, 3 ES and 6 Wilms Tumor (WT). We decided to introduce in this study also WT to evaluate a possible immunological approach in an intermediate-risk solid tumor setting. We set up primary tumor cell lines and we characterized them first immunohistochemically, with specific tumor markers to evaluate the % of neoplastic cells, and phenotypically by flow cytometry. We created a specific panel of 22 markers: 5 hematopoietic markers as negative controls (CD45, CD14, CD34, HLA-DR, CD56), 7 mesenchymal (CD133, CD44, CD29, CD73, CD105, CD90, HLA-ABC), 1 endothelial (CD31) and 9 tumoral (CD271, CD71, MIC-A/B, CD155, CD112, CD9, CD58, CD99, Myogenin). Finally, we analyzed the HLA of patients whose culture was evaluated with good % of neoplastic cells and the alloreactivity of donor NK cells against patient’s blasts and primary neoplastic cell lines. We performed 2 HSCT by selection of
CD34 þ cells and we studied the patient’s post-transplantation follow up. Results: We obtained about 60% of primary culture evaluated as neoplastic with immunoistochemical analysis. The phenotypic evaluation analysis shows high expression of mesenchymal markers, particularly CD44, CD105 and CD90, while CD73 has an heterogeneous expression, and a low expression of CD45 (always less than 1%). Tumor markers, such as CD10, CD58, CD9, CD271, have a variable expression. The evaluation of HLA allowed us to find 3 patients missing of a receptor: 2 missing Bw4 and 1 missing C1. 2 patients underwent haploidentical HSCT (Table). Conclusion: The high expression of mesenchymal markers confirms what reported in literature: mesenchymal markers are often adhesion molecules physiologically expressed on the membrane of normal cells and involved in processes such as migration or cell-cell interaction, proliferation or differentiation, but on tumor cells can be up-regulated and act as positive regulators for tumor progression and metastasis processes. Solid tumors haven’t hematopoietic origin, so the negativity for the hematopoietic markers makes the great first screening of hematopoietic tumors and not hematopoietic ones. We found heterogeneity in the expression of tumoral markers; this can be explained by the fact that the expression of some cell surface receptors may represent different stages of maturation of tumor as well as different expressions related to different tissues and organs. Thanks to our work we document the importance of have an in vitro model that better mimics what occurs in vivo and consequently improves the opportunities and criteria for the selection of the best potential haploidentical alloreactive donor. Disclosure of Interest: None declared. P372 Biosimilar filgrastim in the treatment and prevention of chemotherapy-induced neutropenia in patients with solid tumours: a sub-analysis of the NEXT study F. Maloisel1,*, S. Lepreˆtre2, D. Kamioner3, C. Berthou4, H. Albrand5 1 Sainte-Anne Clinic, Strasbourg, 2Henri Becquerel Hospital, Rouen, 3Hoˆpital Prive´ de l’Ouest Parisien, Trappes, 4Hoˆpital Morvan, Brest, 5Hospira France, Meudon La Foret, France Introduction: The safety and efficacy of biosimilar filgrastim (Nivestimt, Hospira Ltd), a granulocyte-colony stimulating factor licensed for the treatment of chemotherapy (CT)induced febrile neutropenia (FN), were assessed post-market in the NEXT study. Here we present the results for patients (pts) receiving CT for solid tumours. Materials (or patients) and methods: The NEXT study was a prospective, observational, non-interventional, longitudinal, national, multicentre study, which recruited 2114 adult pts undergoing cytotoxic CT and receiving biosimilar filgrastim as primary or secondary prophylaxis, or as curative treatment. The primary objective was to evaluate the safety of biosimilar filgrastim. Secondary objectives were to evaluate the efficacy of biosimilar filgrastim, to describe characteristics of patients treated with biosimilar filgrastim, and patterns of use of biosimilar filgrastim.
[P371]
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Data collected included pt characteristics, biosimilar filgrastim treatment-related data and treatment emergent AEs. Pts were followed up for a maximum of six CT cycles at three visits: inclusion, during treatment, and following CT. The current subanalysis includes pts receiving CT for a solid tumour. Results: Of 2114 pts enrolled in the NEXT study, 2102 were included in the overall analysis. Mean age ± standard deviation (SD) was 63.5±12.7 years. On inclusion, 90% of the pts had a performance status of 0 or 1. The majority (98.2%) of pts received prophylactic biosimilar filgrastim (primary prophylaxis: 91.0%; secondary prophylaxis: 7.3%). Of the pts receiving prophylactic biosimilar filgrastim, 79.9% received a dose of 30 MIU and therapy was administered subcutaneously in 99.4% of these. In total, 1579 (75.1%) pts had solid tumours. Of these, 29.3% had breast cancer, 18.8% had lung cancer, 12.5% had colorectal cancer (colon-rectum-anal canal), 9.6% had urological cancer, 7.9% had a gynaecological cancer, 6.8% had pancreatic cancer, and 6.4% had ear, nose and throat cancer. 50.6% of pts were receiving metastatic CT (44.4% adjunctive; 5.1% other). The majority of pts were receiving first-line CT for their solid tumour (89.0% of pts with non-metastatic disease and 62.7% of pts with metastastic disease). During the study, 21.1% of pts experienced Z1 AE. 12.4% of pts reported muscle or bone disorders. The other most common AEs (Z2.0% incidence) included gastrointestinal disorders (6.2%), nausea (3.6%), diarrhoea (2.8%), nervous system disorders (2.6%) and headache (2.0%). 76.3% of pts were treated with an adequate dose according to their weight. In total, 4.1% of pts with a solid tumour experienced FN. In addition, 2.2% were receiving concomitant anti-infective prophylaxis (compared with 51% of pts with haematological malignancies). Conclusion: Biosimilar filgrastim (Nivestimt) was effective and well-tolerated in pts undergoing cytotoxic CT for solid tumours. Muscular and chest pain were the main expected adverse events. Disclosure of Interest: F. Maloisel Funding from: Hospira, Sandoz, Pfizer, S. Lepreˆtre: None declared, D. Kamioner: None declared, C. Berthou: None declared, H. Albrand Employee of: Hospira SAS. P373 Busulfan and treosulfan have a comparable efficacy in patients with Ewing sarcoma – retrospective analysis of Polish pediatric centers K. A. Drabko1,*, A. Raciborska2, K. Bilska2, M. Ussowicz3, J. Styczynski4, A. Zaucha-Prazmo1, M. Pogorza"a4, R. Chaber3, E. Gorczynska3, J. Musia"3, J. Kowalczyk1 1 Department of Pediatric Hematology, Oncology and Transplantology, Medical University, Lublin, 2Department of Surgical Oncology for Children and Youth, Institute of Mother and Child, Warsaw, 3Department of Pediatric BMT, Hematology and Oncology, Medical University, Wroc"aw, 4Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland Introduction: Megachemotherapy (MCT) with autologous stem cell transplantation has been used in clinical practice in high risk Ewing sarcoma for 30 years. Recent registry study confirms efficacy of this procedure however suggest possible lower survival after treoseulfan based conditioning when compare to busulfan based regimen. Aim of the study: Comparison of busulfan and treosulfan based MCT results in patients treated in Polish pediatric centers for high risk Ewing sarcoma. Materials (or patients) and methods: Between 1999 and 2013 in 11 pediatric centers 207 patients with Ewing sarcoma were treated according to EuroEwing protocols. Among this group 49 (24%) children (24 females) underwent MCT after pre-operative chemotherapy (VIDE, median 6 cycles) and local therapy. Median age of the patients was 13 years (range 2.6-19.2). busulfan based MCT was administered to 34 and treosulfan based in 15 children. In busulfan group 23 patients had metastatic disease at presentation and 11 were transplanted due to poor histological
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response or non-resectable tumor. In treosulfan group 12 patients had metastases and 3 poor histology. Results: Median follow up of 49 transplanted patients was 39 months (range 11-176 months); 53 months in busulfan group and 31 in treosufan group P ¼ 0.08. 3-years overall survival (OS) in busulfan group was 67% as compare to 64% in treosulfan group and 5-years 63% and 52% respectively (P ¼ 0.31). Disease free survival (DFS) at 3 years was 56% in busulfan and 32% in treosulfan group while 5-years DFS was 53% and 32% respectively ( P ¼ 0.09). Median follow up in non-transplanted patients with high risk features (n ¼ 97) was 20 months (range 2141 months). OS all transplanted patients (n ¼ 49) was 66% at 3years and 58% at 5-years as compared to 30% and 19%, respectively, in non-transplanted patients (P ¼ 0.001). DFS was also significantly better in transplanted patients as compared to other with high risk factors (3-years 49% vs 30%; 5-years 46% vs 15% respectively (P ¼ 0.003). Conclusion: Our retrospective analysis does not confirm lower efficacy of treosulfan based MCT as compared to busulfan based in patients with high risk Ewing sarcoma. We concluded that patients with high risk features have significantly higher probability of survival, when treated with MCT (busulfan or treosulfan). Further studies are necessary to compare efficacy of busulfan and treosulfan in patients with high risk Ewing Sarcoma. Disclosure of Interest: None declared. P374 Retrospective comparison of treosulphan/melphalan versus carboplatin/etoposide/melphalan as preparative regimen for autologous transplantation in pediatric neuroblastoma R. Khismatullina1, M. Maschan2, D. Balashov 2, J. Skvortsova2, D. Shasheleva2, G. Novichkova2, D. Kachanov3, E. Skorobogatova4, T. Shamanskaya5, G. Muftakhova6, K. Kirgizov7, S. Varvolomeeva5, L. Shelikhova8,*, A. Maschan2, 1 hematopoietic stem cell transplantation, FEDERAL CENTER FOR PEDIATRIC HEMATOLOGY, 2Hematopoietic stem cell transplantation, 3Oncology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, 4Hematopoietic stem cell transplantation, Russian chilren’s hospital, 5Oncology, FEDERAL CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, 6Oncology, FEDERAL CENTER FOE PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, 7Hematopoietic stem cell transplantation, Russian children’s hospital, 8 Hematopoietic stem cell transplantation, DMITRIY ROGACHEV FEDERAL CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: High-dose chemotherapy with autologous stem cell transplantation is a standard component of multimodality treatment for high-risk neuroblastoma. Common high-dose preparative regimens for neuroblastoma include busulphan/ melphalan (Bu/Mel), carboplatin/etoposide/melphalan (CEM)
and TBI-based combinations. Definitive comparison of alternative preparative regimens has not been performed thus far. In the current study we analyse retrospectively the results of autotransplantation with two regimens, CEM and treosulfan/ melphalan (Treo/Mel), used consecutively for conditioning in patients with high-risk neuroblastoma. Materials (or patients) and methods: Fifty three patients with high-risk neuroblastoma were treated in the Federal Center for pediatric hematology and Russian children’s hospital since April 2012 till September 2014. All patients were treated according to the NB-2004 protocol, excluding MIBG therapy, which was not available. Two types of preparative regimens were used. Twenty nine (f-8, m-21, median age 2,3 years) recieved CEM regimen (since September 2012 till June 2013): Melphalan 45 mg/m2/day, day -8,-7,-6,-5, Etoposide 40 mg/kg, day -4, Carboplatin 500 mg/ m2/day, day -4,-3,-2, G-CSF 10 mcg/kg/day IV since day þ 5 till WBC 4 5 x 109/l. Twenty four patients (f-12, m-12, median age 2,8 years) recieved Treo/Mel regimen (since July 2013 till September 2014): Treosulfan 14 g/m2/day, day -5,-4,-3, Melphalan 140 mg/m2, day -1, G-CSF 10 mkg/kg/day since day þ 5 till WBC 4 5 x 109/l. The median dose of infused CD34 þ cells was 10 (3,5-26) x 106/kg and 8,2 (1,3-31) x 106/kg respectively. The groups were balanced for disease stage, response to induction therapy and bone marrow involvement. There was a trend towards overrepresentation of MYC-N amplification (58% vs. 29%, p - 0,073) in the CEM group. Results: Primary engraftment was achieved in 50 of 53 pts, the median time to neutrophil and platelet recovery was 10 and 15 days in the CEM group, 10,5 and 13,5 days in the Treo/Mel group, respectively. In the CEM group 3 pts died of bacterial sepsis before 100-day after SCT, 1y. pTRM - 10,6% (95%CI: 3,7– 30,1). None of the patients died of transplant complications in the Treo/Mel group, 1y. pTRM 0%. In CEM group (median followup 1,9y) at 1 year cumulative incidence of relapse is 41,4% (95%CI: 26,8-63,8), pEFS is 48,3% (95%CI: 30,1-66,5), pOS - 69% (95%CI: 52,1-85,8). In the Treo/Mel group (median follow-up 0,67y) 1 year cumulative incidence of relapse is 11,2% (95%CI: 341,5), pEFS is 88,8% (95%CI: 74,2-100), pOS - 93,8% (95%CI: 81,9100). There is a trend towards improved event-free (p - 0,035) and overall survival (p - 0,07) in the Treo/Mel group. Conclusion: In this retrospective analysis Treo/Mel preparative regimen was associated with improved short-term results in comparison to the more conventional CEM regimen in highrisk neuroblastoma. Further observation, matched pair analysis and prospective testing will be performed to confirm these results and account for potential bias. Disclosure of Interest: None declared. P375 High-dose chemotherapy and autologous hematopoietic stem cell transplantation as adjuvant treatment in highrisk breast cancer: data from the EBMT registry 1995-2005 M. Martino1,*, P. Pedrazzoli2, A. Zambelli3, U. de Giorgi4, F. Arpaci5, D. Blaise6, R. Leno Nu´n˜ez7, H. Schouten8, M. Badoglio9, M. Bregni10, C. Bengala11, F. Lanza12 on behalf of on behalf of the European Society for Blood and Marrow Transplantation, Solid Tumors Working Party (EBMT-STWP) 1 Azienda Ospedaliera BMM, Reggio Calabria, Hematology and Stem Cell Transplant Unit, Reggio Calabria, 2Dept. Onco Hematology, Fondazione IRCCS Policlinico S. Matteo - Pavia, 3 Fondazione S.Maugeri, Pavia, 4Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Mendola, Italy, 5GATA BMT Center, Gu¨lhane Military Medical Academy, Ankara Etlik, Turkey, 6Institut Paoli Calmettes, Inserm UMR 891, Marseille, France, 7Hospital Universitario de Salamanca, Servicio de Oncologı´a Me´dica, Salamanca, Spain, 8University Hospital Maastricht, Maastricht, Netherlands, 9Paris EBMT Study Office, Paris, France, 10Medical Oncology, di Circolo Hospital, Busto Arsizio, 11Medical Oncology, Misericordia Hospital, Grosseto, 12 Hematology Institute, Hospital of Cremona , Cremona, Italy Introduction: The efficacy of high-dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation
(AHSCT) for high-risk breast cancer (HRBC) patients has been an area of intense debate and controversy among the medical oncology community. Although limited informations are available on efficacy of HDC in subgroups of patients with different biologic breast cancer subtype, data from modern phase III studies suggest that HDC may still have a role in selected patients. Materials (or patients) and methods: The aim of this retrospective, multicentre study was to assess toxicity and efficacy of this procedure in a large cohort of operable HRBC treated with adjuvant HDC and transplanted between 1995 and 2005 in Europe. The outcomes that we analyzed were probabilities of DFS, OS, and TRM. Stratifying parameters included menopausal status, age, hormone receptor status (estrogen- or progesterone-receptor positive versus both negative), HER2 status (positive versus negative), number of positive lymph nodes, primary and grading tumor categories. Results: We have analyzed the EBMT registry and a total of 583 patients (43 positive nodes) were eligible for survival analysis. All patients received surgery before transplant and 55 (9.5%) patients received a neo-adjuvant treatment before surgery. The median age was 47 years, 66.9% of patients were premenopausal at treatment, 66.7% had endocrine-responsive tumors, 38% had a human epidermal growth factor receptor 2 (HER2) positive tumor and 72.7 % had 4 10 positive lymph nodes at surgery. Eighty percent received a single HDC procedure. The source of stem cells was peripheral blood in the vast majority of patients. Transplant-related mortality was 1.7%, whereas secondary tumor-related mortality was 0.91%. With a median follow-up of 120 months, OS and DFS at 5 and 10 years in the whole population were 75/61 and 64/44%, respectively. Subgroup analysis demonstrated that OS was significantly better in patients with endocrineresponsive tumors, less than 10 positive lymph nodes and smaller tumour size. HER2 status did not affect survival probability. Conclusion: Adjuvant HDC with AHSCT has a low mortality rate and provides impressive long-term survival rates in patients with HRBC. Our results suggest that this treatment modality should be proposed in selected HRBC patients and further investigated in clinical trials. Disclosure of Interest: None declared. P376 The efficacy of tandem stem cell transplantation in patients with high-risk neuroblastoma M. Sugiyama1,*, Y. Terashita1, J. Ohshima1, Y. Cho1,2, A. Iguchi1 1 pediatrics, Hokkaido University Graduate School of Medicine, 2 pediatrics, Hokkaido Cancer Center, Sapporo, Japan Introduction: Despite of high-dose chemotherapy (HDC), stem cell transplantation (SCT) and advanced therapies, such as 131I-MIBG, 13-cis-retinoic acid and anti-GD2 antibody, the prognosis of patients with high-risk neuroblastoma remains poor. Thus, we have analysed tandem SCT, including allogeneic SCT, in patients with high-risk neuroblastoma to improve their prognosis. Materials (or patients) and methods: We retrospectively reviewed medical records to evaluate the efficacy and safety of tandem SCT for patients with high-risk neuroblastoma between January 1998 and November 2013. We analysed 33 consecutive SCT for 27 patients (M/F ¼ 15/12) with advanced stage neuroblastoma (stage 4). Their median age was 3 years (range: 1-10 years). Twenty patients received SCT during first complete remission or very good partial response and 7 received SCT during second remission or non-remission: all of them were included in this study. Twenty-one patients received autologous peripheral blood SCT (PBSCT) or bone marrow transplantation (BMT), 8 received un-related cord blood transplantation (CBT) and 4 received related PBSCT or BMT. Five patients received tandem SCT: among them, 3 received auto-allo SCT: HDC and auto-PBSCT in addition to
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reduced-intensity conditioning (RIC) and allogenic CBT and 2 received tandem-auto-SCT: double HDC and double autoPBSCTs. HDC comprised melphalan (LPAM), etoposide (VP-16), and carboplatin in 8 patients, busulfan and LPAM in 7 patients and total body irradiation (TBI), LPAM/Cyclophosphamide, and VP-16 in 3 patients. RIC comprised of TBI, LPAM, fludarabine and anti-thymocyte globulin in 3 patients. Results: In all patients, the 4-year estimated overall survival (OS) and disease-free survival (DFS) rates were 54.5% ± 9.8% and 40.0% ± 9.6%, respectively. In patients receiving single HDC and SCT, OS and DFS were 41.6% ±11.0% and 28.6% ±9.9%100%, respectively. In contrast, in patients with tandem HDC and SCT, OS and DFS were 100% and 83.3% ± 15.2%, respectively (P ¼ 0.029 and P ¼ 0.034). There was a tendency that SCT during first remission or very good partial response had better outcome (P ¼ 0.059). There were no significant differences in age, sex, conditioning regimen and stem cell source for OS and DFS. Conclusion: Although it was a small scale analysis, significant favourable outcome has been achieved in patients receiving tandem SCT. Not only tandem auto SCT but also auto-allo SCT was effective. This suggests that auto-allo SCT is an alternative treatment to reduce late side-effects, and CBT as a second SCT could increase allo-reactive anti-tumour effects without severe graft-versus-host disease. Moreover, in advanced neuroblastoma, early tandem SCT should be performed because the prognosis of the patients with relapse or beyond second remission was extremely poor. Disclosure of Interest: None declared. P377 Autologous peripheral stem cell transplantation in testicular cancers: single institution experience N. Soyer1,*, A. F. Yilmaz1, R. Uslu2, A. P. Gorcegiz2, G. Saydam1, F. Sahin1, F. Vural1 1 Hematology, 2Medical Oncology, Ege University Medical Faculty, IZMIR, Turkey Introduction: Germ cell tumors are curable disease even in the presence of metastasis. Patients with relapsed or refractory tumors are candidates for salvage therapy. Salvage chemotherapy consisted of cisplatin, ifosfamide, vinblastine, paclitaxel or high-dose chemotherapy with autologous stemcell transplantation (ASCT) but the effectiveness of ASCT is not documented clearly. Materials (or patients) and methods: We retrospectively analyzed patients diagnosed as testicular cancer and treated with ASCT between January 2004 and November 2013. There were 28 ASCT transplantations of 19 patients, 9 of them were tandemly transplanted. Results: The characteristics of the patients and their response to chemotherapy before ASCT were shown at table 1. BEP regimen (bleomycin þ etoposide þ cisplatin) was given to patients with testicular cancer as first line treatment. Patients with severe lung involvement treated with BEP chemotherapy without bleomycin. Salvage chemotherapies were consisted of VIP- VeIP (etoposide þ ifosfamide þ cisplatin - vinblastin þ ifosfamide þ cisplatin) and TIP (paclitaxel þ ifosfamide þ cisplatin) for relapsed and refractory patients with testicular cancer. Median numbers of CD 34 ( þ ) stem cells infused to all transplanted patients were 5,71x106/kg (range; 2,5 – 6,83x106 / kg). Carboplatin and etoposide containing regimen were administered to 18 patients and one patient received ICE chemotherapy (ifosfamide- carboplatin- etoposide) as a conditioning regimen. 9 patients who underwent tandem transplantation received conditioning regimen consisted of carboplatin and etoposide in each transplantation. Complete response and partial response was documented in eleven of patients after ASCT. There was progressive or stabile disease in six patients. Two patients were died during transplantation procedure without response evaluation.Relapsed or progressive disease was documented in five patients with complete or partial response after median of 5 (range; 2- 13 months)
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months. Nine of 19 patients (%) were died because of progressive disease after transplantation. The median overall survival was 21 months (range; 4-264 months). N ¼ 19 Diagnosis Testicular cancer Site of diagnosis of testicular cancer Orchiectomy Liver biopsy Biopsy of mediastinal mass Biopsy of retroperitoneal mass Age (median, range, in years) Metastatic disease yes no Response to first line treatment Complete response Partial response Progressive disease Radiotherapy Yes No
19 16 1 1 1 31,5 (15-44) 18 1 4 8 7 2 17
Number of treatment before ASCT First line Second line Z 3 lines
1 13 5
Response before ASCT Complete/ partial response Progressive / stabile disease
6 13
Conclusion: ASCT transplantation is a treatment option in relapsed and refractory testicular cancers. It is a relatively safe procedure in terms of both mobilization and transplantation. Results of long term follow up were still not favorable. The effectiveness of ASCT should be evaluated in prospective randomized studies with large cohorts. Disclosure of Interest: None declared. P378 Cell therapy with EBV LMP2-specific cytotoxic T-lymphocytes for patients with nasopharingeal carcinoma P. Comoli1,*, S. Basso2, S. Secondino3, A. Gurrado2, P. Morbini 4, M. Benazzo5, G. Quartuccio2, I. Guido2, A. Pellerano2, C. Paglino3, M. Zecca2, P. Pedrazzoli3 on behalf of on behalf of The ‘‘San Matteo’’ Head and Neck Tumor Multidisciplinary Group 1 Pediatric Hematology/Oncology, Fondazione IRCCS Policlinico S. Matteo, 2Pediatric Hematology/Oncology, 3Oncology, Fondazione IRCCS Policlinico San Matteo, 4Pathology, 5Otolaryngology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia, Italy Introduction: Nasopharyngeal carcinoma (NPC) is an EpsteinBarr virus (EBV)-related malignancy expressing a restricted set of viral antigens. The standard of care for locally advanced head & neck cancer is concurrent platinum-based chemotherapy and radiotherapy, with overall response rates exceeding 90% in NPC. However, for patients with local-regional recurrent or metastatic disease, the benefits of combination chemotherapy are short-lived. Onset of these virus-related tumors is favored by immune surveillance defects, and expression of antigenic viral proteins by malignant cells constitutes a good target for immunotherapeutic strategies. Materials (or patients) and methods: Since 2001, we have implemented a T-cell therapy program for patients with NPC failing conventional treatment. Standard EBV-specific cytotoxic T-lymphocytes (EBV-CTLs) have been so far expanded for 28/35 patients, while LMP2-specific CTLs expanded with LMP2-derived 15mer peptide pools could be successfully generated from 15 patients. Eligible patients with stage IV disease in relapse were treated in two sequential trials of cell therapy with autologous EBV-CTLs. In the first set
of patients, 4 escalating doses to a maximum of 8x107 CTL/dose were administered, followed by maintenance infusions in case of response; in the second protocol, the patients received 2 CTL administrations with a median cell dose/infusion of 3.5 x 108 (range 2-4 x 108) preceded by lymphodepleting chemotherapy. Results: We have treated 27 patients with disease resistant/ relapsing after 42 lines of radio-chemotherapy. Overall, the objective control of disease was 57%, with 6/27 patients showing complete (n ¼ 1), partial (n ¼ 4) and minimal (n ¼ 1) responses, and 10/27 disease stabilization (median duration 7 months). No severe adverse events were observed after cell therapy; 4 patients showed an inflammatory reaction at the tumor site. The use of preparative chemotherapy and increased CTL dose did not influence outcome. Importantly, patients showing response to cell therapy showed the emergence of EBV LMP2 antigen-specific T-cells in their peripheral blood. As we did not observe an increase in cell therapy activity, measured as proportion of CR þ PR measured according to the RECIST criteria, we inferred that a more specific T cell product, administered in earlier disease stages, may yield better results. Thus, we started a new trial in patients treated for metastatic disease, with EBV- or LMP2-CTL administered as adjuvant therapy (at completion of first line treatment). So far, we treated 6 patients (3 in each arm). None of the patients treated with EBV-CTL þ LMP2-CTL experienced disease relapse at a median follow-up of 12 months, while 2 of the 3 patients enrolled in the EBV-CTL only arm showed disease relapse at months þ 1 and þ 8, respectively. Conclusion: EBV-specific CTL therapy is safe and associated with clinical benefit in patients with advanced NPC refractory to standard therapies. The use of CTL with higher specificity for the EBV subdominant antigens expressed by the tumor, such as LMP2, at an earlier stage of disease, could further implement the strategy and ameliorate the outcome of patients with relapsing/refractory NPC. Disclosure of Interest: None declared. P379 High dose chemotherapy supported with autologous hematopoietic stem cells transplantation in patients with advanced germ cell tumors who had failed to be cured by standard treatment. Warsaw experience T. Sarosiek1, B. Mlot1, C. Szczylik1, P. K. Rzepecki1,* 1 MILITARY INST. OF MEDICINE, Warsaw, Poland Introduction: About 5 to 50% of all patients with advanced germ cell cancer (depending on the IGCCCG prognostic group) will experience a relapse or a disease progression, despite undergoing standard treatment including chemotherapy and resection of residual lesions. According to the published papers, patients who have failed to be cured with the first line chemotherapy have rather poor prognosis with only 20 to 40% probability of long term survival using the standard rescue treatment. High dose chemotherapy (HDC) followed by autologous haematopoietic stem cell transplantation has already been studied for the last 20 years as a rescue treatment for such patients. To find factors influencing the efficacy and toxicity of the high dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation (BMT) in patients with relapsed or refractory germ cell tumors (GCT). Materials (or patients) and methods: We performed 65 procedures of HDC þ BMT in 51 patients. One (in 37 patients) or two sequential (14 patients) cycles of HDC were used. The median of 2 x 106/kg CD34 þ cells were infused after HDC. Results: Treatment toxicity increased with Beyer prognostic score, number of previous chemotherapy cycles and ECOG performance status. Complete remission has been achieved in 31,37%, partial in 39,22% and stable disease in 4% of patients. 12-month overall survival rate (OS) averaged 58,5% , 24-month
OS was 50,6%. 12- and 24-month progression free survivals (PFS) were 58% and 54,5%. Conclusion: Independent prognostic factors for OS included beta-HCG level above 1000 mIu/ml, elevated AFP level before transplantation, IGCCCG prognostic group at diagnosis. Beyer prognostic score was an independent prognostic factor for PFS. HDC can be curative for some relapsed or refractory GCT patients. Those with upfront poor prognosis (according to Beyer prognostic score), high beta-HCG levels or poor ECOG performance status do not benefit from this kind of treatment. Disclosure of Interest: None declared. P380 Hematopoietic Stem Cell Transplantation in Relapsed or Refractory Extracranial Primitive Neuro-Ectodermal Tumor of Children and Adolescents: a Multicenter Survey by the Turkish Pediatric Bone Marrow Transplantation Study Group (TPBMT-SG) V. Hazar1,*, M. Karakukcu2, A. Kupesiz3, V. Kesik4, E. Unal2, U. Kocak5, N. Eker3, E. Atas4, S. Aksoylar6, N. Tacyildiz7, M. Elli8, S. Yilmaz9, Z. Kaya5, S. Kansoy6, E. Unal7, F. Erbey10, S. Emir11, H. Oniz12, A. Yesilipek13 1 Pediatric Hematology/Oncology and BMT Unit, Medipol University Faculty of Medicine, Istanbul, 2Pediatric Hematology/ Oncology and BMT Unit, Erciyes University Faculty of Medicine, Kayseri, 3Pediatric Hematology/Oncology and BMT Unit, Akdeniz University Facuty of Medicine, Antalya, 4Pediatric Oncology and BMT Unit, Gulhane Military Medical Academy, 5Pediatric Hematologyand BMT Unit, Gazi University Faculty of Medicine, Ankara, 6Pediatric Oncology and BMT Unit, Ege University Faculty of Medicine, ˙Izmir, 7Pediatric Oncology and BMT Unit, Ankara University Faculty of Medicine, Ankara, 8Pediatric Oncology and BMT Unit, On Dokuz Mayis University Faculty of Medicine, Samsun, 9Pediatric Hematologyand BMT Unit, Dokuz Eylul University Faculty of Medicine, ˙Izmir, 10Pediatric Hematology/ Oncology and BMT Unit, Medical Park Bahcelievler Hospital, Istanbul, 11Pediatric Hematology/Oncology and BMT Unit, Childrens Hematology and Oncology Hospital, Ankara, 12Pediatric Oncology and BMT Unit, Tepecik Childrens Hospital, ˙Izmir, 13 Pediatric Hematology/Oncology and BMT Unit, Bahcesehir University Faculty of Medicine, Istanbul, Turkey Introduction: The objective of this study was to evaluate the outcomes experienced by children with relapsed or refractory extracranial Primitive Neuro-Ectodermal Tumor (rr-ePNET) following high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT). Materials (or patients) and methods: We retrospectively analyzed the outcomes of 44 patients with rr-ePNET. Complete remission could not be achieved in 7 patients prior to transplantation due to primary refractory disease. At the time of transplantation, only 66% of the patients were chemosensitive. The majority of patients received busulphan þ melphalan for conditioning (28/44), and peripheral blood (41/44) was used as a source of stem cells. Results: After a median follow-up period of 14 months, 20 patients were alive. At 2 years, the probabilities of overall survival (OS) and disease-free survival (DFS), the relapse rate (RR) and the non-relapse mortality (NRM) rate were 33.3%, 41.4%, 50.0% and 9.1%, respectively. The probability of DFS in chemosensitive and chemoresistant patients at 2 years was 63.8% and 13.3%, respectively (P ¼ 0.004). Patients with only lung metastases had better PFS than those with other or combined metastases (2-year DFS 63.2% vs 31.5%, P ¼ 0.102). Multivariate analysis showed that chemoresistant disease at the time of transplantation was the only factor predicting limited PFS (odds ¼ 2.739, P ¼ 0.026). Conclusion: HSCT increased PFS in a significant proportion of children with rr-ePNET. Survival rates were better for patients with chemosensitive disease at the time of HSCT. Disclosure of Interest: None declared.
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Poster Session 1 Cell therapy/cellular therapy I P001 Abstract Withdrawn
P002 HLA-haploidentical allografting with high-dose posttransplant cyclophosphamide: clinical outcomes and immune-reconstitution A. Busca1,*, E. Maffini1, F. Ferrando1, C. Dellacasa1, R. Pulito1, E. Saraci1, L. Giaccone1, M. Boccadoro1, P. Omede`1, B. Bruno1 1 Division of Hematology, Department of Molecular Biotechnology and Health Sciences, University of Torino, A.O.U. Citta` della Salute e della Scienza di Torino, Torino, Italy Introduction: HLA-haploidentical allografting is a potential cure in patients (pts) without a HLA-identical donor. Materials (or patients) and methods: Between April 2010 and November 2014, 41 pts (median age 50, r 21-71 years) with hematologic malignancies (33 acute leukemias; n ¼ 2 CML; n ¼ 6 lymphomas) were transplanted from a HLAhaploidentical donor. Fifty% had advanced disease at the time of transplant. Eight pts had received a previous allograft. Conditioning was either reduced-intensity (11 pts) with fludarabine (30 mg/m2/day for 5 days), cyclophosphamide (Cy) (14.5 mg/Kg/day for 2 days) and single dose 2 Gy TBI, or myeloablative (29 pts) with thiotepa (TT) (5 mg/Kg/day for 2 days), fludarabine (50 mg/m2/day for 3 days) and i.v. Busulphan (Bu) (3.2 mg/Kg/day for 3 days) or thiotepafludarabine-melphalan (140 mg/m2/day) (1 patient), followed by T-cell replete bone marrow (34 pts) or peripheral blood stem cells (7 pts). Post-transplant immunosuppression consisted of Cy, 50 mg/Kg/day on days þ 3 and þ 4, followed by mycophenolate mofetil and FK-506. G-CSF was administered from day þ 5 until engraftment. In 22 (54%) pts, T-cell reconstitution and thymic activity were studied for the first 2 years post-transplant. T-cells subsets in both the CD4 þ and CD8 þ T cell compartments were evaluated by flow-cytometry: naı¨ve (CD45RO- and CD27 þ ); central memory (CD45RO þ CD27 þ ); effector memory (CD45RO- CD27-); and revertant (CD45RA þ CD45RO þ ). Thymic function through T-cell receptor rearrangement excision circles (TRECs) was measured by RT quantitative PCR on DNA extracted from peripheral mononuclear cells and from sorted CD4 þ and CD8 þ T cells. Here we present preliminary data on thymic output and naı¨ve CD4 þ T cell recovery Results: Neutrophil recovery occurred at a median of 17 days. Incidence of acute grade II-IV and III-IV GVHD was 28,2% and 7,6%. Blood stream infections were documented in 13 pts (7 gram þ bacteria; 6 gram- bacteria). Six had probable invasive fungal infection, in 2 cases after prophylaxis with micafungin and in 4 with azoles. CMV reactivation occurred in 21 and EBV reactivation, successfully treated with rituximab, in 1. At a median follow-up of 501 days (r 18-1278 days), 19 pts (67%) are alive, 11 (58%) in complete remission. Median OS and DFS at 2 years were 46% and 34%. Overall, 14 pts (34%) died of relapse and 8 pts (19%) of transplant related mortality (TRM). The probability of TRM was 16% at 100 days and 26% at 1 year.
(CD4 þ CD27 þ CD45RA þ ) Naı¨ve T cells decreased from a median of 23.92/ul at day 28 to 1.8/ul at day 90, then gradually increased to 3.8/ul, 3.9/ul, 8.7/ul at 6, 12 and 18 months, respectively. Median TREC copies/100 ng DNA from sorted CD4 þ cells decreased from 14.5 (r 0-202.2) pre-transplant to 4.4 (r 0-49.2) at day 90, than increased to 9.5 (r 0-134.2), 24.8 (r 2.7-83), 41.5 (r 5.5-102.2) and 47.41 (r 31.5-584.3) at 6, 12, 18 and 24 months, respectively. In healthy donors, median TREC copies/100 ng DNA was 74.5 in sorted CD4 þ cells. Conclusion: Feasibility of HLA-haploidentical transplantation with high-dose post-transplant Cy in heavily pretreated pts was confirmed. Thymic activity and immune recovery of naı¨ve CD4 þ T cells were slow and less robust when compared with transplants from HLA identical donors. Longer follow-up on a larger series of pts is needed to evaluate long-term outcomes and correlate clinical issues with immunological data. Disclosure of Interest: None declared. P003 Cost analysis of dendritic cell therapy for acute myeloid leukemia patients A. Van de Velde1,*, P. Beutels2, S. Anguille1, A. Gadisseur1, W. Schroyens1, A. Verlinden1, E. Smits3, V. Van Tendeloo4, G. Nijs3, S. Dom5, I. Cornille5, Z. Berneman1 1 Department of Hematology, Antwerp University Hospital, 2 Centre for Health Economics Research & Modeling Infectious Diseases, University of Antwerp, 3Centre for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, 4Vaccine & Infectious Disease Institute, University of Antwerp, 5Business Intelligence, Antwerp University Hospital, Antwerp, Belgium Introduction: Treatment of acute myeloid leukemia (AML) is a significant economic burden for patients, insurance companies, and society and is doubled over 10 year. Hospitalization costs are the key drivers. Most costs are associated with remission induction treatment. When relapse occurs, high costs are incurred again, particularly when another round of chemotherapy is given. Findings suggest that savings to the healthcare system could be achieved by sustaining complete remission (CR) status for longer periods. Materials (or patients) and methods: Between January 2005 and December 2010, a total of 55 adults with AML received antileukemic therapy at the Antwerp University Hospital (UZA). The analysis excluded five patients because of incomplete cost data about remission-induction therapies in their referring hospitals. A total of 50 patients were included in the analysis. Fifteen patients were treated with chemotherapy alone (group 1), 25 patients with chemotherapy followed by allogeneic hematopoietic stem cell transplantation (HSCT) (group 2) and 10 patients with chemotherapy followed by immunotherapy using dendritic cells engineered to express the Wilms’ tumor (WT1) protein (group 3). (Van Tendeloo, 2010). The Ethics Committee at UZA approved this cost study and patients in group 2 and 3 provided signed, informed consent respectively for their allogeneic HSCT and for their enrollment in the immunotherapy study. Data regarding patient characteristics were obtained from patient’s medical records. Professional and facility charges were collected using electronic billing information. Drug costs and drug administration costs were based on list prices published by the Belgian reimbursement authority (RIZIV/INAMI). Room costs and care at the hematology ward, pharmacy, intensive care unit, laboratory tests and medical imaging were analyzed. The cost of dendritic cell vaccine preparation was h 20,450 per patient.
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Results: The median cost in group 1 (15 patients) was h 32, 648 (range: h 4,759 - h 140,383). Only 2 patients in group 1 went into remission after induction therapy and received consolidation therapy. The median post-consolidation treatment cost in group 1 was h 35,581 (range: h 30,069 - h 41,093). All patients in group 1 died within 5 year after diagnosis, 13 patients died within 1 year and 5 died within 1 month. The median cost in group 2 (25 patients) was h 185,212 (range: h 87,932 - h 449,155). The median post-consolidation treatment cost in group 2 was h 117,241 (range: h 31,364 - h 304,366). Five-year survival in group 2 was 19%. Seventeen patients in group 2 died within 1 year after HSCT. The median cost in group 3 (10 patients) was h 114,505 (range: h 45,114 - h 207,731). The median post-consolidation treatment cost in group 3 was h 40,748 (range: h 26,907- h 156,870). Five-year survival in group 3 was 30%. Four patients in group 3 died within 1 year after vaccination. Conclusion: It is critical to focus on developing new therapies that can prevent relapse and maintain AML patients’ CR status to maximize their survival. Immunotherapy using dendritic cells engineered to express the Wilms’ tumor protein is one of these new therapies and in this study has 1/3 of the post consolidation costs of HSCT. Disclosure of Interest: None declared. P004 Generation of HLA*A02:01 restricted CD19 specific T cells in CD19 non-tolerant hosts A. Maurberger1,*, H. Balzer1, E. Roth2, E. Kieback3, A. Kremer1, A. Mackensen1, H.-M. Ja¨ck2, W. Uckert3, A. Gerbitz1 1 Medicine 5, Haematology/Oncology, Erlangen University Hospital, 2Division of Molecular Immunology, Department of Internal Medicine III, Friederich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Erlangen, 3Max-Delbru¨ck-Center for Molecular Medicine, Humboldt-University Berlin, Berlin, Germany Introduction: Targeting the B cell specific antigen CD19 by antibodies or CAR-modified T cells appears to be very effective in treatment of leukemias and lymphomas. However, downregulation of surface CD19 renders target cells invisible for antibody/CAR mediated therapies making it also an attractive target for TCR-transduced T cell therapy. One major obstacle for the generation of high avidity T cells with specificity for self antigens is circumvention of central tolerance. Materials (or patients) and methods: CD19 deficient mice were crossbred onto an HLA*A02:01 transgenic HHD2 background to generate CD19 non-tolerant animals. CD19-/-HHD2 mice were immunized against human CD19 using peptidepools containing HLA*A02:01 binding fragments of the huCD19 protein. Immunogenic peptides were identified by ELISA and peptide-specific T cells were enriched and expanded by restimulation in vitro. TCRs from specific T cells were identified by nested PCR and cloned into a pMP71 vector. Human T cells were retrovirally transduced to express a CD19 specific TCR. Results: Using repetitive peptide pool immunization we identified a dominant CD19 derived peptide and isolated specific HLA*A02:01 restricted CD8 þ T cells. When restimulated with the specific peptide in vitro T cells displayed proliferation and showed a strong secretion of IFNg. After four rounds of in vitro restimulation flow cytometric T-cell receptor analysis revealed a TCRva4.3 and TCRvb4 restriction which was confirmed by nested PCR. Repetitive stimulation in vitro resulted in enrichment of CD8 þ T cells binding peptide loaded HLA multimers from 2% up to 20%. CD19 specific T cells could be further enriched by flow cytometry based on peptide loaded HLA multimer. A purity of 498% was achieved. After cloning of both TCR chains into an MP71 vector backbone human T cells were retrovirally transduced to express a CD19 specific TCR. TCR transduced T cells were enriched by flow cytometry using antibodies specific for TCRvb4 (498% purity) and expanded by CD3/28 bead stimulation.
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Conclusion: Our results demonstrate that human CD19 serves as a T-cell antigen in the context of human HLA*A02:01. By utilizing a xenogenic approach central tolerance was circumvented and human CD19 specific T cells were generated and expanded. Retroviral transduction of the identified TCRs into human T cells will allow for testing this approach in preclinical models. Disclosure of Interest: None declared. P005 Optimised protocol for clinical scale isolation and expansion of invariant NKT cells for prevention of aGVHD A. Rotolo1,*, S. Loaiza1,2, S.-A. Finn2, E. Bray2, O. Pello2, S. Uddin2, A. Bradshaw2, J. F. Apperley1, A. Chaidos1, A. Karadimitris1 1 Centre for Haematology, 2John Goldman Centre for Cellular Therapy Hammersmith Hospital, Imperial College London, London, United Kingdom Introduction: Acute graft–versus-host disease (aGVHD) severely limits the wider applicability of allogeneic HSCT. iNKT cells, a rare subset of CD1d-restricted, glycolipid-specific T cells, may be crucial in the regulation of aGVHD. In line with animal models showing a role of donor iNKT cells in protecting/abrogating aGVHD, we found that a high donor CD4– iNKT cell dose in the graft is associated with a 4x reduction of the relative risk of clinically significant aGVHD. Therefore, we hypothesise that in vitro expanded donor iNKT cells adoptively transferred to allogeneic HSCT recipients receiving an iNKT-poor graft will prevent occurrence of clinically significant aGVHD. Towards this goal, we propose to develop a clinical scale GMP-compliant protocol to isolate and expand donor CD4– iNKT cells. Materials (or patients) and methods: iNKT cells were positively selected from fresh PBMC/frozen lymphapheresis products using iNKT magnetic beads (6B11mAb) and expanded with irradiated autologous PBMCs, IL2 and aGalCer (aGC) or aCD3/CD28-coated beads. For the GMP-compliant protocol, the CliniMACS device and GMP-grade TexMACS medium and IL2 were used. Expanded cells were characterized by flow cytometry-based cytokine profile. Results: First we optimised a protocol for iNKT isolation and expansion aiming to preserve the pre-selection relative frequency of CD4– cells in the post-expansion cell product. Using iNKT-specific magnetic beads, iNKT cells were effectively selected from fresh PBMCs as well as frozen lymphapheresis products with high yield and purity (Table 1). We tested 3 different post-selection expansion protocols, using aCD3/CD28 beads, autologous aGC-loaded PBMCs or aCD3/CD28 beads in the presence of autologous PBMCs (n ¼ 3). iNKT cells were expanded 4450x by all 3 methods in o3 weeks. However, the aGC-based protocol was less dependent on the initial iNKT purity and it was the only one that preserved the relative frequency of the CD4– cells. Next we adopted the aGC-based
approach for clinical scale iNKT selection and expansion from donor frozen lymphapheresis products using the CliniMACS apparatus and GMP-grade reagents (TexMACS medium and IL2). In 2 experiments, pre-selection iNKT cells were 0.13% of T cells in donor 1 (1.4x106,B0.03% of TNC) and 0.009% of T cells in donor 2 (1.6x104,o0.002% of TNC). Post CliniMACS selection, iNKT cells were 94% and 36% of T cells respectively. In both cases cells were effectively expanded, achieving B700x increase by day 18 from donor 1. Analysis of the Th1/2/ 17 cytokine profile showed that clinical scale expanded iNKT cells displayed a preferential Th1-like profile. Conclusion: We optimised a protocol for in vitro iNKT cell isolation and expansion, which ensures preservation of the relative frequency of the CD4– fraction that likely imparts protection from aGVHD. This and the 4100x expansion by 4 weeks using GMP-compliant reagents and processes provides proof-of-principle that clinical scale iNKT isolation and expansion is feasible. Notably, the process is efficient for iNKT-rich as well as iNKT-poor donors. These results show that with availability of GMP-grade iNKT selection beads and aGC the process of clinical scale iNKT selection and expansion can be easily rendered fully GMP-compliant and therefore suitable for clinical use. References: Chaidos et al, Blood 2012. Kuwatani et al, Immunol Lett 2006. Leveson-Gower et al, Blood 2011. Yang et al, Transfusion 2010. Disclosure of Interest: None declared.
HLA identical family members (n:5) or alternative donors (n:3) at Hacettepe University (n:4) or Bahc¸es¸ehir University (n:4). Of these 8 children who underwent HSCT, 4 received Busulfan (Bu) and Fludarabin (Flu) and 4 received Bu, Flu and Thiothepa as a conditioning regimen. Engraftment was achieved in 7/8 children at median day þ 14 and day þ 23 for neutrophils and platelets; respectively. The median chimerism level was 96% (82-100%) at 1 month after HSCT. Veno-occlusive disease developed in 2 children, pulmonary hypertension was seen in 2, and post-transplant hypercalcemia was seen in 1 child during HSCT. All complications related to HSCT were successfully treated. The mean follow up duration was 20.8±4.5 months (2 months-60 months). During the follow up period, 12 of 14 children are alive. Among 8 children who underwent HSCT; 7 are alive with no complications related to HSCT. However, only 1 child who received HSCT from HLA 6/10 identical mother (haploidentical HSCT) had engraftment failure and HLA identical unrelated donor search has been continued for 2nd HSCT. Conclusion: In this study; among 8 children who underwent MSC-HSC co-transplantation; 7 achieved engraftment and they are alive with no HSCT related complications. In conclusion; this pilot study suggests that MSC-HSC co-transplantation may be useful to decrease early morbidity and mortality in children with MIOP. However, MSC administration alone does not appear to change the clinical course of the disease. Disclosure of Interest: None declared.
P006 Mesenchymal Stem Cell Therapy in Malign Infantile Osteopetrosis: Hacettepe Experience From Turkey B. Tavil1,*, G. Karasu2, B. Kus¸konmaz1, F. V. Okur1, M. O. Kaynak1, M. Çetin1, D. Uc¸kan1 1 Hacettepe University Faculty of Medicine, Pediatric Hematology Unit, Ankara, 2Medicalpark Go¨ztepe Hospital, Bahc¸es¸ehir University, ˙Istanbul, Turkey
P007 Post-thaw DMSO depletion using a cGMP-compliant spinning-membrane separation device B. Calmels1,2,*, E. Guyon1, C. Wegener3, G. Bouchet1, J. Couquiaud1, L. Regimbaud1, C. Lemarie1,2, P. Houze4, K. Min3, C. Chabannon1,2 1 centre de therapie cellulaire, 2INSERM CBT-1409, institut paolicalmettes, marseille, France, 3Fresenius Kabi USA LLC, Lake Zurich, United States, 4laboratoire de biochimie, hopital saintlouis, paris, France
Introduction: Malignant infantile osteopetrosis (MIOP) is an autosomal recessive fatal disorder. Even if hematopoietic stem cell transplantation (HSCT) is performed from an HLA identical donor, morbidity and mortality of HSCT is very high in MIOP. This study aims to apply mesenchymal stem cell (MSC) to the children with MIOP to decrease the morbidity and mortality of HSCT, to decrease the complications related to MIOP, and to support engraftment. Materials (or patients) and methods: This study included 14 children with the diagnosis of MIOP referred to Hacettepe University Faculty of Medicine, Pediatric Bone Marrow Transplantation Unit between December 2012 and December 2014. Detailed laboratory and imaging studies were performed in our hospital. Ethical approval was obtained from local ethical committee of Ankara University (Number: 29-589) and from the HSCT scientific advisory committee of Turkey Ministry of Health. Mesenchymal stem cell suspensions were prepared at GMP Laboratories of Ati-gen cell in Trabzon and applied by intravenous infusion. This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK 1001 project, 112S127). Results: The mean age of 14 children (7 boys/7 girls) with MIOP was 8.0±6.7 months (1-24 months). Homozygous mutation was detected in TCIR gene for 7 children, in ClCN7 gene for 5 children, in STX gene for 1 child, and in RANK gene for 1 child. Among these 11 children; 8 received MSCs as MSCHSC co-transplantation and the other 3 children received only MSCs while waiting for HSCT, but they were not received HSCT because neuropatic form of the disease (n:2) or his parents were not give permission (n:1). A total of 18 MSC infusions were performed for 11 children with MIOP. The mean MSC dosage was 3.4±1.3 x106/kg (median: 3.5 x106/kg; range: 2-5.4 x106/kg). MSC donors were family members who were HLA 510/10 identical with the children. HSCTs were performed from
Introduction: Cryopreserved stem cell grafts are still widely infused to many patients, both in the autologous or allogeneic settings. Cryopreserved grafts can be thawed at the bedside or thawed and washed at the cell therapy laboratory. We recently published that post-thaw washing did not impair hematopoietic engraftment, in a cohort of 2,930 autologous transplanted patients receiving either unwashed or washed grafts (Calmels B et al, 2014). Post-thaw washing can be implemented using various methods such as manual centrifugation, automated centrifuge-based or spinning-membrane devices such as the Lovo (Fresenius Kabi). Materials (or patients) and methods: We here report the pre-clinical evaluation of the Lovo for washing thawed stem cell grafts. We took advantage of 15 apheresis products intended for destruction and cryopreserved in 2 identical bags; after dry-thawing (PlasmaTherm, Barkey), bags were connected to the Lovo, diluted at 20 ml/min with þ 4-81C 6% hydroxyethylstarch 130/0.4 (Voluven, Fresenius Kabi) and processed using 2- or 3-cycles reduction (a cycle referring to one pass through the spinning membrane). Results: After washing, CD34 and CD45 absolute counts and viability were evaluated (Stem-Kit, Beckman Coulter) and DMSO was quantified by capillary zone electrophoresis (P/ACE, Beckman Coulter). Lengths of procedures were 15 min and 18 min for 2- and 3-cycles, respectively. Post-wash data show consistent viability, effective DMSO depletion and relevant CD34 recovery. Conclusion: We conclude that this new spinning-membrane device enables for high efficiency DMSO depletion while preserving optimal CD34 viability and recovery. Moreover, the length of procedures does not significantly delay the process, as compared to bedside thawing. Post-thaw washing using automated cell processing systems have thus to be preferred since they provide multiple benefits such as DMSO and cell
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debris depletion, precise determination of infused CD34 cell dose, increased cellular stability as well as adjustable washing efficiency and final volume. Disclosure of Interest: B. Calmels: None declared, E. Guyon: None declared, C. Wegener Employee of: Fresenius Kabi, G. Bouchet: None declared, J. Couquiaud: None declared, L. Regimbaud: None declared, C. Lemarie: None declared, P. Houze: None declared, K. Min Employee of: Fresenius Kabi, C. Chabannon: None declared P008 Adoptive Immunotherapy of refractory systemic adenovirus infections after allogeneic umbilical cord blood (UCB) or Peripheral Blood stem cell transplantation C. Qian1,*, V. DECOT2, Y. WANG1, H. L. CAI3, V. VENARD4, H. JEULIN4, M. DE CARVALHO BITTENCOURT3, J. H. DALLE5, C. POCHON6, B. BRUNO7, C. PAILLARD8, S. VIGOUROUX9, C. JUBERT10, P. CEBALLOS11, A. MARIE CARDINE12, C. GALAMBRUN13, L. CLEMENT6, D. BENSOUSSAN1 1 UTCT, Cell Therapy Unit , 2UTCT Cell Therapy Unit , 3Plateforme Nancytomique, 4Laboratoire de Virologie , CHU de Nancy, VANDOEUVRE LES NANCY, 5Service d’he´mato-immunologie , Hoˆpital Robert Debre´, Paris, 6UTMA , CHU de Nancy, VANDOEUVRE LES NANCY, 7Service d’He´matologie pe´diatrique CHU de LILLE, CHU de LILLE, Lille, 8Service d’He´matologie Pe´diatrique, CHU de Strasbourg, STRASBOURG, 9Service de transplantation me´dullaire, 10Servide d’He´matologie Pe´diatrique, CHU de Bordeaux, BORDEAUX, 11Service de transplantation me´dullaire, CHU de Montpellier, MONTPELLIER, 12Service d’He´matologie Pe´diatrique, CHU de Rouen, ROUEN, 13Service d’He´matologie Pe´diatrique, CHU La Timone, MARSEILLE, France Introduction: Adenovirus (ADV) systemic infection refractory to antiviral treatment after allogeneic stem cell transplantation is associated with a high mortality rate up to 50%. Adoptive transfer of ADV-specific T cells (ADV-STs) is becoming an alternative treatment that has already proved feasible, safe and helpful in viral clearance and immune reconstitution related to an in vivo expansion of ADV-STs leading to clinical improvement. Materials (or patients) and methods: We previously demonstrated the feasibility of isolating ADV-STs from healthy donors using the IFNg-capture system from Miltenyi followed by immunomagnetic selection on the CliniMACS device (AissiRothe´ et al, 2010). We have now included 13 patients among 14 in a multicentric phase I/II clinical trial consisting in infusion of polyclonal ADV-STs generated by a 6-hour ex vivo stimulation with Pept-ADV5 hexon (peptide pool from the immunodominant Hexon protein, Miltenyi Biotec) of leukapheresis collected from their original stem cell donor or from third party haploidentical donors, followed by isolation of IFNg producing cells. Results: We report on the generation of 12 ADV-ST preparations and the infusion of 10 pediatric and adult patients (8/2) with ADV infection (n ¼ 5; blood and/or stool viral load) or ADV disease (n ¼ 5). Five patients had experienced grade II GVHD controlled by immunosuppressive treatments before infusion. They received a mean of 5.84 103 CD3-IFNg þ cells/ kg (range: 0,25 to 31.78 103) 1 to 9 months after HSCT. In vivo expansion of transferred ADV-STs was observed in 9 of 10 patients from day 14 to 60 following adoptive transfer infusion associated with ADV load decrease or clearance for 9 of them (90%) but one patient experienced a
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secondary increase. Neither de novo GVHD, nor side effects were observed. Reactivation of GVHD occurred in 3 patients and could be controlled by immunosuppressive treatments in 2 patients. In one patient, with stabilized GVHD grade II before infusion who experienced a modulation of immunosuppression before ADV-VSTs infusion, GVHD worsened in grade III with cutaneous, hepatic and intestinal manifestations responsible for the patient death. Two other patients died, one related to ADV disease (ADV and EBV meningitis), but none of the responders had ADV-associated mortality. Conclusion: Adoptive transfer of ADV-STs is a feasible and well-tolerated therapeutic option, representing a fast and efficient procedure to achieve reconstitution of antiviral T-cell and decrease or clearance of ADV viral load. We expect that complete analysis of all the clinical data from the trial will allow attributing GVHD reactivations either to VSTs infusion or modulation of immunosuppressive drugs. References: Aissi-Rothe´ L, Decot V, Venard V, Jeulin H, Salmon A, Clement L, Kennel A, Mathieu C, Dalle JH, Rauser G, Cambouris C, De Carvalho M, Stoltz JF, Bordigoni P, Bensoussan D. Rapid generation of full clinical grade human anti-adenovirus cytotoxic T cells for adoptive immunotherapy. J Immunother. 2010; 33: 414–424. Disclosure of Interest: None declared. P009 Mononuclear cell collection for extracorporeal photochemotherapy by using the ‘‘off-line’’ system: a study comparing an automatic and a semiautomatic apheresis device in graft versus host disease E. Gonzalez-Arias1,*, C. Pascual1, A. Perez-Corral1, L. Solan1, S. Redondo1, M. Bastos1, D. Serrano1, J. Gayoso1, M. Kwon1, J. Anguita1, J. Diez-Martin1 1 Hematologia, Hospital Gregorio Maran˜on, Madrid, Spain Introduction: Extracorporeal photochemotherapy (ECP) is an effective cell therapy employed in several diseases, including graft-versus-host disease (GVHD) after stem cell transplantation (SCT). When ECP is performed using the off-line technique, optimal mononuclear cells (MNC) collection by leukapheresis (LA) is necessary for further manipulation. The objective of our study was to compare the semiautomatic COBE Spectra (Terumo BCTs, Lakewood, CO, USA; version 7.0) and the new automated Spectra OPTIA (Terumo BCTs v.5.0) in terms of efficacy and safety in ECP. Materials (or patients) and methods: All patients with refractory acute and chronic GVHD in treatment with ECP were retrospectively studied, from June 2014 (when OPTIA device was introduced into our service) to November 2014. Patients were alternatively assigned with either apheresis device. Peripheral blood samples were obtained before and after each LA procedure, along with a sample of each product just before reinfusion. The cell blood count (CBC) was perfomed in all samples with Unicell DHX 800 (Beckman Coulters). Collection efficiency (CE) of white blood cell (WBC) and total mononuclear cells (TMNC) and platelet loss were calculated as follows: WBC CE% ¼ [productWBCx106/mL x product volume* mL/ preapheresis NC (x106/mL)x(Vol processed Ml-AC volume Ml)]x100. TMNC CE% ¼ [product MNCx106/mL x product volume* mL/ preapheresis MNC (x106/mL)x (vol processed mL-ACvolume mL)]x100.
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Platelet loss ¼ [Preapheresis PL count-Postspheresis PL count/ Preapheresis PL count] x100. *In all cases we completed with saline solution to a total volume of 300ml. We compared patient’s CBC before and after the LA, the CBC of the product and the procedure variables with both devices, using non-parametric methods (U Mann-Whitney). Results: One hundred fourteen procedures (63 in COBE and 53 in OPTIA) were carried out in 9 patients (8 with chronic GVHD and 2 with acute GVHD). The median age was 39 years old (range: 18-56). Central venous access was used in 8/9 (88,8%) patients. Both devices were safe, and there were no significant adverse events recorded. CBC results before and after LA are detailed in Table 1. The processed volume and the amount of ACD used were less in the Optia group. Hemoglobin and hematocrit product levels were higher in the Optia group, and TMNC count was similar with both devices. Notably, TMNC CE, WBC CE and platelet loss was better with OPTIA device. There was no difference in the time of the procedure. Further details are shown in Table 2. Conclusion: In our study, the Spectra Optia has proven to be more effective in collecting MNC and WBC than COBE device. The lower platelet loss is an important aspect because these patients often have low platelet counts. Fewer ACD used in the Optia group may be an advantage that may result in a lower symptoms of hypocalcemia. In our short experience, OPTIA has proven to be a safe and very effective choice for MNC collection ECP in patients with EICH after SCT. Furthermore, it has the advantage of being fully automatic and therefore independent of the technical skills. References: C. del Fante et al. Transfusion 2013; 53: 2027– 2033. Disclosure of Interest: None declared.
P010 Treatment of Secondary Graft Failure after Hematopoietic Stem Cell Transplantation with Alpha/Beta T-Cell Depleted Grafts as Booster Infusions E. Rådestad1,2,*, H. Wikell1,3, M. Engstro¨m1,2, E. Watz1,3, B. Sundberg1,2, S. Thunberg3, M. Uzunel3, J. Mattsson1,2, M. Uhlin1,2 1 Department of Oncology-Pathology, Karolinska Institute, 2Center for Allogeneic Stem Cell Transplantation, 3Clinical Immunology and Transfusion Medicine, KAROLINSKA UNIVERSITY HOSPITAL, Stockholm, Sweden Introduction: Infusions of extra lymphocytes from the original stem cell donor can be used as a treatment after hematopoietic stem cell transplantation (HSCT) for, e.g., relapse, poor immune reconstitution or secondary graft failure. In peripheral blood, 95% of T-cells express the ab T-cell receptor and the remaining T-cells express the gd T-cell receptor, which are not dependent upon human leukocyte antigen (HLA)- molecules for activation. As ab T-cells are the primary mediators of graft versus host disease (GVHD), depleting them from the graft should reduce this risk, leaving gd T-cells as primary T-cell type in the booster infusion. Materials (or patients) and methods: In this pilot study, five patients transplanted with HLA-matched related or unrelated donors were treated with ab T-cell depleted stem cell boosters, with secondary graft failure as primary indication. Secondary graft failure was in this study defined as initial signs of engraftment with subsequent development of bone marrow hyperplasia requiring frequent transfusions beyond day 60 as well as prolonged neutropenia and thrombocytopenia. All patients also suffered from infectious complications prior to the infusion, which is likely to have been associated with the poor graft function and development of secondary graft failure. The ab T-cell depletion was performed using a CliniMACS system (Miltenyi Biotech). Analysis of the booster infusion fraction as well as blood samples from the patients was performed by using flow cytometry and evaluation of chimerism as well spectratyping of gd-chains by using PCR. Monitoring of white blood cells (WBC), platelets, and granulocytes was carried out repeatedly during 30 days post infusion.
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Results: The majority of gd T-cells in the boosters expressed Vd2 and/or Vd9. Median log depletion of gd T-cells in the products was 3.72, and the mean yield of gd T-cells was 84.4%. Most patients receiving ab-depleted stem cell boosters increased their levels of WBC, platelets, and/or granulocytes 30 days post infusion. Regarding the infectious complications, the stem cell booster had a positive effect for four out of five patients. The most obvious response could be observed in the three patients with the best-combined response in WBC and granulocyte count. The cell characterization by flow cytometry on peripheral blood samples 3-7 months post infusion showed that the gd T-cell population was similar and had normalized in the four out of five patients that were characterized, regardless of the spread observed in the gd T-cell repertoire in the characterized booster infusion fractions. Conclusion: In conclusion, we describe the use of ab T-cell depleted grafts as stem cell booster in five patients suffering from infectious complications due to secondary graft failure after HSCT with encouraging results. No signs of GVHD or other side effects were detected and four out of five patients were alive six months after booster infusion. The individual contribution of gd T-cells, NK-cells, or CD34 stem cells cannot be determined based on this pilot study. A larger pool of patients with longer follow-up time is needed to confirm the data in this study and compare the treatment modality to other therapy options. Disclosure of Interest: None declared. P011 Preemptive cytokine-induced killer (CIK) cell infusions sustained remission in high-risk leukemia patients after allogeneic stem cell transplantation E. Rettinger1,*, S. Huenecke1, V. Pfirrmann1, M. Merker1, A. Willasch1, A. Jarisch1, J. Soerensen1, H. Boenig2, G. Bug3, T. Klingebiel1, P. Bader1 1 University Hospital Frankfurt, Goethe University, Department for Children and Adolescents, Division for Stem Cell Transplantation and Immunology, 2German Red Cross Blood Donor Service Baden-Wu¨rttemberg-Hessen, Division for Cell Processing, 3University Hospital Frankfurt, Goethe University, Department of Medicine II, Hematology, Oncology, Rheumatology and Infectious Diseases, Frankfurt/Main, Germany Introduction: Allogeneic stem cell transplantation (allo-SCT) is an established treatment option for high-risk leukemia patients, but relapse limits the therapeutic success. Relapse can be treated by donor lymphocyte infusion (DLI), but T cells within DLI raise the risk for acute graft versus host disease (aGvHD). Cytokine-induced killer (CIK) cells, mainly T cells with characteristics of natural killer (NK) cells, demonstrate nonmajor histocompatibility complex-restricted cytotoxicity against hematological malignancies with negligible alloreactivity and therefore were licensed as an immunotherapy. Materials (or patients) and methods: Here we report our experience of repetitive, dose-escalating CIK cell infusions in 14 leukemia patients with risk of relapse (n ¼ 11) or overt relapse (n ¼ 3) showing no signs of aGvHD 4grade I after allo-SCT. Results: Between Aug 11, 2011 and Nov 30, 2014 14 patients (o18yr, n ¼ 13, median age 9, range 1-17yr; 418yr, n ¼ 1, age 69yr) with hematological malignancies (AML, n ¼ 7; ALL, n ¼ 6; CML, n ¼ 1) received a total of 58 CIK cell infusions (median number 3, range 1-10 per patient) from matched unrelated (n ¼ 6) or haploidentical (n ¼ 8) stem cell donors at a minimum of 3 weeks after allo-SCT and an interval of 4-6 weeks between infusions (median follow up after 1st CIK cell infusion 213 days, range 56-802 days). 6 patients received CIK cell infusions based on minimal residual disease or chimerism status, 5 patients, most of whom weren’t in remission at the time of allo-SCT, were treated prophylactically, and 3 relapsed patients received CIK cell infusions with or without chemotherapy. Clinical batches of CIK cells were generated from peripheral blood mononuclear cells of the original stem cell donors under good manufacturing practiceconditions in the presence of interferong, anti-CD3 antibody,
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interleukin (IL)-2 and IL-15. Cellular composition of CIK cell infusions showed that CD19 þ B cells, CD14 þ monocytes and CD3-CD56 þ natural killer (NK) cells were quantitatively depleted while conventional CD3 þ CD56- T cells and CD3 þ CD56 þ T-NK cells made up the bulk of the CIK cell product. T cells, evenly distributed between the CD4 þ and CD8 þ compartment, were activated, which was shown by CD25 þ expression and were mostly TCRa/b type.Starting T cell dose within CIK cell infusions was 1x106 T cells/kg body weight. Regardless of donor type, dose escalation continued up to 1x108 T cells/kg body weight if no signs of aGvHD 4grade I occurred. Median doses of infused T cells were 6x107 in the matched unrelated and 1x107/kg in the haploidentical transplantation setting. Mild signs of aGvHD occurred in 2 patients (14%). Another 3 patients (21%) developed aGvHD grade III. In 6 patients (43%), 3 with overt and 3 with impending relapse, CIK cell infusions provided transient remission (median 218 days, range 56-563 days), followed by another allo-SCT in 2 cases. Deaths of 2 heavily pretreated AML-patients (14%) who remained in remission after CIK cell infusions were not CIK cell-related. Another 6 leukemia patients (43%), 3 of whom were not in remission at the time of SCT, are still in complete remission. All of them were offered CIK cell infusions from haploidentical donors. Conclusion: Allogeneic CIK cell infusions seemed to be very promising and may improve immune cell-based therapies especially in leukemia patients with impending relapse after allo-SCT. Disclosure of Interest: None declared. P012 Donor lymphocyte infusion (DLI) as salvage treatment for refractory or relapsed lymphoid malignancies after allogeneic stem cell transplantation: Experience from a single centre G. Martin Sanchez1,*, A. Insunza1, L. Yan˜ez1, A. Bermudez1, M. Colorado1, M. Lopez Duarte1, C. Amunarriz2, O. Irati1, B. Lopez Pereira1, C. Richard1, J. L. Arroyo2, E. Conde1 1 Hospital Universitario Marques de Valdecilla, 2Banco de Sangre y Tejidos de Cantabria, Santander, Spain Introduction: DLI has been used during the last decades for relapsed hematological malignancies after allogeneic stem cell transplantation, with different results. We present the outcomes and complications of patients with relapsed or refractory lymphoid malignancies treated with DLI. Materials (or patients) and methods: We retrospectively reviewed the 30 patients who received DLI between 1999 and 2014 after T cell-replete allogeneic stem cell transplantation. The median age at DLI was 42 (range: 8-69) years old. Diagnosis was multiple myeloma (MM) in 9 patients, Hodgkin´s lymphoma (HL) in 7, chronic lymphocytic leukemia (CLL) in 6, acute lymphoblastic leukemia (ALL) in 6 and non-Hodgkin´s lymphoma (NHL) in 2. All patients were off immunosuppressive treatment and with no active GVHD. Median time from progression or relapse to the first DLI was 63 (17-315) days, and 16 (53%) patients were additionally treated with chemotherapy or radiotherapy. A total of 71 DLI doses were infused, with a median of 2 (1-6) doses per patient. The maximum DLI dose varied between patients with an HLA matched donor (n ¼ 27) and mismatched (n ¼ 3, including one haploidentical) donor, with a median CD3 dose of 7,5x107/kg and 0,2x107/kg respectively. Relapses and responses were defined by disease-specific criteria based on flow cytometry, cytogenetic and imaging studies. Acute and chronic graft versus host disease (GVHD) was classified according to the Glucksberg and NIH criteria respectively. Results: Eleven (36,7%) patients achieved a complete response (CR), which lasted a median of 12 (1-61) months. Analyzing patients by diagnosis we observed a higher CR rate in ALL patients (4/6) followed by MM (3/9), CLL (2/6) and HL (2/7). All patients with MM and ALL relapsed within a median of 6 (1-12) and 8 (1-21) months, respectively, but none of the 4 patients with HL and CLL relapsed after a median follow-up of 33 (10-61) months (Table 1). Five (16,7%) patients
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(MM: 2, CLL: 1, HL: 1, NHL: 1) achieved a partial response, which was transient in all cases, with a median duration of 3 months. Overall 14 (47%) patients developed GVHD, which was acute in 4 cases (13%) and chronic in 10 (34%). Median time since the first DLI dose to GVHD onset was 33 (28-56) days in acute and 116 (89-223) days in chronic forms. Only 2 (7%) patients developed grade III-IV acute GVHD and 5 (17%) moderatesevere chronic GVHD. Skin was the most commonly affected organ in both acute and chronic GVHD. After a median follow-up of 20 (1-129) months for the entire cohort, patients who achieved CR after DLI had a significant higher overall survival at 2 years, 66% vs 44% (P ¼ 0,018). Disease progression was the main cause of death (n ¼ 16) and none of the patients died because of GVHD. Conclusion: We observed a low incidence of severe acute and chronic GVHD after DLI (7% and 17% respectively). More than half of the patients achieved partial or complete response (n ¼ 16). CR lasted more than 10 months in 7 (23%) cases, with no relapse at last follow-up in 4 of them (HL:2, CLL:2). DLI was a safe treatment and provided durable responses in a subgroup of patients with relapsed or refractory lymphoid malignancies. Disclosure of Interest: None declared. P013 Immunemodulation of blasts in AML-patients (pts) with clinically approved response modifiers to improve antileukemic T-cell reactivity: An ex vivo simulation of the clinical situation H. Schmetzer1,*, Z. Stankova1, D. Deen1, A. Hirn-Lopez1, Y. Vokac1, A. Rabe1, T. Stoschek1, T. Kroell1, C. Wendtner2, C. Schmid3, J. Tischer1 on behalf of Christoph Schmid and Johanna Tischer contributed equally 1 Dept for hematopoetic cell transplantations, University Hospital Munich Grosshadern, Munich, 2Dept for Hematology and Infectiology, Municipal Hospital Munich-Schwabing, Munich, 3 Dept for hematopoetic cell transplantations, Med2, Municipal Hospital Augsburg, Augsburg, Germany Introduction: Background: Antileukemic T-cell-reactivity has to be improved/re-established in AML-pts in vivo to prevent relapses. Ex vivo leukemia-derived DC (DCleu) are the most effective antileukemic T-cell-stimulators. Aim: To develop and test combinations of clinically approved immune modifiers that convert blasts into a ‘DCleu-vaccine‘, that induces specific antileukemic T-cell response and Tcell memory without induction of blast proliferation. Materials (or patients) and methods: Methods: A culture system using heparinised whole blood (‘WB‘, to simulate the in vivo situation) was developed to generate DCleu ex vivo from AML blasts from 52 AML-pts in active stages of the disease. 11 KITS containing selected combinations of 7 clinically approved compounds (GM-CSF, INTRON,
CALCIMYCIN, PICIBANIL, PGE1, PGE2, TNFa) were tested for their capability to generate DCleu and different DC-subtypes, as well as their potential to induce an (unwanted) blast proliferation. Further, the capability of DCleu generated from WB to stimulate antileukemic activity of T-cells from pts before or after SCT in vitro was evaluated. Results: Results: 1. Generation of DC (DCleu ) from WB: Our culture system qualified to test the convertibility from blastcontaining WB to DCleu using 11 different KITS of clinically approved immune modifiers. Average results of 7 Kits were comparable to those generated with standard methods, the remaining KITS did not produce efficient amounts of DC. Moreover patient-individuell variations were observed. 2. Some KITS induced ex vivo blast proliferation in individual pts‘ WB samples. 3. A ranking list allowed the evaluation of the best 5 of 11 blast modulating KITs. 4. Antileukemic reactivity of T-cells stimulated with KIT-treated WB could always be improved compared to controls, although with variations. 5. Blast lysis correlated with higher proportions of DC-subtypes (DC, DCleu), with proportions of CD8 þ Tcells detectable after mixed lymphocyte culture, IL-12 and IFNg concentrations and lower concentrations of IL-6 and -8 in culture supernatants. Conclusion: Conclusion and next steps: KITs can create DCsubtypes and especially DCleu from blasts in WB cultures. Their blast-proliferation-stimulating potential as well as their capacity to generate antileukemic T-cells can be evaluated. A ranking list based on DC, DCleu subtypes, antileukemic reactivity and cytokine profiles should be created to allow an individualised selection of ’best KIT‘ for in vivo applications in mouse or human. References: (Patent pending: reference number: 102014014993.5). Disclosure of Interest: None declared. P014 Expansion of Bone Marrow Derived Mesenchymal Stem Cells Using a GMP Compliant Closed System Bioreactor and Quality Analysis of the Expanded Cells I. Kozanoglu1,2,*, E. Maytalman3, P. Bilir2, G. Unver2, C. Siber2, C. Boga4, H. Ozdogu4 1 Physiology, Baskent University medical Faculty, 2Cell Processing Unit, BASKENT UNIVERSITY ADANA ADULT BONE MARROW TRANSPLANTATION CENTER, 3Cell Processing Unit, Baskent University Adult Bone Marrow Transplantation Center, 4Clinical Unit, BASKENT UNIVERSITY ADANA ADULT BONE MARROW TRANSPLANTATION CENTER, Adana, Turkey Introduction: The production of MSCs, requires extensive resources, space, labor and Good Manufacturing Practices compliance for clinical uses. In recent years, use of bioreactors have become popular as an alternative to open systems. The aim of this study is to share the quality control results of bone marrow derived MSCs expanded using a bioreactor (Quantum Cell Expansion System, Teruma BCT, Colorado, USA).
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Materials (or patients) and methods: 25 milliliters of bone marrow aspirated from a normal healthy donor, 4 liters of media and 4 liters of PBS was loaded to the Quantum machine using the closed system set. The cells were fed by media according to the results of regular lactate measurements as recommended by the manufacturer. After 14 days of culture, the cells were removed from the system using trypsin and cultured for the second passage. The samples taken from the final product were analysed for viability, cell count, microbial growth, endotoxins and telomerase enzyme activity. Flow cytometric studies, and lymphocyte proliferation inhibition tests were also performed. MSCs were differentiated to adipocytes and osteocytes. Results: Starting with a 25 ml sample of bone marrow, after 21 days of culture, 160 million cells were obtained. Cell viability was shown to be 99%. No microbial growth was observed and endotoxin analysis was negative. The cells were CD45 and CD34 negative but CD73, CD105 and CD90 positive. MSCs cultured with peripheral lymphocytes were shown to inhibit lymphocyte proliferation and the cells were shown to differentiate into adipocytes and osteocytes. Telomerase enzyme activity was determined to be below %1.5 RTA. Conclusion: Some studies have reported that immunomodulatory capacity of MSCs produced in different laboratories, although GMP compliant may not be standard. Moreover, culturing for too many passages and presence of hundreds of manipulations are important factors affecting the standardization of the cells. Bioreactors are advantageous as they provide a closed system (cells do not have any contact with the CO2 in the air), they require minimum air quality for production (GMP Class C is sufficient), more cells are obtained in less time and the product quality is high. Production with bioreactors also help decreasing the cost and time of cell expansion. Disclosure of Interest: None declared.
In T helper cells, compared to unstimulated cells, low-dose IL-2 or IL-15 alone and the combination with TKD peptide induced an up-regulation of KIR2DL2/3 and CD94. NKG2D expression increased after stimulation with IL-2 or IL-15. DNAM1 upregulation appeared only after stimulation with IL-15. NKRP1 was upregulated by IL-2± TKD and by IL-15 alone. Other NK receptors showed no or minimal expression on Th cells. In CTL cells, treatment with interleukines alone or with TKD peptide induced expression of KIR2DL2/3, CD94, DNAM1, NKRP1A. Cell number positive for NKG2D, NKp80, ILT2 and CD16 was increased by IL-2 or IL-15 and CD16 expression was also increased by the combination IL-2 ± TKD peptide. Other NK receptors showed no or minimal expression on CTL cells. In NKT cells, DNAM-1, KIR2DL2/3, KIR2DL4, NKG2D, CD94 and LAMP1 were up-regulated with IL-2 or IL-15 alone or in combination with TKD peptide. The expression of ILT-2 was increased after stimulation with IL-2 or IL-15 and also after stimulation with IL-15 plus TKD peptide. Up-regulation of NKG2C and NKp30 could be detected only after IL-15 treatment. NKRP1A and NKp80 were down-regulated by IL-2 or IL-15 and NKp80 showed even lower expression after stimulation with IL-12 plus TKD peptide. Other NK receptors showed no or minimal expression on NKT cells. Conclusion: The study showed that some activatory and inhibitory NK receptors are also expressed on CD3 þ CD4 þ T cells, CD3 þ CD8 þ T cells, B cells and NKT cells and can be regulated by the treatment with low-dose IL-2 or IL-15 themselves or in combination with TKD peptide under in vitro condition. Disclosure of Interest: I. Hromadnikova Funding from: This work was done under the framework of CellEurope project (FP7-People-2012-ITN, No. 315963) coordinated by Professor Anne Dickinson from University of Newcastle upon Tyne, UK., S. Li: None declared, A. Dickinson: None declared
P015 Influence of in vitro IL-2 or IL-15 alone or in combination with Hsp 70 derived 14-mer peptide (TKD) on the expression of NK cell activatory and inhibitory receptors on peripheral blood mononuclear cells I. Hromadnikova1,*, S. Li1, A. Dickinson2 1 Dpt. Molecular Biology and Cell Pathology, Third Faculty of Medicine, Charles University Prague, Prague, Czech Republic, 2 Institute of Cellular Medicine, Haematological Sciences, The Medical School, Newcastle University, Newcastle Upon Tyne, United Kingdom
P016 In vitro study of peripheral blood mononuclear cell cytotoxicity against Hsp70 positive human leukaemia cell lines in paediatric patients indicated to haematopoietic stem cell transplantation I. Hromadnikova1,*, S. Li1, P. Sedlacek2, A. Dickinson3 1 Dpt. Molecular Biology and Cell Pathology, Third Faculty of Medicine, Charles University Prague, 2Dpt. of Pediatric Hematology and Oncology, University Hospital Motol, Second Faculty of Medicine, Charles University Prague, Prague, Czech Republic, 3 Institute of Cellular Medicine, Haematological Sciences, The Medical School, Newcastle University, Newcastle Upon Tyne, United Kingdom
Introduction: NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. The aim of the current study was to investigate the expression of NK activatory and inhibitory receptors on T helper lymphocytes, cytotoxic T lymphocytes, B lymphocytes and NKT cells after preincubation of PBMN cells of healthy individuals with interleukines (IL-2, IL-15) alone and in combination with hsp70 derived peptide (TKD) using flow cytometry. This is the follow-up study of the expression of NK receptors on NK cells, whose results have already been reported. Materials (or patients) and methods: Flow cytometry was performed on days þ 1, þ 3, þ 5 (stimulated and unstimulated PBMN cells) using a standard direct immunofluorescence technique (anti-human monoclonal antibodies conjugated with FITC, PE, APC) on a FACSCaliburTM. NK activatory receptors (CD16, CD94,NKG2D, NKG2C, NKp46, NKp44, NKp30, NKp80, KIR2DL4, DNAM-1, and LAMP1) and NK inhibitory receptors (NKG2A, KIR2DL2/L3, ILT-2, and NKR-P1A) in healthy individuals were studied. Results were expressed as the percentage of receptor expressing cells on day þ 5. Results: In B cell population, ILT2 and LAMP were up-regulated and NKRP1A was down-regulated after stimulation with IL-2 or IL-5. NKG2D was up-regulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD peptide. Other NK receptors showed no or minimal expression on B cells.
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Introduction: NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. The aim of the study was to investigate the in vitro PBMN cell cytotoxicity after stimulation with IL-2 or IL-15 alone or in combination with hsp70 derived peptide (TKD) against human leukaemia cell lines derived from patients with CML, ALL and AML (UoC-M1, THP-1, TF-1, K562, Jurkat, CCRF-CEM) during the onset and/or relapse of the disease showing high membranebound Hsp70 positivity using ELISPOT. Materials (or patients) and methods: The first set of paediatric patients indicated to haematopoietic stem cell transplantation was analysed to see NK cell response against various leukemia cell line. 13 patients were analysed (1 cALL CR1, 1cALL CR2, 1 preBALL CR2, 1 CML CP1, 4 MDS, 1 SAA, 1 iMPAL, 1 SCID, 1 AML M5 CR1, 1 ALCL). All patients´ fresh peripheral blood samples were analysed in three different effector:target ratios (E/T 3:20, 1:10, 1:20). The constant concentration of target cells (50 000 cells/per well) and descending concentration of effector cells (7500, 5000 and 2500 cells/per well) were used. Results: Patient with SCID had no T and NK cells, which resulted in negative response in ELISPOT assay as expected. Very low response was observed in case of 2 patients with MDS-RAEBt
and MDS 7-. In case of other 2 patients with MDS (both MDS RAEB) significant number of spots was detected, however one patient did not show significant difference between responses to IL-2 or IL-15 with or without TKD peptide and the second patient showed increased response to IL-15 þ TKD compared to IL-15 alone only. The patient with SAA responded quite well in ELISPOT assay mainly to interleukines, but no significant increase of spots was observed when TKD peptided was added. The patient with iMPAL showed the significant increase in lysis of leukaemia cells derived from Jurkat, CCRF-CEM, K562 and UOCM1 leukaemia cell lines after the stimulation with the combination of IL-2 and/or IL-15 and TKD peptide compared with interleukines alone. Similarly the patient with cALL CR2 showed the highest response to leukaemia cell lines (Jurkat, CCRF-CEM, K562 and UOC-M1) after the treatment with IL-15 and TKD peptide. In case of patient (AML M5 CR1) the significant response to UoC-M1 (AML M1) and TF-1 (AML M6) cell line appeared after IL-15 þ TKD treatment compared to IL-15 alone. In case of patients with cALL CR1, CML CP1, preBALL CR2 and ALCL the immunotherapy will not probably be effective, since the combination of interleukines with TKD peptide did not lead to the increase of the PBMN cell response. Conclusion: PBMCs from different patients responded differently to various Hsp70 membrane positive leukemia cell lines after stimulation with low dose interleukines and TKD peptide. In the case of cALL CR2 and iMPAL patients the in vitro assay indicated that the immunotherapy with interleukines and Hsp70 derived peptide may be considered as a treatment option, if the patients´ own leukemic blasts will be highly Hsp70 positive. In the case of patients with cALL CR1, preBALL CR2 and CML CP1 the immunotherapy will not probaly be effective.
Disclosure of Interest: I. Hromadnikova Funding from: This work was done under the framework of CellEurope project (FP7-People-2012-ITN, No. 315963) coordinated by Professor Anne Dickinson from University of Newcastle upon Tyne, UK. , S. Li: None declared, P. Sedlacek: None declared, A. Dickinson: None declared P017 Full donor chimerism without graft-versus-host disease: the key factor for maximum benefit of pre-emptive donor lymphocyte infusions (pDLI) J. Feliu1,*, V. Potter1, J. Clay1, L. Floro1, C. Saha1, L. Barber2, G. Orti3, A. A. Alnagar1, F. Grimaldi1, M. Kenyon1, P. Krishnamurthy4, K. Raj1, D. McLornan1, H. de Lavallade1, A. Pagliuca1, G. Mufti1 1 Haematology Department, King’s College Hospital, 2Department of Haematological Medicine, King’s College London, London, United Kingdom, 3Servei d’Hematologia, Hospital Universitari de la Vall d’Hebron, Barcelona, Spain, 4Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom Introduction: Reduced-intensity conditioned (RIC) protocols and T-cell depletion (TCD) deliver low transplant-related mortality and decreased rates of graft-versus-host disease (GvHD). These advantages may however be mitigated by increased relapse rates and delayed achievement of full donor chimerism (FDC). Pre-emptive donor lymphocyte infusions (pDLI) potentially facilitate conversion of mixed donor chimerism (MDC) to FDC, with the aim of decreasing relapse
[P017]
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risk. Despite many centres adopting this approach, the rutine use of pDLI remains controversial due to a lack of published data on the risk/benefit profile of this intervention. Herein we present the results of a large single centre analysis of the efficacy and toxicity of pDLI delivered via a uniform escalating dose schedule. Materials (or patients) and methods: A retrospective analysis of 119 consecutive patients (72 males/47 females) who received pDLI between Jan 2001 and Dec 2013 was performed. pDLI was delivered after TCD RIC allogeneic haematopoietic stem-cell transplantation (HSCT) for a range of haematological malignancies. Diagnoses included: AML (n ¼ 48), MDS (n ¼ 35), HL/NHL/CLL/ALL (n ¼ 24), MPN (n ¼ 8) and MDS-MPN (n ¼ 4). Donors were 81 identical siblings and 37 unrelated donors (18/37 were 1-2 antigen mismatched). FDC was defined as unfractioned and CD3 chimerism Z95%. Chimerism o95% in either fraction was considered MDC. Indications for pDLI were falling CD3 chimerism, CD3 o50% or mixed XY karyotype/FISH. Results: Median follow-up after DLI was 52.5 months. The 119 patients received 276 pDLI doses. 78 (66%) had a 2nd DLI dose, 40 (34%) a 3rd and 21 (18%) Z4 doses. Median number of pDLI doses per patient was 2 (range 1-7) and median cumulative pDLI dose was 1.56 x107 CD3 cells/Kg (range 5 x105 - 2.8 x108). Median time from HSCT to 1st pDLI was 267 days (range 83-3228). Efficacy: 71 patients (60%) converted to FDC after pDLI, of whom 1 returned to MDC. Relapse occurred in only 11/71 FDC patients (16%) compared to 16/46 MDC patients (35%) (P ¼ 0.024). The group who achieved FDC after pDLI had improved event-free survival (EFS) (P ¼ 0.0002) and overall survival (OS) (P ¼ 0.0003), compared to those with post-DLI MDC (Fig 1). These results did not differ for those with myeloid vs lymphoid malignancies. Interestingly, patients in FDC who developed DLI-induced GvHD showed a similar outcome to those with post-DLI MDC (Fig 2). Toxicity: Cumulative incidence of GvHD at 5 years after DLI was 37% (95% CI, 29% - 48%) and median time to GvHD onset after the 1st pDLI dose was 7 days (range, 1-39). The post-DLI FDC status was associated with the development of DLI-induced GvHD (P ¼ 0.034), but there was no correlation with pre-DLI GvHD, degree of HLA mismatch, T-cell dose or time from HSCT to DLI. Conclusion: A majority of patients who receive pDLI convert to FDC and impressively retain that status, potentially contributing to low rates of relapse and excellent long-term OS. The achievement of FDC after pDLI has an impact on survival, and those patients who achieve FDC without GvHD, experience maximum clinical benefit. Strategies to minimise DLI-induced GvHD should be considered to maximise the therapeutic potential of this intervention. Disclosure of Interest: None declared. P018 Immunosuppressive Properties by Activation of B7-H1 in Human Mesenchymal Stem Cells is mediated through STAT-1 signaling J. E. Park1,*, D.-H. Lee2, I. K. Jang2, K. C. Lee3 1 Pediatrics, Ajou University School of Medicine, Suwon, 2Biomedical Research Institution, Lifeliver Co. Ltd., Youngin, 3Pediatrics, College of Medicine, Korea University, Seoul, Korea, Republic Of Introduction: Mesenchymal stem cells (MSCs) derived from either bone marrow (BM) or Wharton’s jelly (WJ) inhibit proliferation of activated T cells, and interferon-gamma (IFN-g) plays an important role in this process. This IFN-g-licensed veto property is B7-H1-dependent in cell contact inhibition. However, the signaling pathway involved in B7-H1 expression of MSCs remains largely undefined. We demonstrate here a activation of B7-H1 through engagement of Signal Transducer and Activator of Transcription(STAT)-1 signaling in MSCs. Materials (or patients) and methods: Human BM- and WJ-MSCs were isolated and cultured. We compared the immunosuppressive effect of BM- and WJ-MSCs on
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phytohemagglutinin (PHA)-induced T-cell proliferation using direct and indirect co-culture system. B7-H1 expression on BMand WJ-MSC was detected by flow cytometry. The small interfering RNA method was used to knockdown the expression of STAT1. Results: The inhibitory effect of MSCs on T lymphocytes was observed in PHA-induced T cell proliferation assay. B7-H1 expression on human BM- and WJ-MSCs by IFN-g was increased time-dependently by flow cytometry. The inhibitory effect of MSCs on T cell proliferation could be restored when the anti-B7-H1 monoclonal antibody was used to block the B7H1. When the STAT-1 small interfering RNA was used to interfere with its signaling, B7-H1 expression on MSCs treated with IFN-g were decreased and T cell proliferation could be restored. Conclusion: These results show that the activation of B7-H1 by IFN-g in human BM- and WJ-MSCs is mediated through the STAT-1 signaling for immunosuppressive properties. References: 1. In Keun Jang, Hee-Hoon Yoon, Mal Sook Yang, Hyo Eun Kim, Doo-Hoon Lee, Myoung Woo Lee, Dae Seong Kim, Jun Eun Park. B7-H1 Inhibits T Cell Proliferation through MHC Class II in Human Mesenchymal Stem Cells. Transplant Proc. 2014; 46: 1638–1641 2. Chinnadurai R1, Copland IB, Patel SR, Galipeau J. IDOindependent suppression of T cell effector function by IFN-glicensed human mesenchymal stromal cells. J Immunol. 2014; 192(4): 1491–1501. 3. Tipnis S1, Viswanathan C, Majumdar AS. Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells: role of B7-H1 and IDO. Immunol Cell Biol. 2010; 88(8): 795–806. Disclosure of Interest: None declared. P019 Allogenic mesenchymal stem cell transplantation as a rescue therapy for lupus nephritis patient refractory to conventional induction therapy D. Wang1, H. Zhang1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Allogenic mesenchymal stem cells (MSCs) transplantation demonstrated significant clinical efficacy on various autoimmune diseases. Here we analyzed the role of allogenic MSCs transplantation in renal remission in patients with active and refractory lupus nephritis (LN). Materials (or patients) and methods: Eighty-one patients with active and refractory lupus nephritis in China from 2007 to 2010 were enrolled in the study. Allogenic bone marrow or umbilical cord derived MSCs were administered intravenously at the dose of one million cells per kilogram of bodyweight. Then all the patients were followed up for 12 months to evaluate renal remission as well as possible adverse events. The primary outcomes were renal complete remission (CR) and partial remission (PR) at each visit times, as well as renal flares. The secondary outcomes included renal activity score, total disease activity score, renal function and serologic index. Rates of overall survival, renal remission, as well as relapse at different visit times were calculated by Kaplan-Meier method and were statistically tested with the log-rank test. We calculated the HR and their 95% CIs using the univariate Cox proportional hazards model. Results: The overall survival rate during 12 months follow-up was 95% (77/81). The probability of renal remission was 41% (18% CR and 23% PR) at 3 months, 45% (18% CR and 27% PR) at 6 months and 44% (23% CR and 21% PR) at 12 months after allogenic MSCs transplantation. Renal remission was not correlated with age, disease duration, MSCs source and baseline SLEDAI score, but was significantly correlated with baseline proteinuria (P ¼ 0.003, 95%CI 0.336-0.794) and serum creatinine levels (P ¼ 0.047, 95%CI 0.224-0.990) by COX regression analysis. Eleven in 81 (14%) patients underwent renal flare in 12 months follow up after a prior complete or
partial remission. Renal activity evaluated by BILAG score significantly declined after MSCT, in parallel with the obvious amelioration of renal function. Total disease activity evaluation by SLEDAI score also decreased after treatment. Additionally, the doses of concomitant prednisone and immunosuppressive drugs were tapered. Four of 81 patients died of uncontrolled lupus nephritis unrelated to MSCT. Conclusion: Allogenic MSCs transplantation resulted in renal remission within 12 months visit, which could be used early as a potential induction therapy for active and refractory lupus nephritis. Disclosure of Interest: None declared. P020 Therapeutic effects of mesenchymal stem cells, anti-tumor necrosis factor and anti-CD20 On collagen induced arthritis Y. Sun1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Tremendous progress has been made in the development of non-conventional therapies for rheumatoid arthritis (RA). In this study, the effects of mesenchymal stem cells (MSCs) transplantation on established collagen-induced arthritis (CIA) were evaluated and compared with two kinds of biologic agents, anti-tumor necrosis factor (TNF) and antiCD20 antibody. Materials (or patients) and methods: CIA was induced with the immunization of type II collagen (CII) and CFA in DBA/1J mice. Human umbilical cord derived MSCs (5 106), anti-TNF antibody (100ug) and anti-CD20 antibody (200ug) were intraperitoneally injected into 3 groups of mice on day 28 after the immunization. The control group was treated with human fibroblasts (5 106). All mice were sacrificed 3 weeks later and arthritis severity was assessed by clinical and histology scoring. The frequency of CD4 þ T cell subsets, B cells and plasma cells in spleen was analyzed by flow cytometry. Serum levels of autoantibody to mouse CII were determined by ELISA. The ability of MSCs to modulate Treg/ Th17 cell percentages in CD3/CD28 stimulated DBA/1J T cells was assessed in vitro. Results: MSCs treatment significantly decreased the severity of arthritis and pathology scores, which was comparable to anti-TNF or anti-CD20 treatment. Anti-CD20 treatment depleted nearly half of B220 þ cells, and markedly reduced the frequency of plasma cells and serum levels of autoantibody compared to the control group (738±187 vs. 1817±447 U/ml, Po0.001). The decrease of autoantibody level was also detectable in those with anti-TNF treatment (663±336 U/ml) and MSCs treatment (1057±362 U/ml), but neither of these two treatments had an impact on the percentage of B cells or plasma cells. All of the three treatments resulted in a decrease in Th1 subset, but none of them altered the percentage of Th2 subset. Except anti-CD20 treatment, both MSCs and anti-TNF treatment significantly decreased the percentages of Th17 cells. Notably, only MSCs treatment increased the percentages of regulatory T cells (11.39±0.85 % vs 7.37±1.82 % in the control group, Po0.01). In vitro study confirmed that MSCs could induce the generation of Foxp3 þ T cells but reduce the percentages of pathogenic IL-17 þ Foxp3 þ T cells. Conclusion: MSCs exerted comparable therapeutic effects as biological agents on CIA through different mechanisms. MSCs may provide a promising approach for the treatment of RA. Disclosure of Interest: None declared.
Hematopoietic stem cells P021 Haploidentical T-cell replete hematopoietic transplantation conditioned with thiotepa-fludarabinebusulfan (TBF) and followed by graft versus host disease prophylaxis including high-dose cyclophosphamide and tacrolimus: Results from 4 Spanish centers A. Esquirol1,*, M. J. Pascual2, M. A. Cuesta2, J. L. Pin˜ana3, R. Rojas4, M. Alejandra2, R. Martino1, J. Sierra1 1 Hematology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, 2Hematology department, Hospital Regional Universitario Carlos Haya, Malaga, 3Hematology Department, Hospital Clı´nico Universitario de Valencia, Valencia, 4Hematology Department, Hospital Reina Sofia, Cordoba, Spain Introduction: Hematopoietic stem cell transplantation (HSCT) is an effective therapy for a variety of severe haematological diseases. Approximately 40% of patients who need a transplant and lack a related donor has an unrelated donor in due time. Cord blood transplants are another option but hematopoietic and immune recoveries are slow. Haploidentical family transplantation using T-cell repleted grafts and high-dose cyclophosphamide (HAPLO-CY) are increasingly being performed as an alternative. Materials (or patients) and methods: Thirty-four patients without a related or unrelated donor consecutively received an HAPLO-CY between 04/13 and 09/14 in 4 hospitals using the same protocol. High-dose conditioning regimen included thiotepa 5 mg/kg x 2 days (-7 and -6), fludarabine 50 mg/m2 x 3 days (-5, -4 and -3) and busulfan 1 mg/kg/6h x 3 days or its equivalent IV dose (day -5, -3 and -2). In patients older than 55 years busulfan administration was limited to two days. Graft versus host disease (GvHD) prophylaxis consisted of cyclophosphamide 50 mg/kg on day þ 3 and þ 4, and tacrolimus continuous iv infusion from day þ 5 and adjusting for blood levels 5-10 ng/L. Results: Median age of the patients was 49 years (range 5-69), 52.9% male and 47.1% female. Diagnosis distribution was: AML 10 (29.4%), MDS 12 (35.3%), ALL 6 (17.6%), Hodgkin Lymphoma 1 (2.9%), CLL 2 (5.9%) and MM 3 (8.8%). Disease status at transplant was 44.1% in complete remission (23.5% CR1, 14.7% CR2 and 5.9% CR3), 26.4% in partial or very good partial remission, 11.8% had stable disease, 17.7% had progression or refractory disease. 29.4% of patients had received a prior transplant. Median age of the donors was 33 years (range 19-74), being in 35.3% a son, 14.7% a daughter, 11.8% a brother, 14.7% a sister, 8.8% the father and 14.7% the mother. Peripheral blood and bone marrow stem cells source were used in 67.6% and 32.4% of cases, respectively. Median CD34 cell dose infused was 3.7x10e6/Kg. Median times to neutrophil (0.5x109/l) and platelet (4 50x109/ l) recoveries were þ 17 and þ 23 days, respectively (2 patients had graft primary failure, one of them recovered). Median follow up of the series is 181 days (range 40-495). Grades 3-4 gastrointestinal, mucositis and liver toxicities have been observed in 4.7%, 26.4% and 14.7% of cases (only one patient developed hepatic sinusoidal obstructive syndrome). Non-severe hemorrhagic cystitis has been observed in 35.3% of patients. The cumulative incidence of acute GvHD grade 2-4 was 26% and of grade 3-4 was 23%. 22 patients were at risk for chronic GvHD, 3 patients (8.8%) developed limited and 3 (8.8 %) moderate/severe GvHD. The cumulative incidence of non-relapse mortality (NRM) was 21% at one year and the causes were acute GvHD (2), infections (2), idiopathic encephalopathy (2) and graft failure (1). Relapse was observed in 4 patients, 2 of them remaining alive. Overall survival (OS) and progression free survival (PFS) are 71.4% and 70.1% at one year.
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Conclusion: Haploidentical T-cell repleted transplantation conditioned with TBF and followed by cyclophosphamide and tacrolimus as GVHD prophylaxis is a valid option with encouraging OS and PFS at one year. Extrahematological toxicities and NRM were limited. Disclosure of Interest: None declared.
in pediatric matched sibling donor transplants using granulocyte colony-stimulating factor-primed bone marrow and steady-state bone marrow. Pediatr Transplantation 2007: 11: 279–285. Disclosure of Interest: None declared.
P022 Clinical outcomes and graft characteristics in pediatric hematopoietic stem cell transplantation: effect of granulocyte-colony stimulating factor priming A. Fettah1,*, N. Ozbek1, F. Azik1, Z. Avci1, P. Isik1, N. Yarali1, B. Tunc1 1 Pediatric Hematology and Bone Marrow Transplantation Unit, Ankara Children’s Hematology and Oncology Research Hospital, Ankara, Turkey
P023 Important prognostic impact of early monocytes recovery after reduced intensity conditioning double umbilical cord blood allogeneic stem cell transplantation in adults A. Le Bourgeois1,*, T. Guillaume1, J. Delaunay1, P. Perterlin1, V. Dubruille1, S. Le Guill1, P. Moreau1, M. Mohty2, L. Campion3, P. Chevallier1 1 he´matologie clinique, CHU Hoˆtel Dieu, Nantes, 2he´matologie clinique, hoˆpital st Antoine, Paris, 3Institut de Cance´rologie de l’Ouest , Rene´ Gauducheau , St Herblain, France
Introduction: Hematopoietic stem cell transplantation from HLA-matched siblings significantly improved the long-term survival of the children with hematologic malignancies and bone marrow failure syndromes. Traditionally, steady-state bone marrow (S-BM) has been used as the source of stem cells. However, after obtaining that granulocyte colony-stimulating factor (G-CSF) promotes hematopoietic progenitors and increase their numbers both in the bone marrow (BM) and in peripheral blood (PB), transplantation after G-CSF mobilization has gained regard. Results of clinical trials demonstrated that G-CSF primed PB stem cells (G-PBSCs) offer the advantage of higher stem cell dose, accelerated engraftment and shorter neutropenic period compared to those with S-BM. On the other hand, it has been associated with a higher risk of chronic graft versus host disease (GvHD). Studies concerning the use of G-CSF stimulated bone marrow (G-BM) revealed that it was safe and able to produce rapid engraftment as is the G-PBSCs with a lower incidence of chronic GvHD compared to those who received G-PBSCs. In this retrospective study, we compared the clinical outcomes of children who had matched related hematopoietic stem cell transplantation (HSCT) by means of G-BM cells with those who transplanted with S-BM cells. Materials (or patients) and methods: From April 2010 to November 2013, a total of 64 pediatric patient and HLAmatched family donors were enrolled in the study at our Bone Marrow Transplantation Unit. Steady state bone marrow was harvested from 35 donors (S-BM group). Bone marrow priming was performed to 29 donors (G-BM group) when the weight of donor and recipient was discordant. Priming was performed in G-BM group with G-CSF at 10 mg/kg/day for 3 consecutive days, given as a single-dose subcutaneous injection. Results: There were 29 patients (14 male, 15 female; median age 10.5 years, range: 1.8-16.5) in the G-BM group and 35 patients (17 male, 18 female; median age 7 years, range: 1-16.3) in the S-BM group. Mean difference between weight of recipient and donor was smaller in G-BM group compared to those in S-BM group [4 kg (range: -20.5 & 38) vs. 18 kg (range: -10 & 52), P ¼ 0.001]. Granulocyte colony stimulating factor primed bone marrow (G-BM) yielded higher numbers of CD34 þ cells (180/mL vs. 125/mL, P ¼ 0,05), total nucleated cells (7,1x108/kg vs. 4,1x108/kg, P ¼ 0,0001), CFU-GM (13,82x104/kg vs. 7,95x104/kg, p:0,017) compared to those in S-BM. Neutrophil engraftment (14 vs. 15 days, p:0,699), platelet engrafment (25 vs. 24 days, p:0,994), length of stay in hospital (38 vs. 38 days, p: 0,707), overall survival and disease free survival were similiar in G-BM and S-BM groups. Also the cumulative incidence of grade II to IV acute GvHD (%10,3 vs. %11,4, P ¼ 0,809) and chronic GvHD (%20,7 vs. %17,1, p: 0,720) were similiar. It was observed that the use of G-CSF has no effect on risk of acute and chronic GvHD. Conclusion: We concluded stem cell mobilization with G-CSF is an effective and safe method in children without increasing the risk of GvHD. References: 1-Chiang KY, Haight A, Horan J, Olson E, Gartner A, Hartmen D, et al. Clinical outcomes and graft characteristics
Introduction: Little is known regarding the impact of hematopoietic and immune recoveries after double umbilical cord blood (dUCB) allogeneic stem cell transplantation (allo-SCT), especially after the TCF (low dose 2 Grays total body irradiation þ cyclophosphamide 50 mg/Kg 1 day þ fludarabine 200 mg/m2 5 days) reduced-intensity conditioning (RIC) regimen, which is considered as a standard RIC regimen for dUCB allo-SCT in adults. Materials (or patients) and methods: Here we considered a homogeneous cohort of 47 patients (males: n ¼ 24; median age: 55.5 years (range: 17.4-69.1, myeloı¨d disease 55%) who engrafted after a dUCB TCF allo-SCT performed between November 2006 and April 2013 in our department. GVHD prophylaxis consisted of cyclosporine þ mycophenolate mofetyl in all cases. All patients received G-CSF from day 1 until neutrophils recovery. The median nucleated cells dose infused was 4.17 107/kg. The aim of the study was to investigate the impact on outcomes of the recovery of the following cellular subsets: leucocytes, monocytes, lymphocytes, neutrophils at day þ 30 and day þ 42, and CD4 þ , CD8 þ T cells, B and NK cells at day þ 100. Median counts of leukocyte subpopulations were used to separate our patients into two groups of analysis. Results: Median times for neutrophils and platelets recoveries were 17 days (range: 6-59) and 37 days (range: 0-164), respectively. With a median follow-up of 30.4 months (range: 2.8-77.5), the 3-year overall and relapse-free survivals (OS, RFS), relapse incidence (RI), and non-relapse mortality (NRM) were 65.7%, 57.2%, 27.1% and 19%, respectively. The cumulative incidences of grade II-IV and grade III-IV acute GVHD were 38.3% and 10.6%, respectively, while, 3-year incidence of chronic GVHD was 53.5% (limited 42%, extensive 11.5%). In multivariate analysis, early higher monocytes and leukocytes count after transplant were the two independent factors associated with a significantly higher OS (monocytes 4615/ mm3 at day þ 30, HR: 0.07; 95%CI: 0.08-0.57, P ¼ 0.013, leukocytes 4 4250/mm3 at day þ 42, HR: 0.24; 95%CI: 0.0610.098, P ¼ 0.047). Early haematological recovery was predictive of lower NRM (leukocytes 4 2760/ mm3 at day þ 30, HR: 0.10; 95%CI: 0.01-0.91, P ¼ 0.042, lymphocytes 4 395/mm3 at day 42, HR: 0.10, 95%CI: 0.11-0.93, P ¼ 0.044). As well, higher monocytes count at day þ 42 (4830/mm3) remained independently associated with better RFS (HR: 0.18, 95%CI: 0.05-0.68, P ¼ 0.011). Older age and lymphoid disease were other independently factors predicting higher RFS (455 years, HR: 0.90; 95%CI: 0.85-0.96, P ¼ 0.001, lymphoid disease, HR 0.18; 95%CI: 0.05-0.63, P ¼ 0.007). No factor was predictive of grade II-IV or grade III-IV acute GVHD or chronic GVHD. Conclusion: These results suggest that early haematological recoveries (especially higher monocytes count) are predictive of outcome after dUCB TCF RIC allo-SCT in adults. Immune recovery seems to have no impact on survivals in this series while influence of age has to be confirmed by other studies. Thus, our results pave the way for future studies aiming to closely and prospectively monitor the kinetics of hematopoietic and immune recoveries after this type of graft. As all
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patients received G-CSF after transplant, other immunostimulatory cytokines should be tested to ensure sufficient hematopoietic recovery in the setting of adult dUCB TCF RIC allo-SCT. Disclosure of Interest: None declared. P024 Hematopoietic Stem Cell Transplantation in the elderly: nutritional and geriatric assessment A. Z. Pereira1,*, J. Silva2, A. P. Barrere2, M. Tanaka2, F. Lucio2, M. Nicastro1, S. Piovacari2, P. Rodrigues1, L. Koch1, N. Hamerschlak1 1 Oncology, Hematology and Hematopoietic Stem Cell Transplantation, 2Clinical Nutrition, Hospital Israelita Albert Einstein, S.Paulo, Brazil Introduction: Hematopoietic stem cell transplantation (HSCT) may improve outcomes of patients with hematologic malignancies not curable with conventional therapies. Being in some diseases the only curative option. HSCT in elderly patients with good performance status and no comorbidities could, in fact, not only survive the transplant with reasonable risk, but also benefit in the same measure as younger patients. Objectives: To study and correlate nutrition and geriatric assessment in elderly patients undergoing HSCT. Materials (or patients) and methods: A retrospective study of 17 elderly patients (460 years) undergoing HSCT May 2012 to January 2014 in the Hematology-Oncology and Bone Marrow Transplantation Center at Albert Einstein Hospital in Sa˜o Paulo, Brazil. All patients were evaluated approximately one month prior to HSCT. In the geriatric assessment were done hand-grip strength(HGS), questions about mobility and functional limitation. In the nutrition, we studied the Body Mass Index(BMI) (kg/m2), and serum levels of vitamin D, zinc and albumin. Results: 17 elderly patients were observed in this study, mean age was 65,5±3,8 years, BMI was 28±6,0 kg/m2, HGS was 28±8,5 kg; serum levels of albumin 3,2±0,5g/dl (normal:3,55,0); serum level of vitamin D 23,4±14 ng/ml (normal 420); serum levels of zinc 65,5±18 mg/dl (normal: 66-132,5 mg/dl). We found the negative correlation between BMI and HGS (rp ¼ 0,42). There were a significant and positive association between serum levels of zinc and albumin, and HGS and grades of mobility questions (Po0,05). The serum levels of vitamin D weren’t significantly associated with geriatric factors. Conclusion: Our study showed that the obese patients with more risks of complications in HSCT had more functional limitation. Besides low levels of zinc and albumin were associated with worst results in the geriatric assessment. In the elderly the immobility and weakness can increased the complications after HSCT. The geriatric and nutrition assessment are important to improve HSCT results. References: 1. Liu P, Zhang ZF, Cai JJ, Wang BS, Yan X. NRS2002 assesses nutritional status of leukemia patients undergoing hematopoietic stem cell transplantation. Chin J Cancer Res 2012; 24(4): 299–303. 2. Horsley P, Bauer J, Gallagher B. Poor nutritional status prior to peripheral blood stem cell transplantation is associated with increased length of hospital stay. Bone Marrow Transplant 2005; 35(11): 1113–1116. 3. Hadjibabaie M, Iravani M, Taghizadeh M, Ataie-Jafari A, Shamshiri AR, Mousavi SA, et al. Evaluation of nutritional status in patients undergoing hematopoietic SCT. Bone Marrow Transplant 2008; 42(7): 469–473. 4. Iestra JA, Fibbe WE, Zwinderman AH, van Staveren WA, Kromhout D. Body weight recovery, eating difficulties and compliance with dietary advice in the first year after stem cell transplantation: a prospective study. Bone Marrow Transplant 2002; 29(5): 417–424. 5. Kyle UG, Chalandon Y, Miralbell R, Karsegard VL, Hans D, Trombetti A, et al. Longitudinal follow-up of body composition in hematopoietic stem cell transplant patients. Bone Marrow Transplant 2005; 35(12): 1171–1177. Disclosure of Interest: None declared.
P025 Voriconazole as antifungal prophylaxis in allogeneic hematopoietic stem cell transplantation: ten years of experience in a single institution C. CHIC ACEVEDO1,*, M. J. LLAMAS POYATO1, E. GARCIA TORRES1, P. MARI1, G. DIEZ1, J. SERRANO1, J. R. MOLINA1, C. MARTIN1, F. MARTINEZ1, C. HERRERA1, R. ROJAS1 1 HEMATOLOGY, HOSPITAL UNIVERSITARIO REINA SOFIA, CORDOBA, Spain Introduction: The arrival of new active azoles against filamentous fungi has encouraged its use in the prophylaxis of invasive fungal disease (IFD) in patients undergoing allogeneic hematopoietic stem cell transplantation (AlloHSCT). The aim of this study was to analyze the incidence of IFD in patients who received Voriconazole as prophylaxis. Materials (or patients) and methods: In this retrospective study, we analyze 252 consecutive allo-HSCT patients (pediatrics and adults) undergoing this procedure in our institution. Patients were recruited from June 2004 to April 2014. All patients received antifungal prophylaxis with oral Voriconazole 200mg/ 12h (5mg/kg/12h in children) from day 0 to day þ 75. Antifungal prophylaxis was prolonged in patients requiring treatment with high doses of corticosteroids (42mg /kg /day) and/or with two immunosuppressive drugs. IFDs were classified according to the revised criteria of the EORTC/MSG 2008. Only proven and probable IFDs were considered for this study. Drug toxicity was evaluated according to CTCAEv4.1. Fungal free survival (FFS) was calculated early and late in the post-transplant period ( þ 100, þ 360 day respectively). The development of adverse drug reactions (ADRs) was analyzed as well as the most influential prognostic factors in the development of IFD and in overall survival (OS). The incidence of IFD in patients receiving Voriconazole as antifungal prophylaxis was compared to our historical cohorts (98 patients recruited from 2001 to 2004 undergoing Allo-HSCT who were administered Fluconazole as antifungal prophylaxis). Results: 157 patients (61.6%) were male and 95 (37.3%) female. The median age was 35 years (1-68). 29 patients (11.5%) had suffered IFD previously. The incidence of IFD in patients receiving Voriconazole as primary prophylaxis was 7.5% (n ¼ 19) and 3.6% in secondary prophylaxis. These results show a 50% reduction in the incidence of IFD in our historical cohorts. Considering only primary prophylaxis, the median onset of IFD was 115 days after Allo-HSCT. There were 5.9% (n ¼ 15) probable cases of IFD and 1.6% (n ¼ 4) proven cases. 9 patients developed early IFD and 10 late one: the latter group were mainly patients with cGVHD receiving long-lasting corticosteroid treatment. The most common cause of IFD was Aspergillus: (n ¼ 15), followed by Mucor (n ¼ 2), Candida (n ¼ 1) and Scedosporium (n ¼ 1). 81 patients discontinued Voriconazole: 61 (24%) temporarily and 20 (8.3%) permanently. The commonest cause for discontinuation was the development of ADRs in 66 patients (26.2%) being liver toxicity the most frequent (44 patients, 17.4%). OS was 59% with a median follow up of 30 months (1-118m). Global FFS at 100 days and 365 days after Allo-HSCT was 93.7% (88.7-98.7) and 89% (83-95) respectively. We found a relation between treatment with high doses of corticosteroids (42mg/kg/day) and incidence of IFD (P ¼ 0.011), with a FFS of 307 days vs 336 days in those receiving vs not receiving this treatment. This variable lost its significance when included in the multivariate analysis (P ¼ 0.051). We found no links between IFD and CMV infection (P ¼ 0.14) or severe mucositis (P ¼ 0.41). Conclusion: Voriconazole as primary prophylaxis in Allo-HSCT has reduced the incidence of IFD to 50% in our institution. We therefore suggest that it is an effective drug to prevent IFD in Allo-HSCT. Disclosure of Interest: None declared.
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P026 Successful bone marrow processing for transplantation with Spectra Optia cell separator Version 9 C. Wissing1,*, A. Prinsloo1, N. Besson2 1 oncology, Pretoria east Hospital, Pretoria, South Africa, 2 Scientific marketing, TerumoBCT, Brussels, Belgium Introduction: Bone marrow (BM) is one source of hematopoietic stem cells (HSC) transplantation. Before storing of both autologous and allogeneic bone marrow, it is strongly recommended to downsize bone marrow volume in order to reduce the cryogenic storage space but also to prevent DMSO toxicity during infusion. In addition, ABO-incompatible grafts may be RBC depleted to minimise the risk of ABO-induced transfusion reactions. Several methods are available. In our center, erythrocyte depletion of BM harvests has been performed on a continuous flow cell separator because of its high mononuclear cell (MNC) yield, low erythrocyte contamination and closed processing system.This study describes a single center experience in processing 11 harvested bone marrow in the blood separator Spectra Optia version 9 (TerumoBCT). Materials (or patients) and methods: 4 patients received BM transplants form ABO incompatible donor and 7 of the harvests were autologous. Directly after ending of the collection, BM was processed on the Spectra Optia version 9. Briefly, volume of the BM was determined as well as complete blood count. The CD34 þ cell counts were measured by flow cytometry and the 7-aminioactinomycin D (7-AAD) dye was used to exclude non-viable cells. 6-8 cycles were processed at an inlet of 60 ml/ min. Neutrophil engraftment was defined as the number of days after infusion until the circulating peripheral blood count reached 0.5x10E9/l for 3 consecutive days and platelet engraftment as the number of days after infusion to reach peripheral blood count Z50x10E9/L. Results are presented as mean±SD. Results: The initial bone marrow collections had a mean volume 1451±262 mL (20.8±3.7 mL/kg)(cf. Table 1). Following processing on the Spectra Optia, the mean product volume was 155.5±37.2 mL (which means a volume reduction 89.1±2.8%) and final product hematocrit was very low 3.45±0.87%. Red blood cell volume reduction was 98.85±0.45%. The mean CD34 þ cells recovery was high 106.7±32.6%. The mean CD34 þ cell dose infused was 2.3±1.4x10E6/kg; the overall CD34 þ cell viability was 98.7±1.2%. Neutrophil engraftment at 0.5x10E9/l averaged 20.8±3.7 days. Conclusion: Spectra Optia is safe and effective to reduce both the volume and red blood cell content of bone marrow collections while ensuring high CD34 þ cells recoveries. Because of the low residual red blood cell content, ABO incompatibility between donor and recipient can be successfully overcome by BM processing using Spectra Optia. Quality of the graft is not affected by BM processing. Disclosure of Interest: None declared.
P027 Prolonged Regression of Ileocolonic Crohn’s Disease following Autologous Haemopoetic Stem Cell Transplantation C. Hawkey1,* on behalf of On behalf of the ASTIC Trialists 1 NOTTIGHAM DIGESTIVE DISEASES CENTRE, UNIVERSITY OF NOTTINGHAM, Nottingham, United Kingdom Introduction: In the ECCO-EBMT ASTIC trial autologous Haemopoietic stem cell transplantation (HSCT) led to mucosal healing in Crohn’s disease. We now report prolonged regression of ileocolonic disease over 2years. Materials (or patients) and methods: Patients with impaired quality of life from active Crohn’s disease not amenable to surgery, despite treatment with Z3 immunosuppressive agents all underwent stem cell mobilisation before randomisation to immunoablation followed by unselected cyclophosphamide-based conditioning and HSCT after 1 month (Early HSCT)or 1 year (delayed HSCT). They were assessed using Crohns Disease activity index (CDAI), Simple Endoscopic Scores (SES-CD), Inflammatory Bowel Disease Questionnaire (IBDQ) and EuroQoL. Results: Forty four patients with ileocolonic involvement underwent mobilisation before randomisation to Early (n ¼ 23, all treated) or delayed (n ¼ 21) HSCT. One Early HSCT patient died 14 days after HSCT. One randomised to delayed HSCT withdrew immediately, 4 did not proceed because they required surgery (n ¼ 3) or were too well after 1 year. Sixteen underwent delayed HSCT. The CDAI fell from 321 (median, IQR 242-433) to 162 (76-294, P ¼ 0.002) one year after Early HSCT vs 353 (300-462)to 310 (236-403, P ¼ 0.124) over year 1 prior to delayed HSCT. At year 2 the CDAI was maintained at 161 (59232, P ¼ 0.555 vs Year 1) in Early HSCT patients and fell to 165 (88-298, P ¼ 0.022) following delayed HSCT. The SES-CD was 13 (8.5-24.5) at baseline, 3(1-10) after one year (p Conclusion: Improvements gained one year after HSCT are maintained into a second year. HSCT delayed by one year appears to be effective. There is a progressive improvement in quality of life. If improvements continue to be durable, the risks of HSCT might become acceptable for a wider group of patients. Disclosure of Interest: None declared. P028 Mobilization in patients over 65 years of age and patients between 55 to 65 years of age is similar in terms of safety and success in Multiple Myeloma and lymphoma patients: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, C. Bal3, I. Dora1, B. Kosan1, E. Er1, N. Baskan1, B. Saritas1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, Kocaeli, 3 Biostatistics Department, Osmangazi University, Eskisehir, Turkey Introduction: Autologous stem cell transplantation is a standard procedure in relapsed lymphoma patients and in multiple myeloma patients after first line treatment especially in patients less than 65 years old. However, autologous stem
Table 1. n ¼ 11
Mean
Standard deviation
BM volume before processing (mL) BM volume after processing (mL) BM volume reduction (%) CD34 þ cells viability (%) CD34 þ cells 10E6/kg before processing CD34 þ cells 10E6/kg after processing CD34 recovery (%) Red blood cell volume reduction (%) ANC 50 engraftment (days)
1451.5 154.9 89.1 98.7 2.4 2.3 106.7 98.9 19.5
262.6 37.3 2.7 1.2 1.4 1.4 32.6 0.5 8.0
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[P028]
cell transplantation could be applied to patients over 65 years if they fullfill certain criterias . In the present study, we aimed to compare the results of mobilization in multiple myeloma or lymphoma patients who were over 65 years and between 5565 years. Materials (or patients) and methods: There were 40 patients over 65 years and there were 81 patients between 55-65 years. The stem cells were harvested from the peripheral blood. While, 30 of the 81 patients aged between 55-65 were female, 12 of the 40 patients over 65 years were female. 75,6% of the patients aged between 55 and 65 and 72,5% of the patients over 65 years were Multiple Myeloma patients. Chi-square test (continuity correction) was used as the statistical method to compare the results. Characteristics of the patients are shown in Table 1. Results: While, median age of the group of the patients who were between 55-65 years was 60, median age of the patients over 65 years were 67(66-80). There were no significant differences between the 2 groups in terms of mobilization regimens used, febrile neutropeni incidence, transfusion requirements (erythrocyte and thrombocyte) and total CD34 þ cell count (P40,005) (Table 2). Conclusion: Mobilization in patients older than 65 years is as safe and successful as mobilization in patients between 55-65 years. Disclosure of Interest: None declared. P029 First 100 Day Results Multiple Myeloma And Lymphoma Patients Over 65 Years are Similar to Multiple Myeloma And Lymphoma Patients Who are 55-65 Years Old: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, C. Bal3, I. Dora1, B. Kosan1, E. Er1, B. Saritas1, N. Baskan1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, Kocaeli, 3 Biostatistics Department, Osmangazi University, Eskisehir, Turkey Introduction: We aimed to compare results of Autologous Hematopoietic Stem Cell Transplantation (AHSCT) patients who were over 65 years to those between 55 to 65 years. Materials (or patients) and methods: We retrospectively analyzed results of 40 patients who were over 65 years and had AHSCT and 81 patients who were 55-65 years and had AHSCT. Peripheral blood was used as the source of the stem cells. Acyclovir and fluconazole were started at the day of transplantation and piperacillin-tazobactam were started when neutropenia ensues prophilactically. We compared 100th day results. Chi-square test (continuity correction) was
used as the statistical method to compare the results. Characteristics of the patients are shown in Table 1. Results: Characteristics of the patients are shown in Table 1 and clinical results are shown in Table 2. While, median age of the group of the patients who were between 55-65 years was 60, median age of the patients over 65 years were 67(66-80). Median stem cell count infused were similar in the 2 groups (5,1 vs 5,3, P40,05). Median follow-up times of the 2 groups were not significantly different (11 months vs 13 months, P ¼ 0,35). Median time to neutrophil and thrombocyte engraftment, hospitalization durations and bacterial and viral infection rates of the 2 groups did not differ significantly (P40,05). 100th day mortality ratios of the 2 groups were similar (1,3% vs 2,7%, P ¼ 0,241). Conclusion: AHSCT in patients over 65 years is as safe as AHSCT in patients between 55 to 65 years. Disclosure of Interest: None declared.
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P030 Comparative study of two techniques for routine volume reduction of umbilical cord blood D. A. Ivolgin1,2,*, A. B. Smolyaninov3 1 Scientific research laboratory of cells Technologies North Western State Medical , 2Pokrovsky Stem Cell Bank, 3University named after I.I.Mechnicov, Saint-Petersburg, Russian Federation Introduction: The aim of this study is to compare a semiautomated versus an automated method for cord blood volume reduction in terms of efficacy, cost and time required to process units. Materials (or patients) and methods: Umbilical Cord Blood units (UCB) (n ¼ 280) were collected at the maternity clinics of St. Petersburg and Leningrad region. UCB units were processed at the Research Laboratory of cell technologies of Mechnikov NorthWestern State Medical University and Pokrovsky stem cell bank from April to December 2014 for public storage. UCB volume reduction was performed using the automated method Sepax S100 (Biosafe, Switzerland) (n ¼ 140) or the semi-automated method MacoPress Smart (Macopharma, France) (n ¼ 140). The Total Nucleated Cell (TNC) count and the hematocrit rates were analyzed pre-and post-processing using a Coulter AcT diff 2 ("Beckman Coulter", USA)hematology analyzer. The number of CD34 þ cells and cell viability were analyzed using a FC500 flow cytometer (CXP software with the laser wavelength of 488 nm, a set of reagents StemKit, "Beckman Coulter", USA). Statistical analysis was carried out using SPSS. All data are represented as Mean±s.d. The student t-test was used to compare the data between each group. The differences were considered statistically significant at Po0.05. In order to compare both techniques in similar conditions, the use of the semiautomated method was adapted to a final volume of 21 mL. Results: The TNC recovery using the semi-automated and the automated methods was: 81.9±0.7% and 80.38±0.7%, the hematocrit values were: 45.48±1.4% and 36.3±1.1%, the CD34 þ cells count were: 2.95±0.2(.106) and 2.56±0.2(.106) and the cell viability was 89.49±0.93% and 89.54±0.9%, respectively. The comparison was also done per group of volumes as shown in the table below (*Po0.01). The TNC recovery was statistically higher in group 2 when compared to group 1as shown by Student t-tests values calculated in pairs. The initial UCB volumes processed were of importance and led to higher differences in TNC recovery in favor of group 2. By contrast, the hematocrit was found to be statistically lower for group 1 when high volumes were processed; No statistical differences were noticed for lower volumes. The CD34 þ counts were similar for both groups for high volumes while a significant statistical difference was found for volumes lower than 80 mL in favor of group 1. Finally, no significant differences for cell viability levels were found between the two groups. The time required to process one UCB unit was shorter with the semi-automated method when compared to the automated one: 25±3 min and 54±2 min respectively (Po0.05). The price calculation per CBU shown that the
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automated method was 3.75 times more expensive than the semi-automated method. Conclusion: Taken together, the comparison of the two techniques show a higher TNC recovery using the semiautomated method but associated with a higher level of hematocrit in the corresponding processed UCB units. The cost of use was clearly in favor of the semi-automated method. Disclosure of Interest: None declared. P031 Intravenous busulfan in combination with thiotepa and fludarabine (TBF) as preparative regimen for patients with lymphoma submitted to allogeneic stem cell transplantation (HCT) E. Pennese1,*, S. Santarone1, P. Olioso1, P. Bavaro1, G. Papalinetti1, P. Accorsi1, T. Bonfini1, F. Papola2, P. Di Bartolomeo3 1 Department of Hematology, Transfusion Medicine and Biotechnology, Pescara, 2Ospedale San salvatore, Cntro Regionale Immunoematologia, L’Aquila, 3Department of Hematology, Transfusion Medicine and Biotecnology, Pescara, Italy Introduction: Allogeneic HCT is a potentially curative option in relapsed/refractory lymphomas due to either the preparative regimen and the graft-versus-lymphoma effect. Intravenous Busulfan (iv-BU), in association with Thiotepa and Fludarabine (TBF regimen), is an active drug against lymphoid malignancies. Our study was designed to assess the safety and efficacy of TBF regimen in patients with lymphoma undergoing alloHCT. Materials (or patients) and methods: From 2010 to 2014, 10 patients (8 males) with either advanced Hodgkin lymphoma (HL) (n ¼ 7) or non-Hodgkin lymphoma (NHL) (n ¼ 3) received an alloHCT from HLA identical sibling (n ¼ 5), or HLA haploidentical donor (n ¼ 4), or matched unrelated donor (MUD) (n ¼ 1). The median age was 41 yr (range, 23-60). The median number of previous chemotherapy lines for each patient was 5 (range, 2-6). The donor’s median age was 46 yr (range, 22-61). Conditioning regimen was myeloablative (TBFMAC: Thiotepa 5 mg/kg/day i.v. at days -7 and -6, Busulfan 3.2 mg/kg/day in a single i.v. infusion over 3 hours and Fludarabine 50 mg/m2/day i.v. in 1 hour at days -5, -4 and 3) in 6 patients or at reduced intensity (TBF-RIC) by deleting one dose of Thiotepa and Busulfan in 4 patients. Six patients were given peripheral blood stem cells (PBSC) collected from identical sibling donors or MUD and received Cyclosporine (CSA) and short course Methotrexate (MTX) as graft-versushost disease (GvHD) prophylaxis. Four patients transplanted from haploidentical donor received unmanipulated bone marrow stem cells (BMSC) and a combination of 5 drugs (CSA, MTX, Mycophenolate mofetil, anti-CD25 monoclonal antibody Basiliximab and Fresenius Anti-thymocyte globulin) for GvHD prophylaxis. Results: The median dose of CD34 þ and CD3 þ cells infused was 6.0x106/kg (range 3.5-10.7) and 245x106/kg (range 188857) for patients transplanted with PBSC and 2.3x106/kg (range 2.0-2.3) and 25.8x106/kg (range 15.8-41) for patients transplanted with BMSC, respectively. All patients engrafted with a median time of 18 days (range 13-33) and 20 days (range 13-75) to reach Z0.5x109/L neutrophil count and Z20x109/L platelet count. No primary or secondary graft failure occurred. At day 60 post-transplant all patients showed a full-donor chimerism. Grade I-II acute GvHD and limited chronic GvHD occurred in 3 and 4 patients, respectively. No patient showed veno-occlusive disease of the liver. One patient had hemorrhagic cystitis associated to BK virus reactivation. The WHO toxicity was classified as follows: slight increase of total bilirubin and SGOT/SGPT in 3 and 1 patients, respectively; mild or severe oral mucositis in 9; gastrointestinal involvement in 3; impaired renal function in 2. Toxicities resolved in all patients. No patient died of transplant-related cause. One HL patient, transplanted in active disease, showed progression of his disease and died
on day 312. With a median follow-up of 20 months (range 156), overall survival and progression-free survival were 90% and 70%, respectively. Conclusion: Our small series of 10 patients, all of them heavily pretreated and with refractory disease, shows that alloHCT is a feasible procedure in patients with lymphoma. The TBF regimen has been well tolerated with minimal secondary effects. More patients and longer follow-up are needed to assess the TBF regimen efficacy. Disclosure of Interest: None declared. P032 Comparison of different enzymatic and non-enzymatic methods on cell surface expression of CXCR4 by human CD34 þ Hematopoietic Stem Cells and Bone Marrow Mesenchymal Stem Cells G. Aydin1,2, F. Aerts-Kaya1,2,*, D. Uckan-Cetinkaya1,2,3 1 Center for Stem Cell Research and Development, 2Institute of Health Sciences, HACETTEPE UNIVERSITY, 3Department of Pediatric Hematology, Hacettepe University Medical Faculty, Ankara, Turkey Introduction: The most commonly used method to detach adherent cells is trypsinization. Trypsin is a serine protease and acts by cleaving peptide chains at the carboxyl side of the amino acids, Lysine or Arginine. As such, it may cleave cellular antigens, present at the surface of the cell. CXCR4 is an important cell surface molecule involved in homing and engraftment of CD34 þ hematopoietic stem cells (HSCs) and Mesenchymal Stem Cells (MSCs). Trypsin and other enzymatic methods, used to dissociate adherent cells/digest tissue may affect the homing ability of these cells by cleaving specific surface antigens. Here, we compared the effects of different enzymatic and nonenzymatic cell detachment methods on the expression of CXCR4 by CD34 þ HSCs and human Bone Marrow (BM) Mesenchymal Stem Cells (MSCs) and recovery of CXCR4 expression in time and after incubation with different cytokines. Materials (or patients) and methods: Peripheral blood (PB), cord blood (CB) and BM CD34 þ HSCs and BM-MSCs were treated using non-enzymatic dissociation solutions (GIBCO, Enzyme-Free Cell Dissociation Buffer and Biological Industries, Non-enzymatic Cell Dissociation Solution) and enzymatic solutions (0.25% or 0.05% Trypsin/EDTA), Mesencults enzymatic dissociation solution (Stem Cell Technologies), Gibcos TrypLEt Express (Life Technologies) and Accutase cell detachment solution (Capricorn Scientific), for 15 minutes at 371C at 5% CO2. Control cells were collected by pipetting in PBS. PBS þ 2% Fetal Calf Serum (FCS) was used to stop the reactions. Recovery of CXCR4 after 0.25% Trypsin/ EDTA treatment was measured after incubation with PBS þ 2% FCS (control) or different cytokine cocktails for 4 and 24 hours at room temperature using a FACSARIA (Becton Dickinson). Results: CXCR4 surface expression on BM-CD34 þ cells collected with PBS and the non-enzymatic solutions from GIBCO and Biological Industries was 26.7%, 28.3% and 23.2%, respectively. After Trypsin/EDTA 0.25%, Trypsin/EDTA 0.05% and Mesencults enzymatic solutions, CXCR4 levels rapidly decreased to 1.1%, 1.9% and 4.4%, respectively. CXCR4 expression by CD34 þ cells, irrespective of source, was negatively affected by enzymatic solutions. CXCR4 cell surface expression by MSCs was 29.7% in the control group and decreased to 0.9-6.9% after incubation with any solution. Spontaneous recovery of CXCR4 at 2 hours, 4 hours, 8 hours and 24 hours was not observed and incubation in presence of different cytokine cocktails resulted in minimal surface expression of CXCR4, with a maximum of 5.2% after 24 hours. Conclusion: Treatment of CD34 þ cells and MSCs with enzymatic solutions rapidly reduced CXCR4 cell surface levels and may negatively affect the homing and engraftment ability of these cells. Spontaneous recovery was not observed and incubation in presence of different cytokine cocktails did not result in re-expression of CXCR4 on the cell surface. This study
may have important implications for the choice of collection methods used in the preparation of clinical products. To maintain maximum CXCR4 levels and achieve optimal homing/engraftment of cells, the use of non-enzymatic and/or mechanical dissociation methods is considered most appropriate. Disclosure of Interest: None declared. P033 Autoimmune hemolytic anemia after allogeneic HSCT – A case report I. Bodova1,*, S. Sufliarska1, O. Fabri1, Z. Laluhova Striezencova1, P. Svec1, A. Chocholova1, J. Horakova1 1 BMT Unit, Departement of Pediatric Hematology and Oncology, Bratislava, Slovakia Introduction: Autoimmune hematologic disease following HSCT may affect one or more cell lines. Its association with GVHD, T-depletion, unrelated HSCT or serotherapy supports the assumption of its development on the basis of immune dysregulation or incomplete immune reconstitution. Materials (or patients) and methods: Between 11/1995 and 9/2014, 194 allogeneic HSCT were performed in 184 consecutive children at BMTU in Bratislava, Slovakia. 117 patients (64%) were treated for malignant disease and 67 (36%) childrens for non-malignant. There were 107 (58%) boys and 77(42%) girls with a median age 9.7 y (0.2 to 18.5 y). 106 patients (55%) had matched related donor: 100x sibling, 3x matched parent, 3x haploidentical parent). 88 childrens (45%) had matched unrelated donor. Results: Autoimmune haemolytic anaemia occurred in two patients (1,03%). First patient was 5 y girl with ALL in 2nd complete remission after allogeneic unrelated HSCT, from þ 70 D she developed signs of slight autoimmune haemolytic anaemia, after adding prednisolone 2 mg/kg/day to CS-A prophylaxis haemolysis resolved with no need of transfusion. The second patient was 16 y girl after unrelated HSCT for CML in 1st chronic phase. She was treated for diabetes mellitus type 1 and autoimmune thyroiditis. Due to major AB0 incompatibility (patient pre-HSCT 0 Rh positive, PBSC donor AB Rh positive) she was transfused with erythrocytes 0 Rh positive, platelets AB. The titre of the immune anti-A and anti-B antibodies before HSCT was slightly increased. She engrafted þ 9 D, readmitted to the hospital on þ 97 D with acute GVHD – GIT, with diarrhoea and vomiting. She responded very good to adding corticosteroids to CS-A prophylaxis. From þ 104 D developed severe anemia with signs of autoimmune haemolysis with positive free and bound polyspecific antierythrocyte autoantibodies, cross-checks before erythrocyte transfusion had been positive. Erythrocyte transfusions were administered due to vital indications (Hb 34 g/l) after corticoids premedication. Despite high doses of corticosteroids and IVIG, severe hemolysis persisted with need for erythrocytes transfusions (12x). Rituximab 375 mg/m2/day therapy started þ 112 D, first 4 doses weekly and the next doses monthly. Last transfusion was indicated after 3rd dose of Rituximab, hemolysis resolved. In total, 7 doses of Rituximab were administered. Currently, the girl is 11 months after HSCT in a good clinical condition and no signs of GVHD, good function of allogeneic graft with molecular remission of CML. Conclusion: The results of our study are similar to the published data. AIHA is the most frequent autoimmune haematological disease emerging after allogeneic HSCT. Rituximab showed to be effective treatment in AIHA after HSCT refractory to corticosteroids and IVIG. Disclosure of Interest: None declared.
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P034 Clinical relevance of anti-HLA Antibodies and DSA in unmanipulated haploidentical stem cell transplantation L. Laget1,*, J. El Cheikh2, A. Basire1, S. Simon1, P. Ladaique2, C. Lemarie´2, D. Blaise2, C. Chabannon2, C. Picard1,3 1 Laboratoire d’Immunoge´ne´tique et d’histocompatibilite´, EFS Alpes-Me´diterrane´e, 2Institut Paoli Calmettes, 3UMR 7268 ADES, Aix-Marseille Universite´/EFS/CNRS, Marseille, France Introduction: Background: The first clinical results of unmanipulated haplo-identical stem cell transplantation (haploHSCT) using cyclophosphamide after graft are promising. However, the detection in many adult recipients of preformed anti-HLA antibodies (Abs) directed against donor mismatched HLA antigens (DSA) raises the question of their clinical relevance, i.e. risks of graft failure, delayed engraftment and graft versus host disease (GVHD) all of which may contribute to reduced overall survival (OS). Objectives:The main objective of this study is to evaluate the clinical relevance of anti-HLA Abs and /or DSA recipient and donor in unmanipulated Haplo-SCT, including engraftment, the development of GVHD and overall survival. We also evaluated the effectiveness of 3 desensitization protocols for patients with DSA before transplant. Materials (or patients) and methods: The study was conducted in thirty haplotype-mismatched donor – recipient pairs taken care of at a single institution (Institute Paoli Calmettes). Patients were transplanted with T-cell replete blood cells collected by apheresis from G-CSF mobilized donors, following a non-myeloablative conditioning regimen, and received post-transplant cyclophosphamide. Anti-HLA antibodies screening and identification of donor and recipient were performed by luminex assay at Day 0 and at D3, D8, D15 and D30 post-graft and DSA were assigned in all serum samples. Results: The presence of preformed anti-HLA Abs was detected in 50% of recipients and 31 % of donors. Men were predominantly and preferably immunized against HLA class I, probably related to excessive sensitivity of the technique used (Luminex). HLA classes I DSA were detected in 3 recipients. Only one graft failure was observed in a patient with DSA at high levels (Mean Fluorescence Intensity 4 10 000) despite a desensitization protocol. Two out of three desensitization protocols (rituximab þ plasmapheresis/ rituximab þ platelets transfusions) had been effective and had significantly reduced the DSA rates to allow an engraftment. A comparison of three recipient groups (15 Abs negative, 12 Abs positive/DSA negative and 3 DSA positive) showed no significant difference on clinical end-points (table). Anti-HLA Abs donor seemed to be associated with a delayed neutrophil recovery (P ¼ 0.07). Monitoring of anti-HLA Abs after transplantation
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showed the absence of development of DSA recipient and donor, the absence of anti-HLA Abs donor detection and a loss of anti-HLA Abs recipient at D30 in many cases Conclusion: These data suggest that the preformed anti-HLA Abs in the patient and/or donor in unmanipulated haplo-HSCT are not associated with a risk of graft failure, except potentially when DSA is detected at high levels. Nevertheless the donor’s anti-HLA allo-immunization appears to be a parameter to consider when selecting the donor since there are usually several potential haploidentical donors in the patient’s family. However, the presence of DSA should no longer be a barrier to HLA haplo-HSCT, providing that further studies with new desensitization protocols are conducted and successful. Disclosure of Interest: None declared. P035 The prognostic impact of CD34 þ /CD38- cell burden at diagnosis and during disease course in acute myeloid leukemia patients undergoing allogeneic stem cell transplantation M. Jentzsch1,*, M. Bill1, S. Leiblein1, K. Schubert1, M. Ple1, U. Bergmann1, K. Wildenberger1, L. Schmalbrock1, M. Cross1, W. Po¨nisch1, G.-N. Franke1, V. Vucinic1, G. Behre1, D. Niederwieser1, S. Schwind1 1 Ha¨matologie, int. Onkologie und Ha¨mostaseologie, Universita¨tsklinikum Leipzig, Leipzig, Germany Introduction: In acute myeloid leukemia (AML) Leukemia initiating cells (LIC) are enriched within the CD34 þ /CD38bone marrow (BM) compartment. LIC may survive chemotherapy as minimal residual disease & cause relapse. Allogeneic stem cell transplantation (SCT) is a potentially curative consolidation therapy for AML patients (pts). However, relapse remains a common cause of death. Risk stratifying pts is crucial to adjust post-SCT care strategies. High LIC burden at diagnosis is associated with worse outcome in pts receiving chemotherapy, but larger studies on outcome after SCT in which graft-versus-leukemia (GvL) effects contribute to treatment success are missing. Materials (or patients) and methods: Here we analyzed 169 AML pts (median age 62 years [y]; range 19-75y) who received myeloablative (MAC, Cyclophosphamid 120 mg/kg þ 12 Gy total body irradiation [TBI], n ¼ 49) or non-myeloablative (NMA, Fludarabine 90 mg/m2 þ 2 Gy TBI, n ¼ 120) SCT in complete remission (CR). European LeukemiaNet (ELN) genetic classification was: Favorable 28%, Intermediate-1 26%, -2 20%, Adverse 26%. Using flow cytometry percentage of BM CD34 þ /CD38cells at diagnosis & in CR before & at day 28 after SCT was measured. Donors were human leucocyte antigene (HLA)-
matched related (21%) or HLA-matched (56%) or mismatched (4 ¼ 1 antigen; 23%) unrelated. 29% of pts developed acute graft-versus-host disease (GvHD; 4 ¼ grade 2) & 58% chronic GvHD. Median follow-up was 3.4y. The amount of CD34 þ / CD38- BM cells was also measured in 15 healthy donors (median 0.2 [0-0.5]). Results: A cutpoint of ¼ 45% defined a high CD34 þ /CD38burden which associated with lower platelet count (P ¼ .03) at diagnosis. Pts with high CD34 þ /CD38- burden more frequently had a complex karyotype (P ¼ .04), deletion 5 or 5p (P ¼ .009) & more secondary AML (P ¼ .006). Interestingly, no pts with high CD34 þ /CD38- burden was CEBPA mutated. The amount of CD34 þ /CD38- cells in CR was not different to levels in healthy donor BMs regardless of the burden at diagnosis (P ¼ .33 & P ¼ .14 at SCT for pts with high & low CD34 þ /CD38- burden respectively; P ¼ .51 & P ¼ .39 after SCT for pts with high & low CD34 þ /CD38- burden respectively). A high diagnostic CD34 þ /CD38- burden associated with shorter event free (EFS, Po.001, Fig. 1A) & overall survival (OS, P ¼ .004, Fig. 1B). A prognostic impact was also seen when we analysed the NMA (EFS P ¼ .02 & OS P ¼ .002) & MAC subset (EFS P ¼ .05) separately. In multivariate analysis pts with high CD34 þ /CD38-burden had shorter EFS & OS (Hazard Ratio [HR] 2.1, P ¼ .003 & HR 1.7, P ¼ .04). Conclusion: The worse outcome associated with higher CD34 þ /CD38- burden at diagnosis was likely mediated by LIC within the CD34 þ /CD38-population escaping the GvL effect of SCT. Thus LIC persistence at low levels may not easily be overcome by SCT. In the future evaluation of CD34 þ / CD38- cell burden at diagnosis may improve AML pts risk stratification & help to personalize post-SCT care. Furthermore, incorporating LIC targeting therapies may improve survival of AML pts receiving SCT. Disclosure of Interest: None declared.
EORTC/MSG 2008. Drug toxicity was evaluated according to CTCAEv4.1 criteria. Results: We found differences between the 2 groups in terms of: type of graft (bone marrow was more frecuently in fluconazole group), regimen and type of conditioning (reduced-intensity conditioning was more common in voriconazole group) and presence of cGVHD (more frecuenty in voriconazol group). The incidence of IFD was 16.3% in the Fluconazole group (n ¼ 16) vs 7.1% (n ¼ 7) in Voriconazole group (P ¼ 0.046). The median of FFS was 24.5 days and 106 days (P ¼ 0.003). According to the EORTC/MSG criteria,taking into account only patients who developed IFD, the percentage of possible IFD in the first group was 50% vs 14.8% in the second group; Probable: 37.5% vs. 71.4%; Proven: 12.5% vs. 14.28%. We found significant statistical differences at 360 days post SCT (83.7% vs 92.9%;P ¼ 0.034). Deaths caused by IFD were 10.1% in Fluconazole (n ¼ 10) and 3.1% in Voriconazole (n ¼ 3) (P ¼ 0.027). The median follow-up was 34 months. There was no significant difference in overall survival: 52.1% vs 52.6% (P ¼ 0.95). There were no differences in causes of death (P ¼ 0.326). Eleven patients (11.22%) presented adverse effects (AE)in the first group vs 18 patients (18.36%) in the second one (P ¼ 0.065). The most frequent AE for both groups was hepatic toxicity. There were not significant differences in the suspension (temporary and permanent) of the drug between the two groups. Conclusion: This study confirms that the use of Voriconazole as antifungal prophylactic treatment in patients undergoing allo-SCT decreases the incidence of IFD and increases FFS versus Fluconazole. In spite of a greater toxicity in Voriconazole group,this toxicity was non severe and rapidly recovered with the withdrawal of the drug. Disclosure of Interest: None declared.
P036 Voriconazole used as antifungal prophilaxis decreases invasive fungal disease in patients undergoing allogenic stem cell transplantation in our institution (Allo-SCT) M. J. LLAMAS POYATO1,*, C. CHIC ACEVEDO1, E. GARCIA TORRES1, J. SERRANO LOPEZ1, J. R. MOLINA HURTADO1, C. MARTIN CALVO1, F. MARTINEZ GUIBELARDE1, R. ROJAS CONTRERAS1 1 HEMATOLOGY, REINA SOFIA UNIVERSITY HOSPITAL, CORDOBA, Spain
P037 Influence of donor’s KIR2DL1-R 245 allelic snp on patient outcome after haploidentical stem cell transplant M. Bastos Oreiro1,2,*, C. Martinez Laperche1,2, D. Carbonell1, D. Champ1, E. Buces1,2, C. Pascual1, A. Perez Corral1, P. Balsalobre1, M. Kwon1, D. Serrano1, J. Gayoso1, J. Anguita1, I. Bun˜o1,2, J. L. Die´z Martı´n1 1 Hematology, Hospital General Universitario Gregorio Maran˜o´n, 2 Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n, Madrid, Spain
Introduction: Invasive fungal disease (IFD) causes significant morbidity and mortality in patients undergoing Allo-SCT. Some new triazole-drugs with anti-filamentous activity (as Voriconazole) have being used during the last years as antifungal prophylaxis for high-risk patients.The aim of our study was to compare the incidence of IFD in patients undergoing Allo-SCT in our institution using Fluconazole vs Voriconazole as prophylactic treatment. Materials (or patients) and methods: We analyse retrospectively 196 allo-SCT consecutive patients (paediatrics and adults) submitted to this procedure in our center from March 2001 to April 2008. A total of 98 patients received antifungal prophylaxis with Fluconazole and 98 with Voriconazole. All patients received antifungal prophylaxis with oral Voriconazole 200 mg/12 hours (5mg/kg/12h in children) or Fluconazole 200 mg/12 hours (5mg/kg/day in children) from day 0 to day þ 75. Antifungal prophylaxis was prolonged in patients requiring treatment with high doses of corticosteroids (42mg/kg/day) and/or with 2 immunosuppressive drugs. The temporary suspension was applied in those patients with toxicity,without reaching criteria for withdrawal,being able to continue the study drug if the suspected toxicity resolved in 7 days. AGA detection was done twice weekly from year 2004. It was considered positive when the index OD was Z 0.5 in 2 consecutive measurements or Z 0.8 on a single determination. When IFD was suspected a new determination of AGA and a high resolution CT were done as well as a bronchoalveolar lavage. IFDs were according to the revised criteria of the
Introduction: Killer-cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells play an important role in the graft versus tumour effect. KIRs are highly polymorphic. Donor KIR2DL1-R snp has been associated with stronger signalling function than others. Our aim in this analysis is to evaluate this donor polymorphism in the setting of T cellrepleted haploidentical hematopoietic stem-cell transplantation (HSCT) with post-infusion high-dose cyclophosphamide (PiCy). Materials (or patients) and methods: Forty-nine adult patients who underwent T-cell repleted haploidentical HSCT with PiCy in our institution were included. Demographic data, KIR genotype and snp are summarized in Table 1. Donor KIR2DL1 functional allele typing was retrospectively performed using single nucleotide polymorphism assay (Bari et al, Clinical Immunology 2011). KIR genotype was analyzed by PCR (KIR Typing, Miltenyi Biotec) using genomic DNA. DNA was purified by the (Maxwell 16 Blood DNA Kit, Promega) from peripheral blood examples. HLA typing was used to identify HLA class I KIR ligands. Results: We did not find progression free survival (PFS) differences between patients who received a KIR2DL1-R 245 homozygous graft or a KIR2DL1-R 245/KIR2DL1-C245 graft (76% vs. 70% P ¼ 0,7). However, patients who received a KIR2DL1-R 245 graft with HLA-C ligand-receptor mismatch had a marked tendency to better PFS compared with those who received a graft with KIR2DL1-R 245 and no HLA-C ligandreceptor mismatch or KIR2DL1-R 245/KIR2DL1-C245 graft,
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(93% vs. 74% respectively, P ¼ 0.057) one year after transplant (Figure 1). Remarkably, and despite the small number, this trend remains if we perform a sub analysis in Hodgkin lymphoma patients (PFS100%, 47%, p: 0.07). Conclusion: Donor KIR2DL1-R 245 allelic snp associated with KIR HLA-C ligand-receptor mismatch could affects recipient outcomes after T cell-repleted haploidentiacal HSCT with PiCy, especially in HL. If this information is confirmed, these findings could have significant repercussion for donor selection. Disclosure of Interest: None declared. P038 In vitro evaluation of NK cytotoxicity in the setting of T-cell-replete haploidentical transplant M. Bastos Oreiro1,2,*, J. Anguita1, C. Falero1, L. Solan1, A. Perez Corral1, C. Pascual1, C. Martinez Laperche1,2, P. Balsalobre1, C. Mun˜oz1, D. Serrano1, M. Kwon1, A. Perez3, I. Bun˜o1,2, J. Gayoso1, J. L. Die´z Martı´n1 1 Hematology , Hospital General Universitario Gregorio Maran˜o´n, 2 Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n, 3Pediatric hematology, Hospital Universitario La Paz, Madrid, Spain Introduction: NK cells has been identified as an important element for graft vs. tumor effect in allogeneic transplant. Our aim in this analysis was to evaluate, by two different methods,
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the ‘‘in vitro’’ cytotoxicity activity of NK cells and to correlate this with patient’s evolution in the setting of T-cell-replete haploidentical hematopoietic stem-cell transplantation (HSCT) with post-infusion high-dose cyclophosphamide (PiCy). Materials (or patients) and methods: Since 03-2013 to 062014, 16 consecutive patient-donor pairs with haematological malignancies who underwent T-cell-replete haploidentical HSCT with PiCy in our institution were included. Patients demographic data is included in Table 1. The cytotoxicity of NK cells was evaluated by 2 methods: 1) Conventional 2-hour europium-TDA release assay (Perkin-Elmer Wallac, Turku, Finland). K562 (erythroleukemia cell line) were used as the target cell; cytolysis was measured by fluorimetry (VictorTM, Wallac) in resting conditions and stimulated overnight with IL15, as previously described (Blomberg et al, J Immunol Methods, 1995). Percentage of degranulation was grouped in low (less than 25%), medium (25-50%) and high (up 50%). The number of NK cells in donor blood was calculated by flow cytometry, multiplying the lymphocyte counts to the percentage of CD3- CD56 þ NK cells. 2). Degranulation by flow cytometry: the percentage of NK cell degranulation was analyzed through the expression on the cell surface of CD107a. This was determined basal and after 2 hours incubation at 371 with K562 cell line. Results: We found that the correlation between tests used for the cytotoxicity assessment was good (correlation coefficient: 0.88, P ¼ 0,000) (Figure 1). Four patients relapsed (progression free survival (PFS): 86% 1 year after transplant).
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Two patients died for no transplant related causes. Median follow up was 19.8 month (r: 6-22). Five cases presented high levels of cytotoxicity by fluorometric assay, of these, four presented more than 50% degranulation by flow cytometry. One case showed more than 50% degranulation by flow cytometry but low-medium levels of cytotoxicity by fluorometric assay (Figure 1). Higher levels of cytotoxicity evaluated by fluorometric assay as well as by flow cytometry appear to be associated with lower relapse rate (HR 0.419, IC95%: 0.17-1.07, P ¼ 0.053; HR 0.88, IC95%: 0.77-1.01, P ¼ 0.088, respectively). Conclusion: Cytotoxicity evaluated by fluorometry assay as well as by flow cytometry could be a helpful tool for selecting the most alloreactive donor, in the setting of T-cell-replete haploidentical HSCT PiCy. Disclosure of Interest: None declared. P039 Outcome improvement following haploidentical stem cell transplantation in patients with high risk leukemia: A comparison of high dose post-transplant cyclophosphamide (PT-CY) versus prophylactic DLI K. alimoghaddam1, A. ghavamzadeh1, M. barkhordar1,* 1 tehran University of Medical Sciences, Hematology- oncology and SCT research center , Tehran, Iran, Islamic Republic Of Introduction: Haploidentical transplantation has become a clinical option for patients lacking an HLA-matched donor. Multiple methods have been made to improve outcome after haploidentical stem cell transplantation (SCT). High dose cyclophosphamide in the early post transplant period (PTCY) is an effective strategy for GVHD prevention and engraftment facilitation due to immunogenic tolerance. Prophylactic DLI was applied to improve both relapse rate and immunity reconstitution after haploidentical SCT. Here we report the outcome of 40 patients of Haploidentical SCT in our center that randomizeded in two arm of PT-CY versus Prophylactic DLI. Materials (or patients) and methods: From Jun 2010 to August 2014, 40 patients ( 27 male, 13 female) with high risk acute leukemia (29 AML, 11 ALL), whose have not suitable related or unrelated donor underwent haploidentical SCT from family members and randomized in two arms. Median patients age was 26.6 year (17-55 year) in PT-CY arm and27.3 year (13-
58 year) in DLI arm. Identical regimen for conditioning and GVHD prophylaxis were used in two arms . The myeloablative conditioning regimen comprised busulfan and cyclophosphamide and ATG. GVHD prophylaxis consisted of cyclosporine and methotrexate. In PT-CY arm patients undergone HSCT and received additional dose of cyclophosphamide 50mg/kg at þ 3 and in DLI arm after transplantation patients received DLI from the same donor after subcutaneous G-CSF at day þ 30 with average cell dose 1-2 107 mononuclear cells (MNC)/kg. Results: Totally 40 patients enrolled, 20 patients allocated in each arm. Median follow up was 720 days in PT-CY arm and 534 days in DLI arm. 5 patients in PT-CY arm and 7 in DLI arm were died. Cause of deaths were infection in 4 patients of DLI arm and 2 patients of PT-CY arm . disease relaps in 2 patients of PT-CY arm and 1 patient of DLI arm. Acute GVHD (grade 2-3) accur in 10 and 16 patient in PT-CY and DLI arm respectively (P-value ¼ 0.046). Chronic GVHD (moderate and severe) accur in 3 and 7 patient in PT-CY and DLI arm respectively (P-value ¼ 0.20). One year disease free survival was 79% and 84% in PT-CY and DLI arm respectively (P-value ¼ 0.97). One year overall survival was 76% and 71% in PT-CY and DLI arm respectively (P-value ¼ 0.43). Conclusion: The results of this study reveals no statistical difference between two arms in DFS and OS and chronic.GVHD, probably due to small size of patients and short time of follow up. However the acute GVHD was significantly lower in PT-CY arm. Disclosure of Interest: None declared. P040 Favourable aoutcome with donor lymphocyte infusion for secondary graft failure in X-linked Hyper Ig M Syndrome M. Torrent1,*, L. Sisinni1, N. Pardo1, L. Medina2, S. Querol2, L. Alsina3, I. Badell1 on behalf of Pediatric BMT Unit, Hospital Santa Creu I Sant PauUniversitat Auto`noma de Barcelona 1 Universitat Auto`noma de Barcelona, Pediatric BMT Unit. Hospital de la Santa Creu i Sant Pau, 2Banc de Sang i Teixits de Barcelona, 3Allergy and Clinical Immunology Department, Hospital Sant Joan de De´u, Barcelona, Spain Introduction: X-linked hyper-Ig M syndrome (XHIGM) is a rare primary immunodeficiency caused by mutations in the gene encoding CD40 ligand glycoprotein. These patients have low
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levels of IgG and IgA with high or normal levels of IgM. Patients presenting with this disease undergo monthly immunoglobulin treatment. Hematopoietic stem cell transplantation (HSCT) should be considered and performed as soon as possible. Mixed donor/recipient chimerism (MC) is increasingly common in the pediatric HSCT setting due to the increased use of reduced intensity preparative regimens for non-malignant diseases. Interventions such as rapid immunosuppression withdrawal, donor lymphocyte infusion (DLI), or stem cell boost are frequently employed to improve donor chimerism. Materials (or patients) and methods: A one year old boy with XHIGM underwent HSCT in 10/24/2013. Unmanipulated bone marrow from total HLA matched unrelated donor (MUD) was the source. Conditioning regimen was based in intravenous alemtuzumab (0.6 mg/kg), fludarabine (180 mg/m2) and busulfan (14 mg/kg with adjusted dose) HSCT complications were CMV reactivation and grade II acute GVHD (cutaneous þ intestinal) which were solved with methylprednisolone 2 mg/kg/day. Initial chimera study (11/18/2013) showed 94% donor cells in myeloid compartment and 84% in lymphocytes. Progressive decrease in donor engraftment, mainly in lymphocytes, despite of rapid withdrawal of immunosuppression was observed. Donor chimera on 2/10/2014 showed 78% in granulocytes and 11% in lymphocytes so we decided to start DLI to improve donor chimerism. On 3/20/2014 he received the first DLI at a dose of 0.1x106 CD3 þ /kg recipient weight. DLI were administered every four weeks with a progressive increase in number of infused T-cells: 0.5x106 CD3 þ /kg, 1x106 CD3 þ /kg, 5x106 CD3 þ /kg and 10x106 CD3 þ /kg from the fifth and subsequent infusions. Results: A total of 10 DLI were performed at the moment, observing progressive improvement of mixed chimerism, achieving after the 9th DLI (11/12/2014) donor cells 100% in granulocytes and 93% in lymphocytes. DLI were well tolerated. No GVHD reactivation or infection was observed. Conclusion: In our experience it is very important closed chimera monitoring to early detect loss of engraftment and to prevent with therapeutic actions as immunossuprpression withdrawal or DLI. Our patient achieved total donor chimerism after monthly DLI without toxicity. Disclosure of Interest: None declared. P041 Comparison of The Adverse Reactions Due to Hematopoietic Stem Cell Infusion Between The Patients Younger Than And Elder Than 60 Years Old N. Baskan1,*, D. Cekdemir1, E. Birtas Atesoglu1, Z. Gulbas1 1 Hematology, Anadolu Medical Center Hospital, Bone Marrow Transplantation Unite, Kocaeli, Turkey Introduction: HSCT is a therapeutic modality and number of the elderly patients undergoing HSCT has been steadily increasing as number of the elderly patients having diagnosis of hematological malignancies increases. In the present study, adverse reactions due to infusion of HSC were compared between the patients elder and younger than 60 undergoing HSCT. Materials (or patients) and methods: A total of 314 patients including 88 (28%) patients with multiple myeloma (MM), 31 (9,9%) patients with Hodgkin’s lymphoma (HL), 17 (5.4%) patients with myelodysplastic syndrome (MDS), 27 (8.6%) patients with acute lymphocytic leukemia, 47 (15%) patients with acute myeloid leukemia (AML), 53 (16.9%) patients with non-Hodgkin lymphoma, 2 (0.6%) patients with solid tumors, 21 (6.7%) patients with chronic myeloid leukemia, 21 (6,7%) patients with with chronic lymphocytic leukemia (CLL), 5 (1.6%) patients with IMF, and 16 (5.1%) patients with AA who underwent transplantation in the bone marrow transplantation (BMT) unit of Anadolu Medical Center Hospital between 2013 and 2014 were enrolled to this study. The patients underwent autologous, allogeneic, haploidentical, or
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unrelated transplantations. Mean age of the patients was 45 (range: 14 – 80) and 120 (38.2%) of the patients were female and 194 (61.8%) of them were male. Of the patients included in the present study, 262 (83.4%) were younger than 60 and 52 (16.6%) were elder than 60. Of the patients elder than 60, 34 (65.4%) had MM, 1 (1.9%) had MDS, 2 (3.8%) had AML, 12 (23.1%) had NHL, 1 (1.9%) had CML, 1 (1.9%) had CMML, and 1 (1.9%) had AA (Table 2). The patients elder than 60 received infusion of 6.11x106/kg of HSC (range: 2.10x106/kg – 21.30x106/kg) whereas those younger than 60 received infusion of 6.21x106/kg (range: 1x106/kg – 20x106/kg) of HSC. Two hundred and seventeen (69.1%) patients underwent infusion of frozen samples (FS) using DMSO while 97 (30.9%) patients underwent infusion of unfrozen samples (UFS). Results: Of 314 patients receiving infusion of HSC, 139 (44.3%) developed AR while 175 (55.7%) didn’t. ARs were observed in 21 out of 52 (40.4%) patients elder than 60 and in 118 (45%) out of 262 patients less than 60 years old. ARs were not observed in 31 out of 52 (59.6%) patients elder than 60 and in 144 (55%) out of 262 patients less than 60 years old. Hypertension (BP Z140-90 mm Hg) was found in 17.3% of the patients more than 60 years old and in 11.5% of the patients less than 60 years old. Rate of nausea was 14.1% in the patients above 60 years old and 5.8% in the patients less than 60 years old. Conclusion: When all evaluations were considered, nausea, vomiting, tachycardia, and chest pain were more common in the patients less than 60 years old while hypertension and hypoxemia were more common in those younger than 60. Disclosure of Interest: None declared. P042 Outpatient care for leucopenic patients after autologous stem cell transplantation N. Zwinkels-Barendse1,*, P. Ympa1,1 1 HagaZiekenhuis, location Leyweg, The Hague, Netherlands Introduction: Within the hematology department of HagaZiekenhuis, the Hague, Netherland, patients do not have the choice between: outpatient care several times a week or clinical care during the leucopenic phase following an autologous stem cell transplantation (ASCT). The core or central question is: ‘‘Is it feasible to treat patients in an outpatient setting during the leucopenic phase following ASCT, in both a responsible and safe way?’’. The motivation for the nurse practitioner (NP) to investigate this subject was the fact that other Dutch hospitals have already achieved such a clinic to treat their patients. It is of interest to know if this treatment option offers a solution to the shortage of hospital beds caused by the bed reduction, imposed by the government. Materials (or patients) and methods: The research design is a quantitative, not experimental research. The research took place on the hematology department of HagaZiekenhuis. The research population were 41 patients, older than 18 years who were treated for a hematologic oncological disease. They underwent an ASCT between January 2012 and February 2013. The study comprised a review of the patients clinical records and a combined questionnaire, sent to 37 patients ,fluent in the Dutch language. The questionnaire consisted of the Lastmeter (a validated tool to measure the load of all kind of aspects) and several closed and open questions. Results: The data-analysis of the medical records focused on: weight and TPN, fever and antibiotics, transfusions and leucopenia. There were significant differences between two patient groups undergoing different forms chemotherapy: HDM (high dose Melfalan) versus BEAM Carmustine, Etoposide, Cytatabine and Melfalan). The significant differences were mostly unfavorable for the BEAM patient group. The study also focused on the length of hospitalization. Patients who stayed longer in the hospital, lost more weight. Fever and use of
antibiotics lengthend hospitalization. The answers to the combined questionnaires showed that patients experienced suffering during their hospital stay for ASCT. The majority of problems were of a physical nature and some emotional. Because of the nature of the physical complaints, patients assume that hospitalization is required. Most patients were satisfied about the length of the hospitalization. The patients did not feel that they had missed out on the opportunity to choose between a clinical and outpatient treatment option. If given this choice, most of them would choose for hospitalization following ASCT. Conclusion: It is feasible in HagaZiekenhuis for patients undergoing HDM, to be treated in both a safe and responsible fashion in an outpatient setting for a period of time during the leucopenic phase following ASCT. However care plans and protocols are needed to treat patients in their leucopenic phase in an outpatient setting. Data needed to achieve such care plans and protocols are: physical condition at discharge, the home situation, hospital care and outpatient care. Attention is needed for patient education, communication between involved care providers and availability of a (emergency) hospital bed including the financial consequences. Disclosure of Interest: None declared. P043 Donor- Specific anti-HLA Antibodies: an emerging problem in Haematopoietic Stem cell transplantation P. Marenco1,*, G. Grillo1, E. Zucchetti1, B. Forno1, M. L. Pioltelli1, I. Lotesoriere1, G. Cornacchini2, G. Lando2, G. Bertani2, R. Cairoli1 1 BMT unit, 2Department of Transfusional Medicine, Niguarda Hospital, Milan, Italy Introduction: Donor-Specific anti-HLA Antibodies (DSA) are involved in graft failure after transplant (Tx). Complement Dependent Cytotocity (CDC) as a routine pretransplantation test has been debated for a long time and remains controversial. However, the increase of Unrelated Donor and, more recently, haploidentical donor transplants as well as the availability of more sensible screening tests for DSA (solid phase immunoassay Luminex) bring a new challenge to the transplant community. Materials (or patients) and methods: Our standard policy is to test every recipient of an allogeneic transplant for anti HLA Class I and II antibodies and to proceed to CDC crossmatch against the donor in all the Unrelated Donor and haplo Txs,
and for related donor Txs, only in case of positivity for anti HLA antibodies. However, regardless of the result of CDC against donor, all the antiHLA positive recipients are screened for DSA. Median Fluorescence Intensity (MFI) is recorded as titre and reported to BMT Unit physician who is responsible for strategy to reduce DSA before stem cell infusion, taking into consideration: DSA titres, class I or II of antibodies, and a possible titre reduction after conditioning. Our policy is: Plasma Exchange (PEX) (1 or 2 procedures, day -1 and day 0), i.v. Immunoglobulin 1gr/Kg day 0, and, in case of anti HLA class I Antibodies, DSA immunoadsorbtion on HLA selected Platelets day 0. Results: In the last 2 years (2013 and 2014) 63 allogeneic Txs were performed in our Centre. We report here (Table 1) the 4 cases (6,3%) with DSA positivity. 3/3 pts evaluable for engraftment regularly engrafted, one pt is not valuable yet. None experienced secondary graft failure or other DSA related problems. To be noted: in two case DSA identification led to a change in donor selection (1 UD, 1 related haploidentical) in order to avoid donor bearing Antigens or Alleles targeted by DSA at higher MFI levels. Conclusion: DSA are an emerging problem, critical levels for engraftment have still to be identified and best strategy to make transplantation safe has to be found in prospective studies. We already revised our recipient preTx evaluation SOP in order to have DSA results earlier, available for a better donor selection. Disclosure of Interest: None declared. P044 Neutrophil engraftment kinetic relationship to timing of Granulocyte Colony Stimulating Factor (G-CSF) in melphalan-conditioned autologous stem cell transplant for multiple myeloma patient cohorts from three United Kingdom centres R. Krishna1,*, H. A. Abhishekh1, R. Brown2, D. Qadir3, V. Ellis1, A.-M. Sjursen2, A. Drake3, C. Ardern1, J. Thachil1, M. Saif1, F. Dignan1, F. Willis3, A. Bloor2, M. Koh3, E. Tholouli1, M. Klammer3, J. Cavet2 1 Haematology, Manchester Royal Infirmary, 2Haematology, Christie Hospital, Manchester, 3Haematology, St Georges Hospital, London, United Kingdom Introduction: G-CSF is routinely administered after high-dose melphalan-conditioned autologous stem cell transplants
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(SCT) in patients with multiple myeloma (MM) to accelerate neutrophil engraftment. Literature on optimal timing of initiation of GCSF however remains limited. This study was performed to investigate the kinetics of neutrophil engraftment in relation to timing of GCSF in three MM SCT cohorts Materials (or patients) and methods: We retrospectively collected data of 157 MM patients who underwent autologous SCT from three U.K. hospitals with differing protocols for initiation of GCSF: GCSF was initiated per protocol on Day þ 7 (n ¼ 60), Day þ 10 (n ¼ 60), Day þ 3 (n ¼ 37) at each centre, and dates of neutrophil and platelet engraftment were recorded as per EBMT guidelines. Additionally patient characteristics including age, gender, weight, creatinine clearance, transplant characteristics incl. dose of CD34 cells, number of transplant (1st vs 2nd), melphalan dose (140mg/m2 vs 200mg/m2), use of plerixafor during harvest, peak CRP during SCT, and GCSF characteristics incl. type and dose along with day of initiation of G-CSF were extracted from patient records. Data were analysed using SPSS by univariate & forward multivariate regression Results: The mean number of days to engraftment were as follows: 13.58 ( þ 3 group), 12.26 ( þ 7 group), and 12.70 ( þ 10 group), censored by death pre-engraftment (1 patient þ 3 cohort). The median day of neutrophil engraftment was as follows: day 12 (Interquartile Range [IQR] 11-18) for þ 3 group, day 12 (IQR 12-13) for þ 7 group, day 13 (IQR 12-13) for þ 10 group. Neutrophil engraftment kinetic was significantly different across the groups [F: 4.09; P ¼ 0.019]. Although patients in the þ 3 group had delay in the overall mean neutrophil engraftment day due to some late engraftments, there were a significant majority of early neutrophil engraftments [w2 ¼ 73.8; Po0.001], with 75% engrafting by day 13. However, time to platelet engraftment was not affected significantly [F: 0.993; P ¼ 0.373]. Day of neutrophil engraftment correlated with duration of hospital stay [r ¼ 0.339: Po0.001]. There was also correlation between stem cell dose and time to neutrophil engraftment [r ¼ -0.315; Po0.001]. Conclusion: In this study, there was a significant acceleration of neutrophil engraftment in the patient group with earliest
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initiation of G-CSF (Day þ 3) which might suggest benefit; however, a proportion of later engrafting patients in this group also resulted in a longer mean overall, suggesting early GCSF cannot overcome other factors e.g. active MM or sepsis. Higher CD34 cell dose also led to earlier engraftment, which correlated with shorter admissions. Data analysis regarding pharmaco-economics & length of stay is ongoing. This study highlights the need for prospective studies in order to determine the best timing for initiation of GCSF in SCT for MM Disclosure of Interest: None declared. P045 Impact of natural-killer immune reconstitution on the development of cytomegalovirus infection, non-relapse mortality, and graft versus host disease after allogeneic stem cell transplantation R. Rodriguez-Veiga1,*, P. Montesinos2, A. Torres Gomez2, A. Muntasell3, L. Cordon2, A. Sempere2, I. Cano2, D. Martinez-Cuadron2, B. Boluda2, I. Lorenzo2, F. Lopez-Chulia2, M. J. Arilla2, A. Lancharro2, I. Jarque2, J. Sanz2, L. Larrea4, G. Sanz2, M. Lopez-Botet3, M. A. Sanz2 1 Hematology, 2Hospital Universitario La Fe, Valencia, 3IMIM: Institut Hospital del Mar d’Investigacions Me`diques, Barcelona, 4 Centro de transfusiones de la Comunidad Valenciana, Valencia, Spain Introduction: The outcome of patients receiving allogeneic stem cell transplant (alloSCT) is related with their immune recovery kinetics. Many studies showed the influence of B and Tlymphocytes in the prevention of relapse and infections after alloSCT, but the potential impact of natural-killer (NK) cell recovery and maturation has not been established. We aim to analyze prospectively the kinetics of NK and other lymphocyte subpopulations and its relation with the risk of post-alloSCT complications. We will compare the immune recovery between unrelated cord blood (UCBT) and HLA identical peripheral blood (HLA-Id). Materials (or patients) and methods: Data from all consecutive patients with hematologic malignant diseases
undergoing alloSCT in our institution were recorded (including CMV infection, grade 2-4 acute GVHD, non-relapse mortality [NRM], and relapse). From May 2013, we have analyzed prospectively by citometry the immune reconstitution after alloSCT on days þ 28, þ 100 and þ 180, including a basic immune assessment (CD45, CD3, CD8, CD4, CD19, and CD16 þ 56 þ ), and a specific NK-cell analysis (NKG2A and NKG2C). Statistical analyses, including description and comparison of variables, as well as univariate cumulative incidence (CI) method, were performed using R.14.2 version. Continuous variables were categorized using the median value. Results: We have studied 94 alloSCT, with the following main characteristics: 45 UCBT (4/6 and 5/6 HLA compatibility) and 49 HLA-id (5 unrelated and 44 sibling donor), median age 49 years (16-65), male 52 (55%), more frequent underlying disease acute myeloid leukemia 44 (47%), acute lymphoid leukemia 20 (21%), conventional conditioning regimens 54 patients (57%). We found that the absolute number (cell/mL) of NK-CD16 þ 56 þ , NKG2A, NKG2C was similar in UCBT and HLAid at day þ 28 and þ 180, but NK-CD16 þ 56 þ and NKG2A were significantly higher in UCBT vs HLA-id at day þ 100 (301 vs 148, P ¼ .006; 230 vs 73, P ¼ .0002). Additionally, the absolute numbers of T-CD4 þ , T-CD8 þ cells were significantly higher in HLA-id vs UCBT at day þ 28, þ 100, and þ 180, while the B-CD19 þ cells were higher in HLA-id vs UCBT at day þ 28, but not after. In UCBT patients, the 1 year CI of CMV was related with CD3 o31 cell/mL at þ 100 (P ¼ .02), and NKG2C 427 cell/mL at þ 180 (P ¼ .02); NRM with CD3 o63 cell/mL (P ¼ .01), CD4 o42 cell/mL (P ¼ .002), CD19 o20 cell/mL (P ¼ .03) (all at þ 180). In HLA-id patients, the 1 year CI of CMV was related with CD19 o18 cell/mL at þ 100 (P ¼ .04), and NKG2C 421 cell/mL at þ 180 (P ¼ .03); NRM with CD19 o18 cell/mL at þ 100 (P ¼ .04); and acute GVHD with CD19 o18 cell/mL at þ 100 (P ¼ .006). We found no impact of immune reconstitution on relapse. Conclusion: In this preliminary and early prospective analysis performed in a small cohort, we found differences between UCBT and HLA-id patients in the kinetics of reconstitution of NK-, T-, and B-cell subpopulations after alloSCT. As it has been described, the T-cell recovery plays a role in the prevention of CMV infection and may protect against NRM. In addition, the CD19 reconstitution could protect against CMV infection and acute GVHD in HLA-id patients, and it could be associated with less NRM in UCBT and HLA-id. As previous studies have suggested, the CMV infection was associated with NKG2C expansion in alloSCT recipients. Disclosure of Interest: None declared. P046 Peripherally inserted central catheters in stem cell transplantation. Results from a large monocentric study S. Sica1,*, S. Francia2, P. Chiusolo1, L. Laurenti1, F. Sora`1, E. Metafuni1, S. Giammarco1, G. Scoppettolo3, M. Pittiruti4 1 Hematology Department, 2Universita` Cattolica del Sacro Cuore, Rome, Italy, 3Infectious Disease Department, 4Emergy Surgery Department, Universita` Cattolica del Sacro Cuore, Rome, Italy Introduction: Peripherally inserted central catheters (PICCs) are widely used in different clinical situations and may play a role also in patients undergoing hematopoietic stem cell transplantation (HSCT). Materials (or patients) and methods: We have retrospectively reviewed our experience with silicon Groshong closedended valved PICCs (S-PICC) and polyurethane power injectable open-ended non-valved PICCs (P-PICC) used in patients undergoing autologous or allogeneic HSCT. All PICCs were positioned by a team of specifically trained physicians and nurses (adopting a standardized insertion protocol) and utilized by specifically trained nurses of our hematology unit (adopting standardized maintenance protocols for prevention of infective and non-infective complications).
Results: In our hematology unit, in the last seven years (20082014), 285 PICCs were inserted in 280 patients (115 females and 165 males; mean age 49, range 19–68 years): in particular, 185 PICCs were inserted in 183 autologous HSCT patients and 100 PICCs in 97 allogeneic HSCT patients. Both S-PICC and P-PICC were used in autologous and in allogeneic HSCT. No major insertion-related complication occurred. Mean duration of PICCs was 25 days in autologous vs. 52 days in allogeneic HSCT. Most PICCs were removed electively, while 14.6% were removed because of accidental PICC removal (1%) or proven catheter complication (3.8%) or suspected PICC infection (9.8%). Symptomatic catheter-related venous thrombosis occurred in 5.26%. Catheter-related bloodstream infection (CRBSI) was proven in 8 cases (2.8%, 0.8 episodes per 1000 PICC days); 14 PICCs were found to be colonized (4.9%, 1.4 per 1000 PICC days), with no sign of infection; there were 43 cases of non-catheter related bacteremia (15.1%). There was no significant difference between autologous and allogeneic HSCT for any of the above described complications. No significant differences were found between S-PICC and P-PICC in terms of duration or of incidence of thrombotic or infective complications. Though, S-PICC were associated with some minor complications (accidental arterial puncture during insertion, 0.5%; primary tip malposition, 2.2 %; malfunction due to lumen occlusion, 6%), which did not occur with P-PICCs. Conclusion: Our data confirm that PICCs are safe and effective in the management HSCT patients, being associated with a low incidence of both early and late complications: insertion complications are minimal and - adopting proper insertion and management protocols – also the incidence of PICC-related thrombosis and of CRBSI is very low. P-PICCs have some advantages over S-PICCs in terms of cost (being less expensive) and in terms of incidence of mechanical complications (malposition and lumen occlusion). Disclosure of Interest: None declared.
Gene therapy P047 A sensitive and rapid HPLC assay for semi-quantitative analysis of globin chain levels in blood after transplantation of autologous hematopoietic stem cells transduced by a lentiviral ?a-t87q globin vector in ?-thalassemia major and sickle cell disease A. AMIN1,*, P. BOURGET1, B. GOURMEL2, J. MAGALON3, F. TOUZOT3, A. MAGNANI3, L. CACCAVELLI3, F. J. PIERCIEY Jr.4, M. CAVAZZANA3, E. PAYEN5, P. LEBOULCH5,6,7, O. NEGRE4,5 1 Department of Clinical Pharmacy, University Hospital NeckerEnfants Malades, AP-HP, 2Department of Medical Biochemistry, Saint-Louis Hospital, AP-HP, 3Biotherapy Department, University Hospital Necker-Enfants Malades, AP-HP, Paris, France, 4Bluebird bio, Cambridge, MA, United States, 5CEA, Institute of Emerging Diseases and Innovative Therapies (CEA-U. Paris-Sud) and Inserm U. 962, Fontenay-aux-Roses, France, 6Mahidol University and Ramathibodi Hospital, Bangkok, Thailand, 7Harvard Medical School and Brigham & Women’s Hospital, Boston, MA, United States Introduction: In patients (Pts) with b-thalassemia major (b-TM) and sickle cell disease (SCD), gene therapy (GT) strategies involving ex vivo transduction of haematopoietic stem cells (HSC) have the potential: (a) to correct a defective erythropoiesis, (b) to sustainably induce high-level production of modified b-globin in the red blood cell lineage, and ultimately (c) to reduce or stop transfusion needs in Pts. To this aim, a family of human b-globin expressing lentiviral vectors was developed and extensively characterized. The amino-acid
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P048 Establishment of a T cell receptor biobank for patients with acute myeloid leukemia to be individually treated by genetically modified donor lymphocyte infusions after haploidentical stem cell transplantation R. Klar1, E. Bra¨unlein1, S. Audehm1, M. Bassani-Sternberg2, S. Mall1, D. Busch3, M. Mann2, C. Peschel1, A. M. Krackhardt1,* 1 Medizinische Klinik III, Klinikum rechts der Isar, TU Mu¨nchen, 2 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 3Institut fu¨r Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universita¨t Mu¨nchen, Mu¨nchen, Germany
(AA) substitution bA-T87Q from the g and d-globin genes1 was incorporated in the therapeutic b-globin. This AA substitution renders the expressed human b-globin protein strongly inhibitory of HbS polymerization in SCD models2 and detectable by HPLC analysis, differentially from other human b-like globin species in b-TM3. A prior trial (LG001) demonstrated clinical benefits of such a lentiviral vector (HPV569) in b-TM. A further improved bA-T87Q vector (LentiGlobin BB305) with both greater transduction efficiency and similar preclinical safety profile was developed; it is under clinical evaluation in the HGB-205 (France) and HGB-204 trials (USA). In this innovative therapeutic context, a reverse-phase HPLC (RP-HPLC) assay was setup to allow quick quantification of globin chains, including the therapeutic chain bA-T87Q. The ion exchange HPLC method that is commonly used to analyze hemoglobin (Hb) is recognized as unable to separate HbAT87Q and HbA. The challenges were: (a) to discriminate b-globin chains with a single AA difference, (b) to isolate the therapeutic globin from both the pathological and normal globins even in Pts receiving transfusions, and (c) to achieve quantification limits as low as possible. Materials (or patients) and methods: In brief, HSC from Pts with b-TM or SCD were harvested, transduced ex vivo with the lentiviral bA-T87Q-globin vector, grown in clonogenic assays, and infused after Bu-based conditioning. bA-T87Q expression was assessed by the RP-HPLC assay on progenitor cells in vitro and on red blood cells from Pts treated by GT3. Clean cell lysates were injected directly onto a C4 (250x4.6 mm) column. Chemical species were detected at 220 nm. Results: After completion of validation stage, the RP-HPLC assay was able to separate and proportionally quantify 9 fractions of Hb in less than 60 min: the heme group, and a, bE, bA, bS, bA-T87Q, gA, gG, and d subunit globin chains, obtained either from a whole blood sample (50 mL) or from in vitro clonogenic cultures with a sensitivity limit of B2,000 erythroblasts (Figure 1). Conclusion: A sensitive and rapid semi-quantitative RP-HPLC assay was developed for the separation of 9 fractions of Hb. It can be applied to: (a) early detection of b-TM or SCD, Hb variants analysis, and various hemoglobinopathies, (b) study of trends and evolution of bA-T87Q blood concentration in Pts undergoing GT, (c) study of bA-T87Q expression on erythroblasts after ex vivo transduction. New clinical and preclinical applications of the method are being explored. References: 1. Takekoshi et al. PNAS 1995. 2. Pawliuk et al. Science 2001. 3. Cavazzana-Calvo et al. Nature 2010. Disclosure of Interest: None declared.
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Introduction: Acute myeloid leukemias (AML) are highly aggressive diseases with often unfavorable prognosis. Allogeneic hematopoietic stem cell transplantation (SCT) and subsequent donor lymphocyte infusions are effective, still treatment failure is frequent. The optimization of haploidentical SCT protocols have led to an enhanced usage of this treatment procedure. The HLA mismatched situation allows redirection of T cells by T cell receptors (TCR) in an HLAmismatched setting. Such genetically modified T cells can be transferred as donor lymphocyte infusions with defined antileukemic specificity. However, target structures are limited and successful treatment will likely imply tools with diverse specificities as well as HLA restrictions. Materials (or patients) and methods: By applying the immunopeptidomic approach on samples of patients with myeloid leukemia and melanoma, we have identified novel HLA ligands that are derived from genes which are restrictedly expressed in hematopoietic cells or overexpressed in malignant cells as well as presented on different HLA molecules. We furthermore optimized a workflow that allows isolation of peptide specific T cells after stimulation of naı¨ve T cells with single HLA-mismatched dendritic cells (DC) with the aim to generate a biobank of leukemia-reactive TCR with specificity for the identified HLA ligands. Results: Using that workflow, T cells with desired specificity were sorted by HLA multimers as well as applying IFN-g capture resulting in several T cell lines and TCR with specificity for diverse hematopoietic differentiation antigens as well as cancer testis antigens as MPO, ITGA2B and PRAME. Within these specificities, a broad spectrum of peptidedependent HLA reactivities as HLA-A1, HLA-A2, HLA-B7, HLAB15, HLA-B42 is covered. Isolated T cell lines and TCR are extensively tested for peptide-dependent anti-leukemic efficacy as well as on- and off-target toxicity. Efficacy has been demonstrated for TCR2.5D6 with specificity for MPO in diverse in vivo models and this TCR is currently prepared for clinical translation. Conclusion: Taken together, we herein show that combination of immunopeptidomics and isolation of peptide specific TCR in the single HLA-mismatched setting is a powerful method to develop therapeutic tools for novel antileukemic immunotherapeutic strategies. The selected TCR build a base for a TCR biobank which can be used in a personalized way in patients undergoing haploidentical SCT for AML. Disclosure of Interest: R. Klar Conflict with: Patent, E. Bra¨unlein: None declared, S. Audehm: None declared, M. Bassani-Sternberg: None declared, S. Mall: None declared, D. Busch: None declared, M. Mann: None declared, C. Peschel: None declared, A. Krackhardt Conflict with: Patent P049 Abstract Withdrawn
P050 A clinically feasible protocol of TCR gene editing to treat Multiple Myeloma without inducing GvHD S. Mastaglio1,*, P. Genovese1, Z. Magnani1, B. Camisa1, E. Landoni1, G. Schiroli1, E. Provasi1, A. Lombardo1, A. Reik2, N. Cieri1, M. Ponzoni1, F. Ciceri1, C. Bordignon3, M. Holmes2, P. Gregory2, L. Naldini1, C. Bonini1 1 San Raffaele Scientific Institute, Milan, Italy, 2Sangamo Biosciences, Richmond - CA, United States, 3Molmed, Milan, Italy
Camisa: None declared, E. Landoni: None declared, G. Schiroli: None declared, E. Provasi: None declared, A. Lombardo: None declared, A. Reik Employee of: Employment at Sangamo BioSciences , N. Cieri: None declared, M. Ponzoni: None declared, F. Ciceri: None declared, C. Bordignon: None declared, M. Holmes Employee of: Employment at Sangamo BioSciences , P. Gregory Employee of: Employment at Sangamo BioSciences , L. Naldini: None declared, C. Bonini: None declared
Introduction: Gene transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach of adoptive immunotherapy for cancer patients. However, T cells transduced with an exogenous TCR produced suboptimal clinical results. This could be due to the dilution of the tumor specific TCR that competes with endogenous TCRs for its expression on the T cell surface. Moreover, mispairing between endogenous and exogenous TCR a and b chains could generate new TCRs with unpredictable and potentially harmful specificities. To overcome these issues, our group developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to a and b chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes by lentiviral vectors Provasi (et al, Nature Medicine 2012). Materials (or patients) and methods: The complete editing procedure requires multiple manipulation steps involving repeated cell activation cycles and 4 transduction procedures. We recently developed the ‘single TCR editing’ (SE), based on the disruption of the single endogenous TCR a chain, followed by transfer of both the a and b chains of the tumor specific TCR, that generates redirected T cells fully devoid of their natural TCR repertoire, in a single round of cell activation. The aim of this project is to determine efficacy and safety of SE T cells in vitro and in vivo. We validated the SE protocol exploiting an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by 60% of high risk multiple myeloma). Results: The SE strategy allowed the rapid production of high numbers of tumor specific T cells, enriched for cells with an early differentiated phenotype. When tested against the U266 myeloma cell line (NY-ESO-1 þ HLA-A2 þ ) in different functional assays (co-culture, g-IFN and 51Cr release), all NY-ESO-1 redirected T cells showed a very high killing potential. However, when we compared the alloreactive potential of the different NY-ESO-1 specific T cell populations in mixed lymphocyte reactions against completely mismatched allogeneic targets, we interestingly observed that the lysis of the allogeneic target by TR cells was significantly higher than that of SE T cells (P ¼ 0.05). These results were validated in NSG mice, engrafted with U266 cells and infused 5 weeks later with NY-ESO-1 specific T cells. All animals treated with tumor specific T cells were completely disease-free at the time of sacrifice, demonstrating the powerful anti-tumor potential of the NY-ESO-1 TCR. However, the overall survival of mice treated with TR vs SE cells was 26% vs 100% respectively (Po0,001), explained by the significant difference in acute and chronic GvHD occurrence (71% vs 0%, Po0,001). This suggests that the residual endogenous polyclonal TCRs and mispaired TCRs expressed on the cell surface of TR T cells can lead to offtarget recognition, while SE T cells are devoid of such reactivity. Conclusion: The single gene editing protocol enables the rapid generation of clinically relevant doses of highly performing tumor specific T cells, fully devoid of their endogenous TCR repertoire, and thus particularly appealing for a future clinical translation. Donor-derived SE T cells, with a significantly reduced alloreactive potential, could be especially suitable to treat patients with minimal residual disease after allogeneic hematopoietic stem cell transplantation. Disclosure of Interest: S. Mastaglio: None declared, P. Genovese: None declared, Z. Magnani: None declared, B.
P051 Abstract Withdrawn
Non-hematopoietic stem cells P052 Tissue-resident stem cells – natural distribution, isolation and biological characteristics A. Klimczak1,*, T. Jurek2, U. Kozlowska1, M. Czuba2, M. Rorat2, M. Paprocka1 1 Institute of Immunology and Experimental Therapy Polish Academy of Sciences, 2Department of Forensic Medicine, Medical University, Wroclaw, Poland Introduction: Natural distribution and biological characteristics of multipotent stem cells responsible for tissue regeneration in human organs is still under investigation. The aim of this study was to verify wheatear it is possible to propagate in vitro, from different human tissues obtained from deceased donors, a population of cells that represent stem cells, and to characterize biologic properties of these cells. Materials (or patients) and methods: Tissue samples were collected from 16 deceased donors (age 9 months to 48 years) with approval by the local Bioethics Committee. Samples of bone marrow (BM), skeletal muscle, heart, liver, and skin were harvested from12,5 h to 48 h after dead. BM was collected to the heparinized tube and mononuclear cells were isolated in the ficoll gradient. Tissue samples were divided and prepared for routine formalin-fixed paraffin block or placed in PBS supplemented with antibiotics for cell isolation. After tissue enzymatic digestion with trypsin, long-term cultures in standard DMEM medium (tissues) or in alpha MEM medium (bone marrow) supplemented with 10% FBS and antibiotics, at 37oC and 5% CO2 was performed. Culture medium was exchanged every 3 days until cells reached 60-70% confluence. Tissue distribution and the phenotype of the cultured cells were characterized by flow cytometry and immunocytochemistry for: CD45, CD34, CD117, CD56, PAX7, CD73, CD90, CD105. Results: Common feature of tissue localized stem cells was expression of c-kit (CD117), CD34 and CD56. Skeletal muscle progenitor cells exclusively express transcriptional factor PAX7. CD73 and CD90 were present on single stem cells of examined tissues and were localized in specific tissue compartments: between the basal lamina and sarcolemma of myofibres of the muscle, in the periportal area of the liver, in the basal layer of epidermis and in the epithelium of adnexal structure of the skin, and were connected to myocytes and fibroblasts in the cardiac niches. We obtained live adherent cells after culture of BM, skeletal muscle and skin progenitor cells. After P1 cell expansion BM-, muscle- and skin-derived live adherent cells exhibit similar phenotype (CD90 þ , CD73 þ , CD105 þ ) characteristic for naı¨ve mesenchymal stem cells (MSC). Muscle progenitor cells (but not from BM or skin) after culture additionally expressed CD56 and CD34, and this observation suggest that those population of MSC is muscle-specific. All
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cultured cells of BM-origin were negative for hematopoietic markers CD45, CD34 and CD117. We were not able to obtain live cells from liver and heart. Conclusion: Stem cells with MSC characteristics can be generated in vitro from several human tissues including BM, skeletal muscle, and skin up to 48 h after dead. The isolation and expansion of progenitor cells from human tissues is an option to obtain a population of stem cells with naı¨ve MSC phenotype. Thus, the opportunity to expand live tissue-origin MSC from deceased donor might have important clinical alternative in regenerative medicine for MSC generated from tissues from live donors. Study supported by National Science Center grant N N407 121940. Disclosure of Interest: None declared. P053 Gene expression of Notch receptors and ligands by human Mesenchymal Stem Cells from different tissues G. Aydin1,2, F. Aerts-Kaya1,2,*, A. Gunel-Ozcan1,2, D. Uckan-Cetinkaya1,2,3 1 Center for Stem Cell Research and Development, 2Institute of Health Sciences, HACETTEPE UNIVERSITY, 3Department of Pediatric Hematology, Hacettepe University Medical Faculty, Ankara, Turkey Introduction: Multipotent Mesenchymal stromal cells (MSCs) can be found in bone marrow (BM), placenta and umbilical cord. BM-MSCs also support expansion of Hematopoietic Stem Cells (HSCs) in vitro. The CD271 antigen can be used for prospective isolation of BM-MSCs. CD271bright BM cells display high clonal capacity and differentiation potential, but also support lymphoid differentiation and hematopoiesis in vivo (1). Notch signaling plays an important role in the regulation of T-cell development and stem cell maintenance. Here, we compared gene expression levels of Notch receptors and their ligands in BM-MSCs, CD271 þ MSCs, Placenta amnion (PA), Placenta chorion (PC) and Umbilical cord (UC)derived MSCs, to identify the optimal cell source of MSCs for support of in vitro expansion of HSCs, capable of lymphohemopoiesis. Materials (or patients) and methods: BM-MSCs were isolated from healthy human BM aspirates. CD271 þ MSCs were prospectively isolated by magnetic selection (Miltenyi). PA-
[P053]
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MSCs, PC-MSC and UC-MSCs were isolated by mechanic dissociation and enzymatic digestions, using Trypsin/EDTA, collagenase IV and dispase I. BM-MSCs (n ¼ 6), CD271 þ MSCs (n ¼ 6), PA-MSCs (n ¼ 7), PC-MSCs (n ¼ 5), and UC-MSCs (n ¼ 4) were cultured up till passage 3. MSC immunophenotype was measured using a FACSARIA (Becton Dickinson). Total RNA was purified and 250 ng RNA was used for cDNA synthesis. Gene expression was assessed by real-time qPCR (LightCycler 480II, Roche) using RealTime ready Single Assays (Roche). All target genes were normalized against the house keeping gene, ACTB and samples were run at least in triplicate. The crossing point (Cp or Ct) for target and reference genes for each sample was calculated according to Second Derivative Maximum Method using LightCyclers 480 Software (Roche). 2 DDCt method was used for relative quantitative PCR analysis by applying a log2 transformation. Gene expression of Notch-2/3/4, Jag-1/2, DLL1/4 in PA/PC/UC/CD271 þ MSCs was determined relative to BM- MSCs. Results: BM-MSCs ane CD271 þ MSCs abundantly expressed CD29, CD44, CD73, CD90, CD105 and CD166, but lacked expression of CD14, CD45, CD34, CD31. In comparison to BMMSCs, UC-MSCs expressed lower levels of CD44 and CD73 and increased levels of CD200 (Po0.01), whereas PA and PC-MSCs expressed lower levels of CD90 (Po0.01). All groups lacked gene expression of NOTCH4. Relative NOTCH2 and NOTCH3 expression was lower in PA/PC/UC/CD271 þ MSCs compared to BM-MSCs. The lowest expression of NOTCH2 and NOTCH3 was found in PA-MSCs with 3.7 and 5.26 fold decrease, respectively. JAG1 expression was reduced in PA-MSCs (-5.99), PC-MSCs (-3.43) and UC-MSCs (-3.63). JAG2 and DLL1 were expressed at lower level in CD271 þ MSCs and PA-MSCs. PCMSCs and UC-MSCs expressed higher levels of JAG2, DLL1 and DLL4. CD271 þ MSCs and PA-MSCs were almost similar to BMMSCs for DLL4 expression. Among MSCs source types, CD271 þ MSCs showed the closest NOTCH2, NOTCH3, JAG1 and DLL4 gene profile to BM-MSCs. Conclusion: CD271 þ MSCs showed a NOTCH2, NOTCH3, JAG1, DLL4 expression profile similar to BM-MSCs. These two groups differed only in expression of JAG2 and DLL1. PC-MSCs and UC-MSCs displayed higher expression of DLL1 and DLL4, which may be important for in vitro support of lymphopoiesis, T and B cell development. References: Kuc¸i S, et al. Haematologica. 2010 Apr; 95(4): 651-659. Disclosure of Interest: None declared.
P054 Chemical inducers of Endoplasmic Reticulum (ER) Stress suppress proliferation and adipogenic differentiation of human Mesenchymal Stem Cells B. Ulum1, F. Aerts-Kaya1,2,*, H. T. Teker3, G. Balta4, D. Uckan-Cetinkaya1 1 Center for Stem Cell Research and Development, 2Institute of Health Sciences, Hacettepe University, 3Department of Biological Sciences, Middle East Technical University, 4Department of Pediatric Hematology, Hacettepe University Medical Faculty, Ankara, Turkey Introduction: The Endoplasmic Reticulum (ER) plays an important role in lipid and protein synthesis and protein folding. Accumulation of unfolded proteins in the ER lumen or protein production exceeding the capacity of ER, results in ‘‘ER stress’’, which causes a series of complex signal pathways, called the ’Unfolded Protein Response’ (UPR) and ensures ER homeostasis. Different levels of ER stress are required for cell proliferation and differentiation. Mesenchymal Stem Cells (MSCs) can be isolated from bone marrow (BM), adipose tissue, placenta, umbilical cord and differentiate into fat, bone and cartilage cells. Our aim was to investigate the effect of chemicals, that modulate ER stress and asses their effect on proliferation and differentiation of MSCs using inducers of ER stress Thapsigargin (TG) and Tunicamycine (TM) and inhibitors of ER stress TUDCA and 4-PBA. Materials (or patients) and methods: Human BM samples (3-5 mL) were obtained from BM transplantation donors, sceduled for BM harvest. Mononuclear cells were plated in DMEM-LG/MDCB201 in presence of Fetal Calf Serum. MSCs were propagated until passage 3. Cells were seeded in triplicate into e-plate wells at 2000 cells/well (xCELLigence, Roche) to assess real time proliferation using impedance measurements. Cell proliferation was followed for 10 days. For adipogenic and osteogenic differentiation MSCs were maintained in DMEM-LG/10% FCS with different concentrations of the chemicals TG, TM, TUDCA and 4-PBA in presence of adipogenic differentiation medium (1 uM Dexamethasone, 60 uM Indomethacin, 500 uM Isobutylmethylxanthine and 5 ug/mL Insulin) or osteogenic differentiation medium (100 nM dexamethasone, 10 mM b-glycerophosphate and 0.2 mM L-Ascorbic Acid). Semiquantitative analysis of differentiation was performed after three weeks of culture, using spectrophotometric comparison of Oil Red O dye inclusion as a measurement of adipogenic differentiation. Results: Treatment with ER stress inducer TG resulted in complete inhibition of proliferation at all doses tested (50-400 nM), whereas treatment with TM at 100-500 nM resulted in a dose dependent suppression of proliferation. Treatment with inhibitor of ER stress TUDCA, did not affect cell proliferation at doses from 50-200 uM, whereas 4-PBA displayed a dosedependent inhibition of proliferation with maximum inhibiton of B50% at doses of 5 mM. The inhibitory effects of TG and TM on MSC proliferation could not be counteracted by either TUDCA or PBA. TUDCA had no clear effects on MSCs, but treatment with 500 nM TM resulted in loss of cells and cell death within a week. ER stress induction using TG resulted in partial and complete inhibition of adipogenesis at 25 and 50 nM, whereas treatment with TM resulted in a 40% reduction of adipogenic differentiation. In contrast, ER stress inhibitors TUDCA and 4PBA caused a slight increase in adipogenic differentiation (120% in comparison to control). 4-PBA count not restore adipogenesis in cultures treated with TG or TM. Most, likely because 4-PBA and TG/TM affect different ER stress signaling pathways. Conclusion: These data demonstrate that ER stress inducing chemicals suppress MSC proliferation and affect its adipogenic differentiation capacities, which cannot be counteracted by chemical chaperons TUDCA and 4-PBA. Disclosure of Interest: None declared.
P055 Mir-663 impairs the effects of bone marrow-derived mesenchymal stem cells on MRL/lpr mice L. Geng1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Previously we have shown that miR-663 was increased in bone marrow derived mesenchymal stem cells (BMSCs) from SLE patients and involved in the functional deficiency of BMSCs through inhibiting transforming growth factor b1 (TGF-b1) production. This study was undertaken to explore whether the modulation of miR-663 in BMSCs could affect their therapeutic effects on MRL/lpr mice. Materials (or patients) and methods: Negative-control-miR663 (miR-663-C), mimics-miR-663 (miR-663-M) and inhibitormiR-663 (miR-663-I) eukaryotic expression vector were artificially transfected into BMSCs and intravenous injected (1 106) into 20 weeks old female MRL/lpr mice. 8 weeks later, mice were sacrificed, with kidneys, lymph node harvested and spleen weighed. Their serum and urinary samples were collected for the measurement of autoantibodies (including IgG, ds-DNA and ANA) and cytokines (TGF-b1, IL-4, IFN-g, IL-17A and so on) by ELISA, and proteinuria by coomassie blue staining assay. Immune complex deposition including IgG and complement 3 (C3) in kidney sections was performed by immunofluorescence staining. The percentages of Th1, Th2, Th17, regulatory T cells (Treg) and follicular T helper (Tfh) cells in splenic mononuclear cells were detected by flow cytometry. Results: Compared to the miR-663-C and miR-663-M group, miR-663-I transfected BMSCs displayed enhanced therapeutic effects on MRL/lpr mice, as shown by significantly declined spleens and lymph nodes size as well as reduced serum IgG and anti-dsDNA levels. Compared to miR-663-C and miR-663-I group, mice treated with miR-663-M transfected BMSCs presented enlarged glomerulus with hyper cellularity and meningeal expansion, and greater amounts of immune complex deposition including IgG and C3 in the meningeal and peripheral capillary loops. Meanwhile, Treg cell percentages were increased in miR-663-I group compared with those in miR-663-M and miR-663-C group (13.3±1.12% vs. 6.90±0.82% and 8.25±1.07%, overall Po0.05), while Tfh cell percentages were decreased (8.58±1.09% vs. 22.9±4.24% and 12.40±1.61%, overall Po0.05). Conclusion: Inhibition of miR-663 in MSCs enhanced the therapeutic effects of MSC transplantation on MRL/lpr mice. Disclosure of Interest: None declared. P056 UC-MSCs inhibit the differentiation and proliferation of T Follicular helper cell in Rheumatoid Arthritis R. Liu1, X. Li1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: The aim of this study is to elucidate whether umbilical cord derived mesenchymal stem cell (UC-MSCs) has immunomodulatory effect on T follicular helper (Tfh) cell under rheumatoid arthritis (RA) background. Materials (or patients) and methods: PBMCs that isolated from RA patients and healthy contorl (HC) were cocultured with UCMSCs at a ratio of 100:1, 10:1 and 1:1 respectively through cell-tocell contact or in a transwell system at a ratio of 1:10. After 3 days’ coculture, PBMCs were collected and the frequency of CD4 þ CXCR5 þ PD-1 þ T cell was examined by flow cytometry. Purified naı¨ve T cells isolated from PBMC of RA patients were cocultured with human UC-MSCs for 4 days at a ratio of 1:10 under Tfh cellpolarizing condition. To detect the effect of MSC on Tfh proliferation, CFSE-labeled purified CD4 þ T cells isolated from PBMC of RA patients or HC were stimulated with anti-CD3/CD28 and cocultured with UC-MSCs for 5 days. The frequency of CD4 þ CXCR5 þ PD-1 þ T or CD4 þ CXCR5 þ PD-1 þ AnnexinV þ
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T was examined by flow cytometry and the level of IL-21 in the cultured supernatant was measured by ELISA. The mRNA levels of indoleamine 2,3-dioxygenase (IDO), IL-10, PGE2, HGF, TGF-b and HLA-G in UC-MSCs were tested by RT-PCR. IDO activity of was measured by high-performance liquid chromatography (HPLC), and P-STAT-1/3/5 and p-Akt in UC-MSCs were tested by Western blot. IDO inhibitor 1-MT or anti-IL-10 antibody was added into UC-MSCs and Tfh cell differentiation coculture system for 5 days and then the frequency of CD4 þ CXCR5 þ PD-1 þ T was examined by flow cytometry. Results: UC-MSCs were able to suppress the generation of Tfh cell both in RA patients and in HC in vitro, which was dosedependent and not relied on cell-to cell contact. UC-MSCs suppressed the differentiation and proliferation of Tfh cell that was more prominent in samples from RA patients, but had no effect on Tfh cell apoptosis. The level of IL-21 in UC-MSCs and Tfh cell differentiation coculture system was significantly decreased. Dramatic increases of both IDO mRNA expression and IDO enzymatic activity were detected in UC-MSCs after coculturing with naı¨ve T cells under Tfh cell-polarizing condition. Meanwhile, pSTAT-1/3/5 and pAkt in these UCMSCs were increased. The addition of the IDO inhibitor 1-MT, but not anti-IL-10 antibody, could reverse the suppressive effect of UC-MSCs on the differentiation of Tfh cell. Conclusion: UC-MSCs suppress Tfh cell differentiation and proliferation in RA patients, which is partially mediated by the secretion of IDO. Disclosure of Interest: None declared. P057 UC-MSCs inhibit T cell autophagy and apoptosis in patients with systemic lupus erythematosus through mitochondrial transfer J. Chen1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: This study is aimed to investigate the role of umbilical cord derived mesenchymal stem cells (UC-MSCs) on autophagy and apoptosis in T cells from SLE patients, and to explore the underline mechanisms involved in this process. Materials (or patients) and methods: Peripheral blood mononuclear cells (PBMC) were isolated from SLE patients and healthy donors, and cultured under stimulation with anti-CD3/28 antibodies in the presence or absence of autophagy inhibitor 3MA (5mM for 6h) or activator rapamycin (50nM for 48h). Autophagy levels and apoptotic rates were measured by flow cytometry with the detection of LC3IIB and Annexin V respectively. To determine the effects of MSCs on T cell autophagy and apoptosis, UC-MSCs were cocultured with T cells at the ratio of 1:10 directly or in transwell system. To observe the changes of pathways upstream of autophagy after MSC treatment, an AMPK activator was added to the cocultures. Meanwhile, mitochondria transmembrane potential (DCm), which was closely related to APPK activation, was marked and measured in MSCs and T cells by MitoTracker Deep Red (MDR). Results: T cells from SLE patients had both elevated autophagy level and apoptotic rate compared with those from normal controls, which were further increased after antiCD3/CD28 stimulation. Apoptotic rate of T cells significantly correlated with autophagy level (r ¼ 0.570, Po0.0001 for CD4 þ T; r ¼ 0.508, P ¼ 0.0001 for CD8 þ T). Inhibition of autophagy with 3-MA decreased the apoptotic rate of total T cells, whereas activation of autophagy with rapamycin increased the apoptotic rate. UC-MSCs significantly inhibited T cell autophagy and T cell apoptosis after cell-to-cell contact coculturing, yet the effect was diminished in transwell system. When AMPK activator added to the cultures, the ability of UCMSCs to regulated T cell apoptosis was greatly impaired. Mitochondria in UC-MSCs could be transferred to SLE T cells when directly cocultured. Consequently, the elevation of DCm in T cells was downregulated after MSC treatment, along with the reduction of AMPK.
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Conclusion: Autophagy levels are elevated in T cells from SLE patients, leading to aberrant apoptosis. UC-MSCs may inhibit T cell autophagy and apoptosis through mitochondrial transfer. Disclosure of Interest: None declared. P058 Leptin and neutrophil activating protein 2 promote mesenchymal stem cells senescence through the activation of PI3K/Akt pathway in systemic lupus erythematosus H. Chen1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Mesenchymal stem cells (MSC) derived from patients with systemic lupus erythematosus (SLE) displays enhanced senescence. Here the aim is to explore the role and mechanism of SLE inflammatory environment on the regulation of MSC senescence. Materials (or patients) and methods: Mesenchymal stem cells were isolated from fresh umbilical cords or human bone marrow. Cellular senescence was measured by senescenceassociated b-galactosidase (SA-b-gal) staining, and the mRNA and protein levels of cell cycle inhibitors p53 and p21 were detected by real time PCR and western blot analysis. Proteins involved in senescence related signaling pathways, including Jak/stat, Ras/MEK/Erk, NF-kB and PI3K/Akt, were detected by western blot. Human serum collected from healthy controls and SLE patients were applied for the measurement of 80 human cytokines and chemokines by RayBios human cytokine antibody arrays. Then differentially expressed factors were replicated by ELISA using independent samples. Results: SLE serum significantly increased the percentage of SA-b-gal positive MSCs, and upregulated the expression of p53 and p21. Jak/stat1/5, PI3K/Akt and MEK/Erk pathways in MSCs were activated after SLE serum stimulation. Treatment of PI3K/ Akt signaling pathway inhibitor, LY294002, could reverse SLE serum induced senescence, while blocking Jak/stat with AG490 or MEK/Erk with PD98059 did not have an effect. As detected by cytokine antibody arrays and independent sample ELISA, leptin and NAP2 were both significantly elevated in SLE serum, and blocking either of them could partially inhibited MSC senescence. Leptin and NAP2 acted synergistically to promote more senescent cells than leptin or NAP2 alone, which could be completely blocked by PI3K/Akt pathway inhibitor. Conclusion: Elevation of leptin and NAP2 in SLE serum helps to accelerate the cellular senescence of MSCs. PI3K/Akt signaling transduction is important in the senescent effect. Disclosure of Interest: None declared. P059 Mesenchymal stem cells from patients with rheumatoid arthritis display impaired function in inhibiting Th17 cells Y. Sun1, X. Feng1, L. Sun1,* 1 The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China Introduction: Mesenchymal stem cells (MSCs) possess multipotent and immunomodulatory properties and are suggested to be involved in the pathogenesis of immune-related diseases. This study investigates the biological characteristics and immunomodulatory function of bone marrow MSCs from patients with rheumatoid arthritis (RA). Materials (or patients) and methods: In vitro cultures of bone marrow MSCs from RA patients and controls were established and characterized by their immunophenotype and differentiation potential into adipocytes and osteoblasts. We assessed the proliferation ability, cellular apoptosis and migration potency of MSCs from RA patients (RA MSCs). Using a protein array we evaluated the cytokine production of RA MSCs. The immunomodulatory capacity of RA MSCs on
suppressing proliferation of peripheral blood mononuclear cells (PBMCs) and on the distribution of CD4 þ T cell subsets (Th17, Treg and Tfh cells) was also investigated. Results: Although differentiation potential and apoptosis rate were similar to those of control MSCs, RA MSCs showed decreased proliferative activity and aberrant cell migration. No significant differences were observed in cytokine production between RA MSCs and control MSCs. RA MSCs appeared to be indistinguishable from control MSCs in terms of suppressing the proliferation of PBMCs, decreasing the proportion of Tfh cells and inducing the polarization of Treg cells. However, the ability to inhibit the polarization of Th17 cells was impaired in RA MSCs, which may be related to the low expression of CCL2 in RA MSCs after co-culture with CD4 þ T cells. Conclusion: MSCs from RA patients have abnormalities compared with their control counterparts. These aberrations may develop after the abnormal immune responses in RA. The functionally altered MSCs may then contribute to the pathogenesis of RA. Disclosure of Interest: None declared. P060 The oncometabolite R-2-hydroxyglutarate impairs the function of human mesenchymal stromal cells and induces DNA hypermethylation L. Liu1,*, K. Hu1, B. Wang1, Y. Zhang1, S. Fu1, X. Yu1, H. Huang1 1 Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Introduction: Elevated serum and myeloblast R-2-hydroxyglutarate (R-2HG) are specific features of IDH1/2-mutant acute myeloid leukemia. Recent evidence indicated that R-2HG is indeed an oncometabolite capable of blocking the differentiation of hematopoietic cells and altering epigenetic modification. Mesenchymal stromal cells (MSCs) are an integral component of the bone marrow microenvironment. Moreover, marrow aspirate R-2HG is significantly elevated in IDH-mutant patients. In this study, we therefore investigated the effect of R-2HG on the proliferation, differentiation, cytokine expression and the DNA methylation status of bone marrow MSCs. Materials (or patients) and methods: The human MSCs were obtained from bone marrow of healthy adult donors. MSCs were treated without or with R-2HG at concentrations of 0.1 to 1.5mM. The proliferation of MSCs was detected by Cell Counting Kit-8. To determine the effects of R-2HG on MSC differentiation, the cells were cultured in osteoinductive and adipogenic medium. Specific stains were performed and differentiation-related genes were quantified. The mRNA l evels of related cytokines were measured by Real-time PCR. DNA methylation status was explored by Infinium 450 K arrays. Results: Our results indicated that proliferation of MSCs was not influenced by R-2HG. R-2HG (1.0-1.5mM) inhibited the osteoblast differentiation of MSCs as measured by reduced mineralization (Alizarin-red S stain). R-2HG significantly reduced the expression of both early (Runx2 and IBSP) and late (Osterix and BGLAP) osteoblast differentiation-related genes, supporting the results obtained in the functional assays. Moreover, R-2HG (1.0-1.5mM) promoted the adipogenic differentiation of MSCs as measured by increased lipid vacuoles (oil red O stain). The relative mRNA expression levels of adipocyte-specific transcription factors (C/EBPa and Pparg2) and the marker gene (Adiponectin and aP2) were all enhanced by R-2HG. In addition, the mRNA expression of granulocyte colony-stimulation factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly diminished in R-2HG treated MSCs. Furthermore, we used Infinium 450 K arrays to investigate the methylation patterns. Statistical analysis of the methylation data identified 154 differentially methylated CpGs. Interestingly, these CpGs consistently showed pronounced methylation differences between healthy controls and R-2HG treated MSCs. Compared
with controls, we found hypermethylation in 117 genes and hypomethylation in 37 genes in R-2HG groups. Ingenuity pathway analysis implicated several signaling pathways involved in cell differentiation and stem cell maintenance including the WNT and TGF-beta pathways. Conclusion: There is evidence indicated that osteoblast lineage cells have been shown to support HSCs, however, adipocytes play an inhibitory role in HSC maintenance. We show here that the oncometabolite R-2HG inhibited osteoblastic differentiation, promoted the adipogenic differentiation and induced a pronounced DNA hypermethylation status of MSCs. Our results suggest that bone marrow microenvironment may be affected by the metabolite R-2HG, providing a novel insight into the proto-oncogenic role of IDH mutation. Disclosure of Interest: None declared. P061 Analysis of the immunoregulatory activity of Chemerin produced by Mesenchymal Stromal Cells P. Vinci1,*, A. Bastone2, S. Schiarea3, E. Dander1, A. Del Prete4, M. Salmona2, S. Sozzani4, A. Biondi5, G. D’Amico1 1 Centro di Ricerca "M.Tettamanti", Clinica Pediatrica, Universita` degli studi di Milano-Bicocca, Fondazione MBBM/Ospedale S. Gerardo, Monza, 2Dipartimento di Biochimica delle proteine, 3 Dipartimento di Ambiente e Salute, Istituto per le Ricerche Farmacologiche Mario Negri, Milano, 4Dipartimento di Patologia Generale e Immunologia, Universita` degli Studi di Brescia, Brescia, 5Clinica Pediatrica, Universita` degli studi di MilanoBicocca, Fondazione MBBM/Ospedale S. Gerardo, Monza, Italy Introduction: In the last years, several studies have shown that Mesenchymal Stromal Cells (MSC) can be widely used for treating inflammatory diseases. However, the mechanisms underlying their capacity to inhibit inflammation are not fully understood. Chemerin is an immunoregulatory protein with chemotactic activity, secreted as a precursor and converted into its active form through the proteolitic cleavage of the last six-seven amino acids at the C-terminal domain, by several serine and cysteine proteases of the inflammatory cascade. Materials (or patients) and methods: The production of Chemerin by MSC was evaluated by ELISA assays under basal conditions as well as after stimulation with IL6, IL1 and TNFa. In addition, Chemerin proteases expression was evaluated by RT-PCR under the same culture conditions. In order to understand if this protein could be involved in MSC immunoregulatory function, Chemerin produced by MSC (MSC-Chem) was purified from MSC supernatant and functional and biochemical analysis were performed. The chemotactic activity of MSC-Chem was evaluated in the presence or absence of different proteases by using a pre-B cell line expressing the human ChemR23 receptor (L1.2-ChemR23). The biochemical analysis was performed by LC/MS Mass Spectrometry. Results: MSC were able to produce Chemerin and its production was strongly increased after stimulation with inflammatory cytokines. RT-PCR analysis of Chemerin activating serine-cysteine proteases showed that MSC express low levels of Neutrophil Elastase compared to PBMC and its expression did not significantly increase after 24h, 48h or 72h of stimulation with inflammatory cytokines. MSC also expressed Cathepsin K, and its levels did not increase after stimulation with inflammatory cytokines. Functional analysis of MSC-Chem revealed that L1.2-ChemR23 cells were able to migrate in response to rh-Chemerin in a dose-depend manner until the concentration of 10 nM. However, at equivalent concentrations, rh-Chemerin was able to induce a stronger L1.2-ChemR23 migration compared to MSC-Chem, suggesting that in MSC supernatant only a fraction of the protein was in the active form. In accordance, the LC/MS Mass Spectrometry identified the active form of Chemerin (with the last peptide Chem144-Chem157) only in rh-Chemerin but not in MSC-Chem (Chem144-Chem163), confirming that MSC-Chem is mostly
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produced in its inactive form. Interestingly, the migration of L1.2-ChemR23 cells towards MSC-Chem pre-incubated with Neutrophil Elastase and Cathepsin L, resulted increased compared to MSC-Chem alone, indicating that MSC-Chem can be activated by proteases usually involved in inflammation. Conclusion: We demonstrated that MSC were able to produce Chemerin and directly cleave it in its active form, although only partially. We hypothesize that, when infused in vivo during inflammation, MSC produce Chemerin as a precursor, which is then converted in its active form by cysteine and serine proteases, highly expressed at peripheral inflamed tissues. Further in vivo studies are needed to clarify whether Chemerin activation can then induce ChemR23-expressing cells migration towards MSC, to better exert their antiinflammatory activity. Disclosure of Interest: None declared. P062 MSC Cavity Injection Boosts Th1 Subset and has Good Response in extensive cGVHD patients: A Prospective Multicentre Randomized Controlled trial W. Zheng1,*, Y. Luo1, S. Wang2, Q. Han3, M. Guo4, D. Zhou5, L. Yu6, H. Huang1 1 Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 2 Chinese PLA General Hospital, 3The Basic Medical Institute of Chinese Medical Sciences Academy, 4The Affiliated Hospital of Chinese PLA Academy of Military Medical sciences., 5Peking Union Medical College Hospital., 6Bone Marrow Transplantation Center, Chinese PLA General Hospital., Beijing, China Introduction: Since steroid-refractory chronic graft-versushost disease was still a great challenge, we conducted a prospective multicenter phase II study to confirm the efficacy of sequential marrow cavity injection of Mesenchymal stem cells (MSC) in extensive CGVHD patients. Materials (or patients) and methods: We recruited 40 patients from four transplantation centers from Jul. 2010 to Dec. 2014, who suffered extensive cGVHD in skin and/or liver, post allogeneic hematopoietic stem cell transplantation (HCT). 30 patients were in experimental group, who received four weekly sequential marrow cavity injection of MSC. 10 patients were in control group, who received traditional immunosuppressive therapy. MSC were cultured from MHC mismatched donors and offered by the Basic Medical Institute of Chinese Medical Sciences Academy. The count of MSC was 1-4*10E7 weekly, totally 4.8-16*10E7 every cycle (four weeks). Patients continued immunosuppressive therapy during and after MSC transfusion. We assessed the improvement of cGVHD sign/symptom, the relapse of hematological malignancies, the overall survival (OS), the progress free survival (PFS) in 1, 2, 3, 6, 12 months post MSC transfusion. At the same time, we detected T cell subsets of Th1/Th2/Th17, Tregs cells. Results: 1) The average follow-up time of control group was 23.6 months, whose overall response was 30%(3/10), including 1 patient (10%) excellent response. The mortality was 10%. 2) The average follow-up time of experimental group was 13.4 months, whose overall response was 90%(27/30), including 15 pts (50%) excellent response. The mortality was 6.7% (2/30), who died not of MSC transfusion, but of HCT complication. The response rates between experimental group and control group were significantly different. 3) Patients who respected to MSC transfusion showed improvement of skin thick, elasticity and movability. And some of them gain weight greatly. Some patients response to MSC transfusion quickly within 12 hours, the mean time of maximum response was six months. 4) Four patients received two cycles of MSC transfusion, one patient received three cycles of MSC transfusion, which didn’t increase the adverse effect, but positive correlated with the response. 5) Post MSC transfusion, the Th1 subset was increased significantly in response
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patients. The th1/th2 ratio was increased post MSC transfusion, and kept 2 to 6 months depends on response time. But for those three no response patients, (two were over-lap CGVHD), showed no difference between MSC transfusion. 6) Th17 subset was very low and no difference during MSC treatment. Conclusion: MSC transfusion by sequential marrow cavity injection was approved effective in treating extensive cGVHD, the response rate was 90%, increase the transfusion cycles could improved the effect. Th1 subset boost and reactivation post MSC transfusion may contribute to good response of cGVHD patients. Disclosure of Interest: None declared. P063 Human bone marrow adipocytes negatively regulate hematopoietic stem/progenitor cells through secreting adiponectin W. Lu1,*, W. Wang1, Y. Zhang1, S. Wang1, Y. Feng1, K. Liu1 1 Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Institute of Hematology, Peking University People’s Hospital, Beijing, China Introduction: Fatty bone marrow, in addition to hematopoietic hypocellularity, is a pathologic characteristic observed in Aplastic Anemia(AA), high-dose chemotherapy, poor graft function and other clinical hematological disorders. Recently, it is proved that bone marrow fat serves as a negative regulator in hematopoiesis. However, the exact role of adipocytes in the regulation of hematopoiesis remains a controversial subject and the mechanism by which adipocytes act on hematopoietic stem cell is still unclear. In our study, we set out to test the hypothesis that bone marrow adipocytes could affect hematopoietic stem/progenitor cells (HSPC) by adiponectin. Materials (or patients) and methods: A direct cell contact or transwell co-culture system of mobilized peripheral blood (mPB) CD34 þ with mesenchymal stem cells (MSC) or adipocytes was established. After 7 days of co-culturing, cell phenotype and cell cycle of HSPC was assessed by flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCT) analysis was performed to detected the mRNA levels of adiponectin, TNF-a, leptin and neuropilin-1. Meanwhile, the protein level of adiponectin was detected by Elisa. To further examine the role of adiponectin, an antiadiponectin antibody(15ug/ml) was added to the culture medium and further assessed the proliferation, differentiation of HSPC. Results: After co-culturing of mPB CD34 þ with MSC or adipocytes for 7 days, we first found that the fold expansion of total nucleated cells was significantly decreased after seeded on adipocytes compared to MSC [(17.76±2.88) vs. (26.15±2.09), Po0.05, n ¼ 4]. Moreover, the lower absolute levels and relative numbers of CD34 þ CD38 þ HPC was also decreased. Next, we tested the effect of adipocytes on HSPC in stromal transwell co-cultures. Consistently, we found that the proliferation and proportion of HPC was also decreased significantly in adipocyte group. Moreover, we found fewer cells in the G0 phase in CD34 þ HSPC co-cultured with adipocytes compared with control. To uncover the mechanism mediated in this process, we tested the expression of adipokine which was known to impact the expansion of HSPC by qRT-PCR. We found that the expression of adiponectin was increased by 60-fold in adipocytes compared with MSC (Po0.05). However, the expression of TNF-a, leptin, neuropilin-1 between adipocytes and MSC revealed no significant difference. Furthermore, the adiponectin protein level was also proved increased in the culture medium of adipocytes group. Studies by other groups proved that adiponectin is a negative regulator of myeloid progenitor cells and is critical for the maintenance of HSC. To further comfirmed the effect of adiponectin in the regulation of HSPC, CD34 þ HSPC was co-cultured with adipocytes in the absent or present of anti-adiponectin antibody (15ug/ml). Interestingly, adipocytes decreased the proliferation and
proportion of HSPC after co-culture for 7days, an effect that was partially reversed by addition of the antiadiponectin antibody. Conclusion: In conclusion, our study demonstrated that bone marrow adipocytes is more than just a tissue filler. It could negatively regulate the proliferation and differentiation of HSPC through secreting adiponectin. Furthermore, it also suggested that the hematopoietic inhibition imposed by fatty bone marrow may be improved by targeting adiponectin. Disclosure of Interest: None declared.
Stem cell donor I P064 Abstract Withdrawn
P065 The Impact of HLA Matching on the Clinical Outcome of Hematopoietic Stem Cell Transplantation from Volunteer Unrelated Donors According to the Uniform, Prospective Transplant Program of the Rome Transplant Network A. Picardi1,*, L. Cudillo1, I. Mangione1, G. De Angelis1, B. Mariotti1, F. Di Piazza1, M. Cantonetti2, E. Ceresoli1, C. Rapanotti3, P. De Fabritiis4, T. Dentamaro4, M. C. Tirindelli5, O. Annibali5, A. Mengarelli6, F. Marchesi6, A. Chierichini7, B. Anaclerico7, E. Montefusco8, A. Ferrari8, M. Andreani9, M. Testi9, R. Cerretti 1, W. Arcese 1 on behalf of on Behalf of Rome Transplant Network. 1 Hematology Division - Stem Cell Transplant Unit, 2Hematology Division, 3Laboratory Medicine, TOR VERGATA UNIVERSITY OF ROME, 4Hematology, S.Eugenio Hospital, 5Hematology, University Campus Bio Medico, 6Hematology, Regina Elena National Cancer Institute, 7Hematology, S.Giovanni Addolorata, 8Hematology, S.Andrea Hospital, Sapienza University, 9Laboratory of Immunogenetics and Transplant Biology, IME Foundation, Rome, Italy Introduction: The impact of HLA matching in unrelated Hematopoietic Stem Cell Transplant (HSCT) is still subject of debate. All the available results come from retrospective, registry based meta-analysis on large cohorts of patients (pts), but not homogeneous for policy of donor selection, conditioning regimen or GVHD prophylaxis. Herein, the clinical outcome of allogeneic HSCT from volunteer unrelated donor (VUD) has been analyzed according to a prospective and uniform transplant platform predefined in terms of donor selection, conditioning regimen, GVHD prophylaxis and time to transplant. Materials (or patients) and methods: In order to identify an alternative donor for patients lacking an HLA identical sibling, the Rome Transplant Network (RTN) has adopted a unique searching strategy following a hierarchical algorithm (1st VUD; 2nd Cord Blood; 3rd Haplo) on the basis of established selection criteria. According to this policy, VUD was identified for at least 8/10 high resolution A-B-C-DRB1-DQB1 matching within 3 months. The same conditioning regimen consisting of myeloablative (MAC) or reduced intensity (RIC) version of the Thiotepa, Busilvex and Fludarabine combination was used. GVHD prophylaxis is modulated according to the type of donor, but identical for each transplant category. From December 2007 to date, 87 consecutive pts, with a median age of 42 yrs (range, 18-66), affected by malignant haematological diseases underwent HSCT(MAC ¼ 64 and RIC ¼ 23pts) from VUD in early (n ¼ 49) or advanced (n ¼ 38) stage. GVHD prophylaxis consists of the CSA þ MTX association þ ATG at total dose of 20 mg/kg. The donor-recipient pairs were divided
according to the HLA matching with equivalence between antigenic and allelic level. Pts characteristics were not statistically different in the 3 groups with 0, 1 or 2 HLA disparities. Two analysis were performed: HLA matched (n ¼ 46) vs all mismatched (n ¼ 41) pairs and HLA matched vs 1 (n ¼ 27) vs 2 (n ¼ 14) mismatched pairs. Results: With a median follow up of 31 months (range, 3-74), the 5-yrs probability of overall (OS) and disease free survival (DFS) for all pts was 57±6% and 41±6%, respectively. By comparing matched vs mismatched HSCT, no significant difference in terms of 5-yrs cumulative incidence (CI) of TRM (28±7% vs 31±8%), CI of grade III-IV aGVHD (4±3% vs 5±3%), CI of extensive cGVHD (11±6% vs 5±4%), OS (56±8% vs 57±9%) and DFS (37±8% vs 45±9%) was observed. By comparing matched vs 1 vs 2 mismatched HSCT, the results were significant better in terms of CI of TRM (28±7%, 48±11% and 0%; P ¼ .02), OS (56±8%, 38±11% and 92±7%; P ¼ .03) and DFS (37±8%, 30±10% and 76±12; P ¼ .03) for pts receiving graft with 2 HLA disparities, for whom the relapse rate was lower without statistically significance. No significant difference was observed in terms of engraftment, aGVHD and cGVHD. Furthermore, the analysis showed that differences at DQB1, alone or combined, did not have any impact on HSCT outcome. Conclusion: Despite the small number of pts, these results confirm the effectiveness of the uniform program of the RTN policy for VUD transplants. The analysis on a larger number of pts will better clarify reasons of the particularly encouraging results observed in 2 HLA disparities subset. To date, the transplant platform for Z8/10 HLA antigen criteria of donor selection remains unchanged. Disclosure of Interest: None declared. P066 Improving Selection for Bone Marrow Donors with a Predictive Collection Score in HLA-Haploidentical Transplantation with Post-Infusion Cyclophosphamide A. Vai1,*, S. Bramanti1, R. Crocchiolo1, B. Sarina1, L. Morabito1, L. Giordano1, I. Timofeeva1, R. Capizzuto1, A. Santoro1, L. Castagna1 1 Humanitas Cancer Center, Rozzano (MI), Italy Introduction: Many transplantation outcomes have been demonstrated to be associated with the infused total nucleated cell (TNC) dose/kg of recipient body weight (BW). Donor bone marrow density (BMD) is directly related to the TNC dose/kg of recipient BW. Materials (or patients) and methods: We retrospectively analyzed the clinical data of 65 consecutive haploidentical (haplo) bone marrow (BM) donors at our center between 2009 and 2013. The target cell dose for haplo T-cell replete transplantation with post-infusion cyclophosphamide was 4x108 TNC/kg of recipient BW. Linear regression model was adopted and a recursive tree was estimated to build up a prognostic score.
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Table 1.
Donors’ characteristics
n ¼ 65 Median age Gender M/F Donor type Sibling Parent Child Cousin Median BMI Median WBC count (x103/mm3)
46 (18-71) 39/26
27 20 17 1 25.1 7.2
(42%) (31%) (26%) (2%) (17.6-37.9) (4.7-14.5)
Results: Donors’ characteristics are shown in table 1. Body mass index (BMI) and white blood cell (WBC) count were directly associated with BMD (Po0.001). 3 mayor prognostic categories were identified. As shown in the image, the probability to obtain a collection with a high BMD was firstly predicted by BMI (BMI Z30, BMD mean ¼ 25.8 TNC/mL x106). Secondly, it was possible to split donors with BMI o30 into two categories according to WBC count (WBC o8 x103/mm3: BMD mean ¼ 18.4 TNC/mL x106; WBC Z8 x103/mm3: BMD mean ¼ 23.1 TNC/mL x106). Moreover, BMD of the first collected bag directly correlated with overall BMD (R2 ¼ 0.69, Po0.001). Conclusion: This retrospective study on 65 haplo BM donors was aimed to identify donor-related variables associated with high BMD, in order to build up a predictive score for optimal collection. BMI Z30 and WBC count Z8 were the only variables independently related to donor BMD. Although in the clinical practice BMD is not a commonly used parameter, it is a valid surrogate of donor TNC content in the harvest. Given the impact of infused TNC dose on many transplantation outcomes, donor-related features affecting TNC quantity obtainable with the harvest could support the choice of the best-suitable donor, after selection on the basis of HLAmatching, gender, age, CMV serostatus and body weight ratio between recipient and potential donor. Disclosure of Interest: None declared.
Materials (or patients) and methods: We analysed 161 transplants, 121 for acute myeloid leukemia and 40 for acute lymphoblastic leukemia. Disease status at transplant was high-risk 1st to 3rd complete remission in 111 patients and chemo-resistant relapse in 50. Patients received TBI-based conditioning regimens and haploidentical, T-cell-depleted peripheral blood hematopoietic stem cells. No post transplant pharmacologic immune-suppressive GvHD prophylaxis was given. Results: In the absence of donor-versus-recipient NK cell alloreactivity, donor activating KIRs had no effects on outcomes. In the 69 transplants with donor-versus-recipient NK cell alloreactivity, transplantation from donors with KIR2DS1 and/or KIR3DS1 was associated with reduced risk of nonrelapse mortality, largely infection-related, (KIR2DS1 present versus absent: HR: 0.25, P ¼ .01; KIR3DS1 present versus absent: HR: 0.18, P ¼ .006) and better event-free survival (KIR2DS1 present versus absent: HR of death: 0.31, P ¼ .011; KIR3DS1 present versus absent: HR of death: 0.30, P ¼ .008) in multivariate analyses. After transplantation from donors with KIR2DS1 and/or KIR3DS1, the infection rate in the first six months after transplant was B50% lower (P ¼ .005). In vitro analyses showed that KIR2DS1 triggered NK cells to release IFN-g upon interaction with KIR ligand-mismatched, HLA-C2/ C2 DCs that were pulsed with LPS or Aspergillus Fumigatus. IFN-g release was inhibited when KIR2DS1-HLA-C2 interaction was blocked. Conclusion: 1) In vitro analyses suggest that activating KIR engagement by HLA ligands enhanced NK cell IFN-g production and this might improve immunity to infections. 2) Clinical data show that transplantation from NK alloreactive donors with the activating KIR2DS1 and/or KIR3DS1 genes reduces infection rates and infection-related mortality and improves survival. In T-cell depleted haploidentical transplantation specific donor factors were shown to impact on survival: KIR ligand mismatches and maternal donors. The present study identifies yet another: NK alloreactive donors carrying KIR2DS1 and/or KIR3DS1. As they constitute B40% of the NK alloreactive donor pool, searching for them may become an additional criterion in donor selection. Disclosure of Interest: None declared.
P067 Haploidentical hematopoietic transplantation from KIR ligand-mismatched donors with activating KIRs reduces non-relapse mortality A. Mancusi1,*, L. Ruggeri1, E. Urbani1, A. Pierini1, M. S. Massei1, A. Carotti1, A. Terenzi1, F. Falzetti1, A. Tosti1, F. Topini1, M. Stern2, F. Aversa3, M. F. Martelli1, A. Velardi1 1 University of Perugia, Perugia, Italy, 2University of Basel, Basel, Switzerland, 3University of Parma, Parma, Italy
P068 Passive transfer of male microchimeric cells from female donors to female HSCT patients: a case report A. van Halteren1,*, J. van der Griendt2, B. Kemps-Mols3, M. Eikmans3, E. Marijt2, T. Netelenbos3, M. Griffioen2, A. Lankester1, M. van Tol1 1 pediatrics, 2hematology, 3Immunohematology & Blood Transfusion, LEIDEN UNIVERSITY MEDICAL CENTER, Leiden, Netherlands
Introduction: In T cell-depleted peripheral blood stem cell haploidentical transplants, donor NK cells play a beneficial role in outcomes. NK cell function is regulated by inhibitory receptors (KIRs) for HLA class I allele groups (KIR ligands), that is, HLA-C group 1, HLA-C group 2 and HLA-Bw4 alleles. Because of KIR ligand mismatches in the GvH direction, donor NK cells are released from inhibition by recipient HLA and mediate donor-versus-recipient NK cell alloreactions which control leukemia relapse and improve survival. Homologues of the inhibitory KIRs, with shorter cytoplasmic tails, activating KIRs transduce signals which activate NK cells. Activating KIRs are heterogeneously expressed in the population. Approximately 25% of whites are homozygous for Group A haplotypes which predominantly contain inhibitory KIR genes. The others are either heterozygous or homozygous for Group B haplotypes that also carry combinations of activating KIR genes (KIR2DS1, 2, 3, 5 and 3DS1). Little is known about their ligands except that KIR2DS1 binds HLA-C group 2 molecules (HLA-C2). We investigated the role of donor activating KIRs in haploidentical T cell-depleted transplantation for acute leukemia.
Introduction: Male microchimerism is frequently acquired by female donors through feto-maternal or trans-maternal cell exchange. We earlier assessed a high prevalence of male CD34 þ stem cells and myeloid-committed cells in female peripheral blood stem cell (PBSC) grafts and in lymphocytes collected after PBSC donation for the purpose of donor lymphocyte infusion (DLI). The impact of passively transferred chimeric cells on HSCT outcome is unknown. Antibodies against Y-chromosome encoded proteins are often induced in male recipients of female grafts. Unexpectedly, we also measured a strong antibody response against a Y chromosome-encoded protein (DDX3Y) in a female AML patient who underwent myeloablative conditioning (TBI/cyclo) followed by infusion of T-cell depleted PBSC (20 mg Alemtuzumab injected into the collection bag) and two consecutive DLI from her HLA identical sister. We analysed whether the DDX3Y-specific antibody response in this female HSCT patient could be explained by the co-presence of passively acquired male microchimerism. Materials (or patients) and methods: The level of male DNA was determined by sensitive real-time PCR specific for a
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multicopy locus on the Y chromosome (DYS-1, detection limit: 1 male cell in 100.000 female cells) in longitudinally collected peripheral blood (PB) and bone marrow (BM) samples as well as in lymphoid and myeloid cells derived thereof. Standard clinical monitoring was used to assess post-HSCT hematopoietic recovery which was registered in the patient’s electronic files. Results: Male DNA was detected in two or more hematopoietic subsets derived respectively from donor PB and BM samples collected before the start of G-CSF treatment and from the DLI product. Multilineage male microchimerism was also detected in flow-separated blood cells collected from the patient at d þ 42 after PBSC infusion. From d þ 47 on, PB lymphocyte counts increased and the level of male microchimerism started to decline until male DNA became undetectable at d þ 196. The patient developed skin Graftversus-Host Disease (GVHD) around d þ 30 which required prolonged topical treatment and a short pulse of orally applied steroids to resolve around d þ 150. After DLI at d þ 272 and d þ 399 respectively, male cells re-appeared for a short while but without any clinical signs of GVHD. Again, male microchimerism became undetectable which was followed by the appearance of DDX3Y-specific IgG antibodies in the patient’s serum. The alloreactive nature of this the novo antibody response is supported by the observation that 1) DDX3Yspecific antibodies were not detected in patient or donor serum samples collected before HSCT and 2) DDX3Y-specific antibodies did not cross react with the homologue X chromosome-encoded DDX3X protein. Conclusion: We showed for the first time that male microchimerism and DDX3Y-specific antibodies can be detected during the course of a HLA identical female-tofemale HSCT procedure. Using PCR specific for unique HLA or other genetic markers which have been selected according to the genotyping results of the entire family, we are currently generating more evidence that the male microchimerism associated with anti-male alloreactivity in this female HSCT patient is passively acquired from her female sibling donor. Disclosure of Interest: None declared. P069 Second or further donations of hematopoietic stem cell for volunteer donors: the experience of the Italian Bone Marrow Donor Registry (IBMDR) A. Vassanelli1,*, N. Sacchi2, S. Pollichieni2 1 transfusion medicine, azienda ospedaliera univeristaria integrata verona, Verona, 2IBMDR, Ospedale Galliera, Genova, Italy Introduction: The Italian Bone Marrow Donor Registry (IBMDR), established in Italy in 1989, with the role of providing an unrelated volunteer to haematological patients waiting for a transplant and who do not have the ideal donor (an identical sibling), ensure that the TC activates a search exclusively for those patients where a HSCT represents a medically acceptable procedure, for pathologies that are defined, with annual revisions, by the EBMT. Haematopoietic Stem Cell donation can be carried out from both bone marrow (BM) and peripheral blood stem cell (PBSC) after administration of G-CSF, for a single recipient only. The TC may ask for the HSC collection only if the transplant is immediately performed, not for cryopreservation In some circumstances, an additional donation for the same patient may be required: a mononuclear cell collection from peripheral blood (for DLI), in the case of relapse, or HSC in the case of non-engraftment or of poor graft function. The volunteer, who has already donated peripheral HSC should, preferably, donate marrow HSC and, in any case, can only be submitted again for growth factor stimulation for HSC mobilization, if at least 12 months have passed since the first donation. A second HSC donation and a second or further lymphocyte donation requests are submitted to the SIMTI Committee
(Committee of Societa` Italiana Medicina Trasfusionale e Immunoematologia, a "second opinion" committee designated by the pertinent scientific society, with the aims to protect donor welfare and safety). A donor follow-up, even if no particular events have occurred, is performed at established times (up to ten years for bone marrow and PBSC donation and up to 6 months for lymphocyte donation). Materials (or patients) and methods: We evaluated all the requests of second or further HSC or lymphocyte donation, analyzing the experience of Italian Registry and the incidence of a second donation request Results: From 1989 to 2013, ‘‘first’’ HSC products donated by Italian donors were 2999 (2160 BM and 839 PBSC): 5% of these donors were asked for a ‘‘second’’ donation (55% DLI, 45% HSC). After PBSC donation the incidence of a ‘‘second’’ donation is 8,3% (87% for DLI, 13% for HSC: BM or second PBSC), after BM donation the incidence of a ‘‘second’’ donation is 2,7% (73% asked for PBSC donation, 22% for DLI and 5% for BM). On 20% of the lymphocyte donation, donor were asked to donate a further lymphocyte donation (31 donation for these donors); all of these requests were authorized by SIMTI Commettee. No donors were authorized for more than two HSC donation Conclusion: Analyzing Italian Bone Marrow Donor Registry experience, after HSC donation, the incidence of a ‘‘second’’ donation request is 5%, more after PBSC donation than after BM donation (8,3% vs 2,7%); when PBSC donation is a first donation, the request of a’’second’’ donation is more frequently for DLI (87% of requests), and when first donation is BM, donor is asked for a second donation less frequently (2,7%), but this request is usually for PBSC (73% of requests). Disclosure of Interest: None declared. P070 Haploidentical TCRab and CD19 Depleted Stem Cell Transplant for Primary Immunodeficiency – 6 cases B. Morillo-Gutierrez1,*, Z. Nademi1, J. Dunn2, D. Barge3, S. Hambleton4, M. Abinun5, T. Flood5, A. J. Cant5, A. Gennery4, M. Slatter5 1 Department of Paediatric Immunology, Great north Children´s Hospital, 2Hematopoietic Stem cell Laboratory, 3Regional Immunology Laboratory, 4Institute of Cellular Medicine. Newcastle University, 5Department of Paediatric Immunology. Great North Children´s Hospital, Newcastle Upon Tyne, United Kingdom Introduction: For many patients with Primary immunodeficiency (PID), Hematopoietic stem cell transplant (HSCT) is the only curative treatment available. If there is no HLA identical donor the use of HLA-haploidentical family donors is problematic due to the increased occurrence of graft versus host disease (GVHD) and graft rejection particularly for patients with forms of PID other than severe combined immune deficiency. Various T-cell depletion strategies have been used, but some can lead to slow immune reconstitution with the consequent risk of morbidity and mortality from infection. Depletion of T cell receptor alpha and beta (TCRab) and CD 19 þ cells, with selection of TCRgd T-cells and preservation of NK, myeloid and plasmacytoid dendritic cells which aid engraftment and also decrease the risk of infection, has been shown to be a promising new technique. Materials (or patients) and methods: Prospective collection of the data from the paediatric patients with different PIDs treated using this technique in our HSCT unit at Great North Children´s Hospital, Newcatle Upon Tyne, UK. Results: 6 patients were treated in our unit from September 2012 to July 2014. All had a successful haploidentical TCRab and CD19 depleted HSCT. All 6 had viral reactivation but are alive and well 4-24 months post transplant, with chimerism 490% and good immune reconstitution. See table for more details.
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Table 1. Diagnosis
Conditioning
1.WAS 12m Father 2.Artemis 18m 2nd HSCT Mother 3.DOCK 8 7y Father 4.CGD 3y Father
ATG Bu/flu/TT Campath T/Flu/TT
5.CID 12m Mother 6. CgC 6m Father
GVHD Neut GVHD prophylaxis
Viral
þ 15 None
CMV
CsA/MMF
Tacro/MMF þ 12 Skin I
Adeno
Adeno CMV
Campath T/Flu/TT
CsA/MMF
þ 12 None
Campath T/Flu/TT
CsA/MMF
þ 14 Skin I Adeno,CMV EBV,HHV6
T/Flu/TT
CsA/MMF
None
CsA/MMF
þ 11 Skin II NA None
Multivariate analysis of Survival
Follow up
N
RR (95% CI)
P-value
2 yr 100% 1 yr 100%
HLA Match 10/10 Match 1 mm 41 mm
878 239 77
1.00 1.22 (1.2-1.5) 1.46 (1.1-1.9)
0.033 0.009
1 yr 100%
Patient Donor CMV Match Mismatch
863 331
1.00 1.37 (1.2-1.6)
o0.0001
Patient age o40y 440y
572 622
1.00 1.26 (1.1-1.5)
0.006
1014 180
1.00 1.36 (1.1-1.7)
0.003
Donor Age o30y 430y
372 822
1.00 1.17 (0.99-1.4)
0.069
ERA 96-99 00-03 04-07 08-11
142 421 345 286
1.00 0.85 (0.7-1.1) 0.80 (0.6-1.1) 0.83 (0.6-1.1)
0.18 0.09 0.21
Disease Risk - EBMT Good Intermediate Poor
557 444 193
1.00 1.34 (1.1-1.6) 1.33 (1.1-1.7)
0.001 0.01
6m CD15 100%,T79% CMV 4m 100% Adeno 4m Rotavirus T 93% CD15 9%
Conclusion: We report here 6 patients who have had a successful haploidentical TCRab and CD19 depleted HSCT. Pending the results of larger series, this could be a promising technique in haploidentical HSCT with early and sustained engraftment, early immune recovery and low risk of GVHD for PID patients. References: Locatelli F, Bauquet A, Palumbo G, Moretta F, Bertaina A. Negative depletion of a/b T cells and of CD19 þ B lymphocytes: A novel frontier to optimize the effect of innate immunity in HLA-mismatched hematopoietic stem cell transplantation. Immunology letters 155 (2013) 21–23 Bertaina A, Merli P, Rutella S, Pagliara D, Bernardo ME, Masetti R, et al. HLA-haploidentical stem cell transplantation after removal of ab þ T and B cells in children with nonmalignant disorders. Blood. July 2014; 124(5): 822–826 Kharya G, Nademi Z, Leahy TR, Dunn J, Barge D, Schulz A, et al. Haploidentical T-cell alpha beta receptor and CD19-depleted stem cell transplant for Wiskott-Aldrich syndrome. J Allergy Clin Immunol. June 2014. Epub Disclosure of Interest: None declared. P071 HLA–DQB1 and –DPB1 mismatches are significantly associated with worse survival in unrelated donor haematopoietic stem cell transplantation B. E. Shaw1,2,*, N. Mayor1, R. Szydlo1, W. Bultitude1, K. Kirkland3, J. Perry3, A. Clark4, S. Mackinnon5, D. Marks6, A. Pagliuca7, M. Potter8, N. Russell9, K. Thomson10, A. Madrigal1, S. G. E. Marsh1 1 Anthony Nolan Research Institute, London, United Kingdom, 2 CIBMTR, Milwaukee, United States, 3BSBMT, London, 4The Beatson, West of Scotland Cancer Centre, Glasgow, 5University College London, Royal Free Campus, London, 6University Hospitals Bristol NHS Trust, Bristol, 7Kings College Hospital, 8 Royal Marsden Hospital, London, 9Centre for Clinical Haematology, Nottingham University Hospital, Nottingham, 10University College London Hospitals, London, United Kingdom Introduction: HLA matching is known to be important in unrelated donor (UD) stem cell transplantation to ensure the best patient outcomes. Historically five HLA loci were tested, HLA-A, -B, -C, -DRB1 and –DQB1. More recently, routine matching for –DQB1 has become less frequent and in some centres is not performed at all. Conversely, -DPB1 typing is much more frequently performed and matching at both an allele and T-cell epitope (TCE) level may be considered. Materials (or patients) and methods: We performed a study to analyse the impact of donor factors (HLA, CMV, age, gender and ABO matching) on survival of patients receiving an UD haematopoietic stem cell transplant. We included 1271 UK recipients transplanted between 1995-2011. All patients
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Previous autos 0 40
had received a transplant for a malignant disease (acute leukaemia/MDS, 63%). Disease type and risk were categorised by the EBMT risk score and 580, 457 and 199 were characterised as good, intermediate and poor risk respectively. The median follow up of 611 surviving patients was 5.5y (range 0.2-15.4). The median age of the cohort at transplant was 40.6y (range 0.9-71.9). 661 and 585 patients had myeloablative and reduced intensity conditioning respectively, with Alemtuzumab in 94%. PBSC were used in 54% of patients. The median donor age was 34.9 (range 19-60.4) with 80% being male and 20% female. The patient/donor CMV status was pos/pos (n ¼ 238), pos/neg (n ¼ 244), neg/neg (n ¼ 647), neg/pos (n ¼ 101). Results: Patients matched for 10/10 HLA alleles had significantly better overall survival than those matched at 9/10 or o9/10 (median survival in years: 2.7 vs 1.2 vs 0.9 respectively, P ¼ 0.001). This finding was retained in multivariate analysis (Table 1). Considering only the 9/10 matched patients, mismatching for HLA-B (P ¼ 0.01) and –DQB1 (P ¼ 0.03) resulted in a significantly worse survival, while mismatching for HLA-A (P ¼ 0.1), -C (P ¼ 0.2) or DRB1 (P ¼ 0.7) resulted in no difference. HLA-DPB1 TCE mismatching showed a significantly worse survival when compared to either DPB1 TCE or allele matched pairs (1.3 vs 2.6 yrs, P ¼ 0.01). Donor gender and ABO matching status did not impact on patient outcome in multivariate analysis. Conclusion: In conclusion, our results show that matching for HLA-DQB1 remains important in this cohort of patients, and that this should be considered pre-transplant. We also reconfirm the importance of matching for DPB1 if a choice of donors is available. Finally, donor CMV status and age should also be considered in the selection. Disclosure of Interest: None declared.
P072 Allogeneic versus Autologous Hematopoietic Stem Cell Transplantation for non promyelocytic Acute Myeloid Leukemia: 30 years experience in a single center C. Montes-Gaisan1,*, A. Bermu´dez1, G. Pe´rez1, A. Cuesta1, C. de Miguel1, C. Richard1, E. Conde1 1 Hematology, Hospital Universitario Marque´s de Valdecilla, Santander, Spain Introduction: The optimal post-remission treatment for Acute Myeloid Leukemia (AML) remains uncertain. Traditional comparisons of Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) versus Autologous Hematopoietic Stem Cell Transplantation (auto-HSCT) noted higher TRM(Treatment Related Mortality) but lower CIR (Cumulative Incidence of Relapse), with discordant results in Overall Survival (OS). Materials (or patients) and methods: Retrospective study of 275 patients diagnosed of AML (Acute Promyelocytic Leukemia excluded) who underwent Hematopoietic Stem Cell Transplantation (HSCT) in our center between 1982 and 2011. We compared OS and Disease Free Survival (DFS), CIR and TRM when receiving allo or auto-HSCT. Kaplan Meier curves and log-rank test were used for OS and DFS whereas cumulative incidences were used for CIR and TRM. Multivariate analysis with Cox Regression was used for controlling confounding factors. Patient and disease characteristics in allo and auto-HSCT were respectively: median age of 37,8(IQR 3052) and 44,5(IQR 36-56), secondary AML in 20,2% and 9,8%, failure after Induction course in 15,8% and 2,8%, complete remission pre-HCST in 87,1% and 97,3% and year of HSCT 42005 in 46,9% and 14,3% .There was no statistical difference in any other characteristic (cytogenetic risk and WBC count at diagnosis, included). Results: The OS in the earliest period (o1997) for allo and autoHSCT was respectively: 39,6% and 60,8% at 1 year and 28,3% and 45,1% at 5 years. And the OS in the latest one (Z1997) was respectively: 65,6% and 70,3% at 1 year and 46,8% and 47,7% at 5 years (image1). Any significant results in the multivariate analysis neither in the log-rank test regarding OS differences between allo and auto-HSCT were observed. Covariates such as elderly, elevated WBC count at diagnosis, high cytogenetic risk, early period of HSCT or secondary AML showed worse prognosis, but only MRD was a strongly outcome predictor. The DFS in the earliest period for allo and auto-HSCT was respectively: 50,0% and 65,2% at 1 year and 37,5% and 45,6% at 5 years. And the DFS in the latest one was respectively: 66,9% and 63,2% at 1 year and 51,7%and 46,5% at 5 years. 74% of the patients who underwent auto-HSCT have ECOG 0 the date of the last follow-up and 6 patients over 14 who relapsed after auto-HSCT were rescued with allo-HSCT. The CIR in allo and auto-HSCT was respectively: 18% and 32% at 1 year and 24% and 50% at 5 years (HR ¼ 0,36, P ¼ 0,01), with no statistical difference if the HSCT was perfomed before or after 1997(start to quantify the pre-HSCT minimal residual disease by flow
citometry). The cumulative incidence of TRM in the earliest period for allo and auto-HSCT was respectively: 30% and 7% at 1 year and 35% and 9% at 5 years. However, the cumulative incidence of TRM in the latest one was respectively: 16% and 2% at 1 year and 25% and 4% at 5 years (HR ¼ 2,87, P ¼ 0,01). Conclusion: In our series, we have found no statistical difference between allo and auto-HSCT in OS and DFS in any period. Despite the higher CIR, we have observed a higher survival in auto-HSCT in the earliest period which turns to get similar nowadays because of the decrease in TRM, more significative in allo-HSCT. References: European groups: HOVON, BGMT, MRC and EORTC. Disclosure of Interest: None declared. P073 JACIE accreditation significantly improves compliance with international recommendations for related donor care in EBMT transplant centres C. Anthias1,*, P. V. O’Donnell2, D. M. Kiefer3, M. A. Pulsipher4, S. C. Goldstein5, B. R. Logan6, D. L. Confer3, B. E. Shaw6 1 Anthony Nolan Research Institute, London, United Kingdom, 2 Fred Hutchinson Cancer Research Center, Seattle, 3(CIBMTR) Center for International Blood and Marrow Transplant Research, National Marrow Donor Program/ Be The Match, Minneapolis, 4 Huntsman Cancer Institute, University of Utah, Salt Lake City, 5 University of Michigan, Ann Arbor, 6(CIBMTR) Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, United States Introduction: Previous surveys in Europe and the US have highlighted discrepancies between the care of unrelated donors (UDs) and related donors (RDs). FACT-JACIE Standards have addressed some of these issues including requirements for independent donor assessment and guidelines for donor care. We hypothesised that changes in FACT-JACIE standards have significantly impacted related donor practices. The Donor Health and Safety Working Committee of CIBMTR undertook a survey of international transplant centres (EBMT and CIBMTR members) to address this issue. Materials (or patients) and methods: An internet-based questionnaire was administered via a secure hyperlink (surveymonkey.com) August-November 2014. Program directors of EBMT and US CIBMTR allogeneic transplant member centres (excluding UK) received an email invitation to participate with a request to forward the survey to whichever physician was responsible for the care of related donors at that centre. Non-responders received 3 further reminders. Centres performing solely paediatric allogeneic
Table 1.
Differences between accredited and non-accredited
centres
Written policy for RD care Recipient is informed of donor HLA results before donor Recipient’s physician is simultaneously responsible for donor Defined donor eligibility criteria exist Policy regarding number of RD apheresis procedures (per donation) Policy regarding volume of marrow to harvest Policy regarding subsequent donations Process for credentialing physicians performing marrow harvests Long term donor follow up
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JACIE
NonJACIE
P
98.4%
83%
0.004 0.038
15%
35%
0.014
93%
75%
0.029
73%
55%
0.057
64%
66%
0.984
45%
35%
0.286
76%
60%
0.014
34%
14%
0.05
transplants were excluded. Based on practice differences, CIBMTR and EBMT centre results were analysed and presented separately. Results: 125 EBMT centres returned evaluable responses. 51% responding centres were fully JACIE accredited. The key findings are shown in Table 1. We found that JACIE accredited centres significantly more frequently complied with international recommended care standards including the existence of a written policy for RD care, defined donor eligibility criteria, and a process for credentialing physicians performing marrow harvests. In other areas JACIE accreditation did not impact practice: In 71% centres BM harvest were performed by the same transplant physicians responsible for the recipient, 35% centres had no defined limit for marrow harvests, and 36% had no limit to the number of apheresis procedures performed. Conclusion: In conclusion, we found significant differences in related donor care between accredited and non-accredited centres. Although accredited centres performed better in some areas, even they failed to meet some standards (eg. in 15% centres care for a donor and their recipient still overlaps). In areas of care not addressed by JACIE standards (e.g. the existence of policies defining limits of donation), both accredited and non-accredited centres failed to meet accepted international UD practice. Through accreditation, centres have improved related donor practice, but there remain clear areas where practice could be improved. Disclosure of Interest: None declared.
not possible in all cases. In addition all centres now reported they had a written policy for the care of related donors. When evaluating areas addressed by other FACT standards, we found that all centres provided RDs with some information prior to HLA typing, however only 52.7% provided written information. 96% centres provided follow-up to 1 week postdonation, but only 13% followed-up RDs to 1 year, and no centre performed follow-up beyond one year. We also addressed other areas, not currently covered in FACT standards but where accepted international unrelated donor guidance provided a benchmark. 32% of transplant centres, did not assess donor health before HLA typing and, where assessed, this consisted of verbal open-ended questions in 65%. 24% centres did not verify that donors were willing to donate before proceeding with HLA typing, 43% centres either disclosed donor HLA results to the recipient first, or had no consistent practice regarding result disclosure. In 47% centres, marrow harvests were performed by the physicians responsible for the recipient. Conclusion: In conclusion, changes to FACT standards have improved practice since 2007. However, related donor followup falls below international recommendations published by the WBMT and this survey identified further important areas in which care received by RDs falls below that of UDs. Efforts are needed to improve these practices in related donor centres, which would be best effected by enhancing regulatory standards. Disclosure of Interest: None declared.
P074 Significant improvements in the practice patterns of related donor care in US transplant centres C. Anthias1,*, P. V. O’Donnell2, D. M. Kiefer3, M. A. Pulsipher4, S. C. Goldstein5, B. R. Logan6, D. L. Confer3, B. E. Shaw6 1 Anthony Nolan Research Institute, London, United Kingdom, 2 Fred Hutchinson Cancer Research Center, Seattle, 3(CIBMTR) Center for International Blood and Marrow Transplant Research, National Marrow Donor Program/ Be The Match, Minneapolis, 4 Huntsman Cancer Institute, University of Utah, Salt Lake City, 5 University of Michigan, Ann Arbor, 6(CIBMTR) Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, United States
P075 Activating KIR2DS1/2 positive receptors on donor NK cells are associated with an improved overall survival and reduced relapse incidence in C1-ligand negative patients with myeloid malignancies after unrelated haematopoietic stem cell transplantation C. Zollikofer1,2,*, D. Fu¨rst2,3, C. Tsamadou1,2, D. Niederwieser4, D. Bunjes5, G. Wulf6, M. Pfreundschuh7, E. Wagner8, G. Stuhler9, B. Glass10, S. Hubert1,2, J. Mytilineos1,2,11 1 Institute of Transfusion Medicine, University of Ulm, Germany , 2 Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service, Baden Wuerttenberg – Hessen, Ulm, Germany, 3Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, Ulm, 4Department of Hematology/Oncology, University of Leipzig, Germany, Leipzig, 5 Department of Hematology/Oncology, University Clinic Ulm, Germany, Ulm, 6Department of Hematology/Oncology, GeorgAugust-University Go¨ttingen, Germany, Go¨ttingen, 7Department Internal Medicine I, Universita¨tsklinikum des Saarlandes, Homburg, Germany, Homburg, 8Department of Medicine III, Johannes Gutenberg-University Mainz, Germany, Mainz, 9Centre for Bone Marrow and Blood Stem Cell Transplantation, Deutsche Klinik fu¨r Diagnostik, Wiesbaden, Germany , Wiesbaden, 10Department of Medicine II, University Hospital Wu¨rzburg, Germany, Wu¨rzburg, 11 DRST – German Registry for Stem Cell Transplantation, Ulm, Germany, Ulm, Germany
Introduction: A survey conducted in 2007 (O’Donnell et al. Blood 2010) investigated practice patterns in the care of related donors (RDs) in the US, and reported a potential conflict of interest in 470% centres (i.e. where a physician had simultaneous responsibility for the care of a RD and their recipient). In addition, 5% centres lacked written policies for RD care. The 5th Edition of the FACT-JACIE Standards addressed these issues. In order to assess the impact of the changes, we undertook a follow-up survey of US transplant centers. We took this opportunity to explore, in both the US and Europe, other areas where we hypothesised that RD care may differ from that of unrelated donors (UDS). Materials (or patients) and methods: An internet-based questionnaire was administered via a secure hyperlink (surveymonkey.com) August-November 2014. Program directors of EBMT and US CIBMTR allogeneic transplant member centres (excluding UK) received an email invitation to participate with a request to forward the survey to whichever physician was responsible for the care of RDs at that centre. Non-responders received 3 further reminders. Centres performing solely paediatric allogeneic transplants were excluded. Based on practice differences, CIBMTR and EBMT centre results were analysed and presented separately. Results: In the US CIBMTR cohort, 76 individual centers responded (response rate 40%), 72 are FACT accredited. We saw significant improvements since the earlier survey particularly with respect to potential conflict of interest in donor-recipient care. Over 93% of centres report that their standard was to specifically separate the care of donors and recipients. However 32% of these centres stated that this was
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Introduction: Natural Killer cells (NK) are lymphocytes that have the potential to recognize and lyse cells with aberrant HLA expression profiles which escaped T-cell mediated lysis. Communication between NK and leukemic cells is facilitated, among others, by killer cell immunoglobulin-like receptors (KIR) on the surface of NK cells and their respective HLA-class I ligands. KIRs influence NK-cell activity by mediating activating or inhibitory signalling, whereas activating signals and missing inhibition both lead to target cell lysis. Activating KIRs may have an immune-modulatory effect on the outcome of HSCT. In this study we show that patients with myeloid malignancies that do not express a C1 ligand (high risk group) suffer from impaired outcome after HSCT. Moreover, we demonstrate the beneficial influence of activating donor KIR2DS1/2 on this high risk group. Materials (or patients) and methods: Patients and donors were high resolution typed for HLA-A, -B, -C, -DRB1, and -DQB1. KIR-typing was performed through PCR-SSP. HLA-C alleles were
grouped in respect to amino acid 80 into C1 (asparagine) and C2 (lysine) ligands. Patient malignancies were AML, CML or MDS. Donors were at least 18 years old, and T-cell replete graft source was either peripheral blood stem cells (95.5 %) or bone marrow (4.5 %). Clinical data for this study where obtained through the German Registry for Haematopoietic Stem Cell Transplantation (DRST). Overall survival (OS), disease free survival (DFS), relapse incidence (RI), and treatment-related mortality (TRM) were assessed using Kaplan-Meier analysis, competing risks regression and extended Cox regression models. Survival endpoints were investigated with respect to HLA-matching grades by separate analyses of 9/10, and 10/10 matched transplant pairs (KIR2DS1 analysis), or by performing stratification for HLA-compatibility (KIR2DS2 analysis). Results: C1 negative patients (n ¼ 200) showed inferior OS (HR ¼ 1.41, CI ¼ 1.14-1.74, P ¼ 0.0012), DFS (HR ¼ 1.27, CI ¼ 1.05-1.53, P ¼ 0.015), RI (HR ¼ 1.33, CI ¼ 1.01-1.75, P ¼ 0.04) and TRM (HR ¼ 1.41, CI ¼ 1.01-1.96, P ¼ 0.04) when compared to C1 carriers (n ¼ 1246). Within this high risk patient group, however, RI was improved if a KIR2DS1 expressing donor was used (HR ¼ 0.26, CI ¼ 0.11-0.63, P ¼ 0.003, n ¼ 74) when compared to a control group of patients transplanted with a KIR2DS1 negative donor (n ¼ 126). Furthermore, in single HLA-mismatched transplants, OS was improved when transplanted with a KIR2DS2 positive donor (9/10: HR ¼ 0.29, CI ¼ 0.09-0.92, P ¼ 0.03, n ¼ 70; 10/10: HR ¼ 1.04, CI ¼ 0.51-2.12, P ¼ 0.91, n ¼ 33). Conclusion: Our findings can be explained by impaired donor derived NK-cell activity in C1-ligand negative patients. As the first KIRs to be expressed are KIR2DL2 and KIR2DL3 which recognize C1-ligands, the early onset of donor NK-cells in these patients is consequently non-reactive against C1-ligand negative recipient cells. Furthermore, C1-restricted NK-cells form bigger populations, react faster to interferon g secretion, and degranulate CD107a more potently. Our study shows, that activating signals derived from KIR2DS1/2 stimulation can overcome this impaired NK-cell response. We therefore suggest inclusion of KIR2DS1/2-genotyping in the unrelated donor search algorithm of C1 ligand negative patients with myeloid malignancies. Disclosure of Interest: None declared. P076 ab/CD19 negative immunoselection for haploidentical transplants in pediatric setting C. Del Fante1,*, M. Zecca2, G. Viarengo1, L. Rubert2, G. Giorgiani2, F. Bonetti2, C. Perotti1 1 Immunohaematology and Transfusion Service, Centre for transplant immunology , 2Oncoematologia Pediatrica, Fondazione IRCCS Policlinico S.Matteo, Pavia, Italy Introduction: Positive CD34 þ immunomagnetic cell selection (ICS) has been used since 90’s to prevent/avoid GVHD in haploidentical stem cell transplant (Haplo-HSCT) and overcome the HLA barrier. Recently, Miltenyi Biotec (Germany) released a new cell depletion system able to remove ab þ /CD19 þ lymphocytes from the graft while retaining TCRgd þ , NK þ and other cell subsets. This platform allows an optimal T cell depletion coupled with a B cell depletion that prevents the onset of post transplant EBV-associated lymphoproliferative disease, in order to maintain anti-tumor and anti-infectious properties. Materials (or patients) and methods: We retrospectively analyzed 18 negative ab þ /CD19 þ and 10 CD34 þ positive ICS (performed on leukapheretic fraction if CD34 þ cell dose/ kg in the graft was o10x106) performed from Oct 2013 to Dec 2014 from peripheral blood (PB) mobilized haploidentical donors (11 mothers, 6 fathers) for hematopoietic stem cell transplantation in children affected with different hematological diseases. ICS (Clinimacs Miltenyi Biotech) was performed according to the manufacturer’s instructions. PB CD34 þ cell number at apheresis collection, cell recovery after immunomagnetic cell selection, total amount of cells infused, engraftment and transplant related mortality were recorded.
Results: CD34 þ cells (mean 86,6/ul, range 35.5-107.2) were collected during 24 leukapheretic procedures from 17 mobilized haploidentical donors. No major side effects were recorded during leukapheretic mobilization and collection. Mean CD34 þ cell recovery from negative ICS was 82.8% (range 64.8-99), mean Log ab/T and CD19 þ /B depletion was 4.46 (range 1.18-7.4) and 5.15 (range 2.1-7.6), respectively. Overall, median graft cell content (x10^6/Kg recipient, considering also CD34 positive ICS when appropriate) was the following: CD34 þ cells 18.24 (min 10.85-max 31.09), ab cells 0.013 (min 0.0001-max 0.14), gd cells 16.21, B lympocytes 0.18 (min 0.0001-max 1.44), NK 36.9 (min 7.61-max 194.4), NK-T 5.58 (min 0.47-66.6). The pediatric HSCT cohort included 11 males and 6 females, median weight 27 kg (range: min 4.7max 80). Recipients’ underlying disease was ALL in 8 pts (47%), AML in 4 (23.5%), SCID in 3 (18%), FA and JMML in 1 pt (6%), respectively. No adverse events upon HSC infusion was recorded. No graft failure was observed. Median days to PMN and PLT engraftment was 14 (min 9-max 23) and 13 (min 9-max 41), respectively. So far, 1 patient developed aGVHD gradeII-IV, that resolved with steroid treatment, 1 patient died before day 100 due to multiorgan failure, and 2 patients experienced leukemia relapse. Conclusion: Our preliminary data show that ab þ / CD19 þ negative ICS is effective in depleting ab þ and CD19 þ lymphocytes and results in a sustained engraftment in pediatric haploidentical setting. A longer pts’ follow-up will be required for a meaningful evaluation of GVHD and infectious disease incidence, anti-tumor efficacy and immune reconstitution. Disclosure of Interest: None declared. P077 In vivo T cell depletion with antithymocyte globulin reduces the incidence of acute graft-versus-host-disease (GVHD) in matched unrelated donor stem cell transplantation without affecting chronic GVHD, relapse risk and survival D. KARAKASIS1,*, I. BATSIS2, F. PANITSAS1, E. XENOU1, Z. BOUSSIOU2, A. XIROKOSTA1, S. KARAKATSANIS1, P. KALOGIANNIDIS2, V. PARDALIS1, A. KAISARI1, D. MALLOURI2, E. GIANNAKI2, A. LOIDORIS1, M. BOUZANI1, C. SMIAS2, I. MARKOU1, J. APOSTOLIDIS1, S. GIGANTES1, I. SAKELLARI2, I. BALTADAKIS1, A. ANAGNOSTOPOULOS2, N. HARHALAKIS1 1 BMT Unit, Evangelismos Hospital, Athens, 2BMT Unit, Papanikolaou Hospital, Thessaloniki, Greece Introduction: There is conflicting data regarding the role of antithymocyte globulin (ATG) in the outcome of matched unrelated donor hematopoietic stem cell transplantation (MUD-HSCT). The aim of this study was to retrospectively analyze the impact of the inclusion of ATG in the conditioning regimen on the outcomes of MUD-HSCT in the era of donor selection by allele-level HLA typing. Materials (or patients) and methods: From 01/2001 until 12/ 2013, 256 adult patients (M/F: 149/107) underwent MUD-HSCT at two Greek transplant centers for myeloid (n ¼ 162) or lymphoid (n ¼ 94) malignancies. The median age of patients was 41 (range, 14-66) years. Donor/recipient HLA allele match was 8/8 (n ¼ 130) or 7/8 (n ¼ 126) at HLA-A, -B, -C, and -DRB1 loci. The conditioning regimen was mainly myeloablative (n ¼ 213), and peripheral blood was the source of the graft in 95% of cases. ATG (Thymoglobulin) was administered in 121/ 256 (47.3%) patients according to each center policy, at a total dose of 2.5-5mg/Kg BW. An ATG vs. no-ATG comparison was made in terms of engraftment, acute or chronic GvHD, relapse, non-relapse mortality (NRM), overall survival (OS) and diseasefree survival (DFS). Results: Patient and transplant characteristics were balanced between the two patient groups (ATG vs. no ATG), except for age (median, 39 vs. 43 years, respectively, P ¼ 0.008) and degree of HLA mismatch (58.7% vs. 40.7%, respectively, P ¼ 0.004). Engraftment occurred at a cumulative incidence
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(CI) of 96.4% without any difference between the two patient groups. ATG administration was associated with a statistically significant reduction in the incidence of grade II-IV acute GvHD (HR: 0.64, 95% CI: 0.44-0.92, P ¼ 0.017). In multivariate analysis, ATG administration was the only additional risk factor besides HLA match for grade II-IV acute GVHD. ATG administration had no impact on chronic GvHD incidence, relapse incidence, NRM, DFS and OS. HSCT-specific comorbidity-age index, degree of HLA match and EBMT score emerged as the major independent risk factors for NRM, OS and DFS in multivariate analysis. Conclusion: Our results do not suggest any considerable effect of in vivo T cell depletion with ATG on survival after MUD-HSCT. However, the observed significant reduction in the incidence of acute GvHD may argue in favor of the use of ATG, especially in the context of HLA mismatched MUD-HSCT. Disclosure of Interest: None declared. P078 Long-term Overall and Event-free Survival after RIC Allogeneic Transplantation in Acute Leukemias in a Single Centre: Impact of Age and Donor Type? S. Wittnebel1, S. Michiels1, S. Anoun1, M. C. Ngirabacu1, N. Meuleman1, G. Claes1, P. Huynh1, P. Crombez1, M. F. Jaivenois1, P. Lewalle1, D. Bron1,* 1 Hematology, INSTITUT JULES BORDET, ULB, Brussels, Belgium Introduction: Between Jan 2000 and Dec 2014, we transplanted 71 patients (Pts) using RIC- conditioning allogeneic transplant for acute leukemias (AML and ALL) and myelodysplastic syndromes (MDS) not eligible for myeloablative transplantation. Clinicians are usually scared to transplant older patients with MUD because of the higher risk of Treatment-related mortality (TRM). We thus reviewed retrospectively the impact of age and donor-choice (familial related donor (RD) versus matched unrelated donors (MUD) ) on the post-transplant outcome of the pts. Materials (or patients) and methods: We retrospectively studied in our cohort of 71 evaluable RIC transplant pts, the overall survival (OS) and event-free survival (EFS), according to ages (r 54yo vs Z55yo) and donor types (RD vs MUD). Conditioning consisted mainly of combinations of BusulfanFludarabin-ATG or Endoxan-Fludarabine-ATG. Immunosuppression was a combination of cyclosporine (CSA)/mycofenolate mofetil (MMF), Tacrolimus (TAC)/ MMF or TAC/ Sirolimus. Chimerism analyses were performed on d30, d90 and d180. Both study-groups were well balanced concerning transplant conditioning regimens, (ATG or not) RD vs MUD donors, type of leukemia and status of disease at transplant. Our local EBMT database was the source for this analysis. Results: 34 de novo AML (20 r54 yr and 14 Z55 yr), 30 secondary AML and MDS (15 r54 yr and 15 Z55 yr) and 7 ALL (5 r54 yr and 2 Z55 yr) were enrolled in our trial. 24 pts received HLA identical RD in the group r54yr and 14 pts in the group Z55 yr. The others pts received MUD transplant from HLA 9 or 10/10 donors (16 in the group r 54yr and 17 in the group Z 55yr). The median follow-up was 22 (3-93) months. Engraftment rate was 100% in the studied cohort. [S1] TRM before d100 was 18,3% (15% r54 yo and 22,6% Z55yyo). Grade II-IV Acute GVHD (aGVHD) at d90 were similar in both age populations (16,6%). Chronic GVHD rates are similar in both age groups (24%). [S2] There was a clear, even though not statistical, significant, difference in 5years OS when stratifying the two subgroups (r54 yr versus Z55 yr) dependent on donor-types (pts r 54yr : RD: 31,6%; and MUD: pts 38%; Z 55yr : RD: 27,8% and MUD 44,2%). This trend in a better OS in older MUD pts could be explained partially by a lower relapse-rate in the group Z 55 yr with an MUD as shown by EFS/5yrs (pts r 54 yr : RD: 32%; r 54yr MUD: 19%; pts Z 55yr RD: 21% and Z 55yr MUD 38,5 %) suggesting the benefit of a greater GVHD/GVL-effect in the MUD transplantation. Conclusion: Our observations confirm that:
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1) RIC transplantation is a curative approach in acute leukemia whatever the age when the pts are fit enough to tolerate the transplant. 2) Despite a higher TRM compared to younger patients, older patients seem to particularly benefit from MUD in terms of OS, probably due to a greater GVHD/GvL effect in the MUD setting leading to an increased EFS. Disclosure of Interest: None declared. P079 Impact of HLA matching on the risk of primary graft failure and acute GVHD in patients after allogeneic unrelated HSCT E. Kuzmich1,*, A. Alyanskiy1, A. Vitrischak1, E. Morozova1, S. Bondarenko1, L. Zubarovskaya1, B. Afanasyev1 1 First Pavlov State Medical University of St. Petersburg, Raisa Gorbacheva Memorial Institute for Pediatric Oncology, Hematology and Transplantation, St-Petersburg, Russian Federation Introduction: Primary graft failure and acute GVHD are the serious complications following unrelated HSCT. The aim of the study was to determine of the immunogenetic risk factors of these complications actual for our cohort patients. Materials (or patients) and methods: We retrospectively analyzed the results of 390 HSCT which were performed to the patients with various hematological diseases between January 2005 and December 2012. All donor/recipient pairs were typed for HLA-A, -B, -C, -DRB1, -DQB1 by PCR-SSP, PCR- SSOP, SBT methods on the high resolution level. Statistical analysis was done by SAS Enterprise Guide v. 5.1. Fischer’s Exact Test was used for comparison of the complications risk in HLAmatched and HLA-mismatched groups. Logistic regression model was used for the analysis of the association between HLA and non-HLA variables and risk of complications. Clinical variables included age of patient (continuous variable), type of disease, risk of leukemia relapse (standard, high, and diseases other than leukemia), donor/recipient gender-matching, donor/recipient CMV status, stem cell source, number of CD34 þ cells, conditioning regimen, aGVHD prevention regimen. Results: 290 patients received a 10/10 allele-matched transplant. 80 patients had a 9/10 allele- matched transplant (single mismatch for HLA-A: 27.5%, HLA-B: 23.7%, HLA-C: 32.5%, HLA-DRB1: 6.3%, HLA-DQB1: 10%). 20 patients received an equal and less than 8/10 allele-matched transplant. The single class I, class II allele mismatches didn’t impact on the frequency of primary graft failure. The multiple class I allele mismatches in the donor/recipient pairs were associated with higher risk of the primary non-engraftment (RR 3.42, CI 1.279.18). Other factors associated with primary graft failure were: using of bone marrow as the source of hematopoietic cells, positive CMV status of the donor in the case of the recipients with negative CMV status, type of disease. The risk of grades III-IV aGVHD was influenced by the number of mismatches, individual class I and class II loci disparities in the recipient. Recipients with single mismatches had an increased hazard of grades III-IV aGVHD compared with matched recipients (RR ¼ 1.98; CI 1.42-2.76; Po0.001). R ecipients with multiple class I, class II mismatches had the highest hazard (RR ¼ 2.71; CI 1.65-4.45; P ¼ 0.007). We analyzed the role of individual class I and class II loci. An increased risk of grades III-IV aGVHD was associated with the single HLADRB1 allele mismatches (RR ¼ 2.89; CI 1.57-5.29; P ¼ 0.03), single HLA-C allele mismatches (RR ¼ 2.36; CI 1.52-3.67; P ¼ 0.004, single HLA-B allele mismatches (RR ¼ 2.16; CI 1.30-3.61; P ¼ 0.02). Other factors associated with severe aGVHD were: using of peripheral blood stem cells as the source of hematopoietic cells, myeloablative conditioning regimens. Conclusion: The study results can be useful for the selection of the optimum donor for carrying out unrelated allogeneic HSCT. Disclosure of Interest: None declared.
P080 Hematopoietic stem cell donors – analysis of motivation and satisfaction throughout the donation process E. Snarski1,*, P. Kawka1, T. Torosian2, M. Achremczyk1, K. Skwierawska1, A. Waszczuk - Gajda1, E. Urbanowska1, W. Wiktor - J˛edrzejczak1 1 Hematology, Oncology and Internal Diseaes, Medical University of Warsaw, 2DKMS Poland, DKMS Poland, Warszawa, Poland Introduction: In recent years the number of Polish potential registered unrelated hematopoietic stem cell donors has increased to over 700 000. This caused substantial rise in the number of collections for recipients in Poland and worldwide. So far there is relatively little experience and knowledge about Polish donors: their motivation, the ways of recruitment, satisfaction during the donation process were never evaluated. Better understanding of these factors can contribute to improvement in methodology of recruitment, information and donation. The aim of our study was to assess these issues by approaching unrelated HSC donors at major centre in Poland. Materials (or patients) and methods: The unrelated HSC donors filled on line questionnaire at the two steps of donation process: first at the Final Clearance visit, and second immediately following the donation. The questionnaire was made available online for the ease of donors. Major questions included in the questionnaire: What are the main motivating factors to become a donor? How did donors register? What are the most important information sources? What are the biggest problems during donation? What are the biggest fears of donors? Results: At the time of abstract submission 93 questionnaires were submitted. The average age of responding donors was 32 years. Sixty six% of the donors registered declared that they wanted to help other people. The most important factors in recruitment were local action (21%), information from the Internet (23%), information from TV (19%) and prior recruitment of family member (15%). The major recruitment took place through the Internet (43%) and in local recruitment actions (39%). The biggest fears prior to the donation were: complications during donation (39%), pain (19%) and problems with work and family life that could be caused by donation (9%). Ninety eight% of donors felt adequately informed prior to donation and stated that the nurse (35%) at the apheresis centre and the qualifying doctor (33%) were the most important sources of information. The donation has been performed in 89% of donors from the peripheral blood and in 11% from the bone marrow. Forty% of donors have been also earlier blood donors. The level of pain during the PBSC donation was 1.2 in 10 point scale. Ninety four% felt that the information received prior to harvesting adequately described procedure. Ninety seven% were satisfied to give stem cells to the other person and 61% felt that the donation will bring a positive change to their lives. Eighty two% of donors expressed desire to meet recipient of their cells in the future, including 20% of donors who wished to meet the recipient immediately after the donation, 20% ready to wait at least for a year, and 23% ready to meet the recipient after 2 years. All declared consent to repeat collection procedure if it was needed. The major complaints concerning the procedure were: need for injections of G-CSF (33%), length of the procedure (30%) and pain (7%). Conclusion: Taken together these results create a positive view of donation process in Poland. The donors are generally well informed, the procedure does not cause significant problems and all the donors declared that they would donate one more time if needed. The major complaints are due to the technical parts of procedure – injections of G-CSF and length of apheresis. Disclosure of Interest: None declared.
P081 Prevalence of donor specific anti-HLA antibody in T cell-depleted haploidentical hematopoietic stem cell transplantation E. S. Choi1,*, H. J. Im1, J. K. Suh1, S. W. Lee1, K. N. Koh1, S. Jang2, H. B. Oh2, J. J. Seo1 1 Department of Pediatric Hematology and Oncology, 2Department of Laboratory Medicine, Asan Medical Center, SEOUL, Korea, Republic Of Introduction: Recently, the role of a donor specific anti-HLA antibody (DSA) in hematopoietic cell transplantation (HCT) has been emphasized as a consequence of the considerably increased cases of HLA-mismatched HCT including cord blood transplantation and haploidentical hematopoietic cell transplantation (HHCT) as well as unrelated HCT. The presence of DSA has been associated with an increased risk of graft failure (GF) after HCT. However, prevalence of DSA and clinically relevant level of DSA for GF in T cell-depleted HHCT remain unknown. Materials (or patients) and methods: We prospectively evaluated anti-HLA antibodies using the Luminex-based single antigen assay for 28 patients with malignant disease undergoing ex vivo T cell-depleted HHCT between Oct. 2011and Aug. 2014. Twenty-five patients had hematologic malignancy (7 ALL, 12 AML, 1 MLL, 1 MDS, 2 JMML, 2 NHL) and 3 had solid tumors. Two (1 ALL, 1 AML) of 28 enrolled patients received two rounds of HHCT for relapse after their first HHCT. Results: Of 30 transplants tested, anti-HLA antibody was detected in 17 (56.7%) patients. Of the positive cases, 4 (4/30, 13.3%) were positive against the donor HLA-antigen. Among the 4 cases with DSA, 2 had antibodies against HLA class I antigen, one against HLA class II, and one against both HLA class I and II. The peak levels of DSA were 1143, 1273, 4422 and 5564 of mean fluorescence intensity (MFI), respectively. None received any treatment in attempt to decrease DSA levels before transplants. Of a total of 30 HCTs, none experienced GF with achieving neutrophil engraftment at a median of 9 days (range, 9-13). In addition, the median times of neutrophil and platelet engraftment were similar regardless of the DSA status (10 versus 9 days for neutrophil, P40.05; 16 versus 18 days for platelet, P40.05). Conclusion: In conclusion, our study demonstrated that DSA up to MFI 5560 was not associated with increased risk of GF even without any interventions prior to T cell-depleted HHCT. However, further study including more cases is necessary to determine the optimal recipient’s MFI cut-off value for haploidentical family donor selection in T cell-depleted HHCT. Disclosure of Interest: None declared. P082 Impact of mismatches in the HLA system on the outcome after unrelated allogeneic stem cell transplantation E. Bojtarova1,*, B. Ziakova1, F. Al Sabty1, L. Sopko1, M. Kusikova1, D. Stemnicka1, M. Mistrik1, A. Batorova1 1 Department of Hematology and Transfusiology, LFUK, University Hospital Bratislava, Bratislava, Slovakia Introduction: Unrelated allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat patients (pts) with malignant and non-malignant hematological disorders, who miss their identical sibling. Better outcome of unrelated HSCT is achieved by knowledge of human leukocyte antigen (HLA) system and fast and precise tissue typisation techniques development. As the ‘10/10 matched’ (that is, high-resolution identity at loci HLA-A, -B, -C, -DRB1 and -DQB1) donor is not always available, alternative donor with single antigen/allele mismatch (9/10) is acceptable and HSCT might be succesfull as well, mainly in young pts. The aim of this analysis is to evaluate, whether the grade of HLA mismatch influenced the outcome of unrelated HSCT at our clinic. Materials (or patients) and methods: We retrospectively evaluated results of unrelated HSCT in 138 consecutive pts between February 1999 and May 2014 and compared grade of
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HLA match between donor and recipient. Results were evaluated to 31st August 2014. 88 pts received graft from 10/10 HLA identical unrelated donors, 50 pts from mismatched unrelated donors: 41pts with 9/10 (i.e. single antigen/allel mismatch) and 9 pts with 8/10 (i.e.two mismatches) HLA mismatch. Patient groups were comparative in age, primary diagnosis and peripheral blood use as the source of stem cells in majority of pts. The median age of pts at the time of HSCT was 37 (range, 18-68) years. AML/ALL was indication for HSCT in 84 pts, CML/MPN in 17 pts, MDS in 18 pts, SAA/PNH in 9 pts, MM/CLL/lymphoma in 10 pts. Median follow-up was 12, 11 or 8 months in ‘10/10 matched’, ‘9/10’ or ‘8/10 mismatched’ groups. Results: Median time to neutrophile engraftment (i.e. neutrophil count exceeding 0.5x10^9/l ) was 18 (range, 11-51) days in ‘10/10 match’, 16 (range, 13-32) days in ‘9/10’ and 20 (range, 14-21) days in ‘8/10 mismatched’groups. Graft failure occurred in 8 vs 0 vs 2 pts in ‘10/10’ vs ‘9/10’ vs ‘8/10’ HLA group. Acute graft versus host disease (GvHD) grades II to IV was observed in 32% of pts in ‘10/10’ vs 54% of pts in ‘9/10’ vs 75% of pts in ‘8/10’ group (P ¼ 0,01), chronic GvHD in 26% vs 41% vs 43% (P ¼ 0,2). Relaps incidence was observed in 24/83 (29%) evaluable pts in ‘10/10’, in 7/39 (18%) evaluable pts in ‘9/ 10’ and in 2/8 (25%) evaluable pts with ‘8/10’groups (P ¼ 0,41). 49/88 (56%) pts transplanted from ‘10/10 matched’ unrelated donors, 18/41 (44%) pts from donor with single mismatch and 4/9 (44%) pts from donor with two HLA mismatches are alive to 31.8.2014. Survival probability in 5 years (according to Kaplan-Meier) was 50%, 40% and 44% (P ¼ 0,254), with median survival of 24 vs 27 vs 19 months. Conclusion: Single or two antigen/allele mismatches between donor and recipient significantly increased the occurence of acute GvHD, non-significantly occurence of chronic GvHD, but didn’t negatively effect the egraftment, relaps incidence and overall survival in pts after unrelated HSCT. Disclosure of Interest: None declared. P083 Prospective study of T-cell -replete Haploidentical matched hematopoietic stem cell transplantation (HSCT) for patients with high risk hematological malignancies G. M. Al Dawsari1,*, S. MOHAMED1, F. ALMOHAREB1, N. CHAUDHRI1, F. ALSHARIF1, H. ALZAHRANI1, W. RASHEED1, A. HANBALI1, M. SHAHEEN1, F. ALFRAIH1, T. ELHASSAN1, M. Aljurf1 1 ONCOLOGY, KFSHRC, RIYADH, Saudi Arabia Introduction: HLA-matched sibling donors have been the standard for allogeneic HSCT, but about 35 %of our patients in Saudi Arabia have no an HLA- matched sibling donors. The chance of finding an HLA-matched unrelated donor from international registries is very low because of different ethnicity. Haploidentical HSCT with modern approach with post- transplant high dose cyclophosphamide (PT-CY) became an attractive approach with promising results, and make transplant available foe almost every patient. Here, we report the outcome results of our initial 11 patients. Materials (or patients) and methods: Analysis of patient’s data who underwent Haploidentical HSCT in prospective phase II study from January 2013 – September 2014. Eligibility include patients with high risk hematological malignancies in complete remission, less than or equal to 55 years of age without a suitable matched related donor , lack of fully matched unrelated donor (MUD) and Karnofsky performance of 70-100 %. Donor selection criteria included age of 18 years or more, a maximum 5 antigen mismatched is accepted and donor were excluded if the recipient’ serum contain anti- donor HLA antibodies. Myeloablative conditioning regimen consisted of total body irradiation (TBI) and fludarabine and thiotepa, busulfan and fludarabine (TBF). GVHD prophylaxis consist of PT-CY (50mg/kg) on day þ 3 and þ 5 and cyclosporine from day ( þ 4 to 180) and mycophenolate mofetil for 28 days. Results: A total of 11 patients received haploidentical HSCT and patients characteristics are shown in Table 1, with a
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median follow up of 6.5 months (range 1.75-16). The median time to both neutrophil and platelet engraftment (ANC40.5 109/L; platelet 420 109/L) were 19.5(15-24) and 30(17-150) respectively. The 100-day cumulative incidence (CIs) of acute GVHD grade II-VI was 59% and CIs of chronic GVHD was 55%.The CI of relapse was 20% with death in remission as competing event. The overall survival (OS) is 36%. Conclusion: Haploidentical HSCT represent a good alternative option for patients with high risk hematological malignancies who do not have suitable HLA- matched donors as it provide good result in term of engraftment of ANC and PLT and a good OS rate in patients with high risk diseases. GVHD remain a major obstacle in this type of transplant in our series when compared with published literature, for which we are currently investigating the addition of small dose ATG to our protocol. Disclosure of Interest: None declared. P084 Observational, prospective study on allogeneic family stem cell donors: results from a multi-center Italian study G. Andreola1,*, M. Martino2, T. Moscato2, M. Vacca3, F. M. Dallavalle4, S. Leoncino4, G. Mancini5, M. Montanari5, A. Babic1, M. Negri1, D. Laszlo1 1 Mobilization & Collection Stem Cell Unit, Division of Hematoncology, Istituto Europeo di Oncologia, Milano, 2Hematology and Stem Cell Transplant Unit, AO ‘‘Bianchi Melacrino Morelli", Reggio Calabria, 3Immunohematology and Trasfusion Unit, AO San Camillo Forlanini, Roma, 4Donation Center and Therapeutic Apheresis Unit, ASO ‘‘SS Antonio e Biagio e C. Arrigo’’, Alessandria, 5Hematology Clinic, Ospedali Riuniti Umberto I, Ancona, Italy Introduction: Allogeneic peripheral blood stem cell (PBSC) transplantation is a more convenient procedure than bone marrow (BM) for both donors and medical teams and shows similar outcomes allowing a faster hematologic recovery in recipients. However, adverse events (AE) and occasionally fatal complications within family donors have been reported. Complications are thought to be more frequent in family donors in respect to donors from international registries for whom suitability criteria and a 10-year(y) follow-up are standardized. Published retrospective studies usually lack long follow-up and prospective studies are urgently needed. We therefore designed a multicenter, observational, prospective study evaluating incidence and type of early and late complications in suitable family donors, in those judged as ‘‘not perfectly suitable’’ but undergoing PBSC collection anyway and in donors Z55 ys old; analysis of criteria excluding donors from PBSC and/or BM donation was also included. A complete blood cell count (CBC) after 7 days from
collection, a medical visit and a CBC after 30 days and a CBC yearly for the following 10 ys were required for each donor. Materials (or patients) and methods: From November 2011 to October 2014, 100 donors in 5 Italian centers were enrolled in the study. There were 51 males and 49 females; median age was 49 (18-70), 29 donors were Z55 ys old. Donors were parents in 8% of the cases, sibling in 80%, children in 12%; 71 donors were HLA-identical, 29 were HLA-haploidentical. A median of 4x106 CD34 þ /kg (range 4-6) of recipient body weight was requested. Results: According to IBMDR criteria, 71 (78%) donors resulted suitable for PBSC donation, 10 (10%) were not suitable for PBSC, 6(6%) were not suitable neither for PBSC nor for BM donation. Ten donors were evaluated as unsuitable for PBSC donation due to autoimmunity (40%), thrombofilic status (29%), not adequate peripheral access (10%), history of cardiac disease(30%); nevertheless 3 of them underwent mobilization anyway because no other donor was available or due to recipient’s related issues. Six donors were judged only partially suitable due to infections in 3, diabetes, splenomegaly and hypertension in the other 3. Lenograstim and filgrastim at 10 ug/kg were used in 96% and in 4% of donors respectively. PBSC collection was started after a median of 5 days (2-8) after GCSF administration. Mobilization-related AE were mostly bone pain (74%), headaches (33%), fatigue (30%), muscle pain (22%), splenomegaly not requiring GCSF interruption in 4%. Pre-leukapheresis white blood cell counts reached values 470.000/mm3 only in 3 donors. Median number of CD34 þ cells collected was 5.7 x106/kg (1.1-16) in a median number of 1 procedure (1-4); the target was reached in 84% of the cases and in 80% of donors Z55 y old. No donor required placement of a central venous catheter, red cell or platelet transfusion. Mild averse events during leukapheresis was reported in 11 donors (13.6%), moderate in 1 and severe in 1 donor Z55 ys. With a median follow-up of 18 months (1-36) no long-term adverse event were registered. Conclusion: PBSC mobilization and collections from family donors is a safe and well tolerated procedure and it allows an adequate collection of stem cells even in donors Z55 ys old as long as IBMDR criteria for donor selection are applied. Further data will be collected in order to reach a 10-y follow-up for each donor. Disclosure of Interest: None declared. P085 Hematopoietic Stem Cell Transplantation in Children Using Preimplantation Genetic Diagnosis for HLA-Matched Donor: a Turkish Multicenter Study ¨ ztu¨rk4, O. Gu¨rsel1, A. E. Ku¨rekc¸i1, A. Ku¨pesiz2, S. Anak3, G. O S. Aksoylar5, T. ˙Ileri6, B. Kus¸konmaz7, I. Eker1,*, M. Çetin7, G. T. Karasu8, Z. Kaya9, T. Fıs¸gın8, M. Ertem6, S. Kansoy5, M. A. Yes¸ilipek10 1 Pediatric Hematology, Gulhane Military Medical Faculty, Ankara, 2 Pediatric Hematology and Oncology, Akdeniz University, Antalya, 3Pediatric Hematology and Oncology, ˙Istanbul University, 4Pediatric Hematology and Oncology, Medikal Park Bahc¸elievler Hospital, ˙Istanbul, 5Pediatric Hematology and Oncology, Ege University, ˙Izmir, 6Pediatric Hematology and Oncology, Ankara University, 7Pediatric Hematology and Oncology, Hacettepe University, Ankara, 8Pediatric Hematology and Oncology, Bahc¸es¸ehir University Medical Faculty, ˙Istanbul, 9 Pediatric Hematology and Oncology, Gazi University, 10On Behalf of, Turkish Pediatric Bone Marrow Transplantation Study Group, Chair, Ankara, Turkey Introduction: Preimplantation genetic diagnosis (PGD) involves the diagnosis of a genetic disorder in embryo obtained through in vitro fertilization, selection of healthy embryos, and transferring them to the mother’s uterus. It has been used not only to avoid the risk of having an affected child, but also by using HLA matching together, it offers preselection of potential HLA-genoidentical healthy donor progeny for an affected sibling, who requires bone marrow
transplantation. Here, we share the hematopoietic stem cell transplantation (HSCT) results of 52 patients with benign or malign hematological diseases, metabolic diseases and immunodeficiencies, whose donors were their siblings borned with this technique in Turkey since 2008. To our knowledge, this is the largest case series in the literature performed to date about the results of the HSCT on the subject, which may thus provide valuable information for clinical outcomes of the transplantations with donors borned via this technique. Materials (or patients) and methods: Fifty two pediatric patients (23 female; 29 male) between 3-16 years old (median 8 years), underwent 53 HSCT at eleven different centers in Turkey, between February 2008 and January 2014. Their donors were their siblings borned via preimplantation HLA typing technique with or without mutation analysis. The most common indication for HSCT was thalassemia major (TM) (42 of all patients; 80%). Other indications for HSCT were acute lymphoblastic leukemia, acute myeloid leukemia, WiskottAldrich syndrome, juvenile myelomonocytic leukemia, Fanconi aplastic anemia, sickle cell disease and adrenoleukodystrophy. Stem cell source in all of the transplantations were bone marrow. In 37 of the transplantation procedures, umbilical cord bloods of the same donor were also used. Results: Except the two cases of TM with primary graft failure, 50 of 52 patients (96%) are primary disease free without any complication with a median follow-up period of 31 months (range 11–81 months). The median number of the IVF cycles, which was resulted with the birth of the donor was 2 (1-8) and the median time between the beginning of the first cycle and the transplantation was 3,7 (1-9) years. The median maternal age on the first IVF cycle was 30,3 (22-39) years. Median cost of the PGD/HLA typing and HSCT was 10.000 (1.600-480.000) and 35.000 (35.000-145.000) euros, respectively. The median total cost (HSCT plus PGD/HLA typing process) was 45.500 (38.000617.000) euros. Conclusion: Our experience showed successful HSCT results and reasonable cost rates for recepients using siblings who are the offsprings of pregnancies utilizing PGD and HLA matching technique. Transplant physicians should be aware of the feasibility and efficacy of the PGD/HLA typing method as well as the indications of its use. After establishing the indication for HSCT, a quick search for matched family donor (MFD) and successive matched unrelated donor (MUD) search in case no MFD available, should be performed. If there is no appropriate donor, either MFD or MUD, or HSCT from MUD carries significantly higher risks than the transplantation from MFD and if there is enough time in terms of primary disease, PGD/HLA typing technology should be considered and discussed with the family. However, it should be performed under strict supervision of national and local ethics committees. Disclosure of Interest: None declared. P086 Haploidentical stem cell transplantation with posttransplant Cyclophosphamide for patients with X-Linked Adrenoleukodystrophy and severe aplastic anemia: retrospective analysis I. Esteves1,*, J. Fernandes1, A. Ribeiro1, F. Santos1, A. Kondo1, C. Bonfim2, F. Kok3, J. Vargas1, L. Fernando Mantovani1, M. Rodrigues1, C. Bonet Bub1, F. Kerbauy1, N. Hamerschlak1 1 Hematology and Stem Cell Transplantation, Hospital Israelita Albert Einstein, Sao Paulo, 2Hematology and Stem Cell Transplantation, Hospital das Clinicas do Parana, Curitiba, 3 Neurology, Hospital das Clinicas de Sao Paulo, Sao Paulo, Brazil Introduction: X-linked adrenoleukodystrophy (X-ALD) and severe aplastic anemia (SAA) are non-malignant disorders. X-ADL is an inherited demyelinating disease caused by the deficiency of the ABCD1 gene and causes neurological disability during the first two decades of life. SAA is a rare acquired disease, commonly idiopathic and characterized by
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bone marrow failure. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only treatment that has shown to change the natural history of the X-ADL and is curative in patients with SAA. In the absence of a matched related or unrelated donor, haploidentical might be an option. Haploidentical HSCT using post-transplant cyclophosphamide (Cy) has been performed in malignant and non-malignant diseases Materials (or patients) and methods: We analyzed 8 patients with X-ALD and 7 patients with SAA treated with HSCT from haploidentical and post-transplant Cy from September 2011 to December 2014. Results: 8 patients with X-ALD (5-18 years) underwent haploidentical HSCT. Two patients received 2 allogeneic HSCT from different related donors and 1 patient received a second transplant after failure of a double cord blood transplant. Pretransplant MRI showed a median Loes score of X (range 2,518), all patients had neuropsychological evaluation with performance IQ above 90. Donors were the father (n ¼ 7), uncle (n ¼ 2) or brother (n ¼ 1). All patients received nonmyeloablative (NMA) regimen: fludarabine 150 mg/m2, Cy 29 mg/kg and total body irradiation (TBI) 2 Gy. Six patients also received rabbit antithymocyte globulin 4,5 mg/kg. Posttransplant immunosuppression consisted of Cy 50 mg/kg/d on days þ 3 and þ 4, tacrolimus and mycophenolate mofetil (MMF) starting on day þ 5. Seven patients engrafted and 1 patient had a primary graft failure and severe progression of disease. Two patients had secondary graft failure with progressive loss of donor chimerism (STR) and were successfully rescued with second haploidentical transplants using different related donors. Four patients had grade II-IV acute graft-versus-host disease and four patients had CMV reactivations (50%). One patient with grade III GVHD showed progression of neurologic symptoms of primary disease. Six patients are alive and engrafted from 3 to 11 months after transplant, with STR from 80 to 100% donor cells. The median of follow up of these patients was 424 days (102-789 days), the OS was 75% and the progression rate was 75%. We analyzed 7 patients with acquired SAA (7-39 years old) submitted to NMA with Flu/Cy/TBI associtated to MMF/tacrolimus/Cy as post transplant immunosuppression. The source was bone marrow for 6 patients and donors were mother (n ¼ 4) and siblings (n ¼ 3). All patients had neuthrophil engraftment and 1 had platelets graft failure and required thrombopoetin receptor agonist with response. Two (28.6%) patients had grade III-IV acute GVHD and chronic GVHD. There was no rejection and STR was 100% donor. The median of follow up was 355 days (111-651 days) and the OS was 71.4% and the DFS was 100%. Conclusion: Haploidentical HSCT with post-transplant Cy is an alternative for X-ALD and SAA lacking a suitable matched donor. Graft failure is still an obstacle that has to be prevented in NMA regimen. Patients with SAA have been submitted to IST before HSCT which has been a reason for low rate of graft failure when compared to X-ADL. Disclosure of Interest: None declared. P087 Haploidentical x Umbilical Cord blood Hematopoietic stem cell transplantation: a comparison between alternative donors I. Esteves1,2,*, F. Pires Santos Souza1, A. Alice Feitosa1, G. Fleury Perini1, R. Helman1, M. Afonso Torres1, A. Tiemi Kondo1, L. Nassif Kerbauy1, J. Folloni Fernandes1,3, F. Rodrigues Kerbauy1,2, N. Hamerschlak1 1 Hematology and Stem Cell Transplantation, Hospital Israelita Albert Einstein, 2Hematology and Stem Cell Transplantation, Escola Paulista de Medicina/ UNIFESP, 3Hematology and Stem Cell Transplantation, Instituto de tratamento do cancer Infantil/ ITACI, Sao Paulo, Brazil Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) from matched donors is used to treat malignant or
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non-malignant disorders. HSCT from Haploidentical donors and umbilical cord blood (UCB) can be an alternative in patients with no matched related donor (MRD) or matched unrelated donors (MUD). Materials (or patients) and methods: From April 2008 to July 2014, we analyzed patients that underwent haploidentical (n ¼ 42) and UCB HSCT (n ¼ 50). Results: Patients’ age ranged from 1-72 years old and most patients in the group UCB HSCT were younger than 18 years old. The median follow up time for UCB HSCT were 178,5 days (12-2183 days) and for haploidentical were 196 days (28-2333 days). In the haploidentical group, 23 patients had nonmalignant and 19 had malignants disorders and UCB group had 19 patients with non-malignant and 31 with malignant disorders. Patients in the UCB group received a nonmyeloablative conditioning regimen with fludarabine, cyclophosphamide (Cy) and TBI 200cGy in 24 patients (57%) and 24 patients (48%) were submitted to a myeloablative regimen (bussulfan and fludarabine or busulfan, fludarabine and thiotepa). The predominant source of cells in haploidentical group was bone marrow (n ¼ 39/ 92.8%) with a median total of infused nucleated cells x 108/Kg of 5.18 (4.367.75 x108/Kg). Patients submitted to UCB received a median total nucleated cell x 107/Kg of 9.5 (3.93 x 107- 4.12 x 107/Kg). Graft versus host disease (GVHD) prophylaxis consisted of tacrolimus/MMF plus post-transplant Cy in 36 (85.7%) patients submitted to haploidentical and calcineurin inhibitor plus MMF (68%) in UCB. Forty (80%) patients had neuthrophil engraftment in UCB group and 36 (85.7%) in haploidentical group. The median time of neuthrophil engraftment in UCB and haploidentical was 16 and 17 days respectively. 14.3% and 20% of patients that received haploidentical and UCB respectively had secondary graft rejection. Two (4.8%) patients developed hepatic synusoidal obstruction syndrome (SOS) in haploidentical group and 8 (16%) in UCB. Acute GVHD was observed in both groups: 28.6% in haploidentical and 46% in UCB. Chronic GVHD was similar in haploidentical and UCB groups (9.5% vs. 8%) respectively. The CMV reativation were similar in both groups (70%). The overall survival (OS) in haploidentical was 53.4% and 46.6% for UCB (P ¼ 0.055). Major cause of death in haploidentical and UCB group was infection in 63.6% vs. 73.9% respectively. The haploidentical and UCB groups relapse rate was 19% and 4% respectively. In multivariate analysis the graft failure and SOS had impact in OS in UCB HSCT (P ¼ 0.001) while graft failure had negative impact in OS in haploidentical group. The time of hospitalization at first year of HSCT was not different between groups. Conclusion: Graft failure was a serious complication in both groups, and conditioning regimen should be reviewed to improve the rates of engraftment. Haploidentical transplant with non-myeloablative regimens showed low rates of acute GVHD but it did not reduce graft failure. References: Chang YJ, Huang XJ. Haploidentical SCT: the mechanisms underlying the crossing of HLA barriers. Bone marrow transplantation 2014. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood 2013; 122(4): 491-8. Koh LP, Chao N. Haploidentical hematopoietic cell transplantation. Bone marrow transplantation 2008; 42 Suppl 1: S60-S63. Disclosure of Interest: None declared.
Graft-versus-host disease – clinical I P088 Platelet Gel for Treatment of Muco-Cutaneous Lesions Related to Graft-Versus-Host Disease: an Update from the Rome Transplant Network A. Picardi1,*, A. Di Veroli 1, A. Ferraro2, A. Lanti2, L. Cudillo1, R. Cerretti1, G. De Angelis1, B. Mariotti1, A. Biagi1, V. Molinari1, F. Trenta1, G. Adorno2, C. Bonchi2, W. Arcese 1 on behalf of Rome Transplant Network 1 Hematology Division - Stem Cell Transplant Unit, 2Transfusion Medicine , TOR VERGATA UNIVERSITY OF ROME, Rome, Italy Introduction: Platelet gel (PLT-gel) has been successfully used for the treatment of muco-cutaneous lesions associated with acute or chronic graft versus host disease (a/cGVHD) in patients undergoing an allogeneic transplant for haematological disorders (Platelet gel for treatment of mucocutaneous lesions related to graft-versus-host disease after allogeneic hematopoietic stem cell transplant. Picardi A, et al Transfusion. 2010 Feb; 50(2): 501–506). The rationale of its use in the transplant setting is based on its well known efficacy in facilitating tissue regeneration of diabetic/ surgical wounds through the releasing of growth factors such as bFGF and PDGF. Herein, we report the results in treating mucocutaneous GVHD associated ulcers with PLT-gel by extending our experience to 20 patients. Materials (or patients) and methods: Twenty patients with median age of 52 (range 38-75) and transplanted for hematological malignant disease developed acute (n ¼ 9) or chronic (n ¼ 11) GVHD complicated by muco-cutaneous lesions: skin grade I (n ¼ 4), skin grade II (n ¼ 5), oral mucosa (n ¼ 10), skin þ mouth (n ¼ 1). The patients were treated with local PLT-gel obtained from allogeneic hemocomponents processed with Vivostat System and applied over the lesions through spray diffusion. At time of PLT gel treatment, 17 out of 20 patients were on immunosuppressive therapy for their GVHD. The response rate was defined as ‘‘complete’’ in cases of total re-epithelization of ulcers and as ‘‘partial’’ when the appearance of granulation tissue on the target sites was not followed by the total re-epithelization, in presence of reduction size of 50% or more of the lesions. Results: Following the second PLT-gel application, the pain disappeared in all cases and the granulation tissue was observed in all patients with grade II lesions. One patient died too early of multiorgan failure and was not evaluable for the response. Fourteen out of 19 (74%) patients achieved a complete response after a median of 5 (range, 1-15) PLT-gel applications, the response was considered partial in 5 (26%) patients, who received a median of 5 (range, 3-9). Neither side effects nor local infections related to the PLT-gel therapy were documented. Conclusion: PLT-gel therapy by inducing pain discontinuation allows strong reduction of the analgesic drug administration and highly ameliorates the quality of patient life. Overall, this update confirms our previous data supporting safety and efficacy of PLTgel in the treatment of muco-cutaneous GVHD lesions. Disclosure of Interest: None declared. P089 Does defibrotide prophylaxis decrease the risk of acute graft versus host disease following allogeneic hematopoietic cell transplantation? A retrospective analysis A. I. E. Tekgu¨ndu¨z1,*, A. H. H. Kaya1, S. C. Bozdag2, S. Kocubaba1, O. Kayıkcı1, S. Namdaroglu1, B. Ugur1, F. Altuntas1 1 Hematology and bone marrow transplant unit, Ankara Oncology Hospital, 2Hematology and bone marrow transplant unit, Ankara University Hospital, Ankara, Turkey Introduction: Both acute (aGvHD) and chronic (cGvHD) graft versus host disease are important complications following allogeneic hematopoietic cell transplantation (allo-HCT)
decreasing survival and quality of life. Defibrotide is widely used agent for prophylaxis and treatment of hepatic venoocclusive disease (VOD). There are some preliminary evidence that defibrotidemay also have a role in decreasing risk of GvHD by preventing tissue damage. We aimed to investigate the role of defibrotide prophylaxis on development of aGvHD. Materials (or patients) and methods: All consecutive patients who received allo-HCT between January 2009 and January 2014 were included in the study. Medical records of patients were retrospectively analyzed.Patients were analyzed in two time frames according to availability of defibrotide. Patients who were transplanted in January 2009–October 2011 (conventional arm) received only low molecular weight heparin (LMWH), ursodeoxycholic acid (UDCA) and N-acetylcysteine (NAC) in terms of VOD prevention. All consecutive patients who undergone allo-HCT in November 2011–January 2014 period (defibrotide arm) received 10 mg/kg intravenous defibrotide prophylaxis for two weeks (from D þ 1 to D þ 14) in addition to conventional approach. Descriptive statistics are presented as median and range. A p value below 0.05 was considered to be statistically significant. Results: A total of 148 patients (conventional arm (n: 58); defibrotide arm (n:90) participated in the study. Median age of study cohort was 36 (17-58).The study groups were comparable in terms of gender, age, intensity of conditioning regimen, GvHD prophylaxis, use of TBI in conditioning and stem cell source. But significantly more patients in defibrotide arm (matched unrelated donor: 5, haploidentical donor: 11) received unrelated or mismatched donor HCT compared to conventional arm (matched related donor: 57; 1 Ag mismatched related donor: 1)(p:0.016). Acute GvHD was observed in 36 (40%) and 27 (47%) patients on defibrotide and conventional arms, respectively (p.0.431). Grade Z2 aGvHD were also similar in treatment arms (defibrotide arm: 15.5%; conventional arm:19%) (p:0.549). Conclusion: Our results indicate that defibrotide prophylaxis seem not to confer an advantage over conventional arm in decreasing aGvHD rate. Before drawing firm conclusions about the role of defibrotide on aGvHD development, some important facts should be bear in mind. First of all, the optimal dose, timing and duration of defibrotide in VOD prophylaxis in adult patients are undefined. The second point is that more patients on defibrotide arm received HCT from unrelated or mismatched related donors. Therefore it can be argued that higher dose and longer duration of defibrotide prophylaxis could reduce aGvHD rate in defibrotide arm if both groups were similar in term of HLA match. On the other hand, it can also be speculated that effect of defibrotide on GvHD may be seen later following HCT, by decreasing cGvHD rate. We hope that, properly designed prospective trials will define the role of defibrotide in GvHD prevention, if any. Disclosure of Interest: None declared. P090 Extracorporeal photopheresis in patients with acute and chronic GVHD modulates the cellular immune system A. Schmitt1,*, L. Wang1, A. Hu¨ckelhoven1, J. Hong1, A. D. Ho1, P. Dreger1, W. Kru¨ger2, S. Michael1 1 University Clinic Heidelberg, Heidelberg, 2University of Greifswald, Greifswald, Germany Introduction: Extracorporeal photopheresis (ECP) constitutes a promising therapeutic approach for both steroid-refractory acute and chronic graft-versus-host disease (GvHD). The mechanism of action in ECP might be elicited by modulation of lymphocyte subsets. We therefore analyzed the clinical and immunological effects during ECP therapy in 10 patients. Materials (or patients) and methods: Six patients with acute GvHD of the gut 1I-1IV received intensive ECP therapy over 12 weeks followed by ECP treatments at a lower frequency for up to 6 months. Despite therapy with steroids, calcineurin inhibitors, cyclosporine A and/or mycophenolate mofetil and pentostatin the patients suffered from extensive diarrhea due
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to histologically proven GvHD with negative microbiological results. Two patients with chronic GvHD of the skin and two patients with cGvHD of the lung with up to a triple immunosuppressive therapy received ECP treatment every 2 to 4 weeks. Immunological analyses of B-, T-, NK(T) cells and myeloid derived suppressor cells were performed. Results: Four of six patients with acute GvHD of the gut 1II-1IV showed a dramatic clinical improvement with reduction of stool volume thus allowing a tapering-out of steroids while on intensive ECP therapy. In parallel immunomonitoring demonstrated an increase of CD4 þ CD25hiFoxP3 þ regulatory T cells and a decrease of CD8 þ CD45RA þ CCR7- effector T cells. Patients with acute GvHD only 1I of the gut did not seem to profit from intensive ECP treatment. Patients with chronic GvHD kept clinically stable on ECP therapy with slow improvement over the time which might require even ECP treatment every four weeks over a period of several years. Conclusion: Intensive ECP treatment in acute GvHD might evoke a change in frequency of effector and regulatory T cells leading to an immune modulation with a subsequent chance to reduce immunosuppressive medication. Taken together, immunomonitoring of T cell subsets can help to elucidate the mechanism of action and might predict the feasibility to taper of immunosuppressive therapy. Disclosure of Interest: None declared. P091 Reduced-intensity conditioning (RIC) with high-dose rituximab and allogeneic stem cell transplantation for relapsed CD20 þ lymphomas: good disease control with low incidence of chronic GVHD A. Dodero1,*, B. Sarina2, G. Milone3, F. Patriarca4, A. Bosi5, A. Dominietto6, L. Farina1, R. Foa’7, E. Terruzzi8, F. Onida9, A. Rambaldi10, P. Corradini1 on behalf of on behalf of GITMO, Gruppo Italiano per il Trapianto di Midollo Osseo 1 Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano , 2Istituto Clinico Humanitas, Rozzano, 3Hematology, Ospedale Ferrarotto, Catania, 4Hematology, Azienda Ospedaliera Universitaria, Udine, 5Hematology, University of Firenze, Firenze, 6 Hematology, Ospedale San Martino, Genova, 7Hematology, Universita’ La Sapienza, Roma, 8Hematology, Ospedale San Gerardo, Monza, 9Hematology, Universita’ di Milano , Milano, 10Hematology, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy Introduction: The best conditioning regimen before allogeneic stem cell transplantation (alloSCT) for relapsed B-cell lymphomas remains to be clarified. The GITMO conducted a multicenter phase II trial (EUDRACT 2007- 003657- 87) to evaluate the effect of high dose rituximab before alloSCT. The primary objective of the study was the PFS at 2-years. Materials (or patients) and methods: 101 patients (pts) affected by relapsed/refractory B-cell lymphomas [n ¼ 57 indolent: 32 follicular lymphomas (FCL), 24 Chronic lymphocytic leukemia (CLL); n ¼ 44 aggressive lymphomas: 24 diffuse large B-cell lymphomas (DLBCL), 20 mantle cell lymphomas (MCL)] were enrolled. Pts received the preparative conditioning regimen consisting of thiotepa/cyclophosphamide/fludarabine and highdose rituximab (500 mg/m2 on day -6). GVHD prophylaxis consisted in cyclosporine and mini-methotrexate; ATG was added to those allografted from class I antigen mismatched sibling or unrelated donors. The proportions of transplantation from matched-related, mismatched-related, matched-unrelated, mismatched-unrelated were 53%, 1%, 34%, 12%, respectively. The majority of the pts were chemosensitive to the last salvage regimen (n ¼ 40 CR, n ¼ 45 PR, 84%). Results: With a median follow-up of 31 months (range,6-79.5 months), the estimated 2-years and 3-years OS and PFS were 74% and 62%, 65% and 48%, respectively. A similar 2-years OS was observed in pts who underwent sibling versus unrelated transplantation (71% versus 63%, P ¼ 0.6). In pts with a diagnosis of FCL, CLL, MCL and DLBCL the 3-years OS were 84%, 67%, 64%, 55%, respectively. The cumulative incidence (CI) of non-relapse mortality (NRM) at 2-years was 13% and
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20% in pts allografted from matched related versus unrelated donors (P ¼ 0.22), respectively. The CI of acute grade II-IV GVHD was not different between sibling and unrelated donors (27% versus 24%, P ¼ 0.91). There was a trend for a higher chronic GVHD CI in pts allografted from matched siblings as compared to unrelated donors (55% versus 29%, P ¼ 0.08). The overall CI incidence of extensive chronic GVHD and of systemic corticosteroid requiring chronic GVHD were 24% and 36% in recipients of sibling graft and 10% and 10% in recipients of unrelated grafts. The survival free of disease and chronic GVHD were 39% at 3 years with the majority of events occurring in the first 24 months post allografting. At 1 year post alloSCT, the administration of Rituximab did not impact T- but only B-cell immune reconstitution (median values CD4 þ , CD8 þ , CD19 þ were 499/ucl, 394/ucl, 88/ucl, respectively). Conclusion: RIC with rituximab is an interesting transplantation platform. In fact, we observed: i) long-term survival in heavily pretreated pts; ii) limited CI of extensive chronic GVHD and low use of systemic corticosteroid. This strategy should be compared with others transplantation platform used for relapsed CD20 þ lymphomas. Disclosure of Interest: None declared. P092 Oral chronic graft-versus-host disease outcomes and resource utilization: a subanalysis from the chronic GVHD Consortium A. Yuan1,2,*, X. Chai3, F. Martins4, C. Cutler5, S. Lee3, N. Treister1,2 1 Division of Oral Medicine and Dentistry, Brigham and Women’s Hospital, 2Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, 3Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, United States, 4Department of Oral Pathology, School of Dentistry, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 5Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, United States Introduction: Chronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. The objective of this study was to evaluate the predictive validity of oral cGVHD consensus assessments for clinical outcomes, including use of clinical services and prescription therapies across institutions with and without oral medicine (OM) centers. Materials (or patients) and methods: Patients enrolled in the cGVHD Consortium with oral cavity involvement were included, and additional mouth-specific cGVHD-associated outcome data was abstracted via retrospective chart review across five transplant centers. Utilizing longitudinal oral cGVHD ancillary therapy data, multivariable linear models were fit correlating mouth-related outcomes with ancillary care category. Results: A total of 79 patients with 656 documented oral visits were observed for a median duration of 7.1 months. Overall mouth scores were ‘‘mild’’ (54%), with ‘‘moderate’’ (15%) and ‘‘severe’’ (4%). The National Institutes of Health (NIH) oral cGVHD severity score corresponded the most with mouth involvement prompting treatment (o0.001). Ancillary therapies for oral cGVHD management were prescribed for 56% of patients for a median duration of 6.9 months. Other oral medicine agents (including sialogogues) were prescribed for 46% of patients, with the majority at OM centers. Compared to patients that did not receive oral ancillary therapy, patients treated with topical therapy were five times more likely to have NIH mouth involvement (Po0.001, OR 5.1, 95% CI: 2.212.0), and almost three times more likely to have mouth pain (P ¼ 0.01, OR 2.6, 95% CI: 1.1-5.8). The odds of receiving topical therapy from OM specialists were estimated to be 52% higher than from transplant physicians (P ¼ 0.03), and 39% higher than from midlevel providers (P ¼ 0.07). Conclusion: Utilization of clinical and prescription approaches for oral cGVHD therapy varies across institutions. Ancillary
therapies were prescribed for the majority of patients, who were more likely to have NIH mouth involvement and mouth pain. OM specialists were more likely to prescribe topical therapy than other providers. References: Rationale and design of the chronic GVHD cohort study: improving outcomes assessment in chronic GVHD. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 2011; 17: 1114–1120. Disclosure of Interest: None declared. P093 Impact of T-cell depletion techniques on post transplant graft versus host disease after allogeneic HSCT with myeloablative conditionning A. P. Grandjean1,*, F. Simonetta1, S. Masouridi-Levrat1, Y. Tirefort1, Y. Beauverd1, A. Simon1, M. Ansari2, C. Dantin1, E. Roosnek1, Y. Chalandon1 1 hematology, 2onco-hematology pediatrics, Geneva University Hospitals, Geneva, Switzerland Introduction: Graft versus host disease (GvHD) is known to be a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloHSCT). Different strategies are developed in attempt to reduce GvHD incidence. T-cell depletion using in vivo ATG or/and in vitro alemtuzumab represent potential strategies. In the present study we evaluated the incidence of GvHD using T-cell depletion in the bag with or without ATG administration as compared to non-Tcell depleted alloHSCT. Materials (or patients) and methods: We performed a retrospective study on 256 patients transplanted at our center from August 1997 to October 2013 with an alloHSCT from identical siblings or HLA 10/10 matched unrelated donors after myeloablative conditionning. Median patients age was 41 years (range 1-63), median EBMT score was 2 (range 0-6). One hundred nine patients received partial TCD (pTCD) grafts, consisting of in vitro alemtuzumab incubation before infusion followed on day þ 1 by an add-back of donor T CD3 þ cells. Thirty-four patients received in vivo ATG administration, consisting of either Thymoglobuline (Genzyme) or ATG-F (Fresenius). Sixty-nine patients had combined ATG and pTCD. Forty-four patients had no TCD. Log-rank Mantel-Cox test was used to compare the 1-year cumulative incidence of clinically significant, grade 2-4 acute GvHD (aGvHD) and 1- and 5-year cumulative incidence of chronic GvHD (cGvHD) as well as 5year transplant related mortality (TRM), relapse rate (RR) and overall survival (OS) in patients receiving partially T-cell depleted alloHSCT with or without ATG administration, with patients receiving non-T cell depleted alloHCST. Results: With a median follow up of 52 months (range 0.07220), grade 2-4 acute GvHD 1-year cumulative incidence was significantly decreased in patients receiving pTCD grafts combined with ATG (11%; Po0.0001) compared to patients receiving non-pTCD alloHSCT (58%). No effect of pTCD (37%, P ¼ 0.0537) grafts or ATG treatment alone (53%, P ¼ 0.9172) was observed on acute grade 2-4 GvHD incidence. Similarly, no significant effect on 1-year and 5-year cumulative incidence of cGvHD was observed for either ATG (15%, P ¼ 0.1132 and 15%, P ¼ 0.0902 repectively) or pTCD alone (27%, P ¼ 0.2631 and 28%, P ¼ 0.1942) compared to non-pTCD (40 % and 44%). Conversely, we observed a highly significant reduction in 1year and 5-year cumulative incidence cGvHD in patients receiving ATG combined with pTCD (11%, P ¼ 0.0010 and 11%, P ¼ 0.0004). 5-year TRM and RR were not significantly different between groups with TRM 8% (P ¼ 0.3569) for ATG and pTCD, 18% (P ¼ 0.6357) for pTCD, 28% (P ¼ 0.1388) for ATG alone compared to 12% for non TCD and RR respectively of 50% (P ¼ 0.7719), 45% (P ¼ 0.3511) and 28% (P ¼ 0.1388), compared to 51%. 5-year OS was found significantly worse with ATG alone (33%, P ¼ 0.0061) with no difference with ATG and pTCD (69%, P ¼ 0.4448) and pTCD alone (61%, P ¼ 0.7743) compared to non-TCD.
Conclusion: pTCD combined with ATG appears to decrease the incidence of acute and chronic GvHD in patients receiving allogeneic HCST with a myeloablative conditioning from sibling or HLA 10/10 matched unrelated donors without affecting 5-year TRM, RR or OS. These results extend our knowledge of effect of T-cell depletion strategies on transplant-related morbidity and mortality. Our findings need to be confirmed in larger prospective studies. Disclosure of Interest: None declared. P094 Cyclosporine Concentration and Incidence of Acute Graft versus Host Disease after Allogeneic Stem Cell Transplantation B. Josu1,*, L. Arrue2, A. Sainz2, I. Urreta3, N. Caminos2, E. Perez2, J. J. Ferreiro2, M. Artola2, I. Ceberio2 1 pharmacy, 2Hematology, 3Biostatistics and Epidemiology, Hospital Universitario Donostia, Donostia, Spain Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for hematological disorders. The combination of Cyclosporine-A (CsA) and metrotexate (Mtx) is the standard inmunosupresive prophylaxis in majority of transplant centers. We report our single-center retrospective analysis of 54 consecutive patients that underwent allo-HSCT with the aim to evaluate the impact of CsA concentration during the first month after allo-HSCT on the incidence of acute graft versus host-disease (GVHD). Materials (or patients) and methods: Between 2009-20013 fifty four patients ( M/F 28/26), median age 50 years (range: 21-72) were transplanted for Myeloid malignancies (78%), Lymphoid malignancies (18%) and Aplastic anemia (6%). at our center. Fourteen male patients underwent a transplant from a female donor. Conditioning regimen was myeloablative (M) in 26 patients and reduced intensity (RIC) in 28. All patients received CsA and Mtx as GVHD prophylaxis regimen. Donors were HLA matched (51) and mismatched (3) related. 85% of the patients received peripheral blood mobilized grafts. Patients who engrafted were evaluable for acute GVHD (aGVHD), and patients surviving at least 100 days were evaluable for chronic GVHD (cGVHD) based on NIH consensus criteria. The mean weekly CsA concentration was calculated using the different concentrations measured during a defined week. CsA doses were adjusted based on drug trough levels between 200-300 ng/mL and toxic side effects like nephrotoxicity. The probability of overall survival (OS) was calculated by the Kaplan-Meier method. Comparison of the groups with grade 0I acute GVHD (aGVHD) and grade II-IV acute GHVD was carried out using the Fisher test for categoric variables and the U-Man Whitney test for continuous data. Results: The median follow up was 1.74 years (range: 0.17-5.64). The median concentration of CsA in the blood at 1, 2, 3 and 4 weeks after allo-HSCT were 270 ng/mL (range: 144-593), 299 ng/mL (range: 165-508), 315 ng/mL(range: 117-814) and 268 ng/mL(range 62-650), respectively. The OS and relapse rates at 1 year after allo-HSCT were 81% and 22%, respectively. Grade II-IV aGVHD occurred in eleven patients (20%) at a median of 51 (range: 14-189) days after allo-HSCT. The incidence of severe grade 3-4 aGVHD and chronic GVHD were 6% and 39%, respectively. Univariate analysis of risk factors for grade II-IV aGVHD developments showed no statistical association between grade II-IV aGHVD and risk factors for aGVHD. Conclusion: It has been already reported that CsA concentrations should be monitored after allo-HSCT, because low CsA concentrations can increase risk of aGHVD. In our study we could not demonstrate a relationship between CsA trough blood concentrations and the risk of aGVHD. This might be because of the relatively limited size of our study
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population and the low incidence of overall and severe aGHVD in our cohort. Some authors have also failed to demonstrate a relationship between trough concentrations and aGVHD. It is not clear which CsA blood-level monitoring is the most appropriate. In the setting of solid organ transplant, some studies have shown that a peak-level (2 hours after and oral dose-C2) is the optimal method of monitoring CsA. Further prospective clinical trials are necessitated to evaluate the most appropriate way of CsA monitoring and its relationship with development of aGVHD. Disclosure of Interest: None declared. P095 Extracorporeal Photopheresis (ECP) As Second Line Treatment For Refractory Graft Versus Host Disease (GVHD): Preliminary Single Centre Experience M. Gonza´lez-Pardo1, B. Aguado1,*, C. Fernandez-Arandojo1, E. Acun˜a1, S. Guijarro1, A. Alegre1 1 Hematology, Hospital Universitario de La Princesa, MADRID, Spain Introduction: Steroid-refractory GVHD is a common cause of morbidity and mortality and remains a challenging therapeutic problem after allogeneic hematopoietic stem cell transplantation (HSCT). There is currently no standard of care for this complication. ECP is an immunomodulatory therapy which has been used in the treatment of acute and chronic GVHD. We present our experience of ECP as second line treatment option for GVHD post-HSCT. The aim of this analysis was to evaluate the clinical effect of ECP, its impact on intensity of immunosuppressive therapy, safety and tolerance of the procedure. Materials (or patients) and methods: Since May 2012 we have performed 197 ECP procedures with the Therakos Cellexs device that is an integrated ‘‘on line’’ system. 11 patients are evaluable with median age 55y (38-65) and weight 61kg (38-80) presented with acute (4 patients) or chronic (7 patients) therapy-dependent/refractory GVHD. Allogeneic HSCT was myeloablative in 9 cases and reduced intensity conditioning in two cases. Six patients were diagnosed of acute myeloid leukemia, two myelodisplastic syndromes, one acute lymphoblastic leukemia, one chronic lymphocytic leukemia and one non-Hodgkin’s lymphoma. The transplant was from an unrelated donor in 6 cases and 5 from a sibling donor, all of them HLA matched except for one with a missmatch in HLA A locus. All patients but one (with exclusive skin presentation) had multiorgan involvement, all suffered from skin changes in combination with liver, lung, oral or ocular disease. As first line treatment they all received esteroids plus cyclosporine or mycophenolate mofetil. Median ECP per patient was 11 (4-43). ECP procedures were performed for two consecutive days, in initial phase weekly (in those with aGVHD), or every two weeks (cGVHD) and then monthly according to clinical response. Median of ECP treatment duration was 3 months (1-30). Response was evaluated by clinical assessment and reduction in immunosuppression. Results: In the cGVHD group response was complete in 3/7 (43%), partial in 4/7 (57%), ECP led to Z 50% improvement in symptoms and signs of cGVHD and significant reduction or even withdrawal of esteroids was observed. The first response was seen in skin and mucosal symptoms. In the aGVHD group 2/4 patients (50%) had complete remission (one died of pneumonia) and 2/4 (50%) did not response. The tolerance to the procedure was excellent and the unique complications seen were related with two central venous catheter infections. Conclusion: In our preliminary experience, the ECP is associated with excellent tolerance, significant response rates and successful reduction of steroids in patients with cGVHD. More experience is required to assess response on aGVHD patients. References: Dignan FL et al. Diagnosis and management of chronic graft-versus-host disease. British Journal of Haematology, 2012; 158: 46–61.
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Merlin E et al. Extracorporeal photochemotherapy as second or first line therapy of acute GVHD?. Bone Marrow Transplantation 2010; 45: 963–965. Disclosure of Interest: None declared. P096 Graft-versus-host disease prophylaxis with high-dose post-transplantation cyclophosphomide in unrelated hematopoetic stem cell transplantation O. Pirogova1, I. Moiseev1, V. Vavilov1, O. Slesarchuk1, S. Bondarenko1, B. Afanasyev1,* 1 R.M. Gorbacheva Memorial Institute of Children Hematology, Oncology and Transplantation, First Saint-Petersburg State Medical University named I.P. Pavlov, Saint-Petersburg, Russia, Saint Petersburg, Russian Federation Introduction: Based on significant improvement in outcomes of haploidentical hematopoetic stem cell transplantation (HSCT) using post-transplantation cyclophosphamide (Cy), we conducted a pilot study to evaluate if this strategy could be translated to unrelated HSCT. Materials (or patients) and methods: The study group (Cy group) included 50 adult patients undergoing unrelated peripheral blood HSCT. Graft-versus-host disease (GVHD) prophylaxis for the Cy group consisted of Cy 50 mg/kg/day on day þ 3 and þ 4, tacrolimus (Tac) starting on day þ 5 and mycophenolate mofetil (MMF) from day þ 5 until day þ 35. Control group was a historical control of 176 patients with antithymocyte globulin (ATGAM) 60 mg/kg, Tac and MMF, or short-course methotrexate, as GVHD prophylaxis. Detailed patients’ characteristics and analysis of groups’ differences are presented in table 1. Results: The median follow-up was 155 days (range, 14 - 493) for the Cy group and 348 days (range, 5 - 1693) for the Control group. Engraftment was achieved in 92% vs 89.2% (P40.05) in the Cy and control groups respectively. In univariate and multivariate analyses Post-transplantation Cy was associated with significantly reduced incidence of grade II-IV (16% vs 46.6%, HR ¼ 0.242, 95% CI 0.120-0.516, P ¼ 0,0001) and grade III-IV (2% vs 23,3%, HR ¼ 0.053, 95% CI 0.007–0.387, P ¼ 0,004) acute GVHD. The cumulative incidence of chronic GVHD was not significantly different between groups (22% vs 39.3% P ¼ 0.27), but in the Cy group there was a significant reduction of severe chronic GVHD (0% vs 20.8%, P ¼ 0.005; the NIH global severity scoring of cGVHD). As a result there was significant reduction of 1-year nonrelapse mortality (10% vs 31.8%, HR ¼ 0.363, 95% CI 0.140–0.942, P ¼ 0.037), and improvement in 1-year overall survival (80% vs 53.4%, HR ¼ 0.448, 95% CI 0,227-0,886, P ¼ 0.021) and event-free
survival (74% vs 43.2%, HR ¼ 0.370, 95% CI 0.202–0.677, P ¼ 0.001). Moreover, the anti-leukemic effect of HSCT was not compromised and 1-year relapse rate was not different between groups (16% vs 24.4%, P ¼ 0.4). The median time to achieve an unsupported absolute neutrophil 4500 and platelet count 425 was not statistically different: 21 (range, 11-33) and 14 (range, 1039) days versus 21 (range, 8-70) and 14 (range, 8-87) days, respectively (P40.05). The toxicity of two GVHD prophylaxis regimens was comparable. There was a trend for a lower probability of fungal infections in the first 6 months posttransplantation in the Cy group (8% vs 18,2%,P ¼ 0,09), but the incidence of viral cystitis was significantly higher (22% vs 10.8%, HR ¼ 2.352, 95% CI 1.110-4.982, P ¼ 0.026). Conclusion: With relatively short follow-up post-transplantation Cy is a promising GVHD prophylaxis for unrelated HSCT. Larger studies with longer follow-up are required to confirm these preliminary results. Disclosure of Interest: None declared. P097 Urinary proteomics for aGvHD-prediction compared to ELISA for aGvHD-related proteins C. Human1,*, J. Metzger2, S. Ehrlich1, P. Schweier1, I. Tu¨ru¨chanow1, A. Ganser1, C. Falk3, E. M. Weissinger1 1 Hematology, Hemostasis, Oncology and Stem cell transplantation, Hannover Medical School, 2mosaiques GmbH, 3Integrated Research and Treatment Centre Transplantation – IFB-Tx, Hannover Medical School, Hannover, Germany Introduction: Graft-versus-host disease (GvHD) is a major complication after hematopoietic stem cell transplantation (HSCT). Prediction of aGvHD can be achieved by monitoring urine for excretion of peptides specific for aGvHD (aGvHDMS17) (1). In an effort to harmonize aGvHD prediction/ diagnosis, we analysed plasma and urine in parallel in more than 60 patients using an ELISA for published proteins(2) and Multiplex analysis of the angiogenesis panel (3). Materials (or patients) and methods: The test set consisted of 11 patients with and 12 without aGvHD, validation was performed on 31 patients. Plasma and urine were collected prospectively from 31 patients (1). While urine can be used as is and data can be normalzied using internal standard
peptides, plasma has to be serial diluted prior to analyses. Two peptides, CD99 and beta-2-microglobulin (b2m) can also be detected in plasma by ELISA. The ELISA-panel consisted of Elafin, IL-2 receptor alpha (IL-2sRa), REG-3a, ST2 and soluble TNF RI; IL-8 and hepatocyte growth factor (HGF) were analysed by multiplex and the outcome of all assays was compared. Results: While aGvHD_MS17 predicted pending aGvHD prior to the clinical diagnosis, most of the plasma biomarkers did not. In the test set IL-2R alpha, ST2 and sTNF RI, beta2-m and CD99 seemed predictive/diagnostic for aGvHD based on receiver operated characteristic (ROC) curves and areas under the curve (AUC) with more than 80% (Po0.01). CD99 (whole protein) was specific for chronic GvHD and could distinguish between patients with acute and chronic GvHD and those without with high sensitivity (70%) and specificity (93%) and AUC was 85% (P ¼ 0.0007). However, in the validation set most of the markers (especially Elafin, REG3-alpha) did not reach significance for prediction or even diagnosis of aGvHD at the time of biopsy proven aGvHD. Both markers are organ-specific – but limiting the analysis to patients with GvHD of the intestine (REG3-alpha) or skin (Elafin) did not increase sensitivity or specificity, albeit the number of patients analysed is still small. aGvHD_MS17 was positive for pending aGvHD, but none of the ELISA markers were reliable for diagnosis (cut off indicated by dotted line). Conclusion: Prediction of pending aGvHD using aGvHD_MS17 is possible with a sensitivity of 82% and specificity of 77% for aGvHD (1). In contrast, plasma biomarkers in our validation set lack sensitivity and specificity. Two biomarkers, CD99 and b2m could not be used to predict aGvHD-development when analysed in plasma. This indicates that the particular fragments excreted in urine are predictive, not changes in plasma protein levels. Taken together our results demonstrate that plasma monitoring of patients post-allogeneic HSCT seems less reliable and more time consuming and costly, as previously envisioned, than urinary proteomics for the prediction of aGvHD. References: 1. Weissinger EM, Metzger J, Dobbelstein C, et al. Leukemia 2014; 28(4): 842-852. 2. Paczesny S, Krijanovski OI, Braun TM, et al. Blood 2009; 8; 113(2): 273–278. 3. Luft T, Dietrich S, Falk C, et al. Blood 2011; 11;118(6): 1685– 1692.
[P097]
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Disclosure of Interest: C. Human: None declared, J. Metzger Employee of: mosaiques GmbH, S. Ehrlich: None declared, P. Schweier: None declared, I. Tu¨ru¨chanow: None declared, A. Ganser: None declared, C. Falk: None declared, E. Weissinger: None declared P098 The impact of donor specific anti-HLA antibodies on the outcome of allogeneic stem cell transplantation C. Kekik1,*, I. Yonal-Hindilerden2, F. Savran-Oguz1, S. Usta1, S. Temurhan1, D. Batu-Demir2, S. Kalayoglu-Besisik2, M. Aktan2, D. Sargin3 1 Istanbul University Istanbul Medical Faculty, Department of Medical Biology, 2Istanbul University Istanbul Medical Faculty, Department of Internal Medicine, Division of Hematology, 3 Medipol University, Department of Internal Medicine, Division of Hematology, Istanbul, Turkey Introduction: Graft failure and graft-versus-host disease (GVHD) are important life-threatening complications after allogeneic hematopoietic stem cell transplantation (AHSCT). In recent studies, GVHD and graft failure have been associated with the presence of donor specific anti-HLA antibodies in patients undergoing AHSCT from HLA-mismatched donors. We aimed to examine the impact of donor specific anti-HLA antibodies detected after AHSCT on the development of acute/chronic GVHD and graft failure. Materials (or patients) and methods: Anti-HLA antibodies were analysed by ELI˙SA/LUMINEX in a total of 74 patients (F/ M:30/44), who were transplanted at the Adult Hematopoietic Stem Cell Transplantation Unit of Istanbul Medical Faculty between 1996 and 2013. Results:
Anti-HLA class I (þ) Anti-HLA class II (þ)
aGVHD (þ) n:20
aGVHD (-) n:54
P value
cGVHD (þ) n:23
cGVHD (-) n:51
P value
4
7
40.05
1
10
40.05
3
4
40.05
1
6
40.05
12.1% of patients had an unrelated donor (10/10 matches, n ¼ 4; 9/10 matches, n ¼ 5) and the remaining patients had a matched sibling donor. Anti-HLA Class I antibodies were detected in 11 of 74 (14.8%) patients, 63% of whom were female. Anti-HLA Class II antibodies were detected in 7 of 74 (9.4%) patients, 71% of whom were female. 5 patients (6.7%) showed the copresence of anti-HLA Class I and II antibodies. The frequency of anti-HLA Class I antibodies was significantly higher than the copresence of anti-HLA Class I and II antibodies (14.8% and 6.7%, respectively; P ¼ 0.04). The frequency of antiHLA antibodies was significantly higher in female patients. Acute GVHD and chronic GVHD were observed in 27% (20/74) and 31% (23/74) of patients, respectively (Table 1). Ten of 74 patients (13.5%) died after AHSCT. Four of the patients carrying anti-HLA Class I antibodies and 2 of the patients showing the copresence of anti-HLA Class I and II antibodies died. Mortality rate was significantly higher in patients carrying anti-HLA Class I antibodies compared to those without antibodies (P ¼ 0.0361). There is no significant difference in mortality rate between patients with and without anti-HLA Class II antibodies (P40.05). Presence of anti-HLA antibodies did not contribute to development of acute/chronic GVHD and graft failure (P40.05). Conclusion: Our data demonstrate anti-HLA Class I antibodies have a significant impact on mortality. Previous studies have shown that presence of anti-HLA antibodies is associated with GVHD and graft failure in HLA-mismatched AHSCT. Our study aimed to evaluate the impact of anti-HLA antibodies on AHCST outcome regardless of HLA matching and consequently donor specific anti-HLA antibody status. In conclusion, our data point
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out that anti-HLA antibodies detected after AHSCT have no significantly effect on the development of GVHD and graft failure. Moreover, our results imply that anti-HLA Class I antibodies may have an influence on overall survival. We suggest that anti-HLA antibodies may play an important role on the outcome of HLA-matched AHSCT as demonstrated previously for HLA-mismatched AHSCT. Further studies including larger number of patients are warranted. References: 1- Taniguchi K, Yoshihara S, Maruya E, Ikegame K, Kaida K, Hayashi K, Kato R, Inoue T, Fujioka T, Tamaki H, Okada M, Onuma T, Fujii N, Kusunoki Y, Soma T, Saji H, Ogawa H. Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors. Bone Marrow Transplant. 2012 Oct; 47(10): 1338–1342. Disclosure of Interest: None declared.
P099 Extracorporeal photopheresis for cGVHD with lung involvement C. Del Fante1,*, P. Bernasconi2, L. Scudeller3, G. Viarengo1, A. A. Colombo2, D. Caldera2, F. Ripamonti2, C. Perotti1 1 Immunohaematology and Transfusion Service, Centre for transplant immunology, 2Division of Haematology, 3Biometry and Statistics, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Introduction: Lung GVHD (LG) is an important cause of morbidity and mortality despite immunosuppressive therapy (IST). Bronchiolitis obliterans syndrome (BOS-obstructive form) and restrictive lung disease (RLD-restrictive form) represent the two forms of lung dysfunction. Spirometry, and in particular Forced Expiratory Volume in 1 second (FEV1), is used to monitor lung function over time. We monitored FEV1 trend over time in 13 patients (pts) with different oncohematological disorders who underwent off-line extracorporeal photopheresis (ECP) for LG after allo-BMT. Materials (or patients) and methods: We retrospectively analyzed 13 pts (mean age 45, 46% females) who developed LG not responsive to standard therapy undergoing off-line ECP from 2012 to 2014. ECP was initially performed 2 times/week for 3 weeks, then 2 times every other week for 3 times, then maintained 2 times/month. Clinical conditions, spirometry and blood biochemistry were checked at least every 3 months, or more frequently whenever clinically indicated. Response was defined as stable/improved FEV1 from baseline. Results: Underlying disease was AML in 6 pts (46%), ALL in 2 (15%), AREB , HL, MF, NHL, THAL in 1 pts each. Cell source was bone marrow in 2 pts (15%) and peripheral blood in 11; donor was related in 7 (54%) and unrelated in 6. Conditioning was myeloablative in 10 and RIC in 3. cGvHD was classic in 7 pts (54%), overlap in 5 (38%) and late acute in 1. Median time from transplant to LG was 14 months. All pts had BOS; grade 3 developed in 8 pts (61%) and grade 2 in 5. Median time from LG to ECP was 3.4 months. Median FEV1 at ECP was 1.76 L (IQR 1.51-2.24). Cumulative follow-up under ECP was 130 pts-months. Median ECP number was 29 (min-max 956). ECP was well tolerated. Ten pts (77%) achieved a response, always preceded by a subjective improvement of dyspnoea. Median time to response was 4.6months (IQR 4.5-8.3). None died. Conclusion: Our preliminary results show that ECP in association to standard IST may be effective in slowing/ stopping lung function decline in pts with cGVHD-related BOS. References: Kooke KR. A window of opportunity for patients with late-onset pulmunary dysfunction after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2014;20:291-94. Disclosure of Interest: None declared.
P100 Parameters of Protein Metabolism and Thyroid Function As Predictors in a Scoring System for Acute and Chronic Graft-Versus-Host Disease C. Skert1,*, A. Turra1, M. Malagola1, S. Perucca1, V. Cancelli1, R. Daffini1, R. Ribolla1, C. Bergonzi1, C. Filı`1, C. Pagani1, A. Di Palma1, F. Cattina1, S. Bernardi1, D. Russo1 1 Chair of Hematology, Bone Marrow Transplant Unit, University of Brescia, AO Spedali Civili Brescia, Brescia, Italy Introduction: Several studies reported ‘‘classical’’ patient-, donor- and transplant characteristics, such as age, gender disparity, donor type, HLA-match, and source of stem cells as predictors for acute and chronic GVHD. However, no studies analysed these ‘‘classical’’ variables together with parameters of metabolic and endocrine functions, which may potentially influence the immune system. Patient-and transplant variables together with index of liver and thyroid function, and some parameters of protein and lipid metabolism were retrospectively evaluated at different time points after transplantation, in order to identify possible predictors of acute and chronic GVHD and to calculate a risk score. Materials (or patients) and methods: Clinical and transplant characteristics, number and type of infections before and after stem cell transplantation (SCT) were analysed in 161 patients. The following variables were also analysed pre-SCT, at day þ 7, þ 14, þ 21, þ 28, at þ 3 and þ 6 months: LDH, parameters of liver function; parameters of protein and lipid metabolism (serum albumin, urea, total protein, cholesterol, triglycerides); thyroid function tests; autoimmune parameters; body mass index. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, in order to solve the problem of the high number of variables in comparison with the relatively limited and heterogeneous pool of patients. Based on the regression coefficient of Cox analysis for each significant predictor, a scoring system for acute and chronic GVHD was calculated. Results: In multivariate analysis, diagnosis of Myelodisplastic Syndrome or Chronic Myeloid Leukemia (P ¼ 0.0004), conditioning regimen including Total Body Irradiation (P ¼ 0.0003), and pre transplantation urea 434 mg/dl with þ 21 day urea 454 mg/dl (P ¼ 0.0008) were evidenced as predictors for acute GVHD. Score values for each factor are 2, 1, and 1, respectively. The probability of acute GVHD ranged from 8% (score 0) to 98% (score 4). Female donor (P ¼ 0.0008), pre-SCT TSH values Z2 m/L with þ 28 day urea Z 39 mg/dl (P ¼ 0.02), þ 6 month total proteino5,5 g/dl with gamma-GT Z347 U/L (P ¼ 0.0001) resulted predictors for moderate/severe chronic GVHD, with score values of 1, 1, and 2 respectively. Risk of chronic GVHD at þ 6,5 month ranged from 3% (score 0) to 97% (score 4). Conclusion: Our study evidenced that factors other than ‘‘classical’’ ones may be associated to GVHD. Our scoring system includes routine-parameters, which are easily available in clinical practice. Urea levels depend on the balance between protein intake, endogenous catabolism and urinary excretion. The inflammatory microenvironment of GVHD promotes muscle catabolism, hence, urea levels do increase. Increased urea levels could be an indirect index of increased uremic toxins as well, which may stimulate the production of pro-inflammatory cytokines and the activation of leukocytes. On the other hand, increased urea levels and uremic toxins could derive from a dysregulated metabolism of the gut microbiome, which may influence immune system. Our findings, which require a prospective validation, suggest the usefulness of a deeper study of the complex network between metabolic/endocrine functions and immune system, in order to develop a holistic approach of the transplant management. Disclosure of Interest: None declared.
P101 Prophylaxis with Rifaximin preserves microbiome diversity in ASCT ¨ fner3, J. Hahn1, K. Peter1, D. Sporrer1,*, J. Ko¨stler2, A. Gessner2, P. O M. Kreutz1, W. Herr1, E. Holler1 1 Internal Medicine III, 2Microbiology, 3Functional Genomics, University Hospital Regensburg, Regensburg, Germany Introduction: Microbiome disruptions seem to be associated with intestinal acute graft versus host disease (aGvHD) and poor outcome after allogeneic stem cell transplantation (ASCT) [1]. In previous studies we found a major impact of broad spectrum antibiotics for prophylaxis and therapy of neutropenic infections on loss of intestinal bacterial diversity as measured by enterococcal overgrowth and low urinary indoxyl sulfate levels (UIS). Therefore, we switched antibiotic prophylaxis to Rifaximin, a non-absorbable gut decontamination, and now retrospectively analyzed its effect on intestinal diversity and clinical risk for neutropenic infections during ASCT. Materials (or patients) and methods: A total of 333 patients undergoing ASCT were included in the analysis. Two hundred patients received the conventional antibiotic prophylaxis with Ciprofloxacin and Metronidazole whereas 133 patients received Rifaximin at a dose of 200 mg twice daily. Prior to admission until four weeks after ASCT stool and urine specimens were collected weekly. Fecal enterococcal concentrations were measured using strain specific PCR and UIS levels were determined using chromatographytandem mass spectrometry. Enterococcal overgrowth was defined as conversion from negative to positive values or increase in copy numbers. Besides, we analyzed the incidence of infections in relation to the type of combined prophylaxis. Statistical analyses were performed using IBM SPSS Statistics 24. Results: In the Rifaximin group conversion to positivity for both germs, Enterococcus (E.) faecalis and E. faecium, was less frequent than in the standard group (P ¼ 0.05). Similarly, during ASCT, we observed higher UIS levels under the use of Rifaximin than under standard prophylaxis (Po0.001) (Table 1). The additional application of systemic antibiotics in neutropenia was similar in both groups with 93.2% of patients with Rifaximin and 91.0% of patients with standard prophylaxis. Even after application of systemic antibiotics, the protective effect of Rifaximin on bacterial diversity seemed to be maintained, as UIS levels were still higher than in patients receiving standard prophylaxis without systemic antibiosis. Regarding overt infectious complications we did not find higher infection rates, neither for gram-negative germs under Rifaximin. Simultaneously to the change of antibiotic decontamination a high dose supplementation with Vitamin (Vit) D had been initiated in our unit. As high Vit D levels seem not only to have tolerogenic immune effects but also to maintain bacterial diversity by induction of antimicrobial peptides, we also analysed the impact of VitD levels on bacterial diversity but did not observe an additional effect beyond Rifaximin. Conclusion: In conclusion, in a large cohort of patients undergoing ASCT we found a correlation between the use of Rifaximin and predictors of bacterial diversity such as lower intestinal enterococcal overgrowth and higher UIS levels.
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Additionally, Rifaximin seemed to exert a protective effect on intestinal microflora and to maintain a complex bacterial diversity despite the use of systemic antibiotics. Randomized studies are needed to define the definitive contribution of Rifaximin versus Vit D and to demonstrate the impact of this approach on overall outcome. References: 1. Taur, Y., et al., The effects of intestinal tract bacterial diversity on mortality following allogeneic hematopoietic stem cell transplantation. Blood, 2014; 124(7): 1174-1182. Disclosure of Interest: None declared. P102 Oral Candida species colonization in chronic Graft-versusHost Disease D. Dusek1,*, M. Mravak Stipetic2, M. Grce3, D. Pulanic4,5, L. Grkovic6, R. Serventi Seiwerth6, T. Klepac Pulanic7, R. Ceovic5,8, S. Bukovski9, M. Jelic9, M. Cindric3, L. Rajic5,10, E. Bilic5,10, Z. Peric5,6, N. Durakovic5,6, A. Vince1,5, R. Vrhovac5,6, S. Z. Pavletic11, D. Nemet5,6 1 University Hospital for Infectious Diseases, 2School of Dental Medicine University of Zagreb, 3Division of Molecular Medicine, Rudjer Boskovic Institute, 4Division of Hematology, Department of Internal Medicine, University Hospital Centre Zagreb, 5Medical School University of Zagreb, 6Division of Hematology, Department of Internal Medicine, University Hospital Center Zagreb, 7 Community Health Center Zagreb East, 8Department of Dermatovenerology, University Hospital Center Zagreb, 9Department of Microbiology, University Hospital for Infectious Diseases, 10 Department of Pediatrics, University Hospital Center Zagreb, Zagreb, Croatia, 11National Cancer Institute, National Institutes of Health, Bethesda, United States Introduction: Colonization with Candida sp has significant role in occurrence and pathogenesis of acute Graft-versusHost Disease (aGVHD) via induction of mucosal innate immunity (Th 17/IL 23 responses). However, data about prevalence and role of Candida sp in chronic Graft-versusHost Disease (cGVHD) are scarce. The aim of this study was to determine prevalence of oral colonization with different Candida sp in patients with cGVHD and possible association with cGVHD characteristics. Materials (or patients) and methods: This study enrolled cGVHD patients who are part of a larger multidisciplinary cGHVD project at the University Hospital Center Zagreb, Croatia. Patients were evaluated for clinical and cGVHD characteristics; severity of cGVHD and oral involvement were determined by using established NIH Consensus criteria. Swabs from oral cavity were collected from all patients. Specimens were analyzed by standard cultivation methods (Sabouraud and blood agar, identification by automated biochemical test). Molecular detection was performed by real-time in house PCR designed to distinguish 12 Candida species. Candida isolates were analyzed at proteomic level by Mass Spectrometry (MS/MS). Data were evaluated descriptively. Fisher’s exact test or Wilcoxon test were used to calculate statistical significance. Results: Twenty-one patient was enrolled, median age was 42 years 38% were male. In 15 patients (71%) underlying disease was AML, ALL or MDS, 3 patients had CML or MPN, 1 patient had severe aplastic anemia and 2 patients had lymphoma. Nine patients (43%) underwent myeloablative conditioning. Majority (71%) received PSBC as stem source. Sixteen patients (76%) had previous aGVHD. Ten patients (48%) had oral cGVHD; 6 patients had moderate or severe NIH oral cGVHD score. Nine patients (43%) had moderate and 12 (57%) had severe NIH global cGVHD score. Six patients (29%) were positive for C. albicans and 3 patients for C. krusei by standard cultivating methods. When using real- time PCR method, 13 patients (61%) were positive for C. albicans, 4 patients for C. krusei, 2 patients with C. dubliensis, 1 patient with C. tropicalis and 1 patient with C. glabrata. Coinfection with 2 or 3
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different Candida sp. was found in 6 patients. Ten samples were analyzed by proteomics with 5 samples confirmed as C. albicans. Although more patients were positive for C. albicans and C. non albicans sp by multiplex PCR than cultivation, this was not statistically significant (P ¼ 0.335 and 0.184, respectively). Nine patients with oral cGVHD (90%) had colonization with at least one Candida sp, while 7 patients without oral cGVHD (64%) had colonization with Candida sp (P ¼ 0.311). Ten patients with global severe cGVHD (83%) and 5 patients with moderate cGVHD (55.5%) had Candida sp colonization (P ¼ 0.331). Conclusion: Significant proportion of patients with cGVHD, especially patients with severe global cGVHD were colonized with Candida sp. Use of molecular methods is important in diagnosing C.non albicans (3 new species identified only by PCR). Majority (90%) of patients with oral cGVHD were colonized with Candida sp, however larger studies are needed to determine association between cGVHD global severity and organ involvement with Candida colonization. Considering significant proportion of cGVHD patients colonized with Candida sp, further studies investigating its possible role in pathogenesis of cGVHD, as well as possible prophylactic/ therapeutic are needed. Disclosure of Interest: None declared.
P103 The use of hyperbaric oxygen therapy in the treatment of hemorrhagic cystitis after allogeneic stem cells transplantation from unrelated donor D. Urbaniak-Kujda1,*, J. Dybko1, M. Biernat1,2, K. Kapelko-S"owik1, M. Laszkowska1, E. Stefanko1, M. Biedron´1, I. Deren´-Wagemann1, T. Wro´bel1, K. Kuliczkowski1 1 Department and Clinic of Haematology, Blood Neoplams and Bone Marrow Transplantation, Wroclaw Medical University, Wroc"aw, 2Deparment of Microbiology, Wroclaw Medical University, Wroclaw, Poland Introduction: Hemorrhagic cystitis (HC) is a diffuse inflammation of the bladder of an infectious or non-infectious etiology, causing bleeding of the bladder mucosa. HC may be caused by different factors including: medications: (busulfan, endoxan, idarubicin, carboplatin), radiation, viruses, and chemicals. Treatment guidelines of HC has not yet been established. Recent studies have demonstrated the efficiency of hyperbaric oxygen therapy (HOT) in patients suffered from HC after allogeneic HSCT (1,2). Materials (or patients) and methods: Patient characteristics is shown in Table 1. Material and methods: We retrospectively analyzed the effectiveness of HOT in 5 patients in the years from 2012 to 2014. Among patients there were 3 men, aged from 31 to 41 years and 2 women aged from 40 to 43 years. Patients characteristics is shown in Table 1. Results: Patient No
1
Age Sex Diagnosis Clinical (years) status
40
F
MDS
CR1
Condidtioning regimen
BuCyTym
2
41
M
AML
CR1
BuCyTym
3
39
M
ALL
CR1
TBI, CyTym
4
43
F
AML
CR1
BuCyTym
5
31
M
AML
CR1
BuCyTym
Source GvHD for prophylaxis alloHSCT PBSC, MUD PBSC, MUD PBSC, MUD PBSC, MUD PBSC, MUD
C, MTX, Gluc C, MTX, Gluc C, MTX, Gluc C, MTX, Gluc C, MTX, Gluc
Abbreviations: C ¼ cyclosporine, Tym ¼ tymoglobuline, Gluc ¼ glucocorticoids, MUD ¼ matched unlrelated donor, MTX ¼ metotrexate,
Results: The median time to onset of HC after allogeneic stem cell transplantation was 22 days (range, 11-55 days). In one patient, the symptoms did not appear until the day 55 after transplantation. Despite immunosuppressive therapy, all patients had macroscopic hematuria and GvHD. The BKV and ADV DNA were detected in urine and plasma samples in 2 patients, ADV DNA was detected in urine and plasma in 2 other patients, and in the one patient, BKV was detected in both samples, respectively. The patients were treated with HOT (2.5 atmospheres for 60 minutes, 5 days per week) after treatment failure of bladder drainage flow, hyaluronic acid administered intravesically, as well as antiviral treatment. All patients showed complete resolution of hematuria and eradicaton of the virus after a median of 13 sessions (range, 11-30) of HOT. Conclusion: HBO may be an alternative and promising therapy in the treatment of severe hemorrhagic cystitis. References: 1.Savva-Bordalo J., Pinho Vaz C., Sousa M., Branca R., Campilho F., Resende R., Baldaque I., Camacho O., Campos A. Clinical effectiveness of hyperbaric oxygen therapy for BKvirus-associated hemorrhagic cystitis after allogeneic bone marrow transplantation. Bone Marrow Transplnatation 2012; 47: 1095–1098. 2. Dellis A., Deliveliotis Ch., Kalentzos V., Vavasis P., Skolarikos A. Is there a role for hyperbaric oxygen as primary treatment for grade IV radiation-induced haemorrhagic cystitis? A prospective pilot-feasibility study and review of literatur˛e. Int Braz J Urol. 2014; 40: 296–305. Disclosure of Interest: None declared.
better OS in comparison to those with lack of response. Also, pts with aGvHD grade IV had significantly worse five-year outcome. Conclusion: aGvHD have significant impact on the morbidity after allo SCT (incidence is 58,5%). Predictors of incomplete response to treatment are advanced grade of aGvHD, PB as a source of SC, sex mismatch (female donor to male recipient), donor- recipient ABO incompatibility and CML at diagnosis. Pts with advanced grade of aGvHD have less probability of OS. Disclosure of Interest: None declared.
P104 Acute Graft- versus-Host Disease predictors and outcomeour experience D. Stamatovic1,*, M. Elez1, O. Tarabar1, S. Marjanovic1, B. Balint2, O. Tasic Radic3, Z. Tatomirovic3, A. Zivanovic Ivic1, L. Tukic1 1 Clinic of hematology, 2Institute for transfuziology, 3Institute for pathology, Military Medical Academy, Belgrade, Serbia
P105 Von Willebrand Factor and Factor VIII as potential biomarkers of chronic Graft-versus-Host Disease D. Pulanic1,*, L. Grkovic2, R. Serventi Seiwerth2, M. Mravak Stipetic3, E. Bilic4, R. Ceovic5, Z. Peric1, L. Rajic6, N. Durakovic1, N. Matic2, T. Klepac Pulanic7, I. Petricek8, T. Vukic9, E. Bilic6, D. Dusek10, E. Prenc11, I. O. Prah11, I. Bojanic12, M. Grce13, R. Zadro14, D. Batinic14, R. Vrhovac1, S. Z. Pavletic15, D. Nemet1 1 Division of Hematology, Department of Internal Medicine, University Hospital Center Zagreb and Medical School University of Zagreb, 2Division of Hematology, Department of Internal Medicine, University Hospital Center Zagreb, 3School of Dental Medicine University of Zagreb, 4Department of Neurology, 5 Department of Dermatology, 6Department of Pediatrics, University Hospital Center Zagreb and Medical School University of Zagreb, 7Community Health Center Zagreb East, 8Department of Ophtalmology, 9Department of Rheumatology and Rehabilitation, University Hospital Center Zagreb, 10University Hospital for Infectious Diseases, 11Medical School University of Zagreb, 12 Department of Transfusion Medicine, University Hospital Center Zagreb, 13Division of Molecular Medicine, Rudjer Boskovic Instute, 14 Department of Laboratory Medicine, University Hospital Center Zagreb and Medical School University of Zagreb, Zagreb, Croatia, 15 National Cancer Institute, National Institutes of Health, Bethesda, United States
Introduction: Acute Graft-versus-Host disease (aGvHD) is an immunologycally mediated inflammatory reaction which remains one of the major limiting factor in succesful allogeneic hematopoietic stem cell transplantation (HSCT). The disease should be defined based on a clinical and pathological features and not solely on the day of its appearence. Standard treatment for aGvHD consist of corticosteroids, although there is a lack of consensus over incidence and predictors of outcome. The goal of our retrospective study were to estimate incidence, type and severity of aGvHD, risk factors for treatment outcome and impact of aGvHD on the overall survival (OS). Materials (or patients) and methods: We have analyzed 222 patients (pts), female/ male ratio 90/132, average age 29 (range 9- 58) with different hematological malignancies (sAA 27, CML 33, AML 65, ALL 70, MDS/ MPN 14, HL/ NHL 6, MM 3, GrSa 3, Thalassemia major 1) in whom we have performed allogeneic HSCT from the identical sibling donors in the previous 15 years. Conditioning regimens were adjusted to the primary diseases and stem cell source was peripheral blood (PB) in 141 and bone marrow (BM) in 81 pts. GvHD prophylaxis was mostly combination of Cyclosporin A (CsA) and Metothrexate (Mtx). Results: aGvHD of any grade was documented in 130 (58,5%) pts, while grade II/IV had 83 (37,4%) pts with onset approximately on the day þ 41. (range 12- 179), while 10 pts (4,9%) had hyper acute form (before day þ 14.). Mostly involved organs were skin (50%), GIT (23,7%), liver (16,2%) or combinations. Predictors of incomplete response to steroid therapy were: advanced grade of aGvHD (II vs. III/IV; P ¼ 0.03), sex mismatch between donor and recipient (female to male vs. other; P ¼ 0.03), stem cell source (BM vs. PB; P ¼ 0.04) and diagnosis (CML vs. other; P ¼ 0.04). Pts with aGvHD that had favorable response to therapy had significantly
Introduction: Predictive, diagnostic, and risk stratification biomarkers of chronic Graft-versus-Host disease (cGVHD) are critically needed to improve treatment of this serious late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Since elevated von Willebrand’s factor (VWF) and factor VIII (FVIII) levels have been described as indicators of endothelial dysfunction and inflammation in different settings, the goal of this study was to assess possible role of VWF and FVIII as potential biomarkers of cGVHD. Materials (or patients) and methods: Multidisciplinary cGVHD clinical infrastructure was organized in 2013 at the Division of Hematology, University Hospital Center Zagreb, Croatia, using established cGVHD-related scales and measurements in collaboration with the National Cancer Institute, National Institutes of Health (NIH), USA. An extensive demography, history, physical and laboratory evaluations were obtained in this cross-sectional prospective study using standardized methods, including measurement of VWF:Ag, VWF:RCo, and FVIII. Descriptive statistic and non-parametric analyses were performed, results with p-valueso0.05 were considered as statistically significant, and variables with Spearman rank correlation coefficient 0.5o|r|o0.7 were considered as moderately correlated. Results: Twenty cGVHD patients were assessed (median age 38.5 years, 55% females). Median time from HSCT to study enrollment was 16.4 months. Median time from HSCT to cGVHD diagnosis was 9.5 months, and median time from cGVHD diagnosis to study enrollment was 5.5 months. Majority of patients (90%) were transplanted for hematologic malignancies. Myeloablative conditioning was used in 50%, 11 (55%) received peripheral blood stem cells, and 12 (60%) transplants were from matched related donors. Eight patients had de novo cGVHD, 5 quiescent, 7 progressive, and 16 (80%)
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classic cGVHD. Ten (50%) patients had moderate and 10 (50%) severe NIH global cGVHD score. Most involved organs were skin, lung, liver, and eyes. At the time of evaluation, clinician’s impression was in 50% that cGVHD was active. Elevated level of FVIII (median 191%, range 52-297%, reference range 50-149%) and VWF:Ag (median 208%, range 108-600%, reference range 50-160%) was found. Median level of VWF:RCo was 138% (range 75-547%, reference range 50-150%). Patients after unrelated HSCT had higher FVIII, VWF:Ag and VWF:RCo. Shorter time from cGVHD diagnosis to study enrollment was associated with higher VWF:Ag (P ¼ 0.015) and VWF:RCo (P ¼ 0.018). NIH cGVHD liver score (P ¼ 0.013), average NIH score (r ¼ 0.535), AST (r ¼ 0.668), ALT (r ¼ 0.603), beta-2 microglobulin (r ¼ 0.510), triglyceride (r ¼ 0.596) and cGVHD activity by therapeutic intent (P ¼ 0.019) were associated with higher FVIII. NIH cGVHD mouth score (P ¼ 0.016), clinician impression of cGVHD activity (P ¼ 0.023) and AST (r ¼ 0.563) were associated with higher VWF:Ag. Ferritin (r ¼ 0.506) was associated with higher VWF:RCo, while cGVHD activity by therapeutic intent, beta-2 microglobulin, D-dimers, and triglyceride were associated with both higher VWF:Ag and VWFR:Co. Other markers of inflammation (C-reactive protein, fibrinogen, erythrocyte sedimentation rate) did not correlate neither with VWF:Ag, VWF:RCo nor with FVIII. Conclusion: Results of this pilot study suggest that VWF and FVIII could represent interesting candidate biomarkers of cGVHD. However, these need to be further investigated in larger studies. Disclosure of Interest: None declared. P106 Protein expression in the setting of GvHD: CXCL9, ELAFIN AND REG3a E. Metafuni1,*, S. Giammarco1, D. De Ritis1, F. Sora`1, L. Laurenti1, S. Sica1, P. Chiusolo1 1 Hematology Department, Universita` Cattolica del Sacro Cuore, Rome, Italy Introduction: Graft-versus-host disease (GvHD) is the major life-threatening complication after allogeneic stem cell transplantation (SCT). There are no validated laboratory tests able to substitute the histopathology in GvHD diagnosis. In the last years, many authors focused their attention on serum protein expression in the setting of GvHD. Materials (or patients) and methods: We enrolled 36 patients (pts), 14F/22M, submitted to SCT in our centre from april 2010 to December 2013. Underlying diseases were: 5 ALL, 1 CLL, 18 AML, 1 CML, 4 NHL, 2 MPN and 5 MDS. Standard conditioning was performed in 12 pts and reduced intensity conditioning in 24 pts. Median number of CD34 þ in the graft was 5.3*106/Kg. Stem cell source were: 34 PB, 1 CB and 1 BM. Donor was sibling in 13 cases and MUD in the remaining 23 pts. GvHD prophylaxis: 28 CSA þ MTX, 7 CSA þ MMF, 1 Tacrolimus þ MTX. ATG was used in MUD
[P106]
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transplants. Statistical analysis was performed using IBM SPSS Statistics 22. Results: Acute GvHD (aGvHD) was detected in 24 pts at a median of 17 days after SCT: 9 grade I, 8 grade II, 5 grade III and 2 grade IV. Organ involvement: skin in 13 pts, skin and liver in 3 pts, skin and gut in 5 pts, skin, gut and liver in 3 pts. Chronic GvHD (cGvHD) was identified in 19 pts: 6 mild, 8 moderate and 5 severe grade. Organ involvement: skin and gut (1 pt), skin (7 pts), skin and liver (2 pts), skin, liver and gut (2 pts), liver (2 pts), gut and liver (1 pt), gut, skin and eyes (1 pt), 1 skin and mouth (1 pt), skin, liver, eyes and mouth (1 pt), skin and lungs (1 pt). CXCL9 and Elafin level were higher in pts who developed aGvHD than in the others: 462 pg/ml (range 2-1820) vs 215 pg/ ml (range 50-422) for CXCL9 (t-test, P ¼ 0.041) and 69000 pg/ ml (range 16000-165100) vs 41782 pg/ml (range 6125-92050) for Elafin (t-test, P ¼ 0.033). Figure 1. Among pts with aGvHD, CXCL9 and REG3a level was higher for skin involvement rather than hepatic or intestinal one: 499 pg/ml (range 2-1820) vs 51 pg/ml (range 6-96) for CXCL9 (P ¼ 0.002) and 1360 ng/ml (range 207-8443) vs 345 ng/ml (range 393-297) for REG3a (ttest, P ¼ 0.018). In the setting of cGvHD, REG3a level was higher for hepatic involvement rather than intestinal or cutaneous one, 791 ng/ml (range 2967-8443) vs 297 ng/ml (range 207-1241) (Mann Whitney, P ¼ 0.0157). Conclusion: CXCL9, Elafin and REG3a serum concentrations showed a correlation with the development of GvHD. Serum levels of these proteins were higher at the onset of aGvHD compared to pts who did not develop this complication. Moreover, we identified an organ-specificity in the setting of GvHD. In particular, CXCL9 and Elafin were overexpressed in pts with skin aGvHD, while REG3a was higher in pts with cutaneous aGvHD and hepatic cGvHD. In conclusion, our results suggest a potential role of these proteins as biomarker of GvHD, both for onset and organ involvement. However, there data need confirmation in a larger cohort of pts. Disclosure of Interest: None declared. P107 Gastro-intestinal microbiome diversity changes after allogeneic stem cell transplantation E. Metafuni1,*, F. Paroni Sterbini2, S. Giammarco1, S. Sica1, L. Masucci2, P. Chiusolo1 1 Hematology Department, 2Microbiology Department, Universita` Cattolica del Sacro Cuore, Rome, Italy Introduction: The immune response induced by gastrointestinal (GI) microbiome translocation through the intestinal barrier after stem cell transplantation (SCT) conditioning chemotherapy plays a key role in the pathophysiology of graft-versus-host disease (GvHD). Severe GI GvHD was closely related to an increased transplant related mortality (TRM). Recently, some data have been published regarding the marked reduction of microbiome diversity after SCT, and its
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relationship with post-SCT outcomes like as GvHD, infections and TRM. Materials (or patients) and methods: The aim of our study was to analyze the microbiome diversity before and after transplantation. We enrolled 8 patient (pts), 3F/5M, with AML submitted to allogeneic SCT (allo-SCT). A myeloablative conditioning was performed in 5 pts, while the other 3 received a reduced intensity regimen. Median dose of CD34 þ was 6.3*106/Kg, obtained in all but one (BM) cases from PB of sibling donor in 4 cases and MUD in the others. GvHD prophylaxis was performed with CSA þ MTX in 6 pts and CSA þ MMF in 2 pts. ATG were added for MUD transplantation. The control group included 3 pts with MM submitted to autologous SCT (auto-SCT). Timing for stool samples collection were: basal, day þ 15 and GvHD onset. Samples were stored at -801C until use. DNA was isolated with QIAamp DNA Stool Mini kit. The hypervariable V1-V3 region of the 16s RNA gene was amplified by PCR and sequenced on 454 GS Junior Titanium. Taxonomic composition was defined according to Greengenes database. IBM SPSS Statistics 22 was used for statistical analysis. Results: Five pts developed acute GvHD with the follow organ involvement: skin and gut (1), gut (1), skin (2) and liver (1). No difference was identified between auto-SCT and allo-SCT basal microbiome. At day þ 15 after auto-SCT, we detected a reduction of bacteroidetes and an increase of Proteobacteria (Wilcoxon matched-pairs signed rank test, P ¼ 0.0071). In alloSCT, at day þ 15 we identified an increase of Bacteroidetes and a reduction of Firmicutes (Paired t-test, P ¼ 0.0023). At the onset of GvHD, pts showed a reduction of Bacteroidetes and an increase of Firmicutes and Proteobacteria (t-test, P ¼ 0.0020). On the contrary, Bacteroidetes were more represented in pts with GI GvHD than in the others (t-test, P ¼ 0.0001). Figure 1. Conclusion: After auto-SCT there was an increase of Proteobacteria (Klebsiella, Proteus, Acinetobacter, Haemophilus, Pseudomonas, Enterobacteriaceae) and reduction of Bacteroidetes (Bacteroides, Saprospirae, Prevotella). After allo-SCT there was an increase of Bacteroidetes and Firmicutes (Bacilli, Lactobacilli, Clostridium, Enterococci, Streptococci). Moreover, pts who developed GvHD harboured more Firmicutes and Proteobacteria and less Bacteroidetes than pts without this complication. However, in pts with GI GvHD
Bacteroidetes were more represented than in pts with liver or skin involvement, although reduced if compared with the basal setting. Disclosure of Interest: None declared. P108 Prospective validation of a chronic GvHD-Specific Proteome Pattern (cGvHD-MS14) Post-Allogeneic Hematopoietic Stem Cell Transplantation E. M. Weissinger1,*, C. Human1, D. Wolf2, J. Metzger3, H. Greinix4, A. Dickinson5, I. Tu¨ru¨chanow1, P. Schweier6, H. Kreipe7, J. Raad3, A. Durban3, D. Ihlenburg-Schwarz1, Z. Kuzmina4, E. Holler2, J. Krauter8,9, C. Koenecke1, M. Stadler1, A. Ganser1 1 Hematology, Oncology, Hannover Medical School, Hannover, 2 University of Regensburg, Regensburg, 3mosaiques, Hannover, Germany, 4Medical University of Vienna, Wien, Austria, 5University of Newcastle, Newcastle, United Kingdom, 6Hematology, Oncology, Medical University of Vienna, 7Pathology, 8Hannover Medical School, Hannover, 9Hematology, Oncology, Klinikum Braunschweig, Braunschweig, Germany Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematologic malignancies and non-malignant hematopoietic disorders, but is associated with significant morbidity and mortality due to acute and chronic graft-versus-host disease (GvHD). Chronic GvHD (cGvHD) occurs with increasing frequency, hampering quality of life of patients post-allogeneic HSCT and leading to increased morbidity and mortality even years after transplant. Diagnosis of cGvHD is based on clinical features and histology. Here we report the generation of cGvHD-specific proteomic pattern (cGvHD-MS14) using capillary electrophoresis and mass spectrometry (CE-MS) in order to diagnose cGvHD, to differentiate acute from cGvHD, and to predict onset and severity of cGvHD prior to its clinical diagnosis as a noninvasive, unbiased laboratory test. Materials (or patients) and methods: cGvHDMS-14 was prospectively evaluated on 487 patients (1198 urine samples) after allogeneic HSCT at MHH and 3 collaborating transplant centres. The majority of the patients had acute leukemias prior to transplantation (n ¼ 304), were not in CR/CP (n ¼ 237) and were transplanted from matched (n ¼ 387) unrelated or
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related donors (MUD n ¼ 317; MRD n ¼ 170). Reduced intensity conditioning regimens were used for 322 patients and the majority (n ¼ 363) received immunosuppressive antibodies as GVHD-prophylaxis prior to transplantation and a calcineurininhibitor based prophylaxis afterwards. Results: Prospective and blinded evaluation revealed the correct classification of patients developing cGvHD with a sensitivity of 78% and a specificity of 71% at time of diagnosis (Figure 1). Differentiation between late onset acute GvHD and cGvHD was achieved in 3 patients in this validation set. Acute GvHD prior to day 100 is not recognized by cGvHD-MS14, since aGvHD-specific peptides had been excluded during cGvHD-pattern generation. The pattern consists of 14 differentially excreted peptides, differentiating cGvHD from immune tolerant patients. Four of 14 peptides have been sequenced to date, 2 are fragments of collagen 1, 1 is from inter-alpha trypsin inhibitor heavy chain 4 and 1 is a fragment of the fibrinogen beta-chain. Conclusion: The proteomic pattern of urine enables diagnosis of cGvHD as well as differentiation of acute versus cGvHD. Further prospective evaluation of the cGvHD-specific pattern cGvHD-MS14 for organ specificity as well as severity prediction is currently ongoing. Taken together our results indicate that diagnosis of cGvHD is possible using CE/MS analysis of prospectively collected urine samples with high sensitivity and specificity. References: Weissinger EM, Metzger J, Dobbelstein C, et al Proteomic peptide profiling for preemptive diagnosis of acute graft-versus-host disease after allogeneic stem cell transplantation. Leukemia. 2014; 28(4): 842–852 Disclosure of Interest: E. Weissinger: None declared, C. Human: None declared, D. Wolf: None declared, J. Metzger Employee of: mosaiques GmbH, H. Greinix: None declared, A. Dickinson: None declared, I. Tu¨ru¨chanow: None declared, P. Schweier: None declared, H. Kreipe: None declared, J. Raad Employee of: mosaiques, A. Durban Employee of: mosaiques, D. Ihlenburg-Schwarz: None declared, Z. Kuzmina: None declared, E. Holler: None declared, J. Krauter: None declared, C. Koenecke: None declared, M. Stadler: None declared, A. Ganser: None declared
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P109 Prospective validation of a chronic GVHD-specific proteome pattern (cGVHD-MS14) post allogeneic hematopoietic stem cell transplantation E. Mischak-Weissinger1,*, C. Human1, D. Wolf2, H. Greinix3, J. Metzger4, A. M. Dickinson5, P. Schweier1, I. Tu¨ru¨chanow1, D. Ihlenburg-Schwarz1, H. Kreipe1, A. Krons4, H. Diedrich1, Z. Kuzmina3, E. Holler6, J. Krauter1, M. Stadler1, A. Ganser1 1 HANNOVER MEDICAL SCHOOL, Hannover, 2Innere Med. III, University of Regensburg, Regensburg, Germany, 3Medical University of Vienna, Wien, Austria, 4mosaiques diagnostics GmbH, Hannover, Germany, 5University of Newcastle, Newcastle, United Kingdom, 6University of Regensburg, Regensburg, Germany Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematologic malignancies and non-malignant hematopoietic disorders, but is associated with significant morbidity and mortality with focus on acute and chronic graft-versus-host disease (GvHD). Chronic GvHD (cGvHD) occurs with increasing frequency, hampering quality of life of patients post-allogeneic HSCT and leading to increased morbidity and mortality even years after transplant. Diagnosis of cGvHD is based on clinical features and histology. Here we report the generation of cGvHD-specific proteomic pattern (cGvHD-MS14) using capillary electrophoresis and mass spectrometry (CE-MS) in order to diagnose cGvHD, to differentiate acute from cGvHD, and to predict onset and severity of cGvHD prior to its clinical diagnosis as a non-invasive, unbiased laboratory test. Materials (or patients) and methods: This pattern was prospectively evaluated on 487 patients (1198 urine samples) after allogeneic HSCT at MHH and 3 collaborating transplant centres. The majority of the patients had acute leukemias prior to transplantation (n ¼ 304), were not in CR/CP (n ¼ 237) and were transplanted from matched (n ¼ 387) unrelated or related donors (MUD n ¼ 317; MRD n ¼ 170). Reduced intensity conditioning regimens were used for 322 patients and the majority (n ¼ 363) received immunosuppressive antibodies as GVHD-prophylaxis prior to transplantation and a calcineurininhibitor based prophylaxis afterwards. Results: Prospective and blinded evaluation revealed the correct classification of patients developing cGvHD with a sensitivity of 78% and a specificity of 71% at time of diagnosis (Figure 1). Differentiation between late onset acute GvHD and cGvHD was achieved in 3 patients in this validation set. Acute
GvHD prior to day 100 is not recognized by cGvHD-MS14, since aGvHD-specific peptides had been excluded during cGvHD-pattern generation. The pattern consists of 14 differentially excreted peptides, differentiating cGvHD from immunotolerant patients. Four of 14 peptides have been sequenced to date, 2 are fragments of collagen 1, 1 is from inter-alpha trypsin inhibitor heavy chain 4 and 1 is a fragment of the fibrinogen -chain. Conclusion: The proteomic pattern of urine enables diagnosis of cGvHD as well as differentiation of acute versus cGvHD. Further prospective evaluation of the cGvHD-specific pattern cGvHD-MS14 for organ specificity as well as severity prediction is currently ongoing. References: Weissinger E, Dobbelstein C, Metzger J, et al., Leukemia 2014 Disclosure of Interest: E. Mischak-Weissinger Conflict with: married with co-owner and founder of mosaiques, C. Human: None declared, D. Wolf: None declared, H. Greinix: None declared, J. Metzger: None declared, A. M. Dickinson: None declared, P. Schweier: None declared, I. Tu¨ru¨chanow: None declared, D. Ihlenburg-Schwarz: None declared, H. Kreipe: None declared, A. Krons Employee of: mosaiques GmbH, H. Diedrich: None declared, Z. Kuzmina: None declared, E. Holler: None declared, J. Krauter: None declared, M. Stadler: None declared, A. Ganser: None declared P110 Genital chronic Graft-versus-Host Disease in women - a population based prospective study E. Smith Knutsson1,*, Y. Bjo¨rk2, A.-K. Broman3, L. Helstro¨m4, M. Nicklasson5, K. Sundfeldt6, M. Brune7 1 Dept of Obstetrics and Gynecology, Nu Hospital Group, Trollha¨ttan, Sahlgrenska Academy, University of Gothenburg, Trollha¨ttan, 2Sahlgrenska University Hospital, Institution of Medicine, Sahlgrenska Academy, University of Gothenburg, Go¨teborg, 3NU-Hospital Organisation, Institution of Medicine, University of Gothenburg, Trollha¨ttan, 4dept of Clinical Science and Education , Karolinska Institutet, Stockholm, 5Sahlgrenska Akademin, Institution of Medicine, University of Gothenburg, 6 Dept of Obstetrics and Gynecology, Sahlgrenska University Hospital, Sahlgrenska Academy, University of Gothenburg, 7 Sahlgrenska University Hospital, University of Gothenburg, Go¨teborg, Sweden Introduction: Typically, chronic Graft-versus-Host Disease (cGvHD) causes inflammation and fibrosis in conjunctival and oral mucosa. In a recent cross-sectional population-based study we accounted for a high prevalence (52%) also of genital cGvHD with 21% cases having vaginal synechiae or stenosis. Concomitant symptoms included dryness, pain, and dyspareunia (Smith Knutsson et al 2014). We hypothesized that frequent gynecological examinations and early intervention with local treatment may improve signs and symptoms of genital cGvHD and reduce the risk of severe genital sequel in allografted women. Here we report preliminary results. Materials (or patients) and methods: In a prospective study in Region Va¨stra Go¨taland, Sweden we included 41 pts, median age 49 (17-66) yrs, i.e. 79% of all allografted women 2005/09 to 2010/02. Thirty pts were followed Z36 months, 11 pts were lost (7 relapsed, 4 other causes). At each visit, pts were asked for genital symptoms and had a structured examination with photo-documentation. In 29 pts 62 biopsies were obtained. Clinical scorings of genital cGvHD were made as per NIH criteria (Filipovich et al 2005), and vaginal strings were considered diagnostic signs. Extra-genital and genital cGvHD clinical scoring were combined for the assessment of global cGvHD. Local immunosuppressive ointments (clobetasol, tacrolimus) were prescribed at a structured scheme modified by each patient´s symptoms and signs. In case of vaginal cGvHD, a dilator was adviced to keep the vagina open. Estrogen was given locally to all pts and systemically in premature
menopause. Systemic corticoid steroids were prescribed by the hematologist. Results: The diagnosis of genital cGvHD was made at least once in 21 pts (51%), at a median 7 (1-30) mo after Tx; the cumulative incidence at 6, 12, 24 and 36 mo being 27%, 51%, 65% and 70%, respectively. Both vulvar and vaginal cGvHD developed in 11 pts, with start in the vulva in 8 pts, the vagina in 2, and at both locals in one pt. Ten pts had genital GvHD only in the vulva (n ¼ 5) or in the vagina (n ¼ 5). Two pts had only genital cGvHD; no other cGvHD. Partial stenosis developed in 3 pts in spite of ongoing systemic prednisone. Twenty pts did not develope genital cGvHD up to 36 mo (n ¼ 11), or up to lost follow-up (4-30 mo). Thirteen pts were treated locally. Median time to treatment was 10 mo. One pt developed a total vaginal stenosis. Conclusion: The aim of this study was to try and reduce signs, symptoms and sequel of genital cGvHD, a frequent complication to alloSCT. In our previous cross-sectional study with a median follow-up of 80 months, 9/42 pts had developed vaginal stenosis before 36 mo post Tx, whereas in this prospective, interventional study we observed on only one stenosis before 36 months. Notably, the high frequency of severe global cGvHD in the previous study was mainly due to severe genital cGvHD, thus illustrating that poor gynecological care may considerably contribute to impaired quality of life in women after alloSCT. From the results of our two studies with a high prevalence and high cumulative incidence of genital cGvHD, we conclude that early and regular gynecological control is important and may reduce the frequency of severe genital cGvHD. Disclosure of Interest: None declared. P111 Impaired NK cell subset reconstitution correlates with development of acute GVHD following allogeneic stem cell transplantation E. Ullrich1,2,3,*, S. Bakhtiar2, E. Salzmann-Manrique2, S. Gerstner1,3, M. Bremm1,2, A. Mackensen3, P. Bader1,2, P. Hoffmann4,5, E. Holler4,5, M. Edinger4,5, D. Wolff4,5 1 LOEWE Center for Cell and Gene Therapy, 2Department for Children and Adolescents Medicine, Division for Stem Cell Transplantation and Immunology, Goethe University Frankfurt, Frankfurt, 3Department of Internal Medicine 5, Hematology & Oncology, Friedrich-Alexander-University Erlangen, Erlangen, 4 Internal Medicine III, Hematology & Oncology, University Hospital Regensburg, 5Regensburg Center for Interventional Immunology, University of Regensburg, Regensburg, Germany Introduction: Allogeneic stem cell transplantation (alloSCT) often remains the only curative treatment for hematological disorders. However, its success is frequently limited by acute and chronic graft-versus-host disease (GVHD) causing significant morbidity and mortality. One of the major challenges of alloSCT is to reduce the incidence and severity of GVHD while boosting the graft-versus-leukemia effect (GVL). In the setting of alloSCT, reconstitution of natural killer (NK) cells is of notable interest due to their known capability to induce GVL without GVHD. This analysis is a single center prospective study performed at the University Hospital Regensburg investigating the possible correlation between the regeneration of NK cell subsets and the incidence of acute GVHD (aGVHD) during the first 200 days following alloSCT with focus on immature NK cells (CD14-CD3-CD56highCD16dim), mature cytotoxic NK cells (CD14-CD3-CD56dimCD16high), and the ratio between these two populations (CD56dim:CD56high). Materials (or patients) and methods: Data were collected from March 2009 to January 2012, and 345 samples of 111 patients were analyzed by FACS. In median 3 samples per patient were analyzed (1 - 9 samples). Median age of the cohort was 50 years (17 – 71 yrs.). Statistical analysis was performed using both B-spline linear and logistic regression.
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Results: While 43 (38.7%) patients did not develop any GVHD during the first 200 days, 68 (61.2%) patients developed aGVHD (grade 1 n ¼ 28, grade 2 n ¼ 23, grade 3 n ¼ 14, grade 4 n ¼ 3) in median at day 28 (9-182) after alloSCT. No differences in NK cell distribution were detectable before alloSCT. In the longitudinal analysis of immune reconstitution after alloSCT, there was a clear association between early expansion of the total NK cell population and lower incidence of aGVHD (P ¼ 0.024). In particular, the onset of aGVHD was accompanied by a lack of the CD56high NK cell subset and reconstitution of CD56high NK cells significantly correlated with a lower incidence of aGVHD (P ¼ 0.0003). Remarkably, we observed a significant inverse correlation between the severity of aGVHD and the frequency of CD56high NK cells during aGVHD. Conclusion: It is well known that NK cell reconstitution following alloSCT goes along with higher numbers of CD56high that further differentiate into cytotoxic CD56dimCD16high NK cells. In our study, aGVHD not only correlates with reduced numbers of total NK cells, but specifically with an impaired early expansion of CD56high NK cells. In sum, these data let suggest a negative impact of aGVHD on early NK cell immune reconstitution, maturation and NK subset distribution. In addition, monitoring of early NK cell reconstitution after SCT may help to identify patients at risk for the development of severe aGVHD. Disclosure of Interest: None declared.
Results: The drug was well-tolerated with no severe side effects: the most frequent side effect was mild hypotension and headache. In 15/19 cases we observed a significant clinical benefit after a median of 6 cycles (range 3-12). The Modified Rodnan Skin score, calculated before and after 1-year treatment, was 27 and 8, respectively. Iloprost courses were given in Outpatient Department for a median of 20 courses, the first 12 every month. All the 15 responsive pts received a maintenance therapy with 3-4 treatments a year. Further improvement was otherwise observed over the subsequent courses. At present, one patient not responsive to Iloprost has died because of sepsis. All the 15 responsive pts are alive in continuous complete remission, with satisfactory improvement of quality of life. Conclusion: In our study Iloprost seems to be one of the most effective drugs in reverting resistant/refractory sclerodermic cGVHD and in reducing the extension of the skin lesions. Improvements were observed in 15/19 pts (79%), after a median of 6 months of treatment. The Rodnan Skin Score is a practical and easy tool to assess skin involvement and to follow clinical outcome. It’s to be noted that the vast majority of the pts of this report received stem cells from an HLA identical sibling, even if in the same period the majority of transplants were done from unrelated donors. Disclosure of Interest: None declared.
P112 Synthetic PGI-2 (Iloprost) treatment in patients with resistant sclerodermic chronic GVHD (cGVHD) after Allogeneic Hematopoietic Stem Cell Transplantation F. Benedetti1,*, M. Sorio1, C. Tecchio1, A. Andreini1, A. Artuso1, D. de Sabata1, G. Turri1, I. Ferrarini1 1 Bone Marrow Transplant Unit, AZIENDA OSPEDALIERA UNIVERSITARIA INTEGRATA VERONA, Verona, Italy
P113 Improved outcome of patients developing Ocular chronic Graft Versus Host Disease and treated with Autologous Platelet-Rich Plasma (PRP) eye drops F. Monaco1,*, F. Zallio2, M. R. Astori3, L. Mazzucco4, G. Drago1, M. Rapetti5, G. Catania1, D. Dolcino3, R. Guaschino4, M. Ladetto1, M. Pini1 1 Hematology, 2AZIENDA OSPEDALIERA SS. ANTONIO E BIAGIO E CESARE ARRIGO, ALESSANDRIA, Alessandria, Italy, 3Opthalmology, 4Transfusion Center, 5Pharmacy Department, AZIENDA OSPEDALIERA SS. ANTONIO E BIAGIO E CESARE ARRIGO, ALESSANDRIA, Alessandria, Italy
Introduction: Scleroderma is a late manifestation of chronic GVHD, presenting as progressive tightness of the skin and hampering patients’ quality of life. Iloprost, a synthetic PGI-2, has been proving effective in the therapy of idiopathic systemic sclerosis, which has a similar pathogenesis to sclerodermic cGVHD. Materials (or patients) and methods: From 2001 to 2013 we observed 19 pts affected by diffuse or limited sclerodermic cGVHD. During the study period 353 allogeneic transplants were performed, 162 from HLA identical sibling and 191 from unrelated donor. The patients (9 AML, 3 MM, 4 ALL, 2 NHL and 1 MFI) were transplanted in 12 cases with classical conditioning regimen (FTBI 1200 cGy plus Cytoxan 120 mg/Kg or BusulfanCytoxan), in 7 cases with reduced intensity conditioning regimen. In 17/19 cases the donor was an HLA identical sibling. The source of stem cells was bone marrow in 3 cases and peripheral blood in 16 cases. Severe cGVHD with scleroderma was observed at a median of 21 months from HSCT (range 8-39), presenting as diffuse skin changes (tightness and thickening of the face, neck, hands, thorax, legs, feet and abdomen), cutaneous dyschromia, digital pitting scars and functional joint impairment. In 15 cases it was classified as de novo cGVHD. Most of the pts showed involvement of organs other than the skin: xerophthalmia in 13, xerostomia in 8 cases, liver in 5 pts, gastrointestinal tract in 4 and lung in 1 patient. In 4 pts these alterations were linked with itching and pain and in 4 cases diffuse scleroderma was associated with severe discomfort in tendons or muscles. In all cases cGVHD severely affected quality of life. The pts failed at least 2 immunosuppressive regimens including CSA, azathioprine, mycophenolate mofetil, steroids, repeated cycles of PUVA with little or no effect on the skin. Iloprost, 50 microgram a day over 8 hours IV continuous infusion for 5 days every month, was started after a median of 6 months from the appearance of scleroderma (range 6-13).
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Introduction: Despite significant advances, graft versus host disease (GVHD) remains the most important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Chronic GVHD is the most common long-term complication, but it is also considered the strongest predictor for improved outcome through a graft-versus Leukemia effect. Ocular involvement occurs in 40-60% of patients with cGHVD and is found to significantly impair the quality of life. Treatment recommendations focus on relieving ocular symptoms. On the basis of encouraging results reported previously with autologous serum eye drops in autoimmune diseases, we started this approach in collaboration with Ophthalmic Division and Transfusion Centre; furthermore we retrospectively investigated the rate of onset of ocular cGVHD in our population submitted to allogeneic transplant and the possible association with improved outcome. Materials (or patients) and methods: The study was conducted at the Hematologic Division of the Hospital of Alessandria, with the collaboration of the Ophtalmological Division and the Transfusion Centre. Since 2005 all patients submitted to alloSCT for hematologic malignancies and with a NIH eye involvement score 41 were treated with autologous Platelet Rich Plasma (PRP) eye drops; ophtalmologist evaluation (at day þ 30, þ 180 and þ 360 from the start of the therapy) included 3 different parameters: SUBJECTIVE SYMPTOMS, analyzed using the NIH Eye Score; OBJECTIVE TEST: Schirmer test, Tear Film Break Up Time and Best Visual Acuity; CORNEAL DAMAGE: anterior segment, fluorescein and lissamine score. PRP eye drops were prepared under laminar flow hood, in sterile single dose, stored at – 201C and instilled 6 times/day for one year.
Results: Between 2005 and 2014, 29 patients developed ocular GvHD. Pretransplantation diagnosis included: acute myeloid leukemia/myelodisplastic syndrome (50%), acute lymphoblastic leukemia (13%), lymphoproliferative malignancies (37%). 25 patients were classified as NIH score 2, and 4 patients as score 3. All but two patients (one interruption for local reaction and one for hematologic progression) completed the one-year schedule. Considering NIH criteria, 10 patients resolved their symptoms, with a NIH score of 0 (zero) at day þ 360. At 30 days 86% and 43% had an improvement of symptoms (S) and ocular signs (T), respectively and 0% had corneal damage; at 180 days, 71% and 57% improved their symptoms (S) and ocular signs (T), respectively and 14% had corneal damage; at 365 days, 86% and 57% kept on an amelioration of their symptoms (S) and ocular signs (T), respectively, while 7% had corneal damage. Compared with a control population submitted to alloSCT in the same period, we found that 5-year overall survival (OS) of the patients with ocular cGVHD was projected at 65% and is significantly better than OS of patients with GVHD other than ocular and without cGVHD (58% and 50%, respectively). Conclusion: Therapy with autologous PRP eye drops shows an improvement of symptoms and corneal damage already since 30 days, and its efficacy is maintained throughout the year of treatment. Therefore autologous PRP eye drops could represent a valid alternative option to conventional therapy in the management of ocular cGVHD. Moreover we found that developing ocular GVHD is a kind of protection for overall survival in patients submitted to alloSCT. Disclosure of Interest: None declared. P114 Neutrophil and monocyte CD64 expression provides limited diagnostic value for detection of Graft-versus-Host disease or viral infections in patients after allogeneic Hematopoietic Stem Cell Transplantation F. Simonetta1,*, A. Pradier1, S. Masouridi-Levrat1, C. Bosshard1, C. Dantin1, C. Hogan1, T. Matthes1, Y. Chalandon1, E. Roosnek1 1 Hematology, Geneva University Hospitals, Geneva, Switzerland Introduction: CD64 expression by neutrophils and monocytes has been proposed as a diagnostic marker of infection or inflammation. In the present study we evaluated the diagnostic power of CD64 levels for identification of viral infections or Graft versus Host Disease (GvHD) in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Materials (or patients) and methods: We performed a prospective study on 295 blood samples obtained at different time points from 80 patients after allogeneic HSCT. We
measured CD64 levels on mature CD10-positive neutrophils (nCD64) and on monocytes (mCD64) as well as plasma levels of C-reactive protein (CRP) in HSCT recipients without complications or with viral infections or GvHD. Receiver operating characteristic curve analysis was performed to determine the discriminatory potential of nCD64, mCD64 and CRP for diagnosis of viral infections and GvHD in HSCT recipients. Results: nCD64 but not mCD64 was significantly up-regulated during early phases after HSCT even in the absence of infectious complications. Levels of nCD64 [MFI: 3.26 (2.07-5.37) vs 2.89 (1.89-4.48), P ¼ 0.0079] and mCD64 [MFI: 38.5 (29.751.5) vs 32.1 (20.2-52.0), P ¼ 0.0078] in patients with GvHD were significantly higher than in patients without but we did not detect significantly higher nCD64 and mCD64 levels in patients with viral infections. Hence, nCD64 and mCD64 expression had only a weak discriminatory power for detection of viral infections or GvHD. nCD64 and mCD64 had an area under the curve (AUC) value of 0.557 and 0.503, respectively, for viral infections and of 0.605 and 0.631 for GvHD. Importantly, nCD64 and mCD64 levels were not more discriminatory than CRP-levels (AUC of 0.570 for viral infections and of 0.629 for GvHD). Conclusion: nCD64 and mCD64 levels are increased during GvHD but have only a low diagnostic potential for the occurrence of GvHD or viral infections and their use for diagnostic purposes cannot be recommended. Disclosure of Interest: None declared. P115 Once weekly extracorporale photopheresis for treatment of steroid-refractory or relapsed chronic graft versus host disease F. Ayuk1,*, M. Christopeit1, M. Guellstorf1, H. Lellek1, T. Wolbers1, U.-M. Von Pein1, C. Wolschke1, N. Kro¨ger1 1 Clinic for Stem Cell Transplantation, UNIVERSITY MEDICAL CENTER HAMBURG-EPPENDORF, Hamburg, Germany Introduction: Extracorporale photopheresis is increasingly used to treat patients with chronic graft-versus-host disease (cGVHD), however published data on its efficacy is limited. While the optimal treatment schedule is unknown, most centres start with a twice-weekly treatment. Materials (or patients) and methods: We performed a retrospective analysis of 78 patients who received ECP at our centre for treatment of steroid-refractory or relapsed cGVHD. Primary end point was overall survival (OS) at 5 years, secondary end point was treatment response at 6 months after start of ECP treatment. Patients received ECP once weekly for at least 4 weeks. Thereafter, responding patients continued ECP every two weeks while non-responding patients continued on the once weekly schedule until 15 weeks. ECP was performed using UVAR-XTS or Cellex machines. Results: In all, 48 of 78 patients (62%) responded to treatment with 46 (59%) PR and 2 (3%) CR. 3 year and 5 year overall survival (OS) were 80% and 66% respectively. Responders had significantly better 5yr OS compared to non-responders (85% vs. 48%, P ¼ 0.01). Platelet counts at start of ECP treatment did not impact treatment response (P ¼ 0.76), however platelet countso100.000/ul vs. 4/ ¼ 100.000 was associated with poorer 5yr OS of 44% vs. 71%, P ¼ 0.01. In multivariate analysis for OS including platelet counts, steroid dose, patient age and cGVHD NIH grade (all at start of ECP), low platelet counts retained negative impact (RR: 0.34, 95% CI 0.11-1.00, P ¼ 0.05) while patient age below median (53yrs) was associated with better OS (RR: 2.8, 95% CI 0.96-8.25, P ¼ 0.06). Cumulative incidence of non-relapse mortality (NRM) at 2 years was higher for patients with low vs. high platelet counts (29% vs. 10%, P ¼ 0.03) and for older vs. younger patients (23% vs. 5%, P ¼ 0.003). Relapse incidence did not differ according to platelet counts (P ¼ 0.53) or patient age (P ¼ 0.1) We identified 9 patients who received ECP as the only immuno-modulatory treatment, 8 of whom attained a PR and none a CR. Seven out
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of 8 patients with eye involvement, 3 out of 5 with skin and none of 3 with joint involvement experienced PR of organspecific symptoms. Conclusion: In this relatively large study we found that the once-weekly schedule of ECP is effective in treatment of patients with relapsed or refractory cGVHD. It is noteworthy that ECP did not overcome the negative impact of low platelet counts on survival. Disclosure of Interest: None declared. P116 Unrelated cord blood (UCB) transplantation for AML: higher incidence of acute GVHD and lower survival in male patients transplanted with female unrelated cord blood. A report from Eurocord and the ALWP of the EBMT F. Baron1,*, M. Labopin2, A. Ruggeri3, M. Mohty4, G. Sanz5, N. Milpied6, A. Bacigalupo7, A. Rambaldi8, F. Bonifazi9, A. Bosi10, J. Sierra11, I. Yakoub-Agha12, J. M. Ribera Santasusana13, E. Gluckman14, A. Nagler15 on behalf of Acute Leukemia Working Party of the EBMT 1 University of Liege, Liege, Belgium, 2EBMT Paris Office, Hospital Saint Antoine, 3Eurocord, Hospital Saint Louis, AP-HP, and IUH University Paris VII, 4AP-HP, He´matologie Clinique et The´rapie Cellulaire, Hoˆpital Saint-Antoine, Paris, France, 5Servicio de Hematologia, Hospital Universitario La Fe, Valencia, Spain, 6 CHU Bordeaux - Hoˆpital Haut-leveque, Pessac, France, 7Department of Haematology II, Ospedale San Martino, Genova, 8 Hematology and Bone Marrow Transplant Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, 9Institute of Hematology & Medical Oncology, Bologna University, S.OrsolaMalpighi Hospital, Bologna, 10BMT Unit Department of Hematology, Ospedale di Careggi, Firenze, Italy, 11Hematology Department, Hospital Santa Creu i Sant Pau, Barcelona, Spain, 12UAM allo-CSH, Hopital HURIEZ, CHRU, Lille, France, 13Hospital Universitari Germans Trias i Pujol, Barcelona, Spain, 14Eurocord, Hospital Saint Louis, AP-HP, and IUH University Paris VII, France Monacord, Centre Scientifique de Monaco, Paris, France, 15 Division of Hematology and Bone Marrow Transplantation, The Chaim Sheba Medical Center, Tel-Hashomer, Israel Introduction: Despite major improvements in the field in the last decade nonrelapse mortality (NRM) has remained the main cause of failure of unrelated cord blood (UCB) transplantation for acute myeloid leukemia (AML). In the setting of allogeneic HLA-matched bone marrow or peripheral blood stem cell transplantation, transplanting male patients with grafts from female donor has been associated with a higher incidence of graft-versus-host disease (GVHD) and of nonrelapse mortality (NRM), and a lower overall survival (OS). This is due to recognition by female donor immune cells of minor histocompatibility antigens encoded by genes on the recipient Y chromosome that are polymorphic to their X-chromosome homologue. The aim of the current analysis was to compare transplantation outcomes in male patients given female UCB versus other gender combinations. Materials (or patients) and methods: Data from 552 consecutive patients with AML given a single UCB transplantation between 2000 and 2014 were included. Among them, 131 patients were male patients given female UCB, 119 patients were male patients receiving male UCB, and 302 were female patients. Male patients given female UCB were less often transplanted following a reduced-intensity conditioning (24% versus 35%, P ¼ 0.03) and received less total nucleated cells (2.47 vs 2.8 x106/Kg, P ¼ 0.001) than other patients. Results: Male patients given female UCB had a trend for a higher incidence of grade II-IV acute GVHD (33% versus 25%, P ¼ 0.08) but for a lower incidence of chronic GVHD (16% versus 25%, P ¼ 0.11), a trend for a higher incidence of NRM (41% vs 33%, P ¼ 0.06), and a lower leukemia-free survival (LFS, 30% vs 41%, P ¼ 0.01) and OS (33% versus 45%, P ¼ 0.008) than other patients. Restricting the analyses to male recipients, those given female UCB versus male UCB had a higher incidence of grade II-IV acute GVHD (33% versus 27%), a
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similar 2-year probability of NRM (40.8 vs 36.6%; P ¼ 0.41), and a suggestion for worse LFS (29.9 vs 40.7%, P ¼ 0.11) and OS (33 vs 42.4%; P ¼ 0.10). In multivariate analyses taking into consideration all patients for which data on HLA-matching and cell dose transplanted were fully available (n ¼ 363), male patients transplanted with a female UCB had a trend for a higher incidence of grade III-IV acute GVHD (HR ¼ 2.0, P ¼ 0.06), a trend for a higher NRM (HR ¼ 1.5, P ¼ 0.06) and a worse LFS (HR ¼ 1.4, P ¼ 0.04) and OS (HR ¼ 1.3, P ¼ 0.06). Conclusion: Our data suggest that male patients transplanted with female cord blood might have higher risk of acute GVHD and of NRM leading to worse LFS and OS. These results should be confirmed in other large cohorts of patients before used for determining the choice of a single UCB unit. Disclosure of Interest: None declared. P117 IL-10 producing B-10 cells can be identified as early as 30 days post an allogeneic stem cell transplant and are enriched in the transitional B-cell compartment G. Chakupurakal1,*, M. Garcia-Marquez1, A. Shimabukuro-Vornhagen1, S. Theurich1,2, C. Scheid1, M. Hallek1, U. Holtick1, M. von Bergwelt-Baildon1 1 Department of Medicine I, Department of Medicine I, University Hospitals Cologne, Kerpener str 62, Germany D 50937, 2MaxPlanck-Institute for Metabolism Research, University Hospials Cologne, Cologne, Germany Introduction: Allogeneic stem cell transplantation (alloSCT) is the curative therapeutic option for a variety of haematalogical malignancies. Graft versus Host disease (GvHD), with an incidence of around 40-60%, remains a major post transplant complication associated with severe morbidity and mortality. The role of B-cells and especially the Interleukin-10 (IL-10) producing regulatory B-cells (B10-cells) in the pathophysiology of GvHD is not clearly understood to date. We studied B cells in allogeneic transplant recipients in the early post transplant phase. Materials (or patients) and methods: We prospectively collected samples, after obtaining informed consent, on 89 patients, transplanted at the University of Cologne on days 30, 90 and 150 post transplant and 10 healthy donors. Patients were transplanted mostly for haematological malignancies (n ¼ 88). Male: Female ¼ 48:41. All received reduced intensity conditioning regimens (90%), 34/89 (38%) received antithymocyte globulin (ATG) in addition to a calcineurin inhibitor with mycophenolate mofetil or methotrexate as GvHD prophylaxis. 41 (46%) had no GvHD, and 9 (10%), 8 (9%), 17 (19%)and 14 (16%) had grade 1-4 GvHD as per the NIH classification respectively. Results: Day 30 post transplant the total B-cell percentages and numbers in transplant recipients were significantly reduced in comparison to the controls (P ¼ o0.0001).The majority of the B-cells had a naı¨ve phenotype, Transitional B-cells were significantly more in transplant recipients (P ¼ 0.02) No differences were observed in the memory B-cell or CD24high CD27high compartments in comparison to controls. B10-cells were studied in the cohort 30 days post transplant and could be demonstrated in transplant recipients (n ¼ 16, 8 with acute GvHD) irrespective of their GvHD status. For data analysis, 4 patients without GvHD had to be discarded due o0.1% B-cells. The percentages of B10-cells were significantly reduced in the transplant recipients on comparison with the control cohort (P ¼ o0.0002) (Figure 1a). The CD24high CD27high B10-cell compartment was significantly smaller in the patient cohort (P ¼ 0.054) (Figure 1b). CD24highCD38hightransitional B10-cell subset with a mean percentage of 9.82±9.157 accounted for the largest subset of the cells analysed. Data on chimerism analysis at the time of sampling showed that the majority of patients (n ¼ 15/16; 93%) had 495% donor chimerism. implying that the B10-cells are predominantly donor derived.
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Conclusion: This is the first longitudinal study of B-cells and B-cell subsets including CD24high CD38high transitional B-cells and CD24high CD27high B-cells commencing as early as 30 days post an alloSCT. The contribution of transitional B-cell phenotype to the total number of B-cells ,as early as 30 days post transplant, is a novel observation. This is the first report demonstrating B10-cells in stem cell transplant recipients in the early post transplant (30 days) period and are enriched within the transitional cell compartment.Unlike the observations in mice, the B10-cells are donor derived. The mean percentage value of B10-cells in our patient cohort 1.6± 1.1was similar to that seen in patients with chronic GvHD, by Khoder et al. The extremely low number of B10-cells in this early post transplant phase may explain the lack of significance between the patients with and without GvHD in our cohort. Disclosure of Interest: None declared. P118 Changes in T and B- cells in steroid refractory GvHD patients following treatment with a CD25-antibody G. Chakupurakal1,*, M. Garcia-Marquez1, A. Shimabukuro.vornhagen1, H. Schloesser1, U. Holtick1, C. Scheid1, M. von BergweltBaildon1 1 Department of Medicine I, Department of Medicine I, University Hospitals Cologne, Kerpener str 62, Germany D 50937, Cologne, Germany Introduction: Allogeneic stem cell transplantation is the therapeutic option for a variety of malignant and nonmalignant haematological diseases. Graft versus Host Disease (GvHD) is a common post transplant complication. In 40% of these patients, GvHD is steroid refractory and associated with a mortality of around 60%. Basiliximab is a chimeric murine – human antibody also selective for interleukin -2 receptor (IL2R) with a half life of 7 days. Phase 2 studies have demonstrated its superior efficacy in treating patients with steroid refractory GvHD(1). We administered Basiliximab in 14 patients with steroid refractory GvHD with a median age of 41 (range 20-69). M: F 7:7. We aimed to study the in vivo T- and B-cell changes following Basiliximab administration as this would be an ideal platform to monitor the alterations in the regulatory T and B-cell compartment. Materials (or patients) and methods: All patients but one 13/ 14 received PBSC from unrelated donors and 6/13 had mismatched unrelated donors. 7/14 achieved a complete response to treatment. PBMCs were obtained from all donors, after informed consent, prior to and after weekly administration of Basiliximab 40mg for 4 weeks. Control samples were obtained from patients with steroid responsive acute GvHD. Results: The total number of CD3 þ as well as CD4 þ and CD8 þ T-cells remained constant during treatment and no change was observed on comparison with the controls. The, naive (CD45RA þ , CCR7 þ ), central memory T-cell (CD45RA-,
CCR7 þ ), effector (CD45RA þ , CCR7-) and effector memory (CD45RA-, CCR7-) compartments remained constant, T-cell population decreased, though statistically not significant, during the treatment period. Gagliani et al 4 demonstrated that regulatory type 1 T-cells can be identified by the co-expression of CD49b and Lag3. The % CD49d þ , Lag3 þ T-cells was lower than that identified in the control population and decreased during the treatment period (statistically significant). No changes were seen in the % CD19 þ , CD20 þ B-cells , activated (CD20 þ , CD86 þ ) and anergic B-cell subsets (CD20 þ , CD21-) during the observation period. The % of, CD24high, CD27high regulatory B-cells were found to be twice that seen in the controls. With treatment a decrease was seen in this population.The CD27high, CD38high transitional B-cells were also found to be higher than that seen in the controls. No change was observed in this cohort with treatment. Conclusion: This is the first attempt to study the in-vivo changes induced by a CD25 antibody in patients with steroid refractory GvHD. We conclude that this antibody not only depletes the alloreactive CD25 þ T, B and NK cell population but also alters the regulatory T-cell and B-cell subsets in comparison to patients with steroid responsive GvHD. The role of regulatory B-cells in enabling this need to be studied further. References: 1. Massenkeil G, Rackwitz S, Genvresse I, Rosen O, Dorken B, Arnold R. Basiliximab is well tolerated and effective in the treatment of steroid-refractory acute graft-versus-host disease after allogeneic stem cell transplantation. Bone Marrow Transplant. 2002 Dec;30(12):899-903. 2. Gagliani N, Magnani CF, Huberr S, Gianolini ME, Pala M, Licona-Limon P, et al. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells Nat Med 2013 Jun 19(6): 739-746. Disclosure of Interest: None declared.
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P119 GVHD-Associated Early Impairment of Marrow Function Is Frequent after Allogeneic Transplantation and Is a Predictor of Treatment Related Mortality and Overall Survival G. Milone1,*, G. Avola1, M. G. Camuglia1, A. Di Marco1, S. Leotta1, A. Cupri1, A. Spadaro1, D. Berritta1, A. Romano1, M. Parisi1, G. Tripepi2 1 Hematology and BMT UNIT, Azienda Policlinico Vittorio Emanuele, Catania, 2Istituto di Fisiologia Clinica, CNR, Reggio Calabria, Italy Introduction: We have previously reported that a-GVHD is an important determinant of primary engraftment. Aim of our study was now to measure GVHD-associated marrow impairment and whether ‘‘a-GVHD-associated marrow impairment’’ is useful in predicting transplant outcome. Materials (or patients) and methods: In a prospective series of allogeneic transplants, we determined the frequency of CFU-GM and BFU-E in marrow at day þ 18. Findings were related to a-GVHD and to Treatment Related Mortality (2-yTRM), Relapse Rate (RR) and Overall Survival (OS). So far, 72 patients have been evaluated, the majority were affected by Acute Leukemia (73%), in various phase of disease (advanced 57%). Our series included matched family (62%) or unrelated donors (38%). In MUD transplants, HLA matching was: 8/8: 26% ; 7/8: 52%; other: 22%. Results: Patients who, within day þ 90, were diagnosed with a-GVHD (overall grade ¼ /4 21), had a lower growth of CFUGM (91/10e5 plated cells) in respect to patients without a-GVHD (185./10e5), P ¼ 0.01. Similarly, BFU-E growth was lower in marrow of patients with a-GVHD in respect to patients without a-GVHD, P ¼ 0.0007. In univariate analysis, in addition of a-GVHD, febrile neutropenia was the only other factor associated to a reduction of frequency of marrow CFU-GM in the bone marrow at day þ 18 (P ¼ 0.08) while all other factors tested resulted not important: ‘‘HSC source’’, ‘‘Donor Type’’, ‘‘Dose of CD34 þ cells infused’’, ‘‘Doses of MTX actually administered’’, ‘‘Phase of disease’’. To assess importance of clonogenic cell measurement at day þ 18, on transplant outcome, patients were divided in two groups based on CFUGM growth in respect to median (‘‘CFU-GM below median’’ and ‘‘CFU-GM over median’’). These two groups were comparable for patient’s age (P ¼ 0.6), percentage of patients transplanted in advanced phase (P ¼ 0.8) and percentage of patients who received MUD transplant (P ¼ 0.6). Patients having a frequency of marrow ‘‘CFU-GM over median’’ had a cumulative incidence of TRM significantly lower than those showing ‘‘CFU-GM below the median’’ (respectively, 4.5% vs 30%, Gray test P ¼ 0.01). Cumulative incidence of RR was not significantly different in the two groups ‘‘CFU-GM over median’’ and ‘‘CFU-GM below the median’’ (RR: 31% vs 34%, P ¼ 0.3). In our series, 2-y -TRM was higher also after MUD than after HLA-ID Family donor transplants (TRM: 30% vs 10%), this difference in univariate analysis was not significant (Gray test
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P ¼ 0.2). In univariate analysis, predictors of OS at 2 years were: i) phase of disease at transplant’’: early phase: 80%; advanced phase: 42%, (P ¼ 0.03); ii) donor type (HLA ID Family donor tx OS: 78%; MUD tx OS: 52%, P ¼ 0.10) and iii) Frequency of CFUGM ("CFU-GM greater than median" had an OS of 81%; "CFUGM below the median" had an OS of 40%, P ¼ 0.001). In multivariate Cox proportional hazard analysis, a frequency of marrow ‘‘CFU-GM below median’’ remained an independent factor important in worsening OS (HR:8.4; P ¼ 0.0006), together with MUD donor (HR 5.3; P ¼ 0.002). Phase of disease was also important and Early phase was associated with improved OS (HR: 0.27; P ¼ 0.04). Conclusion: a-GVHD impairs early marrow function and it is the principal determinant of frequency of clonogenic precursors cells found in marrow at day þ 18. Marrow-CFU-GM frequency at day þ 18 may be used an early and simple biomarker for prediction of TRM and OS. Disclosure of Interest: None declared. P120 The beneficial impact of incorporation of low-dose antithymocyte globulin (rabbit) 5mg/kg to standard graftversus-host disease prophylaxis in matched or mismatched unrelated allogeneic stem cell transplantation I. Batsis1,*, D. Mallouri1, Z. Bousiou1, V. Constantinou1, C. Apostolou1, K. Tsirou1, P. Kaloyannidis1, C. Smias1, E. Yannaki1, A. Fylaktou2, A. Anagnostopoulos1, I. Sakellari1 1 Haematology department - BMT unit, George Papanicolaou Hospital, 2Immunology department, Hippokration hospital, Thessaloniki, Greece Introduction: The use of in-vivo T-cell depletion in the setting of unrelated transplants is still controversial since randomised studies have shown a reduction in severe graft versus host disease (GVHD) occurrence but no difference in overall survival. Hence, there is evidence for reduction in late morbidity and mortality. Moreover, the dose of prophylactic anti-thymocyte globulin (ATG) used in various studies ranges and the optimal dose is not established. Materials (or patients) and methods: In a prospective, single centre study, ATG (rabbit) has been used as standardised part of the conditioning in a uniform dose of 5mg/kg (total dose) and was infused on days -2 and -1. We analysed the outcome of all consecutive patients (pts) who underwent matched or mismatched unrelated alloHCT for hematologic malignancies. In total 92 pts (36 female, 56 male) suffering from acute leukaemia (79), MDS/MPDs (6), multiple myeloma (1), lymphoma (4) and CML (1) underwent allogeneic haematopoetic cell transplantation (alloHCT) from matched (8/8, n ¼ 53) or allele or antigen mismatched (7/8, n ¼ 37 and 6/8, n ¼ 2) unrelated donors. The grafts were peripheral stem cells in 84 and bone marrow in 8 pts, with median dose of CD34 þ cells infused 6.4 (0.78-17.74) 106/kg. Disease phase was classified as early in 37, intermediate in 23 and advanced in 32 pts. Anti-
GVHD prophylaxis consisted of cyclosporine or tacrolimus plus methotrexate in myeloablative transplants (85) and mycophenolate mofetil in RIC (7). Results: With a median follow-up of 20 months (0.8-139), overall survival (OS) was 59.8%, relapse rate (RR) 25% and treatment related mortality (TRM) 22.8%. The incidence of acute GVHD was 47.8%, grade Z II 34.7% (matched 32%, mismatched 38.4%, P ¼ 0.5), grade III-IV 14% (matched 9.4%, mismatched 20.5%, P ¼ 0.27) and of chronic 65% (extensive 40.2%, matched 41.5%, mismatched 38.4%, P ¼ 0.2). The matched and mismatched pts were comparable in age, sex and disease phase (x2 test). In univariate analysis no significant difference was found in the two groups in terms of incidence and severity of acute and chronic GVHD. Overall survival was superior in matched alloHCT (69.8% vs 46.1%, P ¼ 0.02) and this was attributed to the higher rate of TRM in mismatched transplants (35.8% vs 13.2%, P ¼ 0.16) while RR was similar (24.5% and 25.6%, P ¼ 0.9). In multivariate analysis incorporating sex, disease phase, mismatched donor, intensity of conditioning regimen and grade Z II acute and chronic GVHD, significant unfavourable factors for OS were grade Z II acute GVHD (P ¼ 0.04), male sex (P ¼ 0.04) and advanced disease phase (P ¼ 0.01). For relapse the most powerful factor was disease phase (early vs any other, P ¼ 0.009) while chronic GVHD was also significant for lower relapse probability (P ¼ 0.01). In terms of TRM, none of the above included factors was statistically significant. Conclusion: Though it is well known that unrelated alloHCT is a higher risk procedure than sibling transplants, the addition of ATG at a relatively low dose of 5mg/kg can lead to a high OS rate and acceptable TRM even in the mismatched transplants. Disclosure of Interest: None declared. P121 Acquired hemophilia A in a patient with chronic GVHD after cord blood transplantation successfully treated with rituximab I. Donnini1,*, C. Nozzoli1, R. Angarano2, C. Bazzini3, F. Bacci3, S. Linari4, S. Guidi1, A. Bosi5 1 Bone Marrow Transplantation, AOU Careggi, Florence, 2Hematology Unit, National Cancer Centre ‘‘Giovanni Paolo II’’, Bari, 3 High Dependency Unit – Internal and Emergency Medicine Department, 4Center for Bleeding Disorders, Department of Hearth and Vessels, 5Hematology Unit, AOU Careggi, Florence, Italy Introduction: Acquired Hemophilia A (AHA) is a rare bleeding disorder related to the development of inhibitor autoantibodies against endogenous factor VIII1 and also described in recipients of allogeneic-Hematopoietic stem cell transplantation (alloHSCT) as expression of Graft versus Host Disease (GvHD)2. Materials (or patients) and methods: We report a case of AHA in a recipient of umbilical cord blood transplantation (UCBT) for acute myeloid leukemia (AML) M2 FAB, NPM1/FLT3 neg/neg. 40-year old male who underwent UCBT with a single cord blood unit 4/6 HLA compatible with total nucleated cells 3x107/Kg and CD34 þ 0.3x 105/Kg. Conditioning regimen consisted of Thiotepa 5mg/Kg on days -7, -6, Busulphan 3.2 mg/Kg on days -5, -4, -3 and Fludarabine 50 mg/m2 on days -5, -4, -3. Anti Thymocyte Globulin, Ciclosporine (CSA) and Mycophenolate Mofetile (MMF) were given for GvHD prophylaxis. Nine months after UCBT the patient experienced cutaneous lichenoid GvHD requiring CSA and steroid treatment. After interruption of steroid treatment, the patient developed a relapse of cutaneous GvHD grade III with eczema feature and started again treatment with steroid and Extracorporeal Photopheresis (ECP). Twenty two months after UCBT the patient experienced sequential spontaneous muscular hematomas in the right gluteus, calf and over the left arm;respiratory/hemodynamic parameters were normal.A thoracic and abdominal CT scan excluded the presence of
muscular and visceral hematomas. Laboratory exams were relevant for normocytic anemia (Hb 8.4 g/dL), prolonged activated partial thromboplastin time (aPTT) (60.5 s, normal range: 22.0-38.0 s), decreased FVIII activity (4%, normal value: 60-150%) and presence of FVIII inhibitors with a low titer of 1,6 Bethesda Units (BU)/mL (normal value o0,6 BU/mL). White blood total, platelet count, prothrombin time, fibrinogen and creatinine clearance were normal; dilute Russell’s viper venom time (dRVVT) for Lupus anticoagulant, antiphospholipid and antinuclear antibodies were negative. No evidence of AML relapse was documented and the chimerism was fully donor. The patient was treated with infusion of plasma derived FVIII (Emoclot D.I., Kedrion) 5.000 International Unit (IU) every 12 hours, associated to methylprednisolone 1 mg/Kg/day. After seven days of steroids treatment without an increase of FVIII activity, we started a second line therapy with Rituximab 375 mg/m2 weekly schedule for a total of 4 infusions. Results: After one week from the first dose there was a marked improvement of aPTT and FVIII activity (Figure 1), with a reduction in hematomas size and related pain. The last evaluation of aPTT, factor VIII and FVIII inhibitor values, performed 6 months after Rituximab was initiated, showed an excellent recovery (respectively 24.8, 168% of activity and 0.1 BU/mL); the patients didn’t experience cGVHD relapse or any clinical complication, included new bleedings. Conclusion: This case report confirms the role of Rituximab in the treatment of AHA as expression of cGVHD and to our knowledge, this is the first case of AHA in recipient UCBT. References: 1. Shetty S, Bhave M, Ghosh K. Aquired haemophilia A: Diagnosis, aetiology, clinical spectrum and treatment options. Autoimmunity reviews 10/2011; 311-316. 2. Wieliczka ML, Ganguly S et al. Rituximab as a treatment for factor VIII inhibitor in a patient with chronic GVHD.Bone Marrow Transplant. 2014 Apr;49(4):588. Disclosure of Interest: None declared. P122 Models for Effective Acute Graft Versus Host Disease Prophylaxis after Allogeneic Bone Marrow Transplantation with Mesenchymal Multipotent Stromal Cells I. Shipunova1,*, N. Petinati1, A. Bigildeev1, N. Sats1, N. Drize1, L. Kuzmina2, E. Parovichnikova2, V. Savchenko2 1 lab. physiology of hematopoiesis, 2Bone Marrow Transplant Department, National Research Center For Hematology, Moscow, Russian Federation Introduction: Graft-versus-host disease (GVHD) is often accompanied allogeneic bone marrow transplantation (alloBMT). As the conventional methods of GVHD prophylaxis are often inefficient new method involving the use of donors’
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multipotent mesenchymal stromal cells (MSC) was developed. In some cases prophylaxis of acute GVHD (aGVHD) failed. The reasons of the failure could be either the result of particular qualities of donor-recipient interaction, patient status or characteristics of MSC samples. The results of the aGVHD prophylaxis with donors’ MSC injections after allo-BMT in patients with hematological malignancies were analyzed. The growth and differentiation characteristics, relative expression levels of different genes were investigated in all MSC samples. Materials (or patients) and methods: The study included 88 patients received allo-BMT from related donors after informed consent. The patients were divided into 2 groups: the first received standard prophylaxis of aGVHD and the second were additionally infused with MSC from the bone marrow of corresponding hematopoietic stem cells donor. MSC were cultivated in aMEM with 4% donors’ platelet lysate. MSC were administered intravenously when the blood counts indicated recovery (peripheral blood leukocytes reached 1x109/l). MSCs and colony-forming unit-fibroblasts (CFU-Fs) from the bone marrow of those donors were analyzed. For this purpose MSC were cultivated in standard conditions (aMEM, 10% fetal calf serum) for 5 passages. Relative expression level (REL) of 30 genes involved in proliferation, differentiation and immunomodulation was estimated by RT-qPCR. Results: The infusion of MSC reduced the incidence of aGVHD 2 times and increased the 5 years overall survival of patients (P ¼ 0.047). Six of 44 MSC samples had been ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index), the MSC properties of these donors found no significant differences between the MSC, effective and ineffective for preventing aGVHD. The combination of predictors that characterize the most suitable for the prevention of aGVHD MSC samples was revealed by multiple logistic regression analysis. A few models calculating the probability of the success of MSC samples application was proposed: logit(P) ¼ a þ bx þ cy þ dz, where logit(P) ¼ ln[P/(1P)], P – probability of successful prophylaxis, x, y and z – REL of corresponding genes in tested MSC sample. These models define 55-71% of effective MSC samples as successful. Threshold for success was considered as 0.9. These models included REL of following genes: FGFR1, PDGFRb, PPARg, IGF1, IDO1, TGFb1 and CHF. Only REL of FGFR1 and PDGFRb were significantly increased in MSC successfully prevented the development of aGVHD comparing with ineffective cases, REL of other genes mattered only in the framework of the models. Conclusion: These data confirm the presence of hierarchy as well as heterogeneity in MSC population. The high variability of all analyzed characteristics among MSC from different donors was shown. The mathematical models revealed the combination of parameters enabling to distinguish effective and ineffective MSC samples. By proposed models ineffective MSC samples could be discharged and replaced. Such strategy hopefully will prevent the development of aGVHD in the maximum number of patients. Disclosure of Interest: None declared. P123 Association of tacrolimus concentrations with incidence of acute graft-versus-host disease and relapse after matched allogeneic hematopoetic stem cell transplantation in adult patients I. Moiseev1,*, N. Senina1, V. Vavilov1, O. Slesarchuk1, S. Bondarenko1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Hematology and Transplantation, First State Medical University named I.P. Pavlov, Saint-Petersburg, Russian Federation Introduction: A number of studies have highlighted the significance of early cyclosporine concentrations for outcomes of allogeneic hematopoetic stem cell transplantation
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(HSCT). Data on the significance of tacrolimus pharmacokinetics is limited, so we retrospectively assessed impact of tacrolimus concentrations on the incidence of acute graft-versus-host disease (aGVHD), 2-year relapse rate (RR), non-relapse mortality (NRM) and overall survival (OS) after HSCT. Materials (or patients) and methods: Concentrations were obtained from 153 consecutive patients who received 10/10 matched related or unrelated allogeneic HSCT between 2010 and 2013. GVHD prophylaxis consisted of tacrolimus, shortcourse methotrexate or mycophenolate mofetil (MMF). In unrelated HSCTs antithymocyte globulin (ATGAM) 60 mg/kg was added. Median follow-up was 342 days (range 28-1250 days). Detailed patient and transplantation characteristics are presented in Table 1. Tacrolimus concentration was measured 2-4 times weekly. Concentrations in the first 50 days after HSCT were analyzed. Results: In the ROC analysis geometric means of concentrations during the first 21 days (conc21Days) after HSCT had the highest predictive values for grade II-IV aGVHD (AUC 0.62) and relapse (AUC 0.58), compared to 30- and 50-day concentrations. The ROC-determined conc21Days cut-offs were o9.1 ng/ml for aGVHD and 413.1 ng/ml for relapse. In the univariate and multivariate analyses patients with conc21Days o9.1 ng/ml had increased incidence of grade II-IV aGVHD (51% vs 25%, HR 0.47, 95%CI 1.01-3.28, P ¼ 0.047) and NRM (31% vs 15%, HR 2.19, 95%CI 1.00-4.80, P ¼ 0.05). On the contrary, conc21Days 413.1 ng/ml was associated with increased RR (65% vs 35%, HR 2.45, 95%CI 1.19-5.06, P ¼ 0.015). In patients with conc21Days between 9.1 and 13.1 the above results translated into better OS compared to the rest of the group (69% vs 47%, HR 0.43, 95%CI 0.23-0.78, P ¼ 0.005). Conclusion: Conclusion: Based on this study, it is recommended to maintain tacrolimus concentrations within 9-13 ng/ml therapeutic range during first 21 days after HSCT. These results should be confirmed in prospective studies. Disclosure of Interest: None declared.
P124 Efficacy of Infliximab for the Treatment of Acute Gastrointestinal Graft-versus-Host Disease in Children after Allogeneic Hematopoietic Stem Cell Transplantation: A Single-Center Experience J. K. Suh1,*, S. W. Lee1, K.-N. Koh1, E. S. Choi1, H. J. Im1, J. J. Seo1 1 Pediatrics, Asan Medical Center Children’s Hospital, Seoul, Korea, Republic Of Introduction: Graft-versus-Host Disease (GVHD) is a major limitation of allogeneic hematopoietic stem cell transplantation (HSCT). Especially, the outcomes of steroid refractory acute GVHD is poor, irrespective of secondary therapy. Infliximab, a chimeric monoclonal antibody against tumor necrosis factor-a, has been reported to have activity against steroid refractory acute GVHD involving gastrointestinal tract (acute GI GVHD). Here we report a single-center experience with using infliximab as a treatment of steroid refractory acute GI GVHD. Materials (or patients) and methods: We retrospectively reviewed the medical records of 16 patients who had been treated with infliximab for stage II-IV acute GI GVHD after alloHSCT at Asan Medical Center from January 2005 to November 2014. The diagnosis of acute GI GVHD was made on clinical grounds with endoscopic biopsy. Severe acute GI GVHD patients, who did not respond to standard steroid therapy (methylprednisolone 2 mg/kg/day), were treated with infliximab (10 mg/kg, weekly). Results: Of total 16 patients who were treated with infliximab for acute GI GVHD, 6 patients (37%) had stage II and 1 (6%) had stage III and 9 (56%) had stage IV acute GI GVHD. Eleven patients (69%) diagnosed with malignancy (5 AML, 4 ALL, and 2 JMML) and 5 (31%) diagnosed with nonmalignancy (2 SAA, 1 MDS, 1 CGD, and 1 IPEX). Acute GI GVHD developed at median 22 days after transplantation (range: 7-54). Median time to start infliximab from onset of acute GI GVHD was 7 days (range 1-28). Median number of infliximab administration was 3 (range 2-10). All the 16 patients responded to infliximab; 13 (81%) achieved complete response (CR) and the remaining 3 (19%) achieved partial response (PR). None of the patients developed therapy-related adverse effects. Eventhough 6 bacterial infection, 3 respiratory viral infection and 8 CMV reactivation were documented after infliximab administration, all the cases were well controlled with standard anti-bacterial and anti-viral therapy. Of the 15 evaluable patients, 6 (38%) had chronic GVHD at a median of 16 months after transplantation (range: 4-52). Three patients died; 1 from chronic GVHD, 1 from acute GVHD and the remaining 1 from disease progression. All the dead patients had stage IV acute GI GVHD. At a median follow up of 24 months (range 4-52), 2 year-overall survival (OS) was 78.7%. In the analysis of responses to infliximab, time to start infliximab from the onset of acute GI GVHD (r10 days) and the stage of acute GI GVHD (r stage III) had positive effect on CR rate (Po0.001). Conclusion: Our study can suggest that the addition of infliximab to steroid refractory acute GI GVHD could be effective to achieve CR without life threatening adverse effect. A multi-center prospective trial is needed to confirm the efficacy of infliximab. Disclosure of Interest: None declared. P125 Impact of GvHD-prophylaxis regimen on incidence and severity of gastrointestinal toxicities and acute GvHD after allogeneic HSCT J. Niestadtko¨tter1,*, L. Mu¨ller1, C. Mu¨ller-Tidow1, T. Weber1 1 Klinik fu¨r Innere Medizin IV, Martin-Luther-Universita¨t Halle-Wittenberg, Halle, Germany Introduction: Besides methotrexate (MTX), two oral mycophenolate formulations, mycophenolate mofetil (MMF) and mycophenolate sodium (EC-MPS) are used for GvHD-
prophylaxis after allogeneic HSCT. Compared to MTX, MMFprophylaxis for GvHD is associated with a lower rate of mucositis but an equal incidence of acute GvHD. Analyses in solid organ transplant show that (1) the frequency of gastrointestinal toxicities (GI-tox) in EC-MPS is lower than in MMF and that (2) gastrointestinal EC-MPS resorption under proton pump inhibitor (PPI) co-medication is higher than of MMF. We hypothesize that this is also the case after allogeneic HSCT, and that GvHD-prophylaxis with EC-MPS will lead to a lower incidence of acute GvHD when compared to MTX and MMF. Materials (or patients) and methods: The rate and severity of GI-tox, acute GvHD, and the frequency of PPI-co-medication as well as dose reductions of methotrexate and mycophenolate formulations were retrospectively analyzed in a cohort of 102 patients after allogeneic HSCT. All patients received GvHDprophylaxis based on cyclosporine A plus MTX or plus one of the mentioned mycophenolate formulations. Patients treated with MMF or EC-MPS and occurrence of severe intestinal mucositis CTC 431 were bridged with MMF i.v. for this time interval. GI-tox CTC Z31 (nausea, emesis, mucositis, diarrhea, abdominal pain) was evaluated until 100 days after allogeneic HSCT or initiation of GvHD-treatment, and the overall incidence of acute GvHD was assessed. Results: In total, 32 patients (31%) received MTX, 40 (39%) MMF p.o. and 30 (29%) EC-MPS p.o. for GvHD-prophylaxis. In the MTX, MMF and EC-MPS-groups, the frequencies of GI-tox CTC Z31 were 31%, 18% and 17% for nausea, 9%, 13% and 7% for emesis, 56%, 10% and 23% for mucositis (MTX vs. mycophenolate formulations Po0.001), 47%, 43% and 33% for diarrhea and 3%, 3 and 17% for abdominal pain (MTX þ MMF vs. EC-MPS P ¼ 0.022). GvHD-prophylaxis was reduced due to GI-tox in 4 MTX and 1 MMF patient but none of the ECMPS treated patients. All patients received PPI-prophylaxis during the entire interval of GvHD-prophylaxis. The incidence of acute GvHD was 66% in the MTX group, 78% MMF group, and 47% in the EC-MPS group (MTX vs. MMF P ¼ 0.299, MTX vs. EC-MPS P ¼ 0.2, MMF vs. EC-MPS P ¼ 0.012, MTX þ MMF vs. ECMPS P ¼ 0.022). Conclusion: GvHD-prophylaxis with mycophenolate formulations was associated with a lower mucositis rate compared to MTX. When compared with MMF, GvHD-prophylaxis with ECMPS did not lead to lower GI-tox but was associated with a significant lower incidence of acute GvHD. This could be due to PPI co-medication lowering the intestinal uptake of MMF but not EC-MPS. Disclosure of Interest: None declared. P126 Incidence of severe chronic GVHD is reduced either by ATG or by statins K. Schmidt1,*, M.-A. Schwarzbich1, N. Lehners1, P. Dreger1, T. Luft1 1 University Hospital Heidelberg, Department of Haematology, Heidelberg, Germany Introduction: Severe chronic graft versus host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic stem cell transplantation (allo-SCT). The pathomechanism of cGVHD is currently not known, however, alloreactive T cells clearly play a role. Nevertheless, severe chronic GVHD poorly responds to immunosuppressive therapy similar to acute refractory GVHD. It has previously been shown that endothelial risk factors are associated with the risk of refractory acute GVHD. These factors include angiopoietin-2 (ANG2), serum nitrates and asymmetric dimethylarginine (ADMA), as well as single nucleotide polymorphisms (SNPs) in the thrombomodulin gene (THBD). In the presence of statins the incidence of refractory acute GVHD in patients with endothelial risk factors is reduced. In the present retrospective study we investigated whether the incidence of severe chronic GVHD (cGVHD) was also influenced by endothelial risk factors and statins.
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Materials (or patients) and methods: Patients were eligible if they were allo-grafted between June 2002 and December 2011 at our institution, and if their blood samples were available for nitrate, ANG2 and/or ADMA measurement at different landmarks (collected immediately before conditioning and on day þ 100 after allo-SCT). Cumulative incidence analysis of cause-specific hazards was performed. The occurrence of cGVHD was evaluated retrospectively by chart review applying clinical and histological criteria developed by the National Institute of Health’s consensus project. Results: Of a total of 329 eligible patients, 167 (51%) fulfilled the criteria for cGVHD, and 47 (14%) of them developed severe cGHVD. There was no correlation between cGVHD and antecedent acute GVHD (P ¼ 0.796). Moreover, there were no significant correlations between serum nitrates, ANG2, ADMA and thrombomodulin-(THBD)-SNPs and the risk of severe cGVHD. Mild and severe cGVHD were associated with a significantly prolonged overall survival (OS) compared to non-cGVHD patients (Po0.001) due to a reduced relapse rate (Po0.001). This effect was stable across all entities. Antithymocyte globulin (ATG), at our institution given in case of unrelated donor, reduced the risk of mild cGVHD (Po0,001), and of severe cGVHD (P ¼ 0,016) without increasing relapse rates. Statins, introduced as part of the supportive treatment for all patients who receive an allo-graft at our institution, increase the incidence of mild cGVHD in patients who had not received ATG (chi2 ¼ 0,039). Moreover, statins reduce the incidence of severe cGVHD in patients who had not received ATG (chi2 ¼ 0,011). Introduction of statins removed the protective effect of ATG on the incidence of severe cGVHD (P ¼ 0,558). Conclusion: Unlike refractory acute GVHD, endothelial risk factors were not associated with severe cGVHD. Our results suggest a different role of the endothelial component in the pathogenesis of acute vs chronic GVHD. Statins reduce the incidence of refractory acute GVHD which has been attributed to modulation of endothelial homeostasis. On the other hand, statins also have an impact on the occurrence of mild and severe cGVHD in the absence of ATG. Thus we hypothesize that the effect in cGVHD is executed by immunomodulation of the T-cell compartment. Within the limits of this retrospective study with consecutive patient cohorts, our data suggest that statins protect from severe chronic GVHD without reducing mild cGVHD. Disclosure of Interest: None declared. P127 Chronic Graft-versus-Host Disease Biomarker Discovery and Replication K. R. R. Schultz1,*, A. Kariminia2, S. Ivison3, S. G. Holton4, M.-J. Hebert5, M. E. Flowers6, P. Martin6, J. Rozmus3, P. Subrt7, B. Storer8, S. J. Lee8 1 Pediatric Hematology/Oncology/BMT, BC Children’s Hospital/ Univ. of BC, 2Pediatric hematology/Oncolcogy/BMT, BC Children’s/Child and Family Research Institute, 3Bc Children’s Hospital/CFRI, Vancouver, Canada, 4University of Minnesota, Blood and Marrow Transplant Program, Minneapolis, United States, 5Hopital Notre-Dae-Pavillion Mailloux, University of Montreal, Montreal, Canada, 6Clinical Research Division, Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, United States, 7BC Children’s Hospital and CFRI, Vancouver, Canada, 8Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, United States Introduction: Chronic graft-versus-host disease (cGVHD) remains one of the most significant long-term complications after allogeneic blood and marrow transplantation. Biomarkers that can act as diagnostic, prognostic and predictive markers for cGVHD are needed to improve the ability to treat cGVHD effectively. Many candidate cGVHD biomarkers have been identified with none validated for use in clinical practice. Larger replication studies are needed.
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Table 1. Univariate replication results for 13 candidate biomarkers Candidate biomarkers sBAFF CXCL10 ICAM-1 CXCL9 Anti-LG32 Gelsolin AGAy
N evaluable
Effect size1
Pvalue
173 173 173 162 173 173
1.2x 1.4x 1.2x 1.5x 0.7x 0.9x
0.11 0.06 0.06 0.03 0.05 0.17
AUC
0.60 0.59 0.61 0.60
1
Fold difference cGVHD vs. control, 2Anti-LG3 has been associated with renal transplant rejection.
Materials (or patients) and methods: We evaluated cGVHD biomarkers in a small adult discovery set (N ¼ 38, 21 cGVHD within one month of diagnosis and 17 controls) followed by a replication attempt in a larger cohort (N ¼ 173, 86 cGVHD and 87 controls). Samples were provided by the Chronic GVHD Consortium. Before proteomic analysis, the plasma samples were depleted for the 13 most common plasma proteins. Samples were tested at the University of Victoria Proteomics Facility (BC, Canada) with a MALDI-TOF-TOF methodology using iTRAQ labels as we have previously done. Multiple Reaction Monitoring-Mass Spectrometry (MRM) was used to measure 71 candidates identified by proteomic studies. Additional Luminex analysis was done for cytokines. Based on these results, as well as leading candidates in the published literature, 16 of the most promising markers were tested in an independent cohort of 86 cGVHD and 87 post BMT controls. Cases and controls were matched on time of sample collection (more or less than 9 months post BMT) and previous acute GVHD. Results: Proteomic analysis of the discovery cohort (N ¼ 38) with confirmation by MRM revealed two proteins of high interest as cGVHD diagnostic markers, aminopeptidase N (sCD13, P ¼ 0.004) and Peptidase Inhibitor 16 (PI-16, P ¼ 0.005) compared to controls. Luminex suggested that IL-2Ra (P ¼ 0.0007), IL-6 (P ¼ 0.05) and IL-8 (P ¼ 0.04) were all of interest. Enzymatic studies confirmed higher levels of aminopeptidase N in cGVHD. Of the 16 candidate biomarkers, assays for 3 biomarkers had technical difficulties and were deferred for later analysis (TNFa, IL-6, IL-8). Results for the other 13 candidates with p values under 0.02 are shown in the Table. Of the other 13 markers, aminopeptidase N, IL2Ra, PI-16, MCP1, Endothelin1, and Kallikerin had p values between 0.23 - 0.52. Results were similar when adjusted for time of sample collection, prior acute GVHD and conditioning with total body irradiation. Four biomarkers, CXCL10, ICAM-1, CXCL9, and antiLG3 (p 0.03 – 0.06) were marginally associated with cGVHD with ROC AUCs of 0.60, 0.59, 0.61 and 0.60, respectively and 0.63 for the four in combination. Conclusion: Despite strong associations observed in a discovery set using proteomic analysis with duplication by MRM as well as by a screening Luminex assay supplemented by enzymatic assays, attempted replication in a larger independent cohort did not replicate the original findings. Three candidate biomarkers could not be analyzed. This analysis confirms the need to replicate results of promising cGVHD biomarkers in larger independent cohorts. Disclosure of Interest: None declared.
P128 The activating NKG2C receptor is significantly reduced in NK cells after allogeneic stem cell transplantation in patients with severe graft-versus-host disease L. Kordelas1,*, P. A. Horn2, D. W. Beelen1, V. Rebmann2 1 Department of Bone Marrow Transplantation, 2Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany Introduction: Natural killer (NK) cells are important players of the innate immune system. The allo-reactivity of NK cells is regulated by a number of receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptor pair, which are both recognizing the non-classical human leukocyte antigen E (HLA-E). In allogeneic stem cell transplantation (alloSCT) donor-derived allo-reactive NK cells are important effectors mediating the killing of leukemia cells and patient’s dendritic cells thereby preventing leukemic relapses or graftversus-host responses. To analyze the contribution of these receptors to NK cell allo-reactivity, the NKG2A and NKG2C expression were monitored in 26 allo-SCT patients. Materials (or patients) and methods: The course of CD94/ NKG2C or CD94/NKG2A expression on NK cells were studied in 26 patients undergoing allo-SCT due to acute myeloid leukemia (AML, n ¼ 20), secondary AML (sAML, n ¼ 4), myelodysplastic syndrome (MDS, n ¼ 1) and T-Non-Hodgkin Lymphoma (T-NHL, n ¼ 1). EDTA samples were serially procured from the patients before (0) and 1, 2, 3, 4, 5, 6, 9 and 12 month(s) after allo-SCT. Receptor expression on NK cells were analyzed by flow cytometer using specific antibodies against human CD56, NKG2A, CD94 and NKG2C. The results obtained were correlated to occurrence of acute or chronic graft-versus-host disease (GvHD) and its severity after alloSCT. Results: The proportion of activating receptor pair CD94/ NKG2C on NK cells was significantly reduced in 10 patients
who had experienced severe acute GvHD (aGvHD) grade 2-4 in the first months after alloSCT (Table 1a, Po0.0001, two-way ANOVA) compared to 16 patients with no or only mild aGvHD. Moreover, the proportion of activating receptor pair CD94/ NKG2C on NK cells was lower in patients with extended chronic GvHD (cGvHD) compared to patients with no or only limited cGvHD (n ¼ 13, Figure 1b, Po0.0001, two-way ANOVA). At contrast there was no difference detectable between the proportion of the inhibitory NKG2A receptor on NK cells and the incidence of acute or chronic GvHD (Figure 1c and 1d). Conclusion: Thus, these results provide substantial evidence that the CD94/NKG2C receptor pair contributes to the alloreactivity of NK cells after allo-SCT and its influence on severe acute and chronic GvHD. Further investigation will have to clarify the role of HLA-E expression, which is the cognate ligand of CD94/NKG2C. References: This study was supported by a research grant by the Deutsche Jose´ Carreras Leuka¨mie-Stiftung e.V. Disclosure of Interest: None declared. P129 Association of NIH organ cGVHD scores with blood lymphocyte subsets – a pilot study L. Grkovic1,*, D. Pulanic1, Z. Peric1, D. Batinic1, R. Serventi Seiwerth1, M. Mravak Stipetic1, E. Bilic1, R. Ceovic1, L. Rajic1, N. Durakovic1, N. Matic1, T. Klepac Pulanic1, I. Petricek1, T. Vukic1, D. Ljubas1, E. Bilic1, D. Dusek 2, E. Prenc1, I. Prah1, I. Bojanic1, M. Grce3, R. Zadro1, R. Vrhovac1, S. Z. Pavletic4, D. Nemet1 1 UHC Zagreb, 2Infectious disease, University Hospital for Infectious Diseases, 3Ruder Boskovic Institute, Zagreb, Croatia, 4 National Cancer Institute, Bethesda, United States Introduction: Immune reconstitution after allogeneic stem cell transplantation is influenced by many factors including
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chronic graft versus host disease (cGVHD). Although cGVHD is a complex multifactorial disease and there is no satisfactory therapy, distinct clinical syndromes with specific organ manifestations may exist, indicating subpathologies, which ultimately may require differently tailored therapies. To address this question, we tested the associations of immunological monitoring parameters with individual organs involvement and other key cGVHD characteristics. Materials (or patients) and methods: After establishment of a multidisciplinary cGVHD team in 2013 at the UHC Zagreb, patients were enrolled into the prospective cross-sectional study that included subspecialists exam, extended clinical assessments, and laboratory data collection at the time of study entry. Five lymphocyte subsets, T-cell (CD3 þ , CD3 þ CD4 þ helper-Th, CD4 þ CD8 þ cytotoxic-Tc) B-cell (CD19 þ ) and natural killer (NK, CD16 þ CD56 þ ) cell were analyzed in peripheral blood by flow cytometry and correlated with demographic, transplant, cGVHD-related and laboratory data. Univariate analyses were undertaken to determine associations between lymphocytes subsets (high/low by median) and outcomes of interest. Results: Thirty adult and 3 pediatric patients were enrolled into the study. The median age was 40 years [9-72], 45% were female. Median time from transplant to study enrollment was 19 months [2-197]. The median time from cGVHD diagnosis to enrollment was 7 months [0.03-178] and the median time from transplant to cGVHD diagnosis was 10 months [1.8-29]. The majority of patients were transplanted for hematologic malignancies (85%). Eighteen patients (55%) underwent myeloablative conditioning and 45% received ATG, 70% had a related donor transplant and 64% received peripheral blood stem cells as graft source. Patients received a median of 2 [0–6] prior systemic therapies, 52% were on systemic immunosuppression, 55% had severe, 45% moderate NIH global score, 76% had previous acute GVHD and 85% were classified as classic. Patients who had previous acute GVHD had lower Th cells (P ¼ 0.02). Patients with progressive cGVHD onset had lower Th (P ¼ 0.009) and B cells (P ¼ 0.0007). Higher Th, B and NK cells were found in patients presenting with classic cGVHD compared to overlap. As expected, patients who were receiving moderate or high immunosuppression had lower B (P ¼ 0.005) and NK cells (P ¼ 0.03). Patients who had documented infections since cGVHD diagnosis had lower Th (P ¼ 0.01) cells. No significant associations were found between cGVHD severity (global NIH score) and activity, and subsets of lymphocytes. However, a statistically significant association was found between moderate or severe organ involvement (NIH score 2-3) and certain lymphocyte subsets in blood: higher NK (P ¼ 0.04) in lung, lower B (P ¼ 0.005) and NK (P ¼ 0.05) in liver, lower B (P ¼ 0.03) in mouth, and higher B cells in skin (P ¼ 0.02) and joint (0.004) cGVHD involvement. No difference was found in T cell subsets depending on different organ involvement. Conclusion: Several individual organs had distinct associations with lymphocyte subset numbers in patients with cGVHD. Immune status is clearly influenced by the intensity of immunosuppression but not by the global NIH severity or activity. These data provide rationale for further studying of chronic GVHD biology in conjunction with distinct clinical syndromes. Disclosure of Interest: None declared.
P130 Multipotent mesenchimal stromal cells as graft-versushost disease prophylaxis: a prospective randomised clinical trial L. Kuzmina1,*, E. Parovichnikova1, M. Drokov1, V. Vasilieva 1, T. Sorokina 1, N. Petinati 2, I. Shipounova 2, N. Drize 2, V. Savchenko1 1 Bone Marrow Transplant Department, 2lab. physiology of hematopoiesis, National Research Center For Hematology, Moscow, Russian Federation Introduction: Acute and chronic graft-versus-host disease (GvHD) develops in more than 50% patients after haemopoietic stem cell transplantation (HSCT) and remains one of the main causes of mortality. So new methods of GvHD prophylaxis are needed. The aim of the study was to investigate the efficacy and safety of multipotent mesenchymal stromal cells (MMSC) administration for graft-versus-host disease prophylaxis. Materials (or patients) and methods: Since February 2009 65 patients received stem cell transplantation from related donors were included in the randomized study (ClinicalTrials.gov:NCTO1941394) , with acute myeloid leukemia – 25, acute myelomonocytic leukemia – 7, chronic myeloid leukemia – 9, acute lymphocytic leukemia – 17, myelodysplastic syndrome – 5, chronic lymphocytic leukemia – 1, T-cell lymphoma – 1. Patients age was 18-63 years. Myeloablative conditioning was used in 43 patients, 22 patients received reduced intensity conditioning. Standard GvHD prophylaxis was cyclosporine A þ methotrexate, cyclosporine A þ methotrexate þ prednisolon, or cyclosporine A þ methotrexate þ mycophenolate mofetil. The observation time was 4-75 months. All patients were randomized in 2 groups: with standard GvHD prophylaxis (n ¼ 33) and those who received MMSC with standard GvHD prophylaxis (n ¼ 32). MMSC were administered in the moment of blood counts recovery (leukocytes more then 109 per liter). The dose was 0,9-1,39 106/kg. The day of administration was 17-54 after HSCT (median þ 24,5 day). MMSC were derived from the bone marrow of haematopoietic stem cells donors at the day of HSCT and cultured for 3-4 weeks in alpha-MEM with 4% human platelet lysate derived from peripheral blood of the same donors. Most of the patients had fever and chills during 24 hours after MMSC administration. There were no other complications. Acute GvHD was monitored during 100 days after HSCT. Results: In standard prophylaxis group acute GvHD II-IV developed in 21,9% patients, in MMSC group – in 9,4% patients (P ¼ 0,06). Chronic GvHD was diagnosed in 33,3% patients in the first group and in 25% patients in the second group. There was no difference in disease recurrence rate and graft failure rate. The overall mortality was 36,4% in standard prophylaxis group compared to 21,9% in MMSC group. Conclusion: The results of our study demonstrate the efficacy and safety of MMSC administration as graft-versus-host disease prophylaxis. Disclosure of Interest: None declared. P131 Tacrolimus (FK) compared to cyclosporine A (CyA) after haploidentical T cell replete stem cell transplantation (Haplo-SCT) with post-infusion cyclophosphamide (PT-Cy): a retrospective analysis L. Castagna1,*, S. Bramanti1, S. Furst2, B. Sarina1, R. Crocchiolo2, L. Giordano1, J. El -Cheikn2, A. Granata2, L. Morabito1, C. Faucher2, B. Mothy2, S. Harbi2, C. Chabannon2, C. Carlo Stella1, A. Santoro1, D. Blaise2 1 Bone Marrow Unit, Humanitas Cancer Center, Rozzano, Italy, 2 Bone Marrow Unit, Institue Paoli Calmette, Marseille, France Introduction: In this retrospective study, we analyzed 100 patients treated with Haplo-SCT with PT-Cy, and receiving FK or CyA.
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Table 2.
Main results in 2 groups FK þ BM vs CyA þ PBSC
CI 30 day ANC 40.5 x109/L CI 60 day PLT 420 x109/L G 3-4 aGVHD cGVHD OS@2 year EFS@2 year Relapse incidence NRM
FK þ BM N 42
CyA þ PBSC N 43
P
79% 76% 2% 13% 65% 22% 25% 17%
93% 85% 12% 10% 70% 29% 13% 15%
0.09 0.54 0.09 0.75 0.65 0.44 0.14 0.68
Materials (or patients) and methods: Conditioning regimen consisting of Fludarabine, Cyclophosphamide, and low dose TBI for all patients. GVHD prophylaxis consisted of Cy 50 mg/ kg on days þ 3 and þ 4, FK or CyA and MMF. FK (at a total dose of 1 mg) or CyA (3 mg/kg/day) were administered as a continuous infusion. MMF (45 mg/kg/day) was administered until day þ 35. FK, CyA and MMF were started on day þ 5. FK and CyA were tapered by day þ 100/ þ 180. Stem cell source was BM or peripheral blood stem cells (PBSC). Results: The cumulative incidence of engraftment was similar between FK and CyA groups: 30-days absolute neutrophil count 40.5x109/L was 79% vs 91% (p 0.3), and unsupported platelet count more than 20 000 at 60 days was 77% vs 84% (p 0.7), respectively. The incidence of grade 2-4 acute GVHD was 33% vs 48% (p 0.1) and the incidence of chronic GVHD was 13% vs 8% (p 0.9), respectively. The 2-year progression free survival (PFS) and overall survival (OS) were 53% vs 64% (p 0.2) and 65% vs 65%, respectively (p 0.7). The 2-year relapse incidence (RI) was 28% vs 12% (p 0.08) and the 2-year non relapse mortality was 16% vs 14% (p 0.7), respectively. In univariate analysis, only disease status (CR vs not CR) impacted the PFS, OS, and RI. Patients were regrouped in 3 cohorts (1 patient excluded because received FK þ PBSC): FK þ BM (n 42), CyA þ BM (n 14), and CyA þ PBSC (n 43). 1-year NRM was not different (17% vs 14% vs 15%, p 0.8) as well as the cumulative incidence of aGVHD and cGVHD. FK þ BM (as standard treatment) was compared to CyA þ PBSC and not significative differences were founded (Table 2). Conclusion: Although this is a retrospective study with potential selection bias, using CyA instead of standard FK did not seem to affect any major clinical outcome, with special interest for aGVHD and cGVHD. The NRM is acceptable in line with that published. Disease status before haplo SCT was the only prognostic factor for survival and relapse. Disclosure of Interest: None declared.
Infectious complications I P132 Immunogenicity of virosomal adjuvanted trivalent influenza vaccination in allogeneic stem cell transplant recipients A. Ambati1,2,*, S. Einarsdottir3, I. Magalhaes1,4, T. Poiret4, R. Bodenstein4, K. LeBlanc1,4, M. Brune3, M. Maeurer1,4, P. Ljungman2,5 1 Centre for allogeneic stem cell transplantation (CAST), Karolinska University Hospital, 2Dept of Medicine Huddinge, karolinska Institutet, Stockholm, Huddinge, 3Sahlgrenska University Hospital, Goteborg, 4Dept of Laboratory Medicine, karolinska Institutet, 5Department of Hematology, Karolinska University Hospital, Stockholm, Huddinge, Sweden Introduction: The immunogenicity after a single dose of adjuvanted trivalent virosomal vaccination Inflexal Vs was
evaluated in a study cohort of 21 hematopoietic stem cell transplant (HSCT) recipients vaccinated during the 2013-14 season. This immunogenicity was compared to a control cohort of 31 HSCT recipients who received a single dose of seasonal subunit vaccination over three seasons from 20102013 at two Swedish transplant centers. Materials (or patients) and methods: Whole blood IFNg (gamma-interferon) release assays were tested both prior to and 30 days after vaccination in response to influenza pandemic (pdm) (A/California/7/2009(H1N1), A/Victoria/210/ 2009(H3N2) and B/Brisbane/60/2008 monovalent bulks. Influenza Matrix 1 HLA-A*02- GILGFVTL and pdm H1 HLA-A*02 RLATGLRNV dextramers, to gauge for the absolute number of antigen-specific CD8 þ T-cells, and pdm 2009 hemagglutinin inhibition (HI) assays, to test for neutralizing serum antibodies, were used as immunological readouts. Results: The pdm HI titers were poor in both cohorts with only 13.3% (4/30) after the seasonal vaccination and 23% (5/21; P ¼ NS) after virosomal vaccination having protective titers (HI Z 40). The increase in IFNg production from pre to post vaccination, i.e the d response was calculated. The d response to pandemic H1N1 influenza (P ¼ 0.005) and influenza B antigens (P ¼ 0.01) were significantly elevated in blood from individuals who received the virosomal as compared to the seasonal vaccine. There was no difference in d IFNg production in response to H3N2 influenza antigens between the cohorts. The IFNg response to pdm H1N1 was stronger after vaccination with the virosomal (Po0.001) as compared the seasonal vaccine in patients vaccinated more than 6 month post HSCT. We detected a significant increase in the frequency of matrix 1 (GILGFVTL) dextramer specific CD8 T-cells after the virosomal vaccine (P ¼ 0.01). There were no differences in the hemagglutinin-specific CD8 T cells between the two cohorts. Conclusion: The virosomal vaccine was a stronger inducer of IFNg production after stimulation with pandemic H1N1 and influenza B antigens than the standard seasonal vaccine while there was no difference in the proportion of patients developing protective HI titers. Additional controlled studies are therefore indicated. Disclosure of Interest: None declared. P133 Sequence typing of human adenoviruses isolated from Polish allogeneic haematopoietic stem cell transplant recipients S. Rynans1, T. Dzieciatkowski1, M. Przybylski1, A. Tomaszewska2,*, G. W. Basak3, W. W. Jedrzejczak3, G. Mlynarczyk1 1 Chair and Department of Medical Microbiology, MEDICAL UNIVERSITY OF WARSAW, 2Department of Haematopoetic Stem Cell Transplantation, Institute of Haematology and Transfusion Medicine, 3Department of Haematology, Oncology and Internal Medicine, MEDICAL UNIVERSITY OF WARSAW, Warsaw, Poland Introduction: Human adenoviruses (HAdV) from species A, B and C are commonly recognized as pathogens causing severe morbidity and mortality in haematopoietic stem cell transplant (HSCT) recipients [1,2]. Materials (or patients) and methods: The purpose of present study was to determine adenoviral types that infect HSCT recipients in Poland, and try to specify whether these infections occurred due to primary infection or endogenous reactivation. Analysis of nucleotide sequences was used to type 40 clinical isolates of HAdV obtained from 40 alloHSCT recipients. Results: Using sequencing of hexon gene, we identified six different HAdV serotypes belonging to species B, C, and E. Possibility of HAdV cross-infection was on very low level, because patients infected with the same HAdV types, were not hospitalized at the same time. In almost all patients, anti-HAdV antibodies in IgG class were detected, which indicates a history of HAdV infection in the past. Clinical symptoms of
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respiratory tract infections occurred in 27.5% of individuals, and were mainly caused by HAdV-2 and HAdV-3. Graft-versushost disease (GvHD) was present in patients infected with species B and C, but grade II of GvHD was observed only in patients infected with HAdV-B Conclusion: The predominance of HAdV-C and common presence of anti-HAdV antibodies in IgG class may strongly suggest that most infections in present study were reactivations of HAdV persisting into patient’s mucosa-associated lymphoid tissues. References: 1. Mynarek M, Ganzenmueller T, Mueller-Heine A, Mielke C, Gonnermann A, Beier R, Sauer M, Eiz-Vesper B, Kohstall U, Sykora KW, Heim A, Maecker-Kolhoff B. Patient, virus, and treatment-related risk factors in pediatric adenovirus infection after stem cell transplantation: Results of a routine monitoring program. Biol Blood Marrow Transplant. 2014; 20(2): 250–256. 2. O¨hrmalm L, Wong M, Aust C, Ljungman P, Norbeck O, Broliden K, Tolfvenstam T. Viral findings in adult hematological patients with neutropenia. PLoS One 2012; 7(5): e36543. doi: 10.1371/journal.pone.0036543. Disclosure of Interest: None declared. P134 Human adenovirus infection in Polish patients during the pre-engraftment and the post-engraftment phase after allogeneic hematopoietic stem cell transplantation S. Rynans1, T. Dzieciatkowski1, M. Przybylski1, G. W. Basak2, A. Tomaszewska3,*, W. W. Jedrzejczak2, G. Mlynarczyk1 1 Chair and Department of Medical Microbiology, 2Department of Haematology, Oncology and Internal Medicine, MEDICAL UNIVERSITY OF WARSAW, 3Department of Haematopoetic Stem Cell Transplantation, Institute of Haematology and Transfusion Medicine, Warsaw, Poland Introduction: Human adenoviruses (HAdV) are one of causative agents of emerging infectious complications in immunocompromised patients [1]. The risk factors for HAdV infection include younger age, T-cell depletion and graftversus-host disease. HAdV infections occur mainly in postengraftment phase, however some studies show that HAdV DNA could be detected also in a few days after transplantation. Materials (or patients) and methods: A total number of 1128 serum samples coming from 100 adults, subjected to allogeneic HSCT was tested for presence of HAdV DNA, using real-time PCR assay. Monitoring of clinical status of all the patients and viral load in serum samples covered the period of 100 days after transplantation. The aim of this study was to identify differences between HAdV infections in the preengraftment and the post-engraftment phase after HSCT. Results: HAdV DNA was detected in 32 patients in the preengraftment phase and in 28 patients during post-engraftment period. Patients with HAdV infection in the preengraftment phase were more likely to have received myeloablative conditioning regimen and developed GvHD. Patients with HAdV infection in the post-engraftment phase showed slightly higher average viral load. Statistical significance was found for underlying disease, and younger age of recipients. Conclusion: There is a high frequency of detectable HAdV DNA in allogeneic HSCT recipients in Poland. Adenoviral infections could occur with the same frequency in the preengraftment and post-engraftment phase, therefore routine monitoring of its DNA for at least 100 days after alloHSCT is strongly recommended. References: 1. Matthes-Martin S, Feuchtinger T, Shaw PJ et al European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ECIL-4 (2011). Transplant Infect Dis 2012; 14: 555–563. ˛ 2. Rynans S, Dzieciatkowski T, Przybylski M et al (2014): Incidence of Adenoviral DNAemia in Polish Adults Undergoing
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Allogeneic Haematopoietic Stem Cell Transplantation. Arch Immunol Ther Exp (Warsz): DOI 10.1007/s00005-014-0320-z. Disclosure of Interest: None declared. P135 Amphotericin B lipid complex is efficient and safe when used empirically or preemptively for suspected fungal infections in patients with acute leukemia or undergoing hematopoietic stem cell transplantation : A single center experience R. Mughnieh1, T. Jisr2, A. Mugharbil1, A. Youssef1, R. Jalloul1, G. Nsouli1, A. Ibrahim1,* 1 Internal Medicine , 2Laboratory Medicine and Blood Bank, Makassed University hospital, Beirut, Lebanon Introduction: Patients (pts) with acute leukemia (AL) or undergoing hematopoetic stem cell transplantation (HSCT) carry a high risk for invasive fungal infections (IFI). Amphoterician B lipid complex (ABLC) is effective when used empirically or preemptively for IFI in such pts. Materials (or patients) and methods: In this retrospective study, we aim to evaluate the use of ABLC on an empiric or preemptive basis for the management of IFI in pts with AL or undergoing HSCT. We mainly focused on response rate and tolerability along with the adverse drug events associated with ABLC therapy; in addition to premedication combinations used in the prevention of drug delivery reactions (DDR). Results: In this retrospective study reviewing 3 years’ experience (between January 2011 and January 2014), a total of 78 pts received ABLC therapy for suspected IFI. Sixty-five percent were treated preemptively and 25% were treated empirically.The overall response rate was 56%.Nephrotoxicity occurred in 24%of pts and improved of serum creatinine was in 5.3%. Moderate hypokalemia was present in 60.8% of the pts and severe hypokalemia was present in 10% but was almost correctable. Eleven percent showed hepatotoxicity throughout ABLC therapy. Drug delivery reactions were observed in 27.8% of the pts. There was a tendency to decrease the incidence of infusion reactions upon using a combination of premedication drugs including paracetamol, steroids, pethidine and antihistamines. Overall mortality rate was c20%. Conclusion: This study suggests that ABLC used emptively or preemptively is an effective and safe treatment option of suspected IFI in pts with AL or undergoing HSCT. Disclosure of Interest: None declared. P136 Active surveillance and targeted antimicrobial therapy abate infectious mortality due to carbapenemaseproducing Klebsiella ssp in HSCT recipients: outline of a CPKs outbreak A. Forcina1,*, M. Noviello1, A. Bondanza1, A. Orsini2, C. Soliman1, P. Cichero3, R. Greco4, F. Lorentino1, E. Xue1, A. Biancardi5, P. Nizzero6, F. Giglio1, C. Messina1, M. G. Carrabba1, M. Bernardi1, J. Peccatori2, P. Scarpellini7, N. Mancini8, M. Clementi8, C. Corti2, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 2Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 3 Unit of Microbiology, 4Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, 5 IRCCS San Raffaele Scientific Institute, Milano, 6IRCCS San Raffaele Scientific Institute, 7Unit of Infectious Diseases, 8Unit of Microbiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Carbapenemase-producing Klebsiella ssp (CPKs) are considered emerging killers in immunocompromised pts, such as those undergoing hematopoietic stem cell transplantation (HSCT), accounting for high rates of morbidity and mortality. From literature data, CPKs-related mortality in HSCT is estimated to be in the 40-65% range. The management of
colonized or previously infected pts is therefore challenging in this setting, and is often considered a stringent criteria of noneligibility to the transplant itself. Materials (or patients) and methods: Following a CPKs outbreak in June 2012 at our Center, we surveyed the epidemiology of CPKs infections in a cohort of 430 HSCT pts from Jan 2011 to Oct 2014 (46 months). This included 129/430 (30%) autologous transplants and 301/430 (70%) allogeneic transplants (51% HLA-haploidentical, 29% matched unrelated, 19% HLA-identical and 1% cord-blood grafts). One-hundred seventy out of 301 allogeneic HSCT (56%) pts had active disease at the time of transplantation. After an initial period of observation (18 mo) in 180 pts, we modified our internal guidelines by implementing the following measures in 250 pts from Jul 2012 to Oct 2014 (28 mo): i) active surveillance for CPKs colonization through weekly rectal swabs; ii) reverse isolation of colonized pts; iii) early initiation of a combined empirical therapy with at least two antibiotics among: colistin, tigecyclin, gentamycin and meropenem in all CPKs-colonized febrile pts. Results: During the entire observation period, we recorded 17/430 (4%) CPKs infections (4 sepsis, 2 severe sepsis and 11 septic shocks). The infection was lethal in 9/17 (53%) cases. in case of septic shock day-30 mortality was 64%. All patients experiencing septic shock had grade IV neutropenia and 7/11 (64%) had advanced disease at the time of infection. The rate of CPKs infections was not significantly different between pts analyzed before (10/180; 6%) or after (7/250; 3%) implementing the specific measures (P ¼ ns). However while before June 2012, 8/10 pts had septic shock and 6/10 died (60%), after implementing the preventive measures from Jul 2012 onwards, 3/7 pts had septic shock, but only 1 pt died (14%), resulting in a significant reduction of CKPs-related mortality (P ¼ 0.01). Rectal swabs routinely performed from Jul 2012 onwards evidenced CPKs colonization in 30/250 (12%) pts. Positive rectal swab anticipated the onset of the infection in 4/ 17 pts (25%). Sixteen pts out of 430 (4%) had a positive rectal swab before HSCT. In this subgroup, only one pt developed a CPKs infection after the procedure, showing that having a positive CPKs rectal swab pre-transplant does not increase CPKs-related mortality (P ¼ 0.007). Conclusion: The implementation of an active surveillance through weekly rectal swabs, a prompt initiation of appropriate combined antimicrobial therapy and a timely reverse isolation in case of colonization, abates CKPs-related mortality and may improve the overall outcome of HSCT. CPKs colonization does not appear as a limiting factor in the application of HSCT. Disclosure of Interest: None declared. P137 Active surveillance of multidrug-resistant gram-negative bacterial infections with rectal swabs: analysis of 332 HSCT recipients A. Forcina1,*, A. Orsini2, A. Bondanza3, P. Cichero4, C. Ossi4, R. Greco5, F. Lorentino1, E. Xue1, M. G. Carrabba1, M. Bernardi1, J. Peccatori2, P. Scarpellini6, N. Mancini7, M. Clementi7, C. Corti2, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 2Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, 3Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milano, 4Unit of Microbiology, 5Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 6Unit of Infectious Diseases, 7Unit of Microbiology, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Infections due to multidrug-resistant (MDR) gram-negative bacteria have an increasing trend in hematopoietic stem cell transplantation (HSCT). Since the mortality of these infections in HSCT pts is very high, strategies of active surveillance capable of identifying MDR carrier patients are
warranted. The aim of this retrospective study was to determine the epidemiology of MDR gram-negative infections in a major HSCT clinic in Italy and the value of routine rectal swabs in active surveillance. Materials (or patients) and methods: From Jun 2012 – Oct 2014, we started a policy of active surveillance of MDR gramnegative bacterial infections through 2778 rectal swabs in 332 consecutive pts receiving allogeneic HSCT (190 pts; 57%) autologous HSCT (67 pts; 20%), or chemotherapy alone (75 pts; 23%). Underlying diseases were: 159 acute leukemias (48%), 73 Hodgkin and non-Hodgkin lymphomas (30%), 49 multiple myelomas (14%), 16 myelodysplastic syndromes (1%), 14 myeloproliferative neoplasms (1%), 7 others (o1%). In the case of allogeneic HSCT, 93 were HLA-haploidentical (49%), 57 matched unrelated (30%) and 38 HLA-identical (20%) and 2 cord blood grafts (1%). Pts were also carefully followed for infections, including 3245 blood cultures, and treated according to internal guidelines. Results: During the observation period, 404/3245 (12%) blood cultures were found positive, 65 (16%) for MDR gram-negative bacteria, 76 for gram-negative non-MDR (20%), 244 for grampositive (61%) and 15 for fungi (3%), for a total of 158/332 (46%) pts. MDR gram-negative bacterial infections developed in 36/332 positive pts (11%) pts (8 sepsis, 11 severe sepsis and 17 septic shocks), and were due to the following pathogens: 15 carbapenemase-producing Klebsiella. P. (42%), 7 P. Aeruginosa (20%), 6 ESBL E. Coli (17%), 5 Sten. Maltophilia (14%), 3 other (7%). Despite aggressive antibiotic treatment, 11/36 (31%) pts died. The risk of MDR gram-negative infections was higher in allogeneic compared with autologous HSCT pts (odd ratio: 2,4; confidence interval 0.8 – 7.1), as it was the mortality due to their complications (39% vs 0%). In the allogeneic HSCT group, this risk was not associated with the disease status at time of transplant (P ¼ 0.5) or with the occurrence of acute graft-versus-host disease (P ¼ 0.1). In a total of 114/332 (34%) pts, 296/2778 (11%) rectal swabs were positive for MDR gram-negative bacteria (11%). In retrospect, positive pts had undergone a higher number of swabs (average 10,4 ± 8,3 SD) compared with negative pts (average 7,3 ± 6,2 SD; P ¼ 0.0006), underlying the importance of multiple testing. Swabs were positive in 24/36 pts (67%) developing a MDR gram-negative bacterial infection due to the same pathogen, as documented by blood cultures, compared with 90/296 pts (30%) that did not develop the infection, showing a highly significant statistical association (P ¼ 0.0001). Moreover, positive rectal swabs anticipated sepsis in 11/36 pts (30%), allowing early initiation of antimicrobial targeted therapy. Conclusion: Active surveillance of MDR gram-negative bacterial infections through weekly rectal swabs in HSCT allows identifying pts at high risk for subsequent, potentially lethal, infectious complications. Positive rectal swab significantly associate with the development of MDR infection, allowing the timely application of prophylactic strategies in order to reduce infectious mortality. Disclosure of Interest: None declared. P138 Analysis of risk factors of viral reactivation after haploidentical and matched unrelated hematopoietic stem cell transplantations with TCR alpha/beta and СD19 depletion A. Laberko1,*, M. Maschan1, L. Shelikhova1, D. Balashov 1, J. Skvortsova1, E. Boyakova1, V. Kalinina2, D. Shasheleva1, M. Persiantseva1, G. Novichkova1, A. Maschan1 1 Hematopoietic stem cell transplantation, 2Molecular Biology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: Viral infection is one of the leading causes of morbidity and mortality in patients after T-depleted (both ex vivo and in vivo) hematopoietic stem cell transplantation. Data regarding the rates and risk factors for viral reactivation
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after novel type of selective T-depletion - TCR alpha/beta depletion are limited. Materials (or patients) and methods: In this retrospective study we analyzed the data of CMV and EBV reactivations in children undergoing HSCT with TCR alpha/beta and CD19 depletion in our center during 2012-2013. We performed 137 transplantations, 44 from haploidentical and 93 from MUD for 92 children with malignant disorders (36 ALL, 38 AML, 11 JMML, 4 lymphomas, 3 MDS) and 45 children with nonmalignant disorders (14 SAA, 8 congenital bone marrow failure, 22 PID, 1 B-thalassemia). The median age of patients was 6,8 years, m/f ratio - 93/44. One hundred eighteen of our patients were seropositive (all PIDs despite documentation were considered positive), 18 seronegative, serostatus of 1 patient and 2 donors before HSCT were unknown. Donor/ recipient status was: D-/R- in 11, D-/R þ in 51 , D þ /R þ in 65, D þ /R- in 7. Sixteen patients were EBV- and 107 EBV þ , EBV serostatuses of 14 patients are unknown. Conditioning regimen differed according to diagnosis. Patients with malignant disease and PID recieved treosulfan-based conditioning, patients with BMF - Cph/Fludarabine - based. All patients recieved ATG. The level of CMV and EBV DNA in blood was monitored by PCR during the first 100 days after transplantation weekly and then, depending on immune reconstitution, up to 1 year after HSCT. Preemptive therapy with gancyclovir was used according to standard guidelines. Results: The cumulative incidence of CMV reactivation was 0,53 (95% CI 0,44-0,63), of EBV viremia is 0,4 (95% CI 0,32-0,52). The median time to CMV reactivation is 5 weeks after HSCT, the median duration of reactivation is 3 weeks, the median number of reactivations is 1. There is also no discrepancy in CI of CMV reactivation depending on donor/recipient serostatus, age, sex, kind of donor and diagnosis. The only factor significantly influencing CI of CMV reactivation was acute GVHD with systemic steroid therapy – 0,71 (95% CI 0,58-0,87) for patients with GVHD and 0,42 (95% CI 0,33-0,54) without GVHD. The incidence of CMV disease is 0,05 (4 patients had CMV-pneumonia, 2 CMV-encephalitis and 2 CMV-choreoretinitis, 1 CMV-cystitis). CMV reactivation did not affect survival. The median time to EBV viremia is 13 weeks, the median duration of viremia is 2 weeks, the median number of reactivations is 1. There is also no significant difference in CI of EBV depending on age, sex, donor, diagnosis, GVHD manifestation and donor/recepient serostatus. There were no cases of PTLD, 5 patients had EBV disease (mononucleosis, high EBV DNA load) and received Rituximab treatment. EBV reactivation did not affect survival. Conclusion: Reactivation rate of CMV and EBV after TCR alpha/ beta depleted transplantation was not different from our previous experience in unmanipulated unrelated donor transplantation. Although CMV/EBV reactivations did not influence survival, these infections remain a significant unresolved medical problem in clinical transplantation. Disclosure of Interest: None declared. P139 BK Polyomavirus Nephropathy after allogeneic HSCT A. Schifferli1,*, T. Diesch-Furlanetto2, C. Rudin3, C. A. Levy4, H. H. Hirsch4 1 Paediatric Haematology /Oncology, 2Paediatric Haematology/ Oncology, University Chilren’s Hospital of Basel, 3Paediatric Nephrology, University Chirlen’s Hospital of Basel, 4Department of Biomedicine, University Hospital of Basel, Basel, Switzerland Introduction: BK polyomavirus (BKPyV) reactivation is associated with haemorrhagic cystitis in hematopoietic stem cell transplantation (HSCT) recipients. In kidney transplant recipients polyomavirus-associated nephropathy (PyVAN) is a major cause of renal allograft failure. In HSCT patients just a few cases of PyVAN have been reported so far. In a 2009 review (Lekakis LJ et al) only 6 cases were reported, all with severe renal failure. However in a more recent study of 68 patients by Oshrine et al (2013) BKPyV detection in urine and/or blood was
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not associated with changes in estimated glomerular filtration rate and the need for dialysis or mortality. Given these controversial results we retrospectively reviewed the charts and laboratory results of our own patients in order to analyse the association of BKPyV reactivation and other factors with deterioration of kidney function in the early phase after HSCT. Materials (or patients) and methods: Patients and methods: 21of 22 patients (mean age 14.3 years, range 2-22 years; 12 males) who underwent an allogeneic HSCT between January 2011 and September 2014 at the University Children’s Hospital of Basel (Switzerland) were included. BKPyV load dynamic in blood and urine was correlated with changes in serum creatinine, urine diagnostics, and with cyclosporine through levels for at least 30 days after HSCT. Results: Fifteen patients (71%) showed BKPyV reactivation, with peak urine loads after a mean of 28.6 days (range 12-94) posttransplant. Four patients (4/21) developed grad III-IV haemorrhagic cystitis (19%) after BK reactivation: one had dialysis-dependent kidney failure, two developed a moderate renal impairment and one didn´t show change in renal function, 3 (3/4) died following complications of HSCT. Of the remaining 17 patients, 11 patients had a BKPyV reactivation with no or microscopic haematuria (52%): 7 of them showed a reactivation in blood and urine (33%). In the remaining 4 patients BKPyV was only detected in urine (19%). Ten of these 11 patients with BKPyV reactivation had a relevant increase in serum creatinine (41.5 x baseline, or over the reference value) shortly after reactivation of BKPyV in urine (range days 5-38).The elevated creatinine level persisted for several weeks or months, and in only 2 patients the serum creatinine returned to baseline (minimal follow up of 3 months). Because of the moderate renal insufficiency no biopsy was performed. Two (2/11) patients had an elevated cyclosporine level (4325ug/l, target range:100-200 ug/l) for one respectively two days within the first 4 weeks after transplantation without an elevation of serum creatinine level. Six (29%) patients had no BKPyV reactivation, 2 of them had no impairment of renal function in the early post-transplant phase (4 weeks), 3 had transient elevation in serum creatinine probably associated with drug toxicities (calcineurin inhibitor, voriconazol), one patient had prolonged renal impairment over weeks but returned to baseline during follow up. Conclusion: Discussion: BKPyV reactivation is frequently associated with impaired renal function after allogeneic HSCT. Although drug toxicity is a frequent factor, BKPyV nephropathy is possibly underdiagnosed.To confirm our hypothesis a prospective study is needed. Disclosure of Interest: None declared. P140 Time course and sites of polyomavirus activation in patients following hematopoietic stem cell transplantation (HSCT) A. Chukhlovin1,*, L. Zubarovskaya1, S. Bondarenko1, Y. Eismont1, M. Vladovskaya1, A. Volkova1, B. Afanasyev1 1 R.Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantology, St.Petersburg State I.Pavlov Medical University, St.Petersburg, Russian Federation Introduction: Polyomaviruses comprise a group of latent pathogens that may be activated in immunosuppressed patients, e.g., following conditioning therapy after HSCT transplantation. Generally, BK virus (BKV) reactivation is associated with hemorrhagic cystitis, whereas JC virus (JCV) is thought to cause specific encephalopathies. Hence, the aim of our work was to assess time dependence of BK and JC reactivation post-HSCT. Materials (or patients) and methods: The study included 297 patients, mostly, with oncohematological disorders, 1 to 60 years old who underwent allogeneic HSCT from 2010 to 2013. The treatment was due to ALL (n ¼ 101), AML (n ¼ 105), MDS (n ¼ 31), CML (n ¼ 17), malignant lymphomas (n ¼ 23), aplastic
anemias (n ¼ 12), neuroblastoma (n ¼ 5), inherited metabolic diseases (n ¼ 4). Myeloablative conditioning regimens were applied in 35% of cases. HSCT was performed from related (16%), unrelated HLA-compatible (63%), or haploidentical donors (21%). Bone marrow or peripheral blood stem cells were used as a source of stem cells (resp., 47% and 53% of the cases). Due to sufficient risk of viral infection, the patients were given standard Acyclovir prophylaxis. To prevent and treat acute GvHD, cyclosporin A and methotrexate were routinely applied. DNA diagnostics of BKV and JCV was carried out in various clinical samples using genospecific PCR primers. Sensitivity of these qualitative assays was 41000 gene copies per mL, as compared to the reference data from quantitative PCR. Results: The overall PCR positivity for BKV in the patients at 0.5 to 6 months after HSCT was, respectively, 31%, 72%, 16%, and 21% in leukocytes, urine, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL). Appropriate prevalence rates for JC were 16%, 36%, 2%, and 19%. Strong correlations existed between BKV, JCV, and CMV positivity in the samples. Hence, BKV was most common in the urine sediments. When assessing time dynamics of BKV and JCV occurrence, higher detection rates were registered at 2 to 6 months post-HSCT, thus correlating with increased risk of hemorrhagic cystitis. It should be mentioned that JCV positivity was quite rare in the CSF samples. Meanwhile, JCV is common in BAL samples at later terms (4 to 6 months) following HSCT. Conclusion: Early polyomavirus reactivation post-HSCT is associated with immunocompromised state in the patients, thus causing a well-known BKV and JCV replication, both in urothelial cells and BAL. Later on, high incidence of JCV in bronchoalveolar cells may contribute to development of alveolitis and chronic GvHD, thus deserving further studies in this field. Disclosure of Interest: None declared. P141 Use of low dose Rituximab for treatment of EBV reactivation post allogeneic stem cell transplant: A single centre experience A. Publicover1,*, M. Prichard1, D. S. Richardson1, K. Hill1, C. Hurlock1, L. Jarvis1, H. Launders1, S. Main1, N. McKeag1, J. Newman1, K. H. Orchard1 1 Haematology, University Hospital Southampton, Southampton, United Kingdom Introduction: In patients who have undergone allogeneic stem cell transplantation (allo SCT), reactivation of Epstein-Barr virus (EBV) infected memory B cells, in the absence of EBVspecific T-cells can result in post-transplant lymphoproliferative disorder (PTLD). PTLD is fatal in over 50% of cases. Quantitative PCR can be used to monitor for EBV reactivation, allowing pre-emptive therapy to be given. The most common agent used is the anti-CD20 monoclonal antibody Rituximab, which targets B cells. A negative aspect of this treatment is profound and long-lasting B lymphocytopenia. Rituximab for EBV-reactivation has usually been given at a standard dose of 375mg/m2 for four once weekly doses but the minimum effective dose for control of EBV reactivation has not been determined. Here we compare effectiveness and post treatment immune reconstitution in patients treated with 375mg/m2 versus 100mg total dose. Materials (or patients) and methods: EBV reactivation is routinely monitored post allo SCT by whole blood PCR and treatment initiated if the number of EBV genome copies per ml of blood exceeds a threshold value. We wished to explore whether effective treatment could result from a lower dose of rituximab and consequently reduced treatment from 375mg/ m2 to a total dose of 100mg irrespective of patient size, repeated if necessary based on EBV PCR response. Between December 2009 and October 2014, 43 patients at our institution were treated for EBV reactivation, two of whom had biopsy proven PTLD. 26 received an initial Rituximab dose
of 375mg/m2 (Group A) and 17 an initial dose of 100mg (Group B). The mean age of the patients was 49 years in Group A and 54 years in Group B. The single largest indication for transplantation was AML/MDS in both groups (41% versus 53%). CD4, CD19 and total IgG pre, post, six months and one year post Rituximab were used as markers of immune reconstitution. Results: In Group A 50% and in Group B 35% of patients received more than one dose of Rituximab (mean 2.1 and 1.4 respectively). No patients in Group B have received more than 200mg in total. The mean total dose of Rituximab received was 1107mg in Group A and 135mg in Group B (P ¼ 9.6x10-7). Both patients with proven PTLD were in Group A and responded to Rituximab alone. Neither CD4, nor IgG levels in either group appear affected by Rituximab. Median CD19 levels pre, post, six and twelve months post Rituximab were 0.21x106, 0x106, 0x106 and 0.12x106 in Group A and 0.18x106, 0x106, 0.06x106 and 0.11x106 in Group B (no statistically significant differences between groups at any time point). Conclusion: We have demonstrated that a single dose of 100mg Rituximab reduces the EBV titre to below our treatment threshold for 460% of patients and a second dose of 100mg has proven sufficient for all patients. Immune reconstitution between the groups was not statistically different, but the numbers are small and this requires further follow up. Reducing the initial dose to 100mg would have significant implications for transplant centres both in terms of drug cost and infusion time. Having reduced to 100mg, it would be interesting to test further dose reduction, to establish the minimum effective dose. Our only patient with a CD19 level of 40.1x106 post first dose of Rituximab had a reduced dose due to an adverse reaction, but the EBV titre dropped to below our treatment threshold. Disclosure of Interest: None declared. P142 Molecular monitoring of Herpesviridae family members after allogeneic hematopoietic stem cell transplantation a single center experience A. M. Balan1,2, A. Oprisoni1,2,*, C. Jinca1,2, A. Isac2, A. Pascalau2, M. Serban2, S. Arghirescu1,2 1 Pediatrics, "Victor Babes" University of Medicine and Pharmacy, 2 BMT, Louis Turcanu Emergency Hospital for Children, Timisoara, Romania Introduction: Molecular monitoring and consecutive preemptive therapy after allogeneic hematopoietic stem cell transplantation (HSCT) managed to reduce the incidence and severity of viral diseases due to members of Herpesviridae family, but, unfortunately there are still unsolved issues in this field. Therefore we aimed to analyze the incidence of active Herpesviridae infections as well as the safety and efficacy of pre-emptive therapy. Materials (or patients) and methods: In a prospective observational study, we have performed weekly or once in two weeks quantitative RT-PCR for Herpesviridae family members (HSV1, HSV2, CMV, EBV, VZV, HHV-6 and HHV-7) in 53 allogeneic HSCT procedures. In the majority of HSCT procedures antiviral prophylaxis consisted of Acyclovir (n ¼ 50; 94.33%), whereas only 3 patients (5.66%), at high-risk for active CMV infection, received Ganciclovir (GCV) prophylactically. Thresholds for pre-emptive non-specific (reduction in immunosuppression) and/or specific (antiviral agents, monoclonal antibodies) measures were the following: 500 copies/mL for CMV, 103 copies/mL for EBV and HHV-6, and positivity of RTPCR (100 copies/mL) for the rest of the Herpesviridae viruses monitored. Results: At least one episode of active infection due to Herpesviridae viruses occurred in 34% of the HSCT procedures, more than one active infection being documented in 11.32% of the HSCT procedures with the following distribution: CMV þ EBV þ HHV-6 – 33.33%, CMV þ HHV-6 – 33.33%, CMV þ EBV – 16.66%, EBV þ HHV-6 – 16.66%. CMV was the most
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frequent pathogen detected, with an active CMV infection incidence rate of 26.41%. At the first positivity of CMV RT-PCR, the number of copies exceeded 500 copies/mL in 85.71% from the patients with active CMV infection. Results of pre-emptive therapy with GCV were the following: clearance of CMV viremia after an average time of 24.1 days, low recurrence rate of active CMV infection (16.66%) and a relatively high-rate of GCV-associated neutropenia (41.66%). Active HHV-6 infection was detected in 9.43% of the HSCT procedures, at a median time of 50.2±61.4 days and responded favorably to GCV therapy (given due to concomitant active CMV infection) with the exception of one patient who required administration of Foscarnet. In 13.2% of the HSCT procedures, EBV was detected through RT-PCR, all of the patients with active EBV infection having relatively low viral loads (190-2520 copies/mL) and responding favorably to reduction of immunosuppresion, without concomitant administration of Rituximab. Quantitative RT-PCR did not detect the other members of the Herpesviridae family – HSV1, HSV2, VZV and HHV-7 in blood samples analyzed. Conclusion: None of the patients included in our study developed viral diseases due to Herpesviridae family members and, moreover, overall survival was not influenced by the active Herpesviridae viruses infections. Although the results of our study are limited by the sample of patients analyzed, further evidence is brought to strengthen the safety and effectiveness of pre-emptive therapy after allogeneic HSCT, guided by molecular monitoring of Herpesviridae viruses. Although, generally initiated at low thresholds comparative to other published studies, pre-emptive therapy was not associated with significant adverse events, with the exception of GCV-associated neutropenia. Disclosure of Interest: None declared. P143 Hepatitis C virus infection triggers severe autoimmune hemolytic anemia in a transplanted toddler with HLH in XIAP deficiency: successful treatment with Sofosbuvir A. Jarisch1,*, J. Soerensen1, B. Kronenberger2, M. Meister1, E. Rettinger1, A. Willasch1, A. Berger3, T. Klingebiel1, P. Bader1 1 Pediatric Hematology and Oncology, University Children’s Hospital , 2Department of Medicine I, 3Institute of Medical Virology, University of Frankfurt, Frankfurt, Germany Introduction: Hemophagocytic Lymphohistiocytosis (HLH) is a multisystem inflammatory disorder due to cytokine overproduction from excessively activated lymphocytes and macrophages. X-linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and presents with highly variable clinical symptoms consistent with HLH. Materials (or patients) and methods: We report a Pakistani boy who developed postpartum symptoms of HLH (pancytopenia, hepatosplenomegaly, culture-negative fevers and pulmonary failure), and a XIAP deficiency was diagnosed. Despite treatment regarding HLH 2004 protocol his HLH remains refractory. At the age of 8 month the boy was successfully transplanted from a 10/10 matched unrelated donor using conditioning with fludarabine, melphalan, treosulfan and antithymocyte globulin. Viral screening prior to transplant was negative (especially HCV IgG and IgM). The posttransplant course was uncomplicated. Results: Six month post-transplant the patient developed a severe autoimmune hemolytic anemia (AIHA, minimum Hb 2.4 g/dl) refractory to immunoglobulins, corticosteroids and rituximab. IgG antibodies were present on red cells, also being positive for C3d, ADAMST 13 was normal. This constellation and a newly presenting thrombocytopenia were suggestive of atypical hemolytic uremic syndrome (aHUS). Treatment with eculizumab resulted in better tolerated red blood cell transfusions and an increase of Hb to 5 g/dl for few days, but did not control AIHA.
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Viral testing for Parvo B19, CMV, EBV, HHV-6, Adenovirus remains negative but was positive for HCV genotype 3a (1.040.000 iE/ml, with still negative serological findings in IgG and IgM). Due to previous allogeneic stem cell transplantation and severe autoimmune hemolytic anemia current standardof-care for hepatitis C with pegylated interferons and ribavirin was not feasible. Four weeks after presenting first symptoms of severe AIHA the patient still showed a severe AIHA, additionally an increase of bilirubin levels up to 12 mg/dl and rising HCV levels (26.900.000 iE/ml). At this time Sofosbuvir, a new nucleotide analog, HCV NS5B polymerase inhibitor, has become commercially available. Even though there was no previous experience with this compound in small children, we used Sofosbuvir in this critical situation. After one week of treatment with Sofosbuvir (100 mg/d) the Hb level was stabilized and the HCV level decreased to 40.000 iE/ml (267 IE/ml after 2 weeks of treatment). Ribavarin was added when Hb levels were stabilized 4 10 g/dl and the hepatitis C PCR became negative after 4 months after starting treatment. Sofosbuvir was well tolerated throughout treatment. Conclusion: Retrospective testing of cryopreserved blood samples from the patient and his mother confirmed intrauterine infection with HCV genotype 3a. This may have triggered HLH in this XIAP deficiency patient and may explain the missing response to HLH protocol. Disclosure of Interest: None declared. P144 Features of haemorrhagic cystitis in unmanipulated haploidentical transplant recipients A. Ruggeri1,*, G. Roth-Guepin1, G. Battipaglia1, A.-C. Mamez1, F. Malard1, A. Gomez1, E. Brissot1, R. Belhocine1, A. Vekhoff1, S. Lapusan1, F. Isnard1, O. Legrand1, J. Gozlan1, D. Boutolleau2, T. Ledraa1, M. Labopin1, M. T. Rubio1, M. Mohty1 1 Hoˆpital Saint Antoine, Hoˆpital Saint Antoine, 2Hoˆpitaux Universitaires Pitie´-Salpeˆtrie`re, Hoˆpitaux Universitaires Pitie´-Salpeˆtrie`re, Paris, France Introduction: Haemorrhagic cystitis (HC) is a common complication after allogeneic stem cell transplantation (alloSCT), associated with intensity of conditioning regimen, cyclophosphamide (Cy) metabolites, and BK virus infection. The incidence of HC after unmanipulated Haplo SCT ranges from 31 to 75%. Materials (or patients) and methods: Here, we analyzed 33 Haplo recipients using post Tx-Cy. Patients were transplanted for AML (n ¼ 18), ALL, (n ¼ 7), MDS (n ¼ 4), T cell lymphoma HTLV-I (n ¼ 1), Non Hodgkin (n ¼ 1), Hodgkin Lymphoma (n ¼ 1) and aplastic anemia (n ¼ 1). Results: Most of the patients (n ¼ 19, 53%) were transplanted in advanced disease status. Thirteen patients had a previous transplant (9 allo-SCT and 4 autologous). Median follow-up was 11 (range, 4-18) months. Eleven patients received a MAC with Thiotepa þ Busulfan þ Fludarabine, and patients with active disease received a sequential approach. All patients except 8 had ATG. GVHD prophylaxis consisted of CSA þ MMF and pTx-Cy (17 patients received a single dose of pTxCy on day þ 3 (BM recipients), and 16 had 2 days of pTx-Cy on day þ 3 and þ 5 (PBSC recipients)). All but 1 patient achieved neutrophil recovery at a median of 17 days. Cumulative incidence (CI) of platelet recovery at day 100 was 65%. Two patients had secondary graft failure (day þ 25 and day þ 43 after Haplo, respectively) and were rescued with a 2nd allo-SCT. Four patients had poor graft function and received a CD34 þ cell boost. CI of 100-day grade II-IV and grade III-IV acute GVHD was 44% and 32%, respectively. CI of HC (all grades) at day 180 was 62%. Twenty patients developed HC, in a median time after Haplo of 38 days. Eleven patients (55%) developed HC before platelet engraftment, (median 26 days).
HC was classified as grade 1 in 1 patient (5%), grade 2 in 6 patients (33%), grade 3 in 8 patients (38%) and grade 4 in 5 patients (24%). At the onset of HC BK virus was positive in blood and urine in 91% of patients. Treatment of HC consisted of hyperhydratation, intravenous Ig, antispasmodic and pain management for 54% of patients (n ¼ 11), 9 patients (46%) were treated with continuous bladder irrigation, and 4 received also iv cidofovir. Two patients required clots evacuation under cystoscopy. One patient was treated by intravescical instillation of potassium alum. All HC but 2 resolved. In the univariate analysis factors associated with HC were previous transplant (P ¼ 0.01) and occurrence of CMV reactivation before HC (P ¼ 0.05). Of 20 patients with HC, 15 had a CMV reactivation before HC. Grade II-IV acute GVHD was not associated with HC (P ¼ 0.62), of 20 patients with HC, 10 experienced acute GVHD grade II-IV and in 7 cases aGVHD occurred before HC. CI of day-180 viral infections was 73% (19 patients had CMV reactivation, 15, EBV reactivation). Two patients developed EBV PTLD (1 requiring chemotherapy). Five patients experienced symptomatic adenovirus infection and 1 a resistant HSV infection. HHV6 reactivation was documented in 16 patients, none requiring treatment. Two-year OS was 50%, 18 patients are alive (16 in remission). The occurrence of HC did not impact on OS (P ¼ 0.29). Nine patients relapsed (7 with AML, 1 MPD and 1 HD). Fifteen patients died, 11 of NRM ((GVHD n ¼ 7, infection n ¼ 4) and 4 of relapse. Conclusion: Incidence of HC after Haplo with pTx-Cy is high and is associated with morbidity, especially in the setting of high risk patients, who received a previous transplant, and with impaired immune reconstitution. Disclosure of Interest: None declared. P145 Subcutaneous administration of rituximab in patients with Epstein-Barr virus reactivation after allogeneic stem cell transplantation A. Klink1,*, V. Schmidt1, S. Dornaus1, N. Winkelmann1, H. G. Sayer2, M. von Lilienfeld-Toal1, A. Hochhaus1, I. Hilgendorf1 1 Department for Internal Medicine II (Hematology and Oncology), Jena University Hospital, Jena, 2Department for Internal Medicine IV (Hematology and Oncology), Helios Klinikum, Erfurt, Germany Introduction: Epstein-Barr virus (EBV) reactivation after allogeneic hematopoietic stem cell transplantation (alloHSCT) is most common between engraftment and the first 6 months after transplant and can develop into a life-threatening posttransplant Non-Hodgkin lymphoma. The CD-20 antibody Rituximab (R) as pre-emptive therapy is an important option alongside others such as tapering of immunosuppression and/ or use of adoptive EBV-specific cytotoxic T-cells. Subcutaneous (s.c.) R is approved for the treatment of follicular- and diffuse large B-Cell Lymphoma in Europe since March 2014. One advantage is the shorter time needed for patients with s.c. in the outpatient-setting. In a single center analysis, we report on 11 patients with EBV-reactivation after alloHSCT treated with R i.v. or s.c. Materials (or patients) and methods: Eleven patients (pts) [5 AML, 2 ALL, 2 MDS, 2 sAA] with a median age of 48 years [range, 24 – 59] underwent alloHSCT between 03/2013 and 08/ 2014 after myeloablative conditioning [6 pts] or reducedintensity conditioning [5 pts]. All ten pts with a graft from an unrelated donor (7 x 10/10; 2 x 9/10, 1 x 8/10 HLA-matched) received antithymocyte globulin [4 pts ATG -Freseniuss 10mg/kg x 4, 5 pts Thymoglobulin 2.5 mg/kg x 4, 1 patient Thymoglobulin 1.5 mg/kg x 4] as in vivo T-cell depletion during conditioning. One patient had a HLA-matched family donor and did not receive ATG. Five patients received bone marrow and 6 pts peripheral blood stem cells for transplantation. GVHD-prophylaxis consisted of cyclosprine þ methotrexate in 6 pts or cyclosporine þ mycophenolate in 5 pts EBV-serostatus was positive in all pts before transplantation, and one seronegative donor. Acute GVHD (grade I/II) was
observed in 4 pts. EBV reactivation occurred on median day 33 [range, 22-191]. At time of starting EBV pre-emptive therapy 10 out of 11 pts were still in need of immunosuppressive drugs. Results: Patients with an EBV reactivation of 4 10,000 copies/ ml in the peripheral blood were given pre-emptive R (375 mg/ m2) as i.v. application for the first dose. Thereafter, ten out of 11 patients dropped their EBV-copy load and four pts received a second and 2 pts a third application of R s.c. (1400 mg total dose) whereas seven pts received a second and one patient a third application of R i.v. at weekly intervals. The response to treatment was measured by quantitative determination of the polymerase chain reaction in the whole blood 7 days after each cycle pre-emptive R therapy and 30 days after the last application. If no EBV-Load (cut offo300 copies/ml) was demonstrated after the first dose, pts received only R application as consolidation. No moderate or severe side effects in the six s.c. applications were noted, in one patient a mild skin flair was observed. So far (12/2014) no second EBV reactivation was recorded in the s.c. group, whereas two reactivations occurred in the conventional i.v. group. Conclusion: Subcutaneous administration of R as pre-emptive therapy in EBV reactivation seems to be safe and feasible. The first results show no difference in efficacy between i.v. and s.c. regimen. The implementation of further studies regarding safety, virus reactivation, disease-free survival and overall survival in long-term follow-up is recommended. Disclosure of Interest: None declared. P146 Candida crusei Septisemia Presented with Skin Lesions After Bone Marrow Transplantation In a Patient with Aplastic Anemia N. Y. Ozbek1, A. D. Do¨nmez1,*, Z. Avcı1, S. Kirkiz1, P. Is¸ık1, N. Yaralı1, B. Tunc¸1 1 Pediatric Hematology/Oncology, Ankara Çocuk Sag˘lıg˘ı ve Hastalıkları Hematoloji Onkoloji EA Hastanesi, Ankara, Turkey Introduction: Systemic candidiasis is a serious problem in hospitalizad patients, especially treated with aggresive antimicrobials, chemotherapeutics, immunomodulating and transplantation therapies (1). Diagnosis of this fatal infection is often delayed or missed, leading to high mortality rates. Nonspesific clinical manifestations and absence of diagnostic tools are the most frequent reasons for this delay (2,3). Cutaneous presentation of sistemic candidiasis is rare, but appearence of lesions may help rapid diagnosis. Here we report an immuncompromised case of sistemic candidiasis with cutaneous manifestations. Materials (or patients) and methods: A 9 year old girl, with aplastic anemia, underwent second bone marrow transplantation from her 9/10 HLA-matched sister. Eight days after transplantation she developed fever and mucositis while using prophylactic flucanasol. Two days later, she also developed severe myalgia, arthralgia and cutaneous lesions. The lesions presented as erythematous macules, papules and nodules on the trunk, extremities and scalp (Picture 1). Skin biopsy showed minimal perivascular mononuclear inflammation. On fourth day of the fever, Candida crusei isolated from her periferic blood, Hickman catheter, as well as urine and skin biopsy cultures. Additional investigations did not show any other organ involvement including spleen and liver. The patient was given intravenous liposomal amphotericin B (12 days), caspofungin (18 days), and granulocyte transfusions. Her catheter was removed immediately. Results: After treatment, skin lesions resolved in one week. Control blood and urine cultures were negative. However, she could not achieve engraftment. She underwent a haploidentical transplantation. Conclusion: We would like to suggest that patients with peculiar dermatological findings should be carefully evaluated. Quick diagnosis by means of biopsy and culture may help
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clinicians to early diagnosis, especially in immunocompromised patients. References: 1. Grabowski R, Dugan E. Disseminated candidiasis in a patient with acute myelogenous leukemia. Cutis 2003; 71: 466–468 2. Pedraz J, Delgado-Jime´nez Y, Pe´rez-Gala S, et al. Cutaneous expression of systemic candidiasis. Clin Exp Dermatol. 2009; 34: 106–110. 3. Bae GY, Lee HW, Chang SE,Clinicopathologic review of 19 patients with systemic candidiasis with skin lesions. Int J Dermatol. 2005; 44: 550–555. Disclosure of Interest: None declared. P147 Is Systemic Primary Prophylaxis Against Yeasts Always Suggested after HLA-Identical Allogeneic Hematopoietic Stem Cell Transplantation? A Single Center Experience using Low-Dose Fluconazole as Gastrointestinal Tract Decontamination B. Sarina1,*, A. Vai1, S. Bramanti1, R. Crocchiolo1, L. Morabito1, A. Santoro1, L. Castagna1 1 Humanitas Cancer Center, Rozzano (MI), Italy Introduction: Invasive fungal infection (IFI) is a major cause of morbidity and mortality after HLA-identical allogeneic hematopoietic stem cell transplantation (HLA-id allo-HSCT). In standard risk patients (pts) anti-yeast active agents based primary antifungal prophylaxis is recommended. Materials (or patients) and methods: We analyzed 97 consecutive HLA-id related allo-HSCT recipients who performed transplantation between 1999 and 2013 and received low dose fluconazole (100 mg/day) as gastrointestinal tract (GIT) decontamination, starting from hospitalization until engraftment. The aim was to describe the incidence of fungal infections. Results: Main pts’ characteristics are shown in Table 1. During early phase (r40 days after allo-HSCT), 3 proven/probable (PP) IFIs were diagnosed and during late phase (41-100 days after allo-HSCT) 1 PP-IFI occurred. PP-IFI incidence rate was 3.0% and 1.6% during early and late phase, respectively. 2 patients had invasive lung aspergillosis. Possible IFIs were diagnosed in 8 patients within the first 100 days after allo-HSCT and they were treated with empiric antifungal therapy. After a median follow-up time of 86 months (range: 12-167), the overall survival was 43% and the non-relapse mortality was 16%. 100 days IFI-related mortality was 1.0%. Conclusion: In our series, the incidence of PP-IFI during early (3.0%) and late (1.6%) phase was lower than reported in randomized and prospective studies. These data suggest that systemic anti-yeasts prophylaxis could be avoided in selected HLA-id allo-HSCT recipients, also on the basis of the local
Table 1.
Patients’ characteristics n ¼ 97
Median age Gender M/F
46 (16-66) 59/38
Diagnosis Hodgkin’s lymphoma Non-Hodgkin’s lymphoma Acute leukemia Chronic leukemia Multiple myeloma Myelodisplastic syndrome Solid tumor
21 27 11 4 10 3 21
Conditioning regimen Myeloablative Nonmyeloablative Reduced-intensity conditioning
20 (21%) 6 (6%) 71 (73%)
epidemiology. However, it should be noted that most of pts received reduced-intensity conditioning regimens. Disclosure of Interest: None declared. P148 Impact of linezolid on hematopoietic engraftment in patients after umbilical cord blood stem cell transplantation T. Vilits1, B. Huber-Krassnitzer1,*, W. Zinke-Cerwenka1, P. Neumeister1, F. Quehenberger2, M. Hoenigl3, H. Sill1, A. Wo¨lfler1 1 Division of Hematology, 2Institute for Medical Informatics, Statistics, and Documentation, 3Section of Infectious Diseases and Tropical Medicine, Medical University of Graz, Graz, Austria Introduction: Linezolid is approved for the treatment of infections caused by otherwise resistant Gram-positive bacteria, but has been associated with hematologic toxicity in the general population. Thus, there are concerns about its potential myelotoxicity in the setting of allogeneic stem cell transplantation (SCT). Umbilical cord blood (UCB) SCT is a curative option in patients, who lack a suitable HLA-identical stem cell donor, but it is associated with delayed and sometimes incomplete hematologic recovery due to the low number of hematopoietic stem cells infused. Materials (or patients) and methods: We retrospectively analysed the data of 35 patients having undergone UCB-SCT and evaluated the impact of linezolid treatment on neutrophil and platelet engraftment as well as the occurrence of linezolid resistant bacteria. Results: 35 patients (12 female, 23 male) with underlying malignant hematologic diseases undergoing UCB-SCT were included in this study. 28 patients (80%) were treated with linezolid during the neutropenic phase and 7 patients (20%) received antibiotic regimens not including linezolid. The median number of days on linezolid therapy after UCB transplantation was 16 days (range 3-57 days). Clinical characteristics including the infused dose of CD34 þ cells were well balanced between both groups. Neutrophil and platelet engraftment were similar between both groups and did not differ in the time to engraftment as well as in the percentages of patients achieving predefined neutrophil and platelet counts. For example, in the linezolid group 21 out of 28 patients (75%) were able to engraft to absolute neutrophil counts of 1000/ml (ANC1000) after a median duration of 32 days (range 18- 67 days). In the alternative group 6 out of 7 patients (85.7%) achieved ANC1000 after a median of 35 days (range 25-52 days). Absolute platelet counts of 50.000/ml (PLT50) were reached in the linezolid group in 20 out of 28 patients (71.4%; median duration to engraftment 45 days (range 19- 105 days)), in the alternative group 5 out of 7 displayed PLT50 (71.4%; median duration 43 days (range 31121 days)). Linezolid-resistant enterococci were identified in routine cutaneous swabs of 3 out of 28 patients. Conclusion: Linezolid had no negative impact on neutrophil and platelet engraftment in patients having undergone UCBSCT. Thus, linezolid can be administered safely in such patients. However, the occurrence of linezolid resistant enterococci may be a matter of concern. Disclosure of Interest: None declared. P149 Evaluation of bacteremia characteristics in allogeneic hematopoietic stem cell transplantation without antibiotic prophylaxis B. Lopez Pereira1,*, M. Lopez Duarte1, L. Yan˜ez1, R. Lobeira1, A. Bermudez1, F. Antolin1, P. Rodriguez Cudin1, M. Colorado1, G. Martin Sanchez1, C. Richard1, E. Conde1 1 Hospital Universitario Marques de Valdecilla, Servicio Cantabro de Salud, santander, Spain
(22%) (28%) (11%) (4%) (10%) (3%) (22%)
Introduction: Bacterial bloodstream infections (BBSI) are a common complication in allogeneic hematopoietic stem cell
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transplantation (HSCT) with a high impact on terms of morbidity and mortality. Systematic use of antibiotic prophylaxis remains controversial. Materials (or patients) and methods: We retrospectively analyzed the cumulative incidence (CI) of bacteremia, incidence density of catheter related bacteremia , incidence of grampositive and gram-negative microorganisms (including multidrug-resistant gram-negative bacteria), risk factors associated with BBSI and mortality rate associated in a cohort of 82 patients underwent HSCT in our centre from January 2012 to June 2014 with a median follow-up of 12 months. All patients had a central venous catheter and any of them received antibiotic prophylaxis. Median age was 51 years and 51 patients were male. Main underlying disease was acute leukemia (35). The majority of patients (35%) had received 1 prior line of treatment and 52 patients (64%) were in CR. Sixty-one patients (74%) received a myeloablative-conditionig regimen. Stem cell source was bone marrow in 58 patients (71%). 41 patients (50%) had an unrelated donor and 26 (32%) had HLA mismatch. All patients except one (singenic HSCT) received a calcineurin inhihibitor þ / MTX or MMF. Median days until PMNl Z500/mm3 was 16 (9-33). Sixtyfour patients presented mucositis grade 3-4 and 32 patients needed parenteral nutrition. Forty-three patients developed acute II-IV GVHD (16 grade III-IV) and 43 chronic GVHD (extense 16). The median time of hospital stay during all the follow up were 41 (21-193) days. Results: We found 91 episodes of BBSI in 51 patients. Median bacteremia episodes per pacient was 2,4 (1-7). The CI of patients with bacteremia was 62,2% (if only catheter related bacteremia was considered the CI was 12,2%). With a median follow-up of 12 months, the incidence density of bacteremia was 9,85 per 1000 days of catheter and the incidence density of catheter related bacteremia was 6,6 per 1000 days of catheter . Gramnegative microorganism accounted for 50,5%. The main bacterial isolations were S. epidermidis (27), E. coli (15) and E. cloacae (8), P.aeruginosa (7), E.faecium (4). The incidence of extended –spectrum beta-lactamase-producing bacteria was 9% (1 case of E.Coli and 8 E.cloacae). We can’t consider mucositis, parenteral nutrition, conditionig regimen, acute and chronic GvHD predictive risk factors for bacteremia in our study. In our serie 29/82 (35%) patients died. Among 51 patients with BBSI, bacteremia was the main cause of death in 6 (11,7%). Mortality rate at 7 days after BBSI was 7%. Thirty-one (38%) patients in absence of prophylaxis don’t have any episode of bacteremia. Conclusion: The cumulative incidence of bacteremia is high (62,2%) in our serie with a increased incidence of gramnegative bacteria (50,5%). However, the incidence of multidrug resistant gram negative bacteria (9%) and the mortality rate at 7 days after BBSI (7%) were lower than other studies (1). The incidence density of catheter related bacteremia (6,6 per 1000 days of catheter) is similar to other series (2). References: 1. Bock AM. Bacteremia in Blood or Marrow Transplantation Patients. Biology and Bone of blood and marrow transplantation (2012). 2. Dettenkofer M. Surveillance of nosocomial infections in allogeneic and autologous bone marrow and peripheral blood stem-cell transplantation. Bone Marrow Transplantation (2003). Disclosure of Interest: None declared. P150 A Delphi method-based consensus for the management of IFI in HSCT recipients C. Vallejo1,*, I. Jarque2, J. Fortu´n3, J. Garcı´a4, J. C. Garcı´a5, J. Lo´pez3, M. Rovira6, M. Salavert2, L. Va´zquez7 on behalf of The Study Group of Delphi Method for IFI Management 1 Donostia Hospital, San Sebastian, 2La Fe Hospital, Valencia, 3 Ramo´n y Cajal Hospital, Madrid, Spain, 4La Paz Hospital, Madrid, Saint Kitts and Nevis, 5de Cruces Hospital, Vizcaya, 6H. Clinic i Provincial, Barcelona, 7Clı´nico de Salamanca Hospital, Salamanca, Spain Introduction: incidence and mortality of invasive fungal infection (IFI) in high risk hematologic patients (including
HSCT recipients) are still high. As a consequence, recommendations on strategies for prevention and treatment of IFI continue to be important. Objective: identify key antifungal strategies with high level of agreement among experts to improve the outcome and survival of patients at high risk of IFI. Materials (or patients) and methods: A national survey based on the Delphi technique in one phase was conducted in Spain in November 2014. The key questionnaire was based on a comprehensive list of several strategies on IFI management compiled from national and international guidelines. The study was conducted anonymously, using an electronic platform, by clinical hematologists with experience in IFI from different centers all over the country. High degree of consensus was defined as ‘‘always’’ or ‘‘frequent’’ answers. Results: 58 experts agreed to participate on the survey and of those, 38 (65.5%) completed the questionnaire. Routinary broad spectrum antifungal prophylaxis in patients with active GVHD achieved a consensus of 93% and in umbilical cord transplant of 91%. Liposomal amphotericin B was the treatment of choice for patients at risk of IFI when the pathogen was unknown (empirical) for 85% of the experts and also the drug of choice for the targeted treatment of mucormycosis in 96%. Patients on posaconazole prophylaxis, with fever and 2 consecutive positive AGA should receive liposomal amphotericin B for 95% of the experts. Antifungal combination therapy should be considered in specific clinical settings for 91% of the experts. Conclusion: Proper prevention and therapy of IFI in the HSCT recipients is critical for their outcome. The Delphi method is a robust tool to design suitable strategies for antifungal management in high risk patients. A high number of Spanish hematologists skilled on IFI participated in the project. Their recommendations may help to improve patients’ survival. Disclosure of Interest: C. Vallejo Funding from: This survey has been financed by Gilead, I. Jarque Funding from: This survey has been financed by Gilead, J. Fortu´n Funding from: This survey has been financed by Gilead, J. Garcı´a Funding from: This survey has been financed by Gilead, J. C. Garcı´a Funding from: This survey has been financed by Gilead, J. Lo´pez Funding from: This survey has been financed by Gilead, M. Rovira Funding from: This survey has been financed by Gilead, M. Salavert Funding from: This survey has been financed by Gilead, L. Va´zquez Funding from: This survey has been financed by Gilead. P151 E. coli bacteraemia is more common in autograft patients compared with allogeneic recipients- a single centre analysis C. Saha1,*, A. Kulasekararaj1, J. Wade2, N. Desai3, V. Potter1, A. Pagliuca1 1 Haematology, 2Microbiology, 3Infection Control, King’s College Hospital, London, United Kingdom Introduction: E. coli is one of the commonly isolated species in a clinical haematology unit. There is evidence of increased E. coli bacteraemia in patients receiving chemotherapy compared to that in transplant recipients. Materials (or patients) and methods: A retrospective audit was done on patients with E.coli bacteraemia, either acquired within 48 hours of admission to haematology wards or admitted within 4 days of discharge from hospital, in King’s College Hospital, London. The audit was initially done for the period of April 2013-March 2014 and then expanded retrospectively to August 2011. The data of eligible patients were collected from the electronic patient records. Results: Between April 2013 and March 2014, total 27 patients developed E. coli bacteraemia. 48% received chemotherapy, 44% autologous haematopoietic stem cell transplant (HSCT) and 1 patient had allogeneic transplant. Among all the autologous HSCT recipients during that time in this hospital,
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14% had hospital acquired E. coli bacteraemia. Six patients had carmustine (BCNU), etoposide, cytarabine, melphalan (BEAM) conditioning and 6 patients received high dose melphalan (HDM) conditioning. The onset of sepsis was between 7-9 days post-transplant. Neutropenia (o1x109/L) was found in 88% and lymphopenia (o0.5x109/L) in 85%. Other comorbidities included diarrhoea in 70% and oral mucositis in 30%. None of them received antibiotic prophylaxis for gut sterilisation. No death was reported during this period. During August 2011-March 2014, total 66 patients had hospital acquired E. coli bacteraemia. Of them 39% patients received chemotherapy, 52% had a transplant and the rest had nonmalignant haematological conditions. 73% in the transplant group received autologous HSCT and 27% received allogeneic transplant. Nine patients receiving BEAM conditioning had this complication compared to 16 patients receiving HDM conditioning. There was 1 death in the former group. Death was reported in 2 cases in the allogeneic group, and in 2 cases in the chemotherapy group. In 80% patients receiving autologous HSCT, sepsis onset was between 5-10 days. Risk factors included neutropenia (o1x109/L) in 83.3%, lymphopenia (o0.5x109/L) in 86.4%, diarrhoea with negative stool microscopy and culture in 58%, and oral mucositis in 24%. Seven patients received prophylactic antibiotics for gut sterilisation during the period of neutropenia as a part of allogeneic transplant protocol. E. coli bacteraemia among allogeneic transplant recipients was found to have reduced each year throughout the audit period (25% in 2011-2012, 17% in 2012-2013, 4% in 2013-2014) compared to a rising trend in the autologous transplant recipients (31% in 2011-2012, 35% in 2012-2013, 44% in 20132014). Conclusion: Incidence of E coli bacteraemia in patients receiving chemotherapy is similar to those with transplants, commoner among patients receiving autologous HSCT receiving conditioning regimens including melphalan, compared to those with allogeneic transplants. Diarrhoea was commonest associated symptoms suggesting melphalan induced mucositis of the gut and subsequent translocation of gut bacteria as the possible mechanism. Gut sterilisation with a short course of antibiotic (oral colistin or ciprofloxacin) may be tried, accepting the inherent risks associated with it. Disclosure of Interest: None declared. P152 Cost of CMV infection after allogeneic HSCT: How to anticipate on the cost-efficacy of new antivirals? C. Robin1,*, F. Hemery2, C. Dindorf3, J. Thillard3, L. Cabanne4, R. Redjoul4, F. Beckerich1, C. Rodriguez5, S. Maury1, I. Durand-Zaleski6, C. Cordonnier4,4 1 Hematology, Henri Mondor Hospital and UPEC, 2Medical Information, Henri Mondor Hospital, Cre´teil, 3URC-Eco , APHP ECEVE UMRS 1123 , Paris, 4Hematology, Henri Mondor University Hospital, 5Virology and INSERM U955 Team 18, Henri Mondor Hospital and UPEC, 6Public Health department Creteil and URCEco APHP ECEVE UMRS 1123 , APHP, Cre´teil, France Introduction: CMV infection and disease are main concerns after allogeneic HSCT. Pre-emptive therapy is the most common and easy strategy to reduce the risk of CMV disease. However, new antiviral drugs, CMV-vaccines and specific cellular therapy, may in a near future, encourage prophylactic strategies. In order to anticipate on the cost-efficacy of new anti-CMV compounds, we have estimated the cost of 1st year of transplant according to the pretransplant D/R CMV serologies and to onset of CMV infection. Materials (or patients) and methods: 208 consecutive allogeneic HSCT recipients transplanted in Creteil between 2008-2013 were included. The median follow-up was 18 months. The primary objective was to estimate the costs of the 12 months after HSCT and analyse CMV serology and infection (± disease) as predictor of costs. All patients except D-/Rwere weekly screened with pp65 Ag until Sept. 2009, or PCR
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from Oct. 2009, until d100 or end of immunosuppression. No patient received CMV prophylaxis. CMV-infected patients routinely receive pre-emptive ganciclovir or foscavir for X14 days. Medical data were collected from the original charts. Hospital resource utilization was retrieved via the linked hospital admissions and Diagnostic Related Groups from conditioning to 12 months after HSCT. Patients who needed a 2nd transplant (n ¼ 6) were censored at this date. Costs were estimated from the viewpoint of the payer using national 2013 tariffs. Analyses were performed for the entire population and according to CMV infection (X1 episode), vital status and pretransplant CMV serologies. Dichotomous variables were compared using the Chi-2 test, while continuous variables, described by mean and standard deviation, were assessed with a Student test, with a 0.05 significance level. Analyses were performed using Excel (2010, Microsoft) and SAS (9.3, SAS corp. NC) software. Results: Median age at transplant was 51 years, the sex ratio M/F 1.39. Patients were transplanted for acute leukaemia (69%), lymphoproliferative (22%) or myeloproliferative disorders (6%), or other (3%). The graft was from an HLA-id sibling (40%), unrelated donor (51%) or cord-blood unit (9%). The CMV D/R serology were / (28%), þ / (10%), / þ (28%) or þ / þ (34%). 71 (34%) patients developed X1 episode of CMV infection, at a mean of 85 days (median: 47 (7-802)). Only 1 case occurred after 12 months. 9 patients developed CMV disease. CMV-infection and death were significantly related (P ¼ 0.0389). The mean 1 y-cost of HSCT was 99 kilo-euros (KE) (range: 27-280; median: 92 [IC95%: 94105]). 89.5% of hospital resources were used during the first 6 mo of HSCT. The costs at 12 months did not correlate with D/R serology. When compared to non-infected patients, CMV-infected patients had longer duration of hospitalization (median: 115 [IC 95%: 102-127] vs 87 days [80-95], P ¼ 0.0002), and same duration of ICU stays (9 vs 11 days). Although the 1-year costs were not significantly different, the costs of the 6 first months were significantly higher in CMV infected-patients (97 vs 86 KE) (P ¼ 0.0291). Conclusion: CMV infection after allo-HSCT is significantly associated with a 1 y-longer hospitalization and with an increased cost of 11 KE at 6 months post-transplant. This difference between patients who do and those who do not develop CMV infection could be reduced by efficient prophylactic strategies. However, their choice should be guided by cost-effectiveness. Disclosure of Interest: None declared. P153 The clinical and financial impact of Respiratory Syncytial Virus infection post-Haematopoietic Stem Cell Transplantation C. M. Gorcea1,*, E. Tholouli1, A. Turner2, N. Flaum1, F. Dignan1 1 Department of Haematology, 2Department of Virology, Manchester Royal Infirmary, Central Manchester NHS Foundation Trust, Manchester, United Kingdom Introduction: Respiratory syncytial virus (RSV) is a common cause of respiratory viral infections and is associated with increased morbidity and mortality in patients undergoing haematopoietic stem cell transplantation (HSCT). Little is known about the economic burden associated with RSV infection. We sought to analyse the outcome and costs associated with RSV infection in a single UK centre. Materials (or patients) and methods: We undertook a retrospective case analysis of 48 consecutive adult patients diagnosed with RSV infection post HSCT between December 2010 and April 2014. Patient characteristics: male 28, median age 55 years (range 23 to 71), diagnosis: acute leukaemia 15, multiple myeloma 16, myelodysplasia 6, aplastic anaemia 5, chronic leukaemia 3, other 3. Conditioning regimen in the allogeneic HSCT group: 22 reduced intensity conditioning (RIC) regimen and 13 myeloablative conditioning. Stem cell source:
1 cord blood transplant, 14 sibling allograft, 20 volunteer unrelated donor. RSV infection was diagnosed by polymerase chain reaction (PCR); overall, 35/227 (17%) allogeneic and 13/180 (7%) autologous HSCT recipients were RSV positive. At diagnosis, 20 patients had an upper respiratory tract infection and 28 had a lower respiratory tract infection (LRTI). LRTI was the primary indication for treatment; patients at high risk of progression to LRTI also received therapy. In total, 42 patients received Ribavirin: orally 16; aerosolised 17; aerosolised and orally combined 5; aerosolised and IV combined 4. Six patients received no treatment. The median treatment duration was 7 days (range 5 to 47). Additional therapy: 13 immunoglobulin replacement, 33 concomitant antibiotic treatment and 11 also received antifungal treatment. Results: Twenty patients were admitted for RSV treatment: 6 required intensive care (ICU) admission, 4 requiring invasive ventilation. Fifteen patients were treated as out-patients. An additional 13 patients were diagnosed as in-patients and RSV was not felt to have extended their hospital stay. The median ward stay was 10 days (range 4 to 55) and median ICU stay 5 days (range 4 to 60). After a median follow-up period of 6.5 months (range 0 to 39): 33 patients are alive, 15 patients died. Causes of death: sepsis 3, relapsed disease 3, GVHD 2, gastrointestinal bleed 1, stroke 1. In 5 patients the cause of death was attributed to RSV infection, 4 having had previous ICU stay. No RSV-related deaths were recorded in the group treated with oral Ribavirin. Admission costs were calculated based on the 2014 hospital tariff. The treatment cost was calculated according to pharmacy prices and formulation for a 70 kg adult as follows: oral dose 1000 mg/day, intravenous formulation 33 mg/kg loading dose followed by 16 mg/kg and aerosolised preparation 6 gm/day. The total cost of ICU admission was d148,268 (h 187,146), ward admission cost of d122,100 (h154,116) and total treatment cost was d78,606 (h99,217). The overall cost for our cohort rose to the total sum of d 348,978 (h440,485). Conclusion: RSV in post HSCT patients remains a challenge due to the high frequency of infection and high rates and costs of readmission. Oral ribavirin may be an option for lower risk patients. Prompt initiation of treatment in high risk patients is essential due to increased morbidity and mortality associated with RSV- associated LRTI and may avoid unnecessary hospital admission. Disclosure of Interest: None declared. P154 Monitoring of blood BK virus is useful to predict BKrelated haemorrhagic cystitis in select high-risk postallogeneic transplant recipients C. Phipps1,*, A. Ghosh1, Y. C. Tan 1, A. Ho1, Y. T. Goh 1, S. Gopalakrishnan1, J. J. Lee1, Y. C. Linn1, W. Hwang1 1 Department of Haematology, Singapore General Hospital, Singapore, Singapore Introduction: Haemorrhagic cystitis (HC) in recipients of allogeneic stem cell transplantation (SCT) has been associated with latent BK-virus (BK) reactivation. Current treatment for BKHC is inadequate and relies on supportive care and measures to control bleeding. We initiated a plasma BK monitoring protocol in our allogeneic SCT patients on the basis that early detection or rising BK blood levels may predict for onset of BK-HC. Materials (or patients) and methods: Our primary objective was to determine whether blood BK monitoring would predict for the development of BK-HC. Secondary objectives were to define incidence of HC and BK reactivation, and identify patients at highest risk of BK-HC after allogeneic SCT. The monitoring protocol involved analysis of plasma before SCT and then weekly from day 14 to day 56 or until 2 consecutive negative results using quantitative polymerase chain reaction targeted at VP1 protein gene of BKV. These were paired with
urine microscopy samples. Only grade Z2 HC was considered positive. Results: The monitoring period involved 117 patients, 50 females and 67 males. Median age was 46 years (range 12 – 68) and median follow up in surviving patients, 17 months. Donor types were as follows: sibling, N ¼ 57; unrelated (URD), N ¼ 39; cord, N ¼ 17; haploidentical, N ¼ 4. Graft source was PBSC in 98, bone marrow in 2, cord blood in 17. Conditioning regimens consisted of conventional myeloablative (MAC), N ¼ 50; reduced-intensity (RIC), N ¼ 60; non-myeloablative, N ¼ 7. Antithymocyte globulin (ATG) was given to 42 patients. It was used routinely in all recipients of unrelated donor grafts, excluding cord units. Apart from ATG, post-transplant cyclophosphamide was given as T-cell depletion (TCD) in the haploidentical SCT recipients. BKV was detected in 39 patients (33%) at a similar rate in those receiving MAC and RIC regimens. HC was detected in 24 patients (21%) and was grade 2 in 17, grade 3 in 4, and grade 4 in 3 patients. Blood BK was positive in 15 of 24 patients with HC (P ¼ .009), giving an incidence of BK-HC of 13% . Ten of these were positive before the onset of HC but only 1 patient had rising BK titres coinciding with HC development. HC occurred at a median of 21 days (range 1-73). In non-BK related HC, median onset was 9 days and attributed to chemotherapy and thrombocytopaenia. BK-HC occurred at a median of 30 days (range 6 – 73). Univariate analysis identified ATG use, URD grafts, and TCD as significantly influencing the development of BK-HC. Only TCD was significantly related to BK-HC on multivariate analysis (P ¼ .009). It occurred in 22% of patients with TCD grafts. Conclusion: BK-HC causes significant morbidity and treatment of overt disease is difficult and usually lengthy. Our prospective monitoring of blood BK levels and development of HC revealed that of patients who developed BK-HC, 67% had detectable levels before the onset of overt HC. Furthermore, we identified TCD (in vivo in our series) as being significantly associated with BK-HC. Considering these results, monitoring of blood BK levels may be advisable in patients who have undergone T-cell depletion of their grafts with a view to reduce immunosuppression where possible and initiate treatment with the goal of halting progression to overt HC. Disclosure of Interest: None declared. P155 Multidrug-resistant bacterial pathogens in allogeneic hematopoietic cell transplantation – dismal outcome of multidrug-resistant Pseudomonas aeruginosa colonized patients D. Heidenreich1,*, F. Nolte1, S. Kreil1, M. Reinwald1, W.-K. Hofmann1, S. Klein1 1 III. Medizinische Klinik, UNIVERSITA¨TSMEDIZIN MANNHEIM, Mannheim, Germany Introduction: Multidrug-resistant bacterial pathogens (MRP) such as vancomycin-resistant enterococci (VRE) and extendedspectrum beta-lactamase producing bacteria (ESBL), particularly Pseudomonas aeruginosa are an emerging challenge in allogeneic hematopoietic cell transplantation (HCT). However, there are no data on the impact of MRP on the outcome after HCT in the literature. Thus, it was the aim of this study to analyze the issue of MRP in patients undergoing HCT. Materials (or patients) and methods: A total number of 72 consecutive patients who received the first HCT from 2010 to 2013 at our institution were retrospectively analyzed. The underlying diseases were AML (44), ALL (5), CML (4), MPN (2), lymphoma (5), MDS (9), and multiple myeloma (3). Conditioning was myeloablative in 23 and reduced intensity in 49 patients. Stem cell sources were peripheral blood (69) or bone marrow (3) from matched siblings (19), matched unrelated (45), mismatched (5) or haploidentical donors (3). As baseline investigation patients underwent a screening for MRP i.e. ESBL, VRE and MRSA. Swabs from nose, throat, axilla, urethra, anus,
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stool and urine were collected. Microbiological investigations such as cultures from blood, urine, swabs, stool or central venous catheters were performed whenever clinically needed. ESBL were categorized as multi-resistant gram-negative bacteria (4MRGN, resistant to cephalosporins, piperacillin, fluorochinolones and carbapenems) or as 3MRGN (resistant to 3 of these 4 drug groups). Results: 23/72 patients (32%) were colonized by MRP (MRP þ group) either at baseline (baseline MRP þ group, n ¼ 13, 18%) or at any other time point until day 100. 4 patients were positive for two MRP and 1 patient for 3 MRP. Detected MRP (n ¼ 29) were 3MRGN E. coli or Klebsiella pneumoniae (n ¼ 12), 4MRGN Pseudomonas aeruginosa (P. aeruginosa, n ¼ 4), 3MRGN P. aeruginosa (n ¼ 2), 4MRGN Stenotrophomonas maltophilia (n ¼ 2), 3MRGN Citrobacter freundii (n ¼ 1), VRE (n ¼ 7) and MRSA (n ¼ 1). Out of these 23 patients 8 developed an infection with MRP: Septicemia with 3MRGN E. coli or K. pneumoniae (n ¼ 4), septicemia with P. aeruginosa (4MRGN n ¼ 3) and one patient with VRE septicemia and 4MRGN P. aeruginosa pneumonia. Out of the 4 patients with 4MRGN P. aeruginosa infection 3 died (2 of these patients had been already colonized with 4MRGN P. aeruginosa at baseline). However, 2-year overall survival of MRP colonized versus noncolonized patients was essentially the same (63 vs. 67%, median follow-up 24 months). Day 100 NRM was higher in the baseline MRP þ group and in the entire MRP þ group in comparison with non-MRP colonized patients (23% and 17% vs. 10%, not statistically significant [ns]). Data for 2 year NRM were 33%, 22% and 17% (ns), respectively. The increased NRM of MRP þ patients was mainly due to the high NRM of patients infected by multidrug-resistant P. aeruginosa. Conclusion: Colonization or infection with 3MRGN gramnegative non-P. aeruginosa bacteria or by VRE has no negative impact on the outcome after HCT. Thus, HCT of patients colonized by MRP is feasible. However, patients colonized by multidrug-resistant P. aeruginosa seem to have a dismal outcome. HCT of these patients should be considered with care. We therefore suggest including screening for MRP in the pretransplant recipient work-up particularly identifying patients colonized by multi-resistant P. aeruginosa. Disclosure of Interest: None declared.
or VGV. 55 patients received treatment with CDF either because of failure to respond to GCV or VGV (n ¼ 43), CMV disease (n ¼ 7), or CMV infection occurring prior to neutrophil engraftment (n ¼ 5). The CMV specific CD8 þ T cell response was serially measured by HLA-CMV peptide tetramers in a sub-group of 20 patients. Results: 101/153 patients responded to GCV as first-line therapy. The likelihood of developing CMV disease was increased by use of T-cell depletion (P ¼ 0.033) and CMV / þ (D/R) serostatus (P ¼ 0.035). 44/55 patients treated with CDF achieved a durable response. 28 of 35 evaluable patients (80%) patients treated with CDF for GCV resistant CMV viremia achieved sustained (Z2 weeks) CMV PCR negativity. Responses were rapid with a median time to eradicate viremia of 15 days. CDF was generally well tolerated and only 2 patients experienced Grade 3-4 nephrotoxicity (NCI toxicity criteria 2.0). There was no difference in overall lymphocyte count, CD4, CD8 or gamma/delta T cells at any point post reactivation between patients that responded to GCV and those that did not. The number of CMV-specific CD8 þ T cells at D þ 50, D þ 100 and D þ 150 post transplant was lower in patients who required salvage therapy with CDF than in patients who responded to GCV. Importantly, no patient with undetectable CMV-specific CD8 þ T cells responded to GCV but 4/7 patients with absent CD8 þ T cells responded to CDF. Furthermore, low CMV-specific CD8 þ T cell numbers after the first episode of CMV reactivation are predictive of further CMV reactivations. The median CMV specific CD8 þ T cell count was 16 times higher in patients that experienced only one episode (n ¼ 13) of reactivation versus those that had multiple episodes (n ¼ 7) (P ¼ 0.007). Conclusion: CDF is a well-tolerated and effective salvage therapy for GCV resistant CMV viremia. In contrast to GCV it has the capacity to eradicate CMV infection in the absence of an antigen specific CD8 þ T cell response. CDF may also be used for secondary pre-emptive therapy in patients identified as at high rsk of recurrent CMV reactivation based on measurement of CMV-specific CD8 þ T cells at the end of the first episode of CMV reactivation. Disclosure of Interest: None declared.
P156 Cidofovir Treatment may benefit patients with low CMVspecific CD8 T cell levels after initial CMV reactivation post allogeneic stem cell transplant D. J. Lewis1,2,*, F. Farina1, P. Moss1,2, S. Nagra1, M. Raeiszadeh2,3, F. Chen1,2,3, R. Malladi1,2, C. Craddock1,2 1 Centre for Clinical Haematology, University Hospital Birmingham, 2School of Cancer Sciences, University of Birmingham, 3 R þ D Stem Cells and Immunotherapies, NHS Blood and Transplant, Birmingham, United Kingdom
P157 Peg-Filgrastim Versus Filgrastim In Prevention Of Febrile Neutropenia In Lymphoma Patients After Autologous Peripheral Blood Stem Cell Transplantation D. Radic-Kristo1,*, V. Zatezalo1, K. Misura Jakobac1, N. Gredelj Simec1, S. Jurenec2, N. Pavic´1, I. Mandac Rogulj1, M. Martinovic1, A. Planinc Peraica1, S. Ostojic Kolonic1 1 Department of hematology, 2Transfusion medicine, Clinical hospital ‘Merkur‘‘, Zagreb, Croatia
Introduction: Cytomegalovirus infection (CMV) remains a significant cause of morbidity and mortality following allogeneic stem cell transplantation (SCT). Up to 50% of CMV seropositive patients undergoing RIC have recurrent reactivation, associated with significant post-transplant complications. CMV-specific T cell measurements have been performed in a research setting using specific HLA-peptide tetramers, but are not yet routinely used in a clinical setting. Ganciclovir (GCV), and its orally available prodrug, valganciclovir (VGV) is established as standard of care in patients with CMV reactivation but the optimal treatment strategy for patients requiring second-line therapy such as with recurrent/ refractory viremia remains unknown. Cidofovir (CDF) demonstrates activity in CMV disease but its tolerability and activity in patients with GCV resistant CMV reactivation has not been studied. Materials (or patients) and methods: 397 patients at risk of CMV reactivation after allogeneic HSCT were sequentially monitored over a 7-year period. 156 patients required treatment of whom 152 received initial treatment with GCV
Introduction: Filgrastim and pegfilgrastim are widely used in prevention of febrile neutropenia after stem cell transplantation. The aim of this study is to compare the efficacy in prevention of febrile neutropenia using a single fixed dose of 6 mg of pegfilgrastim versus multiple daily administration of filgrastim 5 mcg/kg and to compare time to engraftment of leukocytes (defined as time to recovery of leukocytes to a value 41x109/L) after autoPBSCT. Materials (or patients) and methods: In this retrospective study we analyzed data of two groups of lymphoma patients who underwent autologous PBSCT. First group consisted of 47 patients (31 male, 16 female; median age 53, range 20-70), transplanted from January 2012 to June 2014, all of whom received pegfilgrastim single 6 mg dose on day 1 after autologous stem cell reinfusion. The second group is a historical cohort of 110 lymphoma patients (56 male, 54 female; median age 45, range 19-71) who had been transplanted between 2003 and August 2009 and received filgrastim 5 mcg/kg daily starting on day 1 until leukocyte
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and neutrophile engraftment was registered. Both groups had similar distribution of lymphoma subtypes; patients with Hodgkin’s disease represented largest subgroup followed by DLBCL, follicular lymphoma, and mantle cell lymphoma while other subtypes were less common. In both groups, all patients, received BEAM as a conditioning regimen before autologous PBSCT. In first group all patients received ciprofloxacin, acyclovir and fluconazole as antiinfective prophylaxis. In second group 54,5% of patients received ciprofloxacin and others received norfloxacin as antimicrobial prophylaxis. Most received fluconazole and ketoconazole (60% and 32% respectively) as antifungal prophylaxis while a few (8%) received other antifungal drugs. All patients received acyclovir. Results: In first group incidence of febrile neutropenia was 57,4%. Of those with febrile neutropenia, microorganism was isolated in 32,65% of cases. Median duration was 3 days (range 1-9), mean 3,18 days. Median time to engraftment of leukocytes was 9 days (4-19), mean 9,85 days. In historical cohort 64% of patients had FN, with median of 2 days ( 1-11), mean 3,36 days. Microorganism was isolated in 31,94% of cases. In this group median time to engraftment of leukocytes was 10 days (6-28), mean 10,28 days.There was no statistically significant difference between the two groups in incidence of FN or its duration, frequency of culture isolation nor in time to leukocyte engraftment. Conclusion: This study has shown no statistically significant differences in incidence nor duration of febrile neutropenia using pegfilgrastim vs filgrastim in lymphoma patients after autologous PBSCT. Disclosure of Interest: None declared. P158 The impact of carbapenem resistant klebsiella pneumonia related infections on the outcome of haematopoietic cell transplant recipients D. Mallouri1,*, E. Tsorlini2, V. Constantinou1, S. Papadimitriou1, M. Kaliou1, V. Kalaitzidou1, K. Panteliadou1, M. Iskas1, I. Batsis1, C. Smias1, D. Apostolou1, D. Sotiropoulos1, I. Sakellari1, A. Anagnostopoulos1 1 Haematology department - BMT unit, 2Microbiology Department, George Papanicolaou Hospital, Thessaloniki, Greece Introduction: Carbapenem resistant klebsiella pneumonia (KP) seems to be an emerging pathogen in haematologic malignancies. Little is known about its epidemiology and deleterious effect on haematopoietic cell transplant (HCT) recipients and potential excess of treatment related mortality. Materials (or patients) and methods: In this retrospective single centre analysis, we reviewed the medical data of all consecutive transplanted patients (pts) from 1/2009-10/2014. The total number of pts were 491; autologous (autoHCT) 266 and allogeneic (alloHCT) 225. Among them we identified 63 pts (21 females, 42 males) diagnosed with at least one KP isolate from the time of disease diagnosis to last follow up. The pts were suffering from acute leukemia (43), lymphoproliferative disease (14) multiple myeloma (3), MDS/MPDs (3) and the disease phase was early in 30, intermediate in 12, advanced in 21 pts, post median lines of treatment 3 (1-11). Twelve pts underwent autoHCT and 51 pts alloHCT (MUD 28, sibling 20, haploidentical 3). Results: In 17 pts (3.4%) a previous to HCT KP colonization was found from either perirectal area (5), urine (8), blood (3) or pancreatic fistula (1). Out of these 17 pts, 10 had infection diagnosed as KP associated that resolved before HCT. From the beginning of the conditioning and afterwards, KP associated infection was diagnosed in 40 pts (total, 8.1%), 4.1% of the autoHCT population (n ¼ 11) and 12.8% of the alloHCT population (n ¼ 29; 10 sibling, 16 MUD, 3 haploidentical). The main source of isolation was blood in 14, urine in 16, sputum in 8 and fecal in 2 pts. Twelve of these pts were previously colonized with KP. The pts developed sepsis (8),
bacteremia (6), pneumonia (10) and urine infection (16). The KP species were mainly refractory to many antibiotics and carbapenems and sensitive mostly to colistin (polymyxin E), gentamycine and tygecycline, while in 11 pts the KP species were refractory in all antibiotics tested. Eventually, 17 pts died from: infectious complications in association with graft versus host disease (GVHD) (4 pts), infectious complications in association with disease relapse (4 pts), refractory GVHD (2), relapsed/refractory disease (7 pts). The early deaths ( þ 100d) were only 2 and TRM was 6/40 pts. Conclusion: Through this time period is obvious that KP is an emerging pathogen and surveillance cultures leading to prophylactic decontamination treatment of infected patients is essential for this high risk population. The pre-transplantation colonization of pts does not seem to be an exclusion criterion and does not predict infection post-transplant. In both transplant modalities KP infection per se was not the sole cause of death since it was associated with heavily immunocompromised pts either due to refractory disease or severe GVHD. Disclosure of Interest: None declared. P159 Hemorragic cystitis after haploidentical stem cell transplantation: predisposing factors analysis D. Champ1,*, J. Gayoso1, M. Kwon1, P. Balsalobre1, D. Serrano1, A. C. Franco1, M. Bastos1, A. Pe´rez-Corral1, C. Pascual1, J. Anguita1, I. Bun˜o1, J. L. Dı´ez-Martı´n1 on behalf of Instituto de Investigacio´n Sanitaria Gregorio Maran˜o´n 1 Hematology, HGU Gregorio Maran˜o´n, Madrid, Spain Introduction: Hemorragic cystitis (HC) is a well known complication after hematopoietic stem cell transplantation related to the toxic effect of the conditioning regimen or viral reactivation/reinfection causing significant morbidity and even mortality to patients. HC has become an increasing problem after the widespread use of haploidentical transplantation (HAPLO) with post transplant cyclophosphamide (PT-Cy) as GvHD prophylaxis, but its real magnitude is not stablished. Our work studied the incidence and predisposing factors for HC in the context of HAPLO with PT-Cy in a single center. Materials (or patients) and methods: We retrospectively analyzed 60 consecutive adult patients receiving an HAPLO with PT-Cy in our center from Dec-2007 to July-2014, either after myeloablative (MAC) or reduced intensity conditioning (RIC). We evaluated patients and donors characteristics, graft source, conditioning regimen, GvHD prophylaxis, HC severity, onset and evolution, microbiologic isolations and HC treatment. Results: HC was diagnosed in 21/60 HAPLO recipients (35%) until day þ 100, median age was 34 years (range: 17-61) and 14/21 were males. HC severity was grade I (microscopic hematuria) in 5 (8%), grade II (macroscopic hematuria) in 6 (10%), grade III (hematuria with clots) in 5 (8%) and grade IV (clots retention, obstructive nephropathy) in 5 (8%). Median days of HC onset was 34 days post-HAPLO (interquartilic range 25-75% (IQR): 28-40) and median days of HC duration was 19 (IQR25-75%: 13-31), without clear relationship with severity. In 18/21 cases (86%) BK virus was isolated in urine samples and no microbiologic isolations were detected in 3/21 (14%). All patients had received fluid overload, diuretics and MESNA prior to PT-Cy and levofloxacin for HC prophylaxis, and received as HC treatment vigorous hydratation, diuretics, hemotherapy, analgesics and bladder irrigation in those with pain or obstruction due to clots. Hyaluronic acid washing was employed in 3, electrocoagulation in 3 and mesenchymal stem cells were used in 4 refractory HC with partial responses only. Antivirals and immunoglobulin replacement were not employed routinely. None of our patients died as direct consequence of HC. None significant relationship was found between patients and donor characteristics, underlying disease, graft source (BM vs PBSC), conditioning regimen
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(RIC vs MAC) or the presence of acute GvHD or steroids use. The only factor probably related to HC development was the presence of CMV reactivations, which was present in 17/21 patients with HC (81%) compared to 27/39 (69%) in patients without HC. The incidence of HC was higher during springsummer (14/21) compared to autumn-winter (7/21), and clustered distribution was observed. Conclusion: HC is a common complication in the setting of HAPLO with PT-Cy, mostly related to BK virus reactivations, that causes significant morbidity but not mortality in our series. Only CMV reactivation was found as a related factor for HC development, but it could also represent another manifestation of the immunosuppression status of the patients and not a real predisposing factor for HC. Epidemiological factors could be responsible for seasonal or clustered distribution. Disclosure of Interest: None declared. P160 Final results of the prospective study on the role of bed side ultrasound (BUS) in neutropenic enterocolites (NEC). Early US diagnosis reduces the mortality and changes the diagnostic criteria with a new discriminant statistical model E. Benedetti1,*, I. Bertaggia1, R. Morganti1, B. Bruno2, P. Lippolis3, F. Caracciolo4, E. Orciuolo1, G. Buda1, F. Cerri5, D. Caramella5, M. Petrini6 on behalf of NEC 1 Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Hematology Unit Ospedale S. Chiara Pisa, Pisa, 2U.O Ematologia Trapianti di midollo, University of Torino, Torino, 3UO Chirurgia D’Urgenza , Azienda OspedalieroUniversitaria Pisana, 4U.O Ematologia Trapianti di midollo, Hematology Unit Ospedale S. Chiara Pisa, 5Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, U.O. Radiologia Universitaria Pisa, 6Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Hematology Unit Ospedale S. Chiara Pisa, Pisa, Italy Introduction: Neutropenic enterocolitis (NEC) is a life threatening complication of leukemic and solid tumors patients (pts) treated with chemotherapy (CHT) with mortality rate up to 50%. NEC is characterized by abdominal pain (AP), fever (F) and diarrhoea (D). Ultrasound (US) was used to evaluate bowel-wall thickening (BWT), and 4 4 mm is considered diagnostic of NEC. Perforation occurs in 5%410% of cases. Early diagnosis is crucial to start conservative medical management (CMM) which appears the optimal strategy for most cases. We evaluated prospectively if Bed-side-US (BUS) can detect early signs of NEC and guide a prompt treatment (CMM or surgical) in order to reduce mortality Materials (or patients) and methods: In the last 7 years all pts admitted in Our Hematology/BMT Unit wards at University of Pisa (Italy), undergoing chemotherapy (CHT), autologous or allogeneic transplant (AutoTx,AlloTx) were enrolled. Abdominal US was performed, baseline before treatment, and as only one symptom (or a combination) appeared within 12h from onset: F and/or D and/or AP in CHT-related neutropenic pts. Results: 76 episodes out of 1680 neutropenic pts were identified (4.7%). Seven pts had 2 separate episodes of NEC. Disease diagnosis were HD (N ¼ 10), ALL (N ¼ 8), AML(N ¼ 21), MM (N ¼ 9) and NHL (N ¼ 28). Treatment received was intensive CHT (N ¼ 35), AutoTx (N ¼ 37) and AlloTx (N ¼ 4). At time of diagnosis (Dx) symptoms were: F þ AP þ D 48%, F þ D 4%, F þ AP 1%, AP þ D 34%,D 3%,AP 9%. F alone were never present at diagnosis of NEC. At Dx, F was absent in 35/76 NEC episodes (46%) and in 17/76 F never developed and 18/76 had delayed onset of F (from 10h to 72 h) from NEC Dx. There is a trend but not a statistical (St) significant difference in mortality among the 3 F groups (P ¼ 0.09). As control group (CG) we considered pts with CHT related mucositis and pts restaged with US during neutropenia in absence of symptoms. A total of N ¼ 509 pts were randomly chosen in the CG. None of them had BWT. Overall 11 pts died (14.5%) without a St difference
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between 1 or 2 episodes (P ¼ 0.309) of NEC. Treatment was CMM in 92% of pts, and was promptly started as BUS diagnosis was made. Mortality in pts treated with CMM was 11.5%. Six pts underwent surgery, guided by US features, during neutropenia, and 50% are alive. Median BWT was 8.6 mm in surviving pts (range 4.2-30mm) and 11mm in deceased (range 9.3-15mm). Authors have suggested BWT to be prognostic of outcome; in our study pts with 410mm had 60% survival. Median time to response from beginning of CMM was 24h and the first sign of was a decrease in AP, while median time to death was 26h (range 10.5-72h). The likelihood of NEC Dx in a discriminant St model (Bayes theoreme) for pts with BWT and AP ¼ 98.8%, AP þ D ¼ 99.9%, AP þ D þ F ¼ 100%, AP þ F ¼ 99.9%, D þ F ¼ 5%. Conclusion: BUS allowed to detect early signs of NEC and to start a prompt treatment, which was CMM in 92% with a 88.5% survival rate. With BUS pts do not live the isolation room. US guided surgical intervention with 50% survival rate. Images of BUS and CT were superimposable. Fever is not a condition sine qua non for NEC diagnosis. A prompt BUS in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical) reducing mortality. Disclosure of Interest: None declared. P161 Biological characterization of cytomegalovirus pp65 and IE1-specific CD4 þ and CD8 þ T cells from healthy subjects unfolds their heterogeneous profiles E. Albiero1,*, E. Amati2, E. Baumeister3, G. Astori2, H. Einsele3, G. U. Grigoleit3, F. Rodeghiero1 1 Hematology Project Foundation Research Laboratories, Department of Cell Therapy and Hematology, San Bortolo Hospital, 2 Advanced Cellular Therapy Laboratory, Department of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy, 3 Department of Internal Medicine II, Division of Hematology and Oncology, Julius Maximilian University Medical Center, Wu¨rzburg, Germany Introduction: Characterization of human cytomegalovirusspecific T cells (CMV-T) is of crucial importance for their potential use in adoptive immunotherapy (AI) after HSCT. In peripheral blood mononuclear cells (PBMCs) from CMVseropositive (CMV þ ) healthy subjects background frequencies of CMV-T are usually very low, hence the requirement for prolonged culture time and multiple stimulations to expand them. The evaluation of the end-culture specificity and composition have been sometimes neglected or difficult to assess in these settings. We explored the identity and the functionality of CMV-T enriched in short-term cultures after ex vivo stimulations with viral antigens (Ags). Materials (or patients) and methods: We induced mixed lymphocyte peptide cultures (MLPCs) to enrich cognate pp65and IE1-specific T cells from PBMCs obtained from CMV þ healthy donors (n ¼ 9) before selection by cytokine capture system or CD154 microbeads. CMV-T were characterized by flow cytometry analysis of T-cell subpopulations, intracellular cytokine staining combined with CD107a degranulation assay, CFSE-proliferation assay and cytokine production. Results: The median frequencies of pp65-specific CD3 þ CD8 þ cells (CTLs) was 0.17% (o0.01%–1.56%) and 0.66% (0.05%–3.61%) of the IE1-counterpart. Median frequency of pp65-specific CD3 þ CD4 þ (Th) cells was 0.34% (o0.012.89%) and 0.17% (o0.01-4.79%) of IE1-specific Th cells. CD107a degranulation by CTLs in PBMCs upon stimulation with pp65 and IE1 was 0.46% (o0.01-6.51%) for the former and 0.13% (o0.01-7.99%) for the latter. There was no correlation between the frequency of CMV-T in PBMCs and their cytotoxic potential. Ag-specific Th cells were negative for CD107a. Cell viability after MLPC_pp65 was 35.4% (6-84.3%) while 21.1% (2.3-81.7%) after MLPC_IE1 in spite of absolute CMV-T number at day 0. The rate of increase of pp65-specific T
cells was 1- to 33-fold while IE1-specific T cells ranged from 1to 961-fold under the same experimental conditions. The final cell culture product was mostly composed by Th cells. Good enrichment of CMV-T upon MLPC was feasible if a frequency of CD3 þ CMV-TZ10% was reached. The isolated CMV-T showed high Ag-specific proliferation (495%). Cytokine repertoire after MLPCs was IFN-g þ TNF-a þ polarized with high levels of IL4 and IL10, too. Expanded CMV-T had significantly lower level of late effector (Temra) CD8 þ cells compared with PBMCs before CMV stimulation (Po.001). Temra CD4 þ cells were less represented upon MLPC_IE1 (Po.05). CMV-T expansion was accompanied by a significant increase of naı¨ve/memory stem (Tn/scm; Po.001) CTLs while the Th counterpart significantly increased upon stimulation with IE1 (Po.005). Conclusion: We improved basis knowledge on CMV-T biology and functionality. Outcome was variable and showed donor and epitope dependency. We used pp65 and IE1 libraries as stimulating Ags to be independent from HLA-type and to be able to investigate T cell responses directed not only against single peptide epitopes. Differences in HLA and epitope dominance and variability in the relative number of CD3 þ effector cells and IFN-g/CD154 expression among healthy donors could reflect the observed individual CMV-specific cellular immunity. This heterogeneity raises points to be considered when approaching AI and should be taken into account in the attempt to define private predictors of putative individual responses. Disclosure of Interest: None declared. P162 Allogeneic Haematopoietic Stem Cell Transplantation in Hepatitis B Virus previously exposed recipients or donors: a safe procedure in antiviral era E. Ravano1,*, B. Forno1, E. Zucchetti1, P. Marenco1, G. Grillo1, I. Lotesoriere1, R. Cairoli1 1 BMT Unit, Niguarda Hospital, Milan, Italy Introduction: A previous exposition to hepatitis B Virus (HBV) is still frequent in donors and patients (pts) who could take advantage from a haematopoietic stem cell transplant (HSC Tx). Viral infection, reactivation or seroreversion can lead to chronic or fulminant hepatitis. Availability of antiviral drugs has certainly reduced risks and has changed standard prophylaxis procedures. Materials (or patients) and methods: To evaluate incidence and outcome of HBV þ Txs in our Centre, all the 161 consecutive pts who underwent an allogeneic HSC Tx between January 2009 and August 2014, were analysed. Those who were positive for HBsAb only were excludes as vaccinated. 29/161Txs (18%) were performed in HBV exposed: 6 in recipients HbsAg þ /HbcAb þ (3,7%), 20 in HbsAg-/ HbcAb þ (12,4%), and 3 in HBV-naı¨ve recipients who received HSC from a donor HBV þ (1 HbsAg þ /HbcAb þ , 2 HbsAg-/ HbcAb þ ). 1/29 died d þ 13 for septic shock, 28 are evaluable surviving over day þ 100 . Anti HBV prophylaxis was started in 27/28 pts before Tx and was stopped not earlier than 6 months after withdrawal of immunosuppression (presently reached only in 3 pts); 23 Txs were performed under Lamivudine, 3 received Entecavir and one Tenofovir. According to our standard follow-up procedure pts were regularly tested for liver function, serology and, when applicable, HBV genome and mutants search. Results: With a median follow-up of 767 days (range: 1811827), 17 pts (62%) are alive. 5 died for relapse, 7 for NRM (none of them for a cause HBV related). The only hepatic complication observed is in a HbsAg-/HbcAb þ pt who experienced a serious acute hepatitis (seroreversion) 6 months after stopping Lamivudine that occurred 1,5 year after Immunosoppression withdrawal; hepatitis resolved after Entecavir therapy. 14 pts are still in prophylaxis: none of them experienced virus reactivation, neither viral hepatitis. To be noted that 1 out of the 3 pts who lost their HbsAb after Tx, was
vaccinated day þ 1035 and reached a good HbsAb titre; so he could stop Lamivudine without any reactivation. Conclusion: With our policy for anti HBV prophylaxis, HBV positivity does not seem an exclusion cause for HSC Tx any longer, at least with our conditioning and GVHD prophylaxis (only 2 Txs were T-depleted with Thymoglobuline, and none received Rituximab). Further studies are needed to define best timing for stopping prophylaxis and a possible role of HBV vaccination after HSC Tx. Disclosure of Interest: None declared. P163 Laboratory surveillance of Herpes and polyoma viruses: results and clinical consequences E. Jaskula1,2,*, J. Lange2, A. Tarnowska2, M. Sedzimirska2, M. Mordak-Domagala2, D. Duda2, K. Suchnicki2, A. Lange1,2 1 L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland, 2Lower Silesian Center for Cellular Transplantation with National Bone Marrow Donor Registry, Wroclaw, Poland Introduction: Viral complications constitute one of the main threats post-HSCT, especially in patients who received transplants from unrelated donors. Therefore, all patients transplanted in our institution were followed for viral reactivation events including HHV6, CMV, EBV, polyoma BK, and JC. Materials (or patients) and methods: This study included 156 unrelated donor HSCTs. 90 and 66 patients received myeloablative or reduced intensity conditioning. All patients except 3 received anti-lymphocyte antibodies (ATG [142 patients] and Campath [21]). The latter patients received ganciclovir as a part of microbial prophylaxis (6 mg/kg b.w. for 5 days). Quantitative detection of Herpes virus DNA copies (real-time PCR), was performed routinely in one-week intervals shortly post-transplant and then in one-month intervals unless required earlier. Results: CMV reactivation events were less frequent shortly posttransplant than at later times ( 14% vs. 27%, P ¼ 0.003, before and after 30 days post-HSCT).Patients positive for CMV DNA copies (detected between 30-100 days post-HSCT) had higher levels of CD8 þ CD57 þ lymphocytes in the blood than counterparts lacking CMV reactivation at any time post-transplant (18.92 þ / 2.14% vs. 8.36 þ / 1.50%, P ¼ 0.0075; 186x106 þ / 41.86x106 vs. 84.15 þ / 32.53x106 cells/L, Po0.001). CMV reactivation was associated with the presence of aGvHD grade II-IV (22/52 vs. 22/89, P ¼ 0.031). In variance to CMV HHV6 reactivation events were rather more frequent early post-HSCT than at later times (15% vs. 6%, P ¼ 0.096). Skin rash was frequent in HHV6-positive patients but encephalitis was the main concern. Out of 12 encephalitis cases, 6 were HHV6-positive as compared to 22 positive cases out of 133 patients lacking encephalitis (P ¼ 0.015). EBV was seen at a similar frequency early and later post-HSCT and resulted in a higher percentage of CD4 þ CD57 þ cells in the blood (3.00 þ / 0.58% vs. 1.42 þ / 0.27%, P ¼ 0.033; 56.045x106 þ / 28.95x10^6 cells/L vs. 8.89x106 þ / 2.39x106 cells/L, P ¼ 0.005). EBV reactivation was seen more frequently in patients with aGvHD (15/52 vs. 13/75, P ¼ 0.044) and in those with encephalitis (6/12 vs. 21/130, P ¼ 0.004) or lymphoproliferative syndrome (7/16 vs. 19/130, P ¼ 0.004). Reactivation of polyoma viruses differed in terms of BK (earlier, median 41 days post-HSCT) and JC (later, median 80 days postHSCT). Conclusion: CMV reactivation seen rather later post-transplant and a lack of apparent clinical symptoms may be related to the effect of prophylactic and pre-emptive ganciclovir treatment. The prevalence of CD8 þ CD57 þ lymphocytes in the blood of CMV-positive cases documented the impact of the infection on the immune system. An increase in the proportions and numbers of CD4 þ CD57 þ lymphocytes is a rather novel observation and demonstrates the profound effect of the EBV virus on the lymph nodes.
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Reactivation of either HHV6 or EBV constituted the main risk factor of encephalitis. EBV is associated with lymphoproliferative syndrome in spite of the fact that EBV DNA positivity is usually pre-emptively treated with anti-CD20 antibodies. Overall survival is only marginally affected by reactivation of Herpes viruses, likely due to the pre-emptive treatment followed according to routine laboratory blood work, which includes viral surveillance. Supported by INNOMED/I/1/NCBR/2014 CellsTherapy NCBiR grant. Disclosure of Interest: None declared. P164 Incidence and outcome of infections after unrelated cord blood transplantation in patients with hematological malignancies: a long-term single center experience E. Scalzulli1,*, W. Barberi1, C. Girmenia1, G. Gentile1, G. F. Torelli1, D. Ballaro`1, A. M. Testi1, M. L. Moleti1, L. Lombardi1, M. Campanelli1, R. Foa`1, A. P. Iori1 1 Hematology, Policlinico Umberto I, Sapienza University Rome, Rome, Italy Introduction: Unrelated cord blood (UCB) has become an alternative stem cell source for patients with hematological malignancies requiring allogeneic transplantation and lacking a HLA-matched donor. Infections after an UCB transplantation (UCBT) represent a leading cause of morbidity and mortality. The aim of this study was to evaluate the incidence, risk factors and outcome of infections occurring after an UCBT. Materials (or patients) and methods: We performed a retrospective study in 75 patients who received a UCBT at our center between July 1995 and July 2013: there were 51 children (median age 9 years, range 1-17) and 24 adults (median age 28 years, range 18-60). Patients were affected by high risk hematological malignancies: 22 had acute myeloid leukemia, 47 acute lymphoblastic leukemia and 6 chronic myeloid leukemia. All patients received a myeloablative conditioning regimen, in vivo T-cell depletion with antilymphocyte serum, and cyclosporine plus corticosteroid as GVHD prophylaxis. Fluconazole, acyclovir and ciprofloxacin were administered as prophylaxis for fungal, viral and bacterial infections, respectively. All patients underwent CMV antigenemia or DNAemia monitoring, while EBV DNAemia monitoring was performed since 2002 for the last 40 patients. Results: The incidences of bacteremia, proven/probable invasive fungal diseases (IFD), CMV infection, EBV infection and herpes zoster disease were 88%, 21%, 61%, 20% and 17% of transplants, respectively. Out of 80 bacterial isolates, 44%
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were Gram negative and 56% Gram positive. IFDs were caused by filamentous fungi in 62% of cases. Gastrointestinal CMV disease was documented in 4% of patients with virus infection. Out of 8 patients with EBV infection, 3 developed a posttransplant lymphoproliferative disorder (PTLD) (fatal in 2 cases). The distribution of the infections in the early (days 140 from transplant), late (days 41- 100), very late (days 101 365) phases and after day 365 are detailed in Figure 1. Bacterial, fungal and CMV infections were documented mainly during the early engraftment phase, while EBV and herpes zoster infections were generally documented after day 100. The duration of pre-engraftment neutropenia did not correlate significantly with an increased infectious risk. Grade II-IV acute GVHD was significantly predictive of the development of an IFD (HR 0.28; IC: 0.09 – 0.87; P ¼ 0.029). At a median follow-up of 10 years, 30 of 75 (40%) patients are still alive. Infections represented the primary cause of death in 24% of patients. The attributable mortality rate for bacterial and fungal infections accounted for 4.5% and 31%, respectively. Conclusion: Our study confirms the high rate of infectious complications occurring after an UCBT. In agreement with other experiences, the early engraftment phase represented the period at higher risk of bacterial, fungal and CMV infections. After this time period, a moderate risk of developing IFDs persists up to 6 months, especially in patients with GVHD. Infections by viruses other than CMV have been more frequently documented after the very late phase posttransplant. These epidemiological findings should be considered for the definition of tailored anti-fungal prophylaxis and virological pre-emptive strategies. Disclosure of Interest: None declared. P165 Micafungin antifungal primary prophylaxis in 40 patients with Acute Myeloid Leukemia candidate to allogeneic tranplantation F. Lorentino1,*, R. Greco1, E. Xue1, A. Forcina1, F. Giglio1, M. Morelli1, C. Messina1, M. G. Carrabba1, M. Bernardi1, J. Peccatori1, S. Paolo1, C. Corti1, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milano, Italy Introduction: Primary prevention of invasive fungal infections (IFI) is a clinically relevant goal in patients with Acute Myeloid Leukemia candidate to allogeneic transplantation. In such population, the greatest proportion of IFI occurs during aplasia following induction chemotherapy (Pagano et al., 2010). Micafungin (MICA) may be an effective antifungal agent since
it has activity against all major Candida spp. and it demonstrates in vitro and in vivo activity against Aspergillus spp. Aim: to investigate the incidence of IFI occurring in pts with newly diagnosed AML undergoing induction chemotherapy (IC) receiving MICA as antifungal primary prophylaxis (APP). Materials (or patients) and methods: Since July 2011 to October 2014, we prospectively treated 40 consecutive pts with newly diagnosed AML: 20/40 pts were treated with ‘‘3 þ 7’’ IC, 12/40 pts with ‘‘FLAI’’ IC, 8/40 pts with ‘‘ICE’’ IC. MICA was started 1 day following the end of IC at the daily dose of 50 mg i.v. MICA was continued until neutrophil engraftment (PMN40.5x109/L for 3 consecutive days). Galactomannan was measured once a week using ELISA test and results were considered positive if Z0.5 in serum. IFI was defined as possible, proven or probable according to 2008 EORTC/MSG definitions. Results: None of our patients discontinued the treatment for drug-related adverse events. 30/40 pts concluded MICA treatment at neutrophil engraftment without signs of IFI. 10/ 40 pts (25%) switched to an alternative antifungal therapy as empirical treatment. Of these, possible (n ¼ 4), probable (n ¼ 3) and proven (n ¼ 1) IFI were diagnosed. Probable IFIs were pulmonary infections, 1 candidiasis and 2 invasive aspergillosis were diagnosed and treated with liposomal B amphotericine or voriconazole. The only proven IFI was a disseminated fusariosis treated with a combination of liposomal B amphotericine and voriconazole. All pts were neutropenic at the onset of IFI with a median duration of neutropenia of 31 days (14–73). All of them resolved the infection at neutrophil recovery after IC. Conclusion: In this 3-year study in 40 patients breakthrough proven or probable IFI occurred in 10%. This result is comparable with randomized clinical trials and several observational studies focused on posaconazole prophylaxis (graded A-I by the European Conference on Infections in Leukemia - ECIL- for antifungal primary prophylaxis during induction chemotherapy) which found breakthrough IFI ranging from 0 to 17%. Micafungin primary antifungal prophylaxis is safe and effective during induction chemotherapy in patients with AML candidate to allogeneic transplantation. Disclosure of Interest: None declared. P166 Incidence and course of clostridium difficile infections after autologous and allogeneic blood stem cell transplantation N. Nagorny1, C. MacKenzie2, E. Rachlis1, M. Kondakci1, A. Dienst1, D. Lopez y Niedenhoff1, T. Schroeder1, R. Fenk1, K. Pfeffer2, R. Haas1, G. Kobbe1,* 1 Department of Hematology, Oncology and Clinical Immunology, 2Department of Medical Microbiology and Hygiene, University Hospital Duesseldorf, Duesseldorf, Germany Introduction: Immunosuppressed individuals are at risk of opportunistic and nosocomial infections. Cancer patients who receive autologous or allogeneic blood stem cell transplantation frequently suffer from infections that may increase morbidity and mortality. The role of intestinal infection with Clostridium difficile (CDI) in this high-risk group has so far not been properly defined. Materials (or patients) and methods: We retrospectively identified all patients who had received a blood stem cell transplant and developed symptomatic CDI (diarrhea and positive test for C.-diff. toxin) after transplantation from a database of the Department of Medical Microbiology and Hygiene. Clinical data was retrospectively collected from the patients’ charts to describe the clinical cause of infection and identify prognostic factors. Results: Between 1/2006 and 10/2014 a total number of 4797 stool specimens were analyzed for C.-diff. toxin from 751 patients with diarrhea who had previously received an
autologous or allogeneic blood stem cell transplant. Sixty-five individuals (MM 25, AML 23, Lymphoma 9, MDS/MPS 7 and 1 germ cell tumor) tested positive, reflecting an infection rate of 8.7% among all transplant recipients with diarrhea during that time period (4,01 % for all autologous and 8,16 % for all allogeneic transplants). Five patients (8%) had a history of CDI before Tx. The detection rate was 6% in 2006, 8.3% in 2007, 10% in 2008, 6.7% in 2009, 5% in 2010, 4.2% in 2011, 4.1% in 2012 and 6.43 % in 2013. Median time from transplant to a positive test was 112 days (median, range 1-5501) in autologous and 155 (range 3-2831, P ¼ ns) in allogeneic recipients. When considering only the 1st year after transplant, infection occurred earlier after autoTx (median day þ 37, range 1-331) than after alloTx (median day þ 85, range 3-364, P ¼ 0.014) and in MM patients (median day þ 19, range 1-331) than in Lymphoma patients (median day þ 93, range 59-159, P ¼ 0.03) which may be the result of metronidazole prophylaxis in allografted patients until day þ 20 after transplant and different previous antibiotic exposure. Of 29 autoTx patients with CDI 21(72%) relapsed with their primary malignancy (11 before, 9 after CDI). Of 36 alloTx patients 18(50%) relapsed (9 before, 9 after CDI). As therapy for CDI 40 patients (61.5 %) received metronidazole, 12 (18.5%) oral vancomycin, 2 (3.1%) fidaxomycin, 7 (10.8%) unknown and 3 (4.6%) no therapy. No patient died of CDI. Sixteen patients (24.6%) had at least one CDI recurrence (12 after metronidazole, 1 after vancomycin, 1 after Fidoxomycin and 2 after unknown therapy). At day þ 30 after the 1st positive CDI test 93.1% and 94.5% of patients were alive after autoTx and alloTx, respectively. While overall mortality in CDI patients was similar after autoTX (55%, median FU 202 days, 4-2361) and alloTx (53%, median FU 365 days, 13223), causes of death differed (TRM 7% and DRM 48% after autoTX vs TRM 17%, DRM 36% after alloTx). Conclusion: Between 2006 and 2013 the rate of CDI among patients with diarrhea after blood stem cell transplantation varied from 4.1 to 10% and was related to diagnosis and type of transplant/prophylaxis. CDI recurrence occured frequently, especially after therapy with metronidazole. While CDI itself was not fatal, patients who developed CDI after blood stem cell transplantation resembled a high risk population with high treatment and disease related mortality. Disclosure of Interest: None declared. P167 False Positive Clostridium Difficile Toxin Testing From Stool Sample by Immunochromatographic Method in a Pediatric Patient With Hemorrhagic Colitis Clinical Findings After Bone Marrow Transplantation I. Eker1,*, O. Gu¨rsel1, O. Babacan1, M. Topdemir2, A. E. Ku¨rekc¸i1 1 Pediatric Hematology, 2Pediatrics, Gulhane Military Medical Faculty, Ankara, Turkey Introduction: Clostridium difficile infection (CDI) is one of the prominent causes of colitis associated with antibiotics in children. Patients, who are required the useage of long term and broad spectrum antibiotics, such as hematopoietic stem cell transplantation (HSCT) conditions, are particularly at risk. Here a pediatric HSCT recipient, who had hemorrhagic colitis clinical findings and false-positive Clostridium difficile toxin test in stool sample by immunochromatographic method is presented and diagnostic approach of pediatric HSCT patients in the suspicion of CDI is discussed. Materials (or patients) and methods: A 6 year old boy had undergone HSCT with the diagnosis of relapsed ALL in our HSCT Center. Full engraftment and donor type chimerism was achieved. But recent after the engraftments, grade IV graftversus-host disease and thrombotic microangiopathy developed. Clinical response was achieved with plasma exchange and immunosuppressive therapy. But during this period, the usage of broad spectrum antibiotics for a very long time was needed. He developed clinical findings presenting like haemorrhagic colitis with fever, abdominal pain, leukocytosis and bloody stools. CDI was thought and C.difficile toxin B was
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detected positive in stool sample with immunochromatographic method. Oral vancomycin therapy was started, but there weren’t any clinical improvement. Results: Although C.difficile toxin B was detected positive again with the same method in a different stool sample, the stool culture was negative. He had undergone endoscopy and colonoscopy, when he was clinically stable. Pathological and microbiological evaluation of biopsy material was normal. Because there were no evidence of CDI, vancomycin therapy was discontinued. The localization of the gastrointestinal bleeding (GIB) couldn’t be identified with nuclear medicine scan and transcatheter arteriography also, but there were seen angiodysplastic lakes in branchs of superior mesenteric artery. With the suspicion of these lakes may be the origin of GIB, superselective embolization was performed and GIB episodes were resolved completely without any complication. Occult GIB was also became negative after ten days of the embolization and he was discharged on the 130th day of HSCT. Conclusion: The gold standard for the diagnosis of CDI is the determination of toxins in cell culture. Tests made with the aim of examining the CDI quickly, detects Clostridium difficile toxin (CDT) or the enzyme glutamate dehydrogenase (GDH) at fecal specimens. Although the sensitivity of these tests is high, specificity is low. Rapid CDT test by immunochromatographic method is based on the principle of binding of the agent toxin to the latex particles coated with monoclonal antibodies. In the literature it is reported that, the positive predictive value of both CDT and GDH positivity in stool sample is 100% and the negative predictive value of both CDT and GDH negativity in stool sample is 95%. Therefore in pediatric HSCT patients, having Clostridium difficile colitis signs and symptoms, the usage of this two step algorithmic approach is recommended. Using of CDT and GDH detection tests together in stool samples for this two step algorithmic diagnostic approach, until the result of the gold standard tests, will reduce the false positivity cases as in our case. Disclosure of Interest: None declared. P168 Successful treatment of sino-orbital mucormycosis after allogeneic transplantation: the role of urgent surgical approach I. Batsis1,*, G. Venetis2, S. Papadimitriou1, Z. Bousiou1, N. Pasteli3, V. Constantinou1, D. Mallouri1,1, I. Sakellari1, A. Anagnostopoulos1 1 Haematology department - BMT unit, 2maxillofacial surgery, 3 Pathology, George Papanicolaou Hospital, Thessaloniki, Greece Introduction: Mucormycosis after allogeneic transplantation is a fulminant, life-threatening infection that has a high mortality rate without prompt combined therapy. Unrelated hematopoietic stem cell transplantation (HSCT), which requires long term immunosuppression for treatment of GVHD, predisposes to fatal infections. Eradication is challenging and needs combination of systemic antifungal treatment and surgical intervention. Materials (or patients) and methods: We present the clinical outcome of a 27 year old male who underwent HSCT from an unrelated donor for chronic myelogenous leukemia in second chronic phase in 29/1/2013 after TBI-Cy plus ATG 5mg/kg. He was treated with high doses of corticosteroids for acute GVHD (grade III), received two preemptive courses of Rituximab for EBV reactivation and 2 further lines of immunosuppression for chronic extensive GVHD. Six months post HSCT he was successfully treated for three months with voriconazole for possible aspergillus lung infection. Two months later he presented with unilateral blurred vision and he was started on Amphotericin B (5mg/kg) with the suspicion of mucormycosis. Results: Imaging study with MRI of the facial skull revealed involvement of all sinuses. After 5 days of Amphotericin B with a stable clinical status, he appeared with rapid progression including mass swelling of the right side of the face, proptosis
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of the orbit and visual loss. MRI scan revealed that the infection was expanded to the orbit and adjacent tissues. An urgent consultation by neurosurgeon and maxillofacial surgeon posed the risk for imminence brain expansion. The patient was then immediately admitted to surgery and sustained exenteration of the right orbit and extensive surgical debridement of necrotic tissues. Debridement was repeated twice and histology confirmed the diagnosis of mucormycosis. He continued receiving Amphotericin B three months after the surgery and at the same time corticosteroid dose was tapered as possible. Six months post-surgery and after resolution of the infection, he received reconstructive surgery with no further complications. Conclusion: We present a case of sinusoidal mucormycosis with a rapid spread to surrounding tissues and all the steps guided by ECIL recommendations that led to a successful management. Eradication of the infection includes surgical debridement, administration of systemic antifungals for sufficient time and an effort to reverse the poor immunological status in the deeply immunocompromised patient. Disclosure of Interest: None declared. P169 Use of micafungin for antifungal prophylaxis in patients undergoing allogenic hematopoietic stem cell transplantation (allo-HSCT) in Spain (GETH-MIC) C. Lo´pez-Sa´nchez1, I. Ruiz-Camps1,*, V. Go´mez2, J. Lo´pezJime´nez3, D. Serrano4, V. Rubio5, C. Solano6, D. Valca´rcel1, L. Va´zquez7 on behalf of Grupo Espan˜ol de Trasplante Hematopoye´tico (GETH) 1 Hospital Universitario Vall d’Hebron, Barcelona, 2Hospital Universitario de La Princesa, 3Hospital Universitario Ramo´n y Cajal, 4Hospital Universitario Gregorio Maran˜o´n, Madrid, 5Hospital Jerez de la Frontera, Ca´diz, 6Hospital Clı´nico Universersitario de Valencia, Valencia, 7Hospital Universitario de Salamanca, Salamanca, Spain Introduction: Echinocandins are highly effective antifungal agents against Candida and Aspergillus spp. Micafungin provides compared to other echinocandins better activity against some Candida spp. (specially C. glabrata) and also Aspergillus spp. All guidelines focused on antifungal prophylaxis recommend its use during neutropenic phase in hematopoietic stem cell transplant (HSCT) recipients. Moreover, its low profile of side effects and drug interactions makes micafungin a good alternative in those patients who need concomitant treatments, present hepatic insufficiency and also in those that do not tolerate oral drug administration. The aim of this study was to describe the experience with micafungin as primary prophylaxis during the neutropenic phase in patients undergoing allo-HSCT in a cohort of Transplant Spanish centres, and to evaluate it’s efficacy and tolerability in this population. Materials (or patients) and methods: retrospective multicentre observational study including all consecutive adult patients admitted for allo-HSCT to haematological units belonging to participating centres of the Grupo Espan˜ol de Trasplante Hematopoye´tico (GETH), from January 2010 to December 2013, which received micafungin as prophylactic treatment during the neutropenic phase. Patients that received less than 5 days of micafungin were excluded from the analysis. Failure of prophylaxis was considered in those patients who developed breakthrough invasive fungal infection (IFI) during prophylaxis with micafungin or early after. Results: Data from 240 patients from 13 HSCT units belonging to the GETH group were collected. Eighty-one patients were excuded from the analysis for different reasons. Finally, 159 patients were included for the analysis. Ninety-four (59%) were men. Mean age was 48 (± 13) years. Underlying disease was leukaemia in 82 (51.6%) patients, lymphoma in 34 (21.4%), myelodysplastic syndromes in 26 (16.4%), multiple myeloma in 8 (5%) and other diseases in 9 (5.7%) patients. One hundred and fifty-one (94.9%) patients had been submitted to
peripheral blood SCT, 6 (3.8%) to bone marrow SCT and 2 (1.3%) to umbilical cord SCT. Mean of says of prophylaxis with micafungin was 19 (± 9) days. Most patients (95.6%) received doses of 50mg per day, while the remaining ones received 100mg per day. During follow-up 7 (4.4%) patients developed breakthrough IFI, 1 proven IFI (Fusarium spp. in skin biopsy) and 6 probable IFI (positive galactomannan with suggestive radiological images), and one patient presented serious drug interactions that obliged micafungin cessation. The median of days of treatment with micafungin in these patients was 14 (10-25) days at a dose of 50mg per day. Four of these patients died: two with multiorganic failure, 1 patient because of venoocclusive disease and 1 because staphylococcal septic shock. Six other patients died because of different complications related to the underlying disease and it’s treatment. Finally, prophylaxis with micafungin was considered effective in 151 (94.9%) of the patients. Conclusion: According to our experience, micafungin is an appropriate alternative for antifungal prophylaxis in patients undergoing an allo-HSCT, because it’s efficacy, it’s low profile of drug interaction and side-effects. This study has been partially funded by Astellas. Disclosure of Interest: C. Lo´pez-Sa´nchez Funding from: Astellas, I. Ruiz-Camps Funding from: Astellas, V. Go´mez Funding from: Astellas, J. Lo´pez-Jime´nez Funding from: Astellas, D. Serrano Funding from: Astellas, V. Rubio Funding from: Astellas, C. Solano Funding from: Astellas, D. Valca´rcel Funding from: Astellas, L. Va´zquez Funding from: Astellas. P170 Fluoroquinolone Prophylaxis Reduces the Incidence of Severe Viral Hemorrhagic Cystitis in Cord Blood plus Third-Party Donor Allogeneic Transplant Recipients I. Sa´nchez-Ortega1,*, G. Bautista2, M. Kwon3, A. Va´zquez2, R. Fores2, A. De Laiglesia2, J. Gayoso3, P. Balsalobre3, D. Serrano3, M. Arnan1, B. Patin˜o1, A. Sureda1, J. L. Dı´ez-Martı´n3, R. Cabrera2, R. F. Duarte1 1 Catalan Institute of Oncology, Hospital Duran i Reynals, Barcelona, 2Hospital Universitario Puerta de Hierro, 3Hospital Universitario Gregorio Maran˜o´n, Madrid, Spain Introduction: Virus-associated hemorrhagic cystitis (HC) is a potential serious complication of allogeneic hematopoietic cell transplants (HCT), most commonly after myeloablative conditioning regimens and in cord blood HCT recipients. Prophylaxis with fluoroquinolones has been shown to reduce BK viral load and the incidence of severe (grades III-IV) HC in HLA matched sibling and unrelated volunteer donor allogeneic HCT. Here, we present our experience on the effectiveness and safety of fluoroquinolone prophylaxis of severe HC in recipients of cord blood supported with thirdparty donor hematopoietic cells (CB-TPD) HCT. Materials (or patients) and methods: 148 consecutive CBTPD HCT recipients from 03/1999 to 07/2014 in three Spanish centers were included in this study: 79 AML/MDS, 48 ALL, 11 CLPD, 8 CMPD and 2 others, 89 men (60%), median age 37 [16 - 64]. Two consecutive cohorts are included, with 28 patients receiving 60 days of sustained post-transplant fluoroquinolone prophylaxis (16 cases with 500 mg/12h oral ciprofloxacin and 12 cases with 500 mg/day levofloxacin) regardless of the initiation of IV broad-spectrum antibiotics, and 120 controls receiving no anti-HC prophylaxis. There were no differences between cohorts in patient demographics and transplant characteristics. Severe HC episodes included grades III (macroscopic hematuria with blood clots) and IV (massive macroscopic hematuria requiring instrumentation of the urinary tract or causing urinary retention) HC [Leung et al, BBMT 2005]. Results: Seventeen patients in the non-prophylaxis group developed severe HC (8 grade III and 9 grade IV) a median of 30 days after HCT, compared with no cases in the fluoroquinolone prophylaxis group (14.2% vs 0, P ¼ 0.043). Severe HC occurred after neutrophil engraftment in 88% of the
cases (15/17). Fifteen cases were related to BK virus, one case to adenovirus and one additional case had negative viruria. Severe HC episodes increased the hospital stay during which patients required supportive therapy (hyperhydratation, forced diuresis), blood and platelets transfusions and up to four lines of treatment including intravesical (n ¼ 3) and IV cidofovir (n ¼ 7), intravesical hialuronic acid (n ¼ 11), mesenchymal stem cells (n ¼ 2), cystoscopic cauterization (n ¼ 2), arterial embolization (n ¼ 1), and immunusoppression switch to sirolimus (n ¼ 2). Eleven patients achieved complete resolution of HC and survived the episode, while 6 died in refractoriness or partial response (significant improvement of symptoms of HC but persistence of gross hematuria). Severe HC-related mortality was 35.3% (6/17), and contributed a 5% to the non-relapse mortality of the cohort without prophylaxis (6/120). In addition to the absence of severe HC, all 28 cases in the fluoroquinolone cohort completed prophylaxis as planned and no fluoroquinolone-related adverse events were reported. Conclusion: Our results suggest that fluoroquinolone prophylaxis may reduce the incidence of severe HC in high-risk cord blood transplant recipients, the morbidity and resource use associated with it and improve patients’ outcome. They also confirm that the maintenance of fluoroquinolone prophylaxis during 60 days after transplantation regardless of IV broadspectrum antibiotics is safe and well tolerated. Disclosure of Interest: None declared. P171 A study of adherence to a vaccination schedule following adult allogenic haematopoietic stem cell transplants in UK transplant centre J. Meiring1,*, T. De Silva1, J. Snowden2 1 Department of Infection and Tropical Medicine, 2Dept of Haematology, Sheffield Teaching Hospitals, Sheffield, United Kingdom Introduction: In the past fifteen years significant advances have been made in the area of preventing infection in haemopoeitic stem cell transplant (HSCT) recipients. Despite this, infection is reported as the primary cause of death in 8% of autologous HSCT patients and 17%420% of allogenic HSCT patients. After HSCT, patients remain immunocompromised and are therefore susceptible to a range of infections. Patients lose vacine-induced immunity they may once have had through childhood vaccination programmes. Therefore in order to restore immunocompetence; exposure to these antigens via a standardised vaccination programme is essential. We assessed adherence to a local vaccination policy following allogeneic HSCT administered via primary care physicians. Materials (or patients) and methods: Between 2008 to 2010 we identified 65 patients that received a HSCT. 30 individuals survived for a period where they should have completed the full vaccination schedule. Primary care physicians were contacted and a full vaccination history attained. Where information was missing we consulted the centralised haematology late effects clinic letters. We also used these to document why vaccines were delayed or not administered. Results: Sufficient data was obtained for 25 patients. The lack of data for 5 patients was due to changes in GP practices with data not following patients and some practices being unwilling to release data to us. Of the 25 patients we did study, only six (24%) patients fully completed their vaccine schedule. Ten had documented reasons for failure, although graft versus host disease (GVHD), disease relapse and donor lymphocyte infusion were the main documented reasons. This left 9 patients who did not complete the schedule, with no documented reason. Looking sequentially through the data, those vaccines given at 12 months like the first DTP, pneumococcal polysaccharide and haemophilus vaccines had adherence of around 70%.
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However, vaccines given at 2 years were considerably lower with only 28% adherence, evident in the third DTP, the second pneumococcal and MMR vaccine. Seasonal influenza vaccine uptake was better with 72% receiving post-HSCT. Conclusion: There were limitations to this study, namely difficulty in accesing accurate data from 30 different sources. This limitation comes directly from the problem with the delivery of de-centralised care. Despite this the data collected showed that the implementation of the vaccine schedule is not effective. The numbers of patients recieving protective levels of vaccine were very low, , partly due to physician perception of GVHD being a contraindication to all vaccines. In light of this a centralised vaccine service that could run alongside a late effects clinic has been proposed with the input of infectious disease specialists. Also, updates to the current schedule have been advised bringing them in line with current EBMT and IDSA guidance. Specifically, using the pneumococal conjugate vaccine, starting schedules at 3 months and for the administration of non-live vaccines dismissing GVHD as a contra-indication to vaccine administration. We hope that these significant developments will improve uptake and ultimately the long term health of transplant recipients. Disclosure of Interest: None declared. P172 Viral Infection and Reactivation in Alemtuzumab Reduced Intensity Stem Cell Transplantation in lymphoid malignancies: a study from the Irish National Adult Stem Cell Transplant (NASCT) Unit J. M. Nolan1,2,*, C. Delaney1, G. Lee1, E. Higgins1, M. Nı´ Chonghaile1, E. Conneally1,2, C. L. Bacon1,2, P. V. Browne1,2, B. Crowley3, E. Vandenberghe1,2 1 Haematology, St James’ Hospital, Dublin 8, 2Haematology, University of Dublin, Trinity College, Dublin 2, 3Microbiology, St James’ Hospital, Dublin 8, Ireland Introduction: Alemtuzumab-based reduced intensity stem cell transplantation (A-RIC-SCT) was adopted by the Irish NASCT unit in 2003 for Chronic lymphocytic leukaemia (CLL), Non-Hodgkins lymphoma(NHL) and Hodgkins Lymphoma (HL). Viral infection-associated morbidity is a concern with this conditioning and we reviewed the risk factors, incidence and morbidity of CMV, EBV and respiratory viral (RV) infections following A-RIC-SCT. Materials (or patients) and methods: Lymphoid patients conditioned with A-RIC-SCT from 2006-2013 considered "at viral risk" were identified and the incidence, risk factors for infections and infection outcomes were reviewed. "At risk" patients included: donor/recipient CMV and/or EBV exposure and were assessed twice weekly by PCR from engraftment to 100 days or cessation of steroid immunosuppression. RVs including influenza, parainfluenza, adenovirus and RSV were detected by PCR on appropriate samples in patients with respiratory symptoms. Risk factors assessed were age, diagnosis, donor type, alemtuzumab dose, disease status at SCT and steroid use for graft versus host disease (GvH) using SPSS (Statistical Package for Social Sciences, version 22) software. Results: Fifty-eight patients (37M:21F) underwent transplantation for CLL (n ¼ 16), NHL (n ¼ 30) and HL (n ¼ 12) at a median of 47.5 years (range 18-60). Donors were HLA-matched siblings (41) 69% and unrelated volunteers (17)31% of whom 2 were mismatched. Alemtuzumab doses (in mg) were: 30(n ¼ 13,22%), 50 (n ¼ 10,17%) and 100(n ¼ 35,60%). Disease status at SCT was: CR(n ¼ 17) 29%, PR(n ¼ 35) 61% and stable disease (n ¼ 6) 10%. Steroid-treated GvH occurred in (24) 41% of patients. 21 patients (36%) were at CMV-risk, of whom 14 (67%) reactivated, prolonging SCT hospital stay in 8 patients for a median of 14 (range 3-21) days. CMV disease affected 2 patients (colitis, pneumonitis) who survived. 56 (91%) patients
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were EBV þ ve and 16(30%) reactivated, 3 required anti-CD20 therapy and one death occurred. RV detected were: adenovirus (13) 22%, RSV/Parainfluenza (15) 26% resulting in 2 death. None of the risk factors assessed were statistically significant. TRM deaths occurred in 10 (17%) patients as follows: bacterial/fungal infection (4), parainfluenza (1), RSV (1), PTLD (1), GvH (2), liver failure (1) with 1 infective death 4100 days. 12 (20%) patients died of disease, giving an overall survival of 62.1% (36 patients) with a median follow-up of 45 (range 11-109) months. Conclusion: CMV reactivation occurred in 2/3 of "at risk" patients causing significantly increased hospital stays, but no deaths. EBV reactivation occurred in 1/3 of "at risk" patients but resulted in 1 death. RV present a challenge for all SCT patients and resulted in 2 deaths in this series. A-RIC-SCT was not associated with an excess viral mortality rate in this series, but strategies to decrease risk include pre-emptive EBV viral reactivation treatment, reduction of alemtuzumab dose (not statistically significant in this small series) and earlier diagnosis and treatment of RV. Disclosure of Interest: None declared. P173 Incidence and outcome of infections in pediatric stem cell transplant centers: comparison with oncohematology patients in nationwide study J. Styczynski1,*, K. Czyzewski1, M. Wysocki1, O. GryniewiczKwiatkowska2, A. Kolodziejczyk-Gietka3, B. Dembowska2, D. Perek2, M. Salamonowicz3, L. Hutnik3, M. Matysiak3, A. Zaucha-Prazmo4, J. Kowalczyk4, L. Che"mecka-Wiktorczyk5, W. Balwierz5, K. Siewiera6, J. Fraczkiewicz6, K. Kalwak6, A. Chybicka6, R. Tomaszewska7, T. Szczepanski7, O. Zajac-Spychala8, J. Wachowiak8, N. Irga-Jaworska9, E. Drozynska9, M. Plonowski10, M. Krawczuk-Rybak10, T. Ociepa11, T. Urasinski11, F. Pierlejewski12, W. Mlynarski12, Z. Gamrot13, M. Woszczyk13, J. Gozdzik5, Z. Malas14, W. Badowska14, A. Urbanek-Dadela15, G. Karolczyk15, W. Stolpa16, G. Sobol16, L. Gil8 on behalf of Polish Society of Pediatric Hematology and Oncology 1 Department of Pediatric Hematology and Oncology, Collegium Medicum, University Hospital 1, Nicolaus Copernicus University, Bydgoszcz, 2Children’s Memorial Health Institute, 3Medical University, Warsaw, 4Medical University, Lublin, 5Jagiellonian University, Krakow, 6Medical University, Wroclaw, 7Medical University, Zabrze, 8Medical University, Poznan, 9Medical University, Gdansk, 10Medical University, Bialystok, 11Medical University, Szczecin, 12Medical University, Lodz, 13Pediatric Center, Chorzow, 14Children Hospital, Olsztyn, 15Children Hospital, Kielce, 16 Medical University, Katowice, Poland Introduction: Infections are the main cause of morbidity and mortality in children undergoing hematopoietic stem cell transplantation (HSCT) or treated for cancer in pediatric hematology and oncology (PHO) centers. The aim of this nationwide study was the analysis of incidence and outcome of bacterial, fungal and viral infections in pediatric HSCT centers, in comparison to PHO centers over a period of 24 months (iPhot-13 project). Materials (or patients) and methods: During analyzed period a total number of 308 HSCTs (232 allo, 76 auto; including 67 ALL, 47 AML, 28 NHL/HD, 60 solid tumors, 39 PID, 38 BMF, 21 other) were performed, and 1768 children were newlydiagnosed for malignancy (430 ALL, 81 AML, 110 HD, 108 NHL, 1012 solid tumors, 27 other) in participating centers. Following episodes were reported: microbiologically documented bacterial infections; proven/probable/possible IFI; latent and sporadic viral infections. Results: The overall incidence of bacterial, fungal and viral infections in HSCT patients was 33.9%, 22.8%, and 38.3%, respectively. The hazard risk of any infection was higher in comparison to PHO patients (HR ¼ 2.7, 95%CI-2.3-3.2, Po0.0001), especially during the first 30 days of therapy (after transplant or cancer diagnosis, respectively). The incidence
was higher in HSCT patients for bacterial (33.9% vs 26.3%, HR ¼ 1.4, P ¼ 0.0046), all fungal (22.8% vs 7.9%, HR ¼ 3.5, Po0.0001), proven/probable IFI (12.3% vs 2.9%, HR ¼ 4.6, Po0.0001), and viral infections (38.3% vs 3.5%, HR ¼ 15.7, Po0.0001). The incidence of IFI in ALL and AML both in HSCT and PHO setting was very high (ALL: 31.3% vs 13.0%, AML: 44.7% vs 43.2%). The incidence was higher in allo- than autoHSCT for bacterial (HR ¼ 1.4, P ¼ 0.2), fungal (HR ¼ 3.2, P ¼ 0.0028), and viral (HR ¼ 15.7, Po0.0001) infections. The rate of resistant bacteria was higher in HSCT patients both for G-negative (72.5% vs 59.2%), and G-positive (41.4% vs 20.5%) strains. The most often detected G-negative bacteria included: E.coli (25.9%), Enterobacter cloacae (22.9%), and Klebsiella pneumonia (16.8%); while Staphylococcus epidermidis (16.5%), Enterococcus faecium (29.6%), and Clostridium difficille (38.6%) among G-positive bacteria. Cumulative incidence of viral infections in allo-HSCT was: 28.0% for CMV, 18.5% for BKV, 15.5% for EBV, 9.5% for adenovirus, 2.6% for VZV, 0.9% for influenza, 0.9% for HHV6, and 0.3% for HBV. Survival rate from infection was lower than in PHO patients in bacterial (96.0% vs 98.2%), fungal (75.5% vs 94.6%), and each viral infection (except influenza). Conclusion: The incidence of infections in allo-HSCT patients is high, while in auto-HSCT is comparable to PHO patients. The rate of multi-drug resistant strains in HSCT patients is very high. The outcome of infections in allo-HSCT patients is still unsatisfactory. Intensified supportive anti-infectious therapy is one of the keys to success in transplant setting. Disclosure of Interest: None declared.
Early complications/late effects & quality of life I P174 Impact of non-transferrin-bound iron (NTBI) in comparison to serum ferritin as prognostic factor for outcome after allogeneic stem cell transplantation (SCT) A. Fritsch1,*, C. Langebrake1,2, C. Wolschke2, J. Kersten3, H. Rohde4, P. Nielsen5, N. Kro¨ger2 1 University Medical Centre Hamburg-Eppendorf, Pharmacy, 2 University Medical Centre Hamburg-Eppendorf, Stem Cell Transplantation, 3University Medical Centre Hamburg-Eppendorf, Department of Medical Biometry and Epidemiology, 4University Medical Centre Hamburg-Eppendorf, Institute of Medical Microbiology, 5University Medical Centre Hamburg-Eppendorf, Department of Biochemistry and Molecular Cell Biology, Hamburg, Germany Introduction: Iron overload (IO) prior to allogeneic SCT has shown to influence negatively outcome after allogeneic SCT, mainly because of a high non-relapse mortality. IO is usually measured by serum ferritin (SF), which however is not specific for IO. We hypothesized that increased risk of infections and tissue damage is due to reactive oxygen species and NTBI is a better marker to predict outcome rather than SF. Therefore, we performed a prospective observational trial evaluating the influence of NBTI in comparison to SF on the outcome of SCT patients, with regard to overall survival (OS), bacteraemia and invasive fungal infections (IFI). Materials (or patients) and methods: 95 consecutive patients who received allogeneic SCT for various different diseases at University Medical Centre Hamburg-Eppendorf between 09/2010 and 09/2011 were analysed. The median follow-up was 19.6 month. NTBI levels, transferrin saturation and SF were determined daily during the conditioning regimen and also on day þ 7 and þ 14 post-SCT. For further analyses, the area under the curve (AUC) was calculated up to the day of transplantation (NTBI-AUC d0) and up to day þ 14 (NTBI-AUC d14). After mobilising free iron with help of
nitrilotriacetic acid and centrifugal filtration, NTBI was measured by graphite furnace atomic absorption spectrometry. Results: Regarding OS the results indicate a nearly 2 fold increased risk of death (HR ¼ 1.729) for patients with a high NTBIAUC d0 compared to patients with a lower iron load, without reaching statistical significance (P ¼ 0.095). Even in multivariate analysis comprising common prognostic factors, patients with a high NTBI-AUC d0 had a nearly 2 fold risk of dying (HR ¼ 1.887), while failing again statistical significance (P ¼ 0.074). Regarding the rate of infection, per one mmol*d/l NTBI-AUC d0 there was a 4% higher risk of bacteraemia (HR ¼ 1.042, P ¼ 0.009) and a 7% higher risk of IFI (HR ¼ 1.070, Po0.001). The NTBI-AUC d14 appears to have none prognostic value. Furthermore, baseline SF had no significant influence on the OS (1000 mg/l cut-off: HR ¼ 1.554, P ¼ 0.283; 2500mg/l cut-off: HR ¼ 1.486, P ¼ 0.389) or bacteraemia (per 1000mg/l: HR ¼ 0.943, P ¼ 0.630), but on IFI (per 1000mg/l: HR ¼ 1.255, Po0.001). It was shown, that only 25% of the patients presented with positive NTBI values prior to their conditioning regimen. However, the reduced intensity regimen containing a preceded chemotherapy had the greatest iron releasing effect. Conclusion: This study provides further hints for the adverse effect of NTBI on OS and the rate of infections of SCT patients. It was shown that NTBI occurs during conditioning regimen even in patients without elevated SF or NTBI at baseline. Furthermore we could not determine a negative prognostic impact of baseline SF on OS or bacteraemia, bur on IFI. This results supports the hypothesis that SF is not an appropriate parameter for estimating iron release in SCT patients. The influence of SF shown in several previous studies is probably rather attributable to other effects than to iron toxicity. Additional studies with a more homogenous group of patients and a larger number of patients are necessary to evaluate the influence of NTBI on the outcome of SCT patients. It remains unclear whether iron is directly affecting the outcome or just reflecting the cytotoxicity as a surrogate parameter. Disclosure of Interest: None declared. P175 Graft-versus-host disease prophylaxis by early commencement of immunesuppression using methotrexate decreases pre-engraftment syndrome and accelerates engraftment after cord blood transplantation A. Iguchi1,*, M. Sugiyama1, Y. Terashita1, J. Ohshima1, T. Sato1, Y. Cho1, T. Ariga1 1 Pediatrics, HOKKAIDO UNIVERSITY, Sapporo, Japan Introduction: Graft failure is one of most serious problems in cord blood transplantation (CBT). The incidence of preengraftment syndrome (PES) after CBT is also high. PES is associated with transplantation-related complications such as acute graft-versus-host disease (GVHD) and respiratory failure. It has been suggested that the incidence of PES after CBT can be decreased by early commencement of immunosuppression after transplantation, but a randomized study has not been carried out. In our institute, GVHD prophylaxis after CBT was changed after January 2011. Between December 1997 and December 2010, 49 patients received prophylaxis against GVHD with cyclosporine (CsA) or tacrolimus (Tac) in combination with methylprednisolone from day þ 7 (mPSL group). Between January 2011 and November 2014, 18 patients received CsA or Tac with methotrexate on day þ 1 and þ 3 (MTX group). Thus, we analyzed engraftment and incidence of PES in CBT recipients in the Department of Pediatrics Hokkaido University Hospital. Materials (or patients) and methods: We reviewed medical records of 67 consecutive child CBT recipients between December 1997 and November 2014. Indications for SCT were hematological malignancies in 41 patients, solid tumor in 12, congenital immunodeficiency in 11, bone marrow failure in 2, and congenital metabolic diseases in one patient. A preparative conditioning regimen consisting of myeloaberative
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conditioning (MAC) was provided to 54 patients, reduced intensity conditioning (RIC) was used for 13 patients. Fortynine patients received prophylaxis against GVHD in mPSL group, and 18 patients in MTX group. PES was defined as noninfectious fever and skin rashes with or without evidence of fluid retention and/or respiratory distress occurring before neutrophil engraftment: an absolute neutrophil count of 40.5x10^9/l. Graft failure was defined as no donor hematopoiesis beyond day þ 28. Results: Engraftment was detected in 56 patients (83.6%). Although there was no significant difference between engraftment rate patients who received MAC and those who received RIC (45/54 (83.3%) and 11/13 (84.6%), respectively), engraftment rate in MTX group was significantly higher than that in mPSL group (18/18 (100%) and 38/49 (77.6%), respectively, P ¼ 0.029). Only four of the 11 patients with graft failure could be rescued by second transplantation. PES developed in 34 patients. The incidence of PES in patients receiving MAC was significantly higher than in the patients receiving RIC (P ¼ 0.043). Moreover, the incidence of PES in mPSL group was significantly higher than that in MTX group (P ¼ 0.029). Additional treatment of corticosteroids were required in 7 patients, but no patient died of PES. Conclusion: Although this study was a small scale analysis, the results showed that increase of engraftment rate and decrease in the incidence of early immune reactions such as PES could be achieved by early commencement of immunesuppression using methotrexate. Disclosure of Interest: None declared. P176 Elevated ferritin level at day þ 100 after allogeneic stem cell transplantation has a higher impact on overall survival and transplant related mortality than the pre-transplant ferritin level A. Casado1,*, M. Sanchez Escamilla1, L. Yan˜ez1, A. Insunza1, A. Bermu´dez1, E. Conde1 1 Hematology Department, Hospital Universitario Marques de Valdecilla, Santander, Spain Introduction: It is well known that iron overload is an important adverse prognostic factor for patients who undergo allogeneic stem cell transplantation (allo-SCT). Therefore, iron chelation before the procedure has been recommended in
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several guidelines. The aim of this study is to analyze the impact of a high ferritin level (Z1000 ng/ml) pretransplantation (Fe-PRE) and at day þ 100 (Fe-100) on overall survival (OS) and procedure related mortality (TRM) at one and two years after transplantation. Materials (or patients) and methods: We have evaluated retrospectively 518 patients who underwent an allo-SCT in our centre between January 2000 and December 2013. Ferritin levels are available pre-transplantation in 312 patients and at day þ 100 in 148 patients. No patient received iron chelation pre-transplantation or before day þ 100. Statistical analysis was performed with the SPSS v 15.0 software. Results: At day þ 100 post-transplantation, the ferritin level and the number of patients with a ferritin level Z1000 ng/ml were higher than before transplantation (2301 ng/ml vs 1437 ng/ml, Po0.001, and 75% vs 54%, Po0.001, respectively). Patients’ characteristics are shown in table 1. Compared with patients with a low ferritin level at day þ 100, patients with a ferritin level Z1000 ng/ml had delayed platelet engraftment (450 x 109/L 26 days vs 17 days, Po0.001), received more red cell transfusions (4 vs 2, Po0.001) and developed more extensive chronic GvHD (37.9% vs 10%, P ¼ 0.024). There were no significant differences in status at transplant, donor type, conditioning intensity, CD34 cell dose, neutrophil recovery or grade III-IV acute GvHD. With a median follow-up of 32 months, Fe-100 Z1000 ng/ml had a more negative impact on OS (54% vs 76%, P ¼ 0.009) than FePRE Z1000 ng/ml (62% vs 55%, P ¼ 0.593). The negative impact of Fe-100 Z1000 ng/ml on TRM at 1 year (17.6% vs 8.1%, P ¼ 0.129) and 2 years (20.8% vs 8.1%, P ¼ 0.068) was also higher than that of Fe-PRE Z1000 ng/ml (15.6% vs 14.4%, P ¼ 0.846, and 18.7% vs 16.1%, P ¼ 0.721, respectively). Conclusion: In our experience, a high ferritin level at day þ 100 showed a more negative impact on OS and on TRM at 1 and 2 years post-transplantation than the pre-transplant ferritin level. These results need to be confirmed in larger studies, and the possible benefit of early post-transplant iron chelation should be evaluated. Disclosure of Interest: None declared.
P177 Chemotherapy-induced nausea and vomiting in adult patients after hematopoietic stem cell transplantation: a survey from the Quality of Life Working Party of the Rome Transplant Network (RTN) A. Tendas1,*, O. Annibali2, F. Sollazzo3, A. D’Apolito1, S. Cacciaraichi4, C. Viggiani5, E. Conte3, V. Molinari6, A. Di Veroli6, A. Chierichini7, M. R. Mauroni6, C. Papa6, D. Saltarelli7, I. Carli7, F. Marchesi5, T. Dentamaro1, P. de Fabritiis1, W. Arcese6 on behalf of Quality of Life Working Party of the Rome Transplant Network 1 Hematology, S. Eugenio Hospital, 2Universita` Campus Bio-medico, Hematology, 3Hematology, Ospedale S. Andrea, 4Hematology, Universita` Campus Bio-medico, 5Hematology, Regina Elena National Cancer Institute, 6Hematology, Tor Vergata University, 7 Hematology, Ospedale S. Giovanni Addolorata, Rome, Italy Introduction: Chemotherapy-induced nausea and vomiting (CINV) has a major impact on quality of life (QoL) of patients undergoing hematopoietic stem cell transplantation (HSCT). Objective: to compare strategies for CINV prevention, monitoring, and treatment adopted in the 6 centers of the RTN. Materials (or patients) and methods: An assessment form had been filled out by each center including the following items: incidence, monitoring, prevention (with the detailed prophylaxis schedule for three broadly used conditioning regimens: MEL200, BEAM and FEAM) and treatment of CINV. Cost-effectiveness ratio of each prophylaxis scheme was represented by measuring the probability of inefficacy and the risk of side effects by comparing each individual prescription as recommended by MASCC/ESMO guidelines. Missing/underdosed prescriptions were counted and scored with a negative score, according to the predictable effectiveness reduction (-1 mild, -2 moderate, -3 strong); scores sum was designated as effectiveness reduction score (ERs). Not required prescriptions were counted and scored, according to different probability and severity of side effects (1 mild, 2 moderate, 3 severe); scores sum was designated as side effects score (SEs). Schedule cost was calculated and compared with the cost of a fully guidelines-compliant one. Relation between CINV incidence estimation and ERs was analyzed. Results: Forms were delivered and filled out on October 2013. Median (range) incidence of CINV was 57% (20-90). All centers declared CINV data recording on medical record (combined with electronic database recording in 1 center); 3/6 centers reported using corticosteroids; 6/6 using a new (1/6) or old (5/ 6) generation 5HTRA; no center commonly used aprepitant. The details of prophylaxis schedules for MEL200, BEAM and FEAM allowed the analysis of costs, effectiveness reduction and risk of side effects. The euro median cost was 74.46 h (28.46 - 128.86) for MEL200, 193.04 h (179.20 - 203.02) for BEAM and 238.84 h (192.62 - 354.64) for FEAM; the cost of a schedule designed on the guidelines amounting to 108.05 h, 68.32 h and 102.16 h for MEL200, BEAM and FEAM, respectively. The median missing/underdosed prescriptions was 6 (3-6), 5 (3-5) and 3 (0-8) for MEL200, BEAM and FEAM, respectively; the median ERs was -17.5 (-9 - -18), -10 (-6 - -14) and -6 (0 - -18) for MEL200, BEAM and FEAM, respectively. Median not-required prescription was 1.5 (1-6), 7 (7-8) and 7 (1-12), for MEL200, BEAM and FEAM, respectively; the median SEs was 3.5 (1-11), 21 (16-22) and 7 (3-26), for MEL200, BEAM and FEAM, respectively. Linear regression analysis showed a strong correlation between CINV incidence estimation and ERs (r40.8); Centers with higher median ERs (more negative) reported a higher estimation of CINV incidence (mean ¼ 83%) than Centers with lower median ERs (mean ¼ 30%). CINV treatment showed a great heterogeneity between the Centers: 5HTRA, metoclopramide and corticosteroids being used in 4 (66%), 6 (100%) and 3 (50%) Centers, respectively. Conclusion: The level of adherence to guidelines showed a wide margin of improvement; strict adherence could provide
direct cost saving as well as prophylaxis efficacy improvement, rescue therapy, indirect costs and, above all, symptom burden reduction. Disclosure of Interest: None declared. P178 Effect of pretransplant body mass index on the clinical outcome after allogeneic stem cell transplantation in patients with hematologic malignancies : a single center experience A. Gokmen1,*, E. Soydan1, M. Kurt Yuksel2, M. Kurdal1, D. Ozbek1, O. Arslan2, M. Ozcan2, O. Ilhan2 1 Department of Hematology, Medicana International Ankara Hospital, 2Department of Hematology, Ankara University, Faculty of Medicine, Ankara, Turkey Introduction: Obesity and overweigt is common in adut patients with hematologic malignancies undergoing hematopoetic stem cell transplantation (HCT). Some studies demonstrate that pretransplant body mass index may affect posttransplant outcome. The aim of the study was to assess the effect of pretransplant body mass index (BMI) on posttransplant outcome in adut patients undergoing HCT for hematologic malignancies. Materials (or patients) and methods: 82 patients ages 23 to 71 with hematologic malignancies who underwent allogeneic hematopoietic stem cell transplantation from matched related donor in 2013 and 2014 were analyzed retrospectively. Diagnosis included acute lymphoblastic leukemia, acut myeloblastic leukemia with first or second remission and active disease, myelodysplastic syndrome and chronic lymphocytic leukemia. Patients were devided into categories based on body mass index (BMI). BMI categories were defined as underweight, BMI o18.5 kg/ m2 ; normal, 18.5 r BMI o25 kg/ m2 ; overweight, 25 r BMIo30 kg/ m2, obese, BMI Z 30 kg/m2. Results: There were 33 patients in normal group ( group 1), 34 patients in overweight group ( group 2), 15 patients in obese group ( group 3). There was no patient who was underweight. Median age and sex were similar in all groups. There were no statistically significant difference among groups in time to neutrophil and platelet engraftment.The median time to neutrophil engraftment was 14 days in group 1, 16 days in group 2, 13 days in group 3 (p: 0.26). The median time to platelet engraftment was 14 days in group 1, 12 days in group 2, 12 days in group 3 (p:0.16). The rate of grade 2-4 acute GVHD was %31 in group 1, %34 in group 2,% 29 in group 3. (p:0.17). Treatment related mortality (TRM) were similar in all groups. TRM was %18 in group 1, %20 in group 2,% 20 in group 3. (p: 0.25). The causes of the death (TRM) were infection (n: 7), GVHD (n: 5), venocclusive disease (1), and pulmonory complication (3). Conclusion: We demonstrated that pretransplant BMI did not affect the major posttransplant clinical outcome (time to engraftment, GVHD, TRM). Prospective studies to fully evaluate the impact of BMI on transplant outcome is warranted. Disclosure of Interest: None declared. P179 Risk factors for early hepatic cytolysis during treatment with anti-thymocyte globulin (ATG) B. Aldweik1,*, R. Redjoul1, S. Bastuji-Garin2, F. Beckerich1, C. Robin1, C. Pautas1, A. Toma1, L. Cabanne1, S. Maury1, C. Cordonnier1 1 Hematology, 2Public Health Department, Henri Mondor University Hospital, Cre´teil, France Introduction: ATG is widely used for treatment of aplastic anemia, and in allogeneic HSCT for conditioning regimens or 2nd line treatment of acute GVHD. Early hepatic toxicity of ATG is poorly reported. Due to several consecutive cases, we aimed
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to assess the risk factors for developing early, acute hepatic cytolysis (HC) under ATG therapy. Materials (or patients) and methods: All consecutive hematology patients receiving ATG (2005-2012), whatever the indication, were retrospectively identified from the local pharmacy files. HC was defined according to the CTCAE v3 as moderate (ALT or AST42.5 and o5 UNL; grade 1-2) or severe (ALT or AST 45UNL; grade 3-4). Data collected from the medical charts included patient and disease characteristics, status, pre-ATG hepatic and renal function, lymphocyte counts, virologic tests, dose and type of ATG, concomitant drugs within the 8 days before and during ATG, immediate tolerance of ATG. Additionally, in case of HSCT, conditioning, donor, graft, donor and recipient CMV serology, and GVHD were collected. ATG was administered according to the official French recommendations, under hyperhydratation, IV dexchlorpheniramine, and methylprednisolone (1mg/kg/d from day-1 until the last day of ATG). Characteristics of the patients developing HC were compared to those of patients who did not, using X2 or exact Fisher test. All significance tests were 2-tailed. Po.05 was considered to be statistically significant. Po0.15 was considered as indicative of a trend. Results: 84 patients received ATG for treatment of aplastic anemia (n ¼ 17, 20%), HSCT conditioning (n ¼ 55, 65.5%) or treatment of aGVHD (n ¼ 12; 14.5%). HC was observed in 24 (28.5%; grade1-2: 9; grade 3-4: 15) patients during or within 10 days following ATG. Maximal ALT/AST levels reached 328xUNL. ATG was stopped during treatment in 3 patients, delayed without changing the total dose in 2, and pursued without change in the other cases. None patient developed hepatic failure. HC evolved favorably in 79% within 10 days and did not impact 3-month survival. However, all the 6 HCpatients who died had grade 3-4 HC. Two patients with HC developed veno-occlusive disease. Both died, one from leukemia relapse, the other one from multivisceral failure. Age, underlying disease, status, indication and type of ATG, pre ATG liver and renal function, immediate side effects, virologic markers, TBI, and concomitant administration of azoles or other hepatotoxic drugs were not associated with the risk of HC. There was a trend for an association between HC and recipient CMV-seropositivity, and liver GVHD. Two factors were significantly associated with a decreased risk of HC: lymphopeniao500/mm3 (6/53 (11.3%) vs 18/31 (58%), Po .001), and conditioning with amsacrine þ cytarabine (0/11 vs 24/73 (33%), P ¼ 0.029), administered in the setting of FLAMSA regimen. Conclusion: In our experience, the incidence of early ATGcytolysis was high (28.5%) and severe in 18%. We were unable to identify any significant risk factor for HC due to ATG. However, the 2 factors associated with a decreased risk (lymphopenia, and amsacrine þ cytarabine in conditioning) suggest that the more immunodepressed patients could be protected of the risk, and therefore that ATG cytolysis could be more an immuno-allergic phenomenon, rather than a direct toxicity. Although this side effect did not impact on survival, we plan to reinforce steroid administration during ATG to decrease this risk. Disclosure of Interest: None declared. P180 Autologuous peripheral blood stem cell transplantation associated with low transplant related mortality rates: Results of a single centre analysis of 856 cases B. Kragl1,*, A. Kraus1, D. Gla¨ser1, C. Groe-Thie1, C. Wittke1, S. Freitag1, M. Leitha¨user1, C. Kahl1, K. Borchert1, M. Freund1, C. Junghan1 1 Department of internal medicin 3, Hematology, Oncology, Palliative Care, University of Rostock, Rostock, Germany Introduction: Autologous peripheral blood stem cell transplantation (PBSCT) is a treatment option for various hematologic diseases such as multiple myeloma and lymphoma. As several new drugs for the treatment of these diseases are
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developed, it is of major importance to investigate continuously morbidities and mortalities associated with autologous PBSCT. Here we report on 856 autologous PBSCTs performed at a single centre academic institution with regards to the early mortality rates. Materials (or patients) and methods: Consecutive autologous PBSCTs performed at a single centre (University of Rostock) over a time period of 20 years were analysed in regards to their 30 day and 60 day mortality rates. Patient characteristics analysed were sex, age, date of transplant, CD34 þ cell number, type of conditioning regimen and underlying disease. Major endpoints were mortality rates at day 30 and day 60 after auto PBSCT. Results: 856 autologous PBSCTs were performed during a time span from 11/1994 to 12/2014. Median age of PBSCT recipients was 53 years (range 16 years to 74 years). Most patients were male (67.1%). Most common underlying diseases were plasma cell diseases (PCDs; n ¼ 419; 49.0% of all), lymphomas (n ¼ 288, 33.6% of all), leukemias (n ¼ 13, 1.5% of all) and others including solid tumours (n ¼ 136; 15.9 of all). Most common conditioning regimen were melphalan based (52.4%), followed by BCNU-based regimen (7.2%) and others including treosulfan based ones (40.4%). 30 day mortality for all entities was low (1.52%). A total of 13 patients died (1 PCD, 9 lymphomas, 3 other). 60 day mortality for all entities was 2.80%. 24 patients died: 4 with PCD, 15 with lymphoma, 5 other. 30 and 60 day mortality rates for the individual underlying diseases were as follows: PCD: 0.12% (1 pt.) and 0.48% (4 pts), Lymphoma: 1.05% (9 pts) and 1.75% (15 pts), Other including solid tumours: 0.35% (3 pts) and 0.58% (5 pts). Most common causes of death within the first 30 days were infections (9/13 pts; 69.23%). Conclusion: Autologous PBSCT is a common procedure in the treatment of PCDs and lymphomas. Treatment associated early mortality rates are low, especially in PCDs. Therefore we conclude that auto PBSCT can be safely administered in PCDs aiming at deeper remission rates. Second, our single center data indicates that mortality rates might be slightly compared to PCDs if PBSCTs are performed in lymphomas which might be due to the fact of the more advanced underlying diseases. References: Disclosure of Interest: None declared. P181 Survival outcome and long-term follow-up of Stem Cell Transplantation (SCT) in Acute Myeloid Leukemia (AML): a single-center experience C. Montes-Gaisan1,*, A. Bermu´dez1, G. Pe´rez1, A. Cuesta1, P. Ibarrondo1, C. Richard1, E. Conde1 1 Hematology, Hospital Universitario Marque´s de Valdecilla, Santander, Spain Introduction: Post-remission treatment for AML is very aggressive and, in many cases, a SCT is needed. Comparisons between Allo-SCT and Auto-SCT have always shown more Transplant Related Mortality (TRM) but less Cumulative Incidence of Relapse (CIR) in the first group. Our study describes, not only the long-term survival outcomes, but also the quality of life in long survivors. Materials (or patients) and methods: Retrospective study of 274 patients diagnosed with non-promyelocytic AML who underwent SCT between 1982 and 2011 in our center. Characteristics in the 162 Allo-SCT and the 112 Auto-SCT groups of patients were respectively: median age of 38 and 45 years old, secondary AML in 20% and 10%, refractory to Induction AML in 16% and 3%, pre-SCT status different from Complete Remission (CR) in 13% and 3% and year of SCT before 2005 in 53% and 86%. No significant differences between both groups were found in other risk factors as hyperleucocytosis at diagnosis or adverse cytogenetics.
Results: With a median follow-up of 55 months [2-316], Overall Survival (OS) until 1997 in Allo-SCT and Auto-SCT were respectively, 40% and 61% at 1 year and 28% and 45% at 5 years, but from 1997, 66% and 70% at 1 year and 47% and 48% at 5 years. TRM until 1997 in Allo-SCT and Auto-SCT were respectively, 30% and 7% at 1 year and 35% and 9% at 5 years, but from 1997, 16% and 2% at 1 year and 25% and 4% at 5 years. Allo-SCT: From the 162 patients, 72(44%) are alive by this moment, 43(60%) with ECOG 0, 21(29%) with ECOG 1 and the other 8(11%) with ECOG 2, basically because of graft versus host disease (GVHD present in 39 patients, 21 steroiddependent and 3 refractory to any treatment). All of them have been in CR during the last 2 years of follow-up. In contrast, 90(56%) patients have died: 52(58%) because of SCT complications (20 infections, 16 GVHD, 8 toxicity and 8 mixed causes), 33(37%) because of disease and 5(5%) because of other causes. With a median follow-up of 43 months [2-316], there have been 4 secondary neoplasm, all of them solid ones, which appeared with a median of 242 months [179-311] from SCT. None of them had previously received radiotherapy. Auto-SCT: From the 112 patients, 43(38%) are alive by this moment, 32(74%) with ECOG 0 and the other 11(26%) with ECOG 1. All of them have been in CR during the last 2 years of follow-up. In contrast, 69(62%) patients have died: 45(65%) because of disease, 14(20%) because of SCT complications and 10(15%) because of other causes. With a median follow-up of 93 months [5-230], there have been 6 secondary neoplasm, 5 of them hematologic ones, which appeared with a median of 90 months [76-115] from SCT. None of them had received radiotherapy, but previously treated hematopoietic stem cells. Conclusion: OS is lower in Allo-SCT during the first years [1982-1996], although it has a tendency towards OS in AutoSCT during the last ones [1997-2011] because of the decrease in TRM, which is more significative in Allo-SCT. Both procedures are efficient to treat AML (near 50% of the patients in both groups are alive at 5 years from SCT in recent years). The decrease in TRM until 2% in Auto-SCT makes it a good choice for older patients without risk factors. The development of secondary hematologic neoplasms is a not inconsiderable fact in Auto-SCT, with an incidence of 10% and a late mortality of 100%. Disclosure of Interest: None declared. P182 Higher Prevalence of HLA-Alloimmunization in Aplastic Anemia and MDS/MPN patients C. Tsamadou1,*, D. Fu¨rst1, C. Zollikofer1, H. Schrezenmeier1, J. Mytilineos1 1 German Red Cross Blood Transfusion Service, Baden-Wuerttemberg – Hessen, Ulm, Germany & Institute of Transfusion Medicine, University of Ulm, Germany, Ulm, Germany Introduction: According to current clinical data, the prevalence of preformed HLA Donor Specific Antibodies (DSA) may lead to graft rejection after Hematopoetic Stem Cell Transplantation (HSCT). The aim of the present study is to investigate the prevalence of HLA antibodies (abs) among different hematological disease groups in terms of identifying patients at higher risk for HLA-alloimmunization. Materials (or patients) and methods: 1405 patients scheduled for a haematopoetic stem cell transplantation (934 males& 471 females) with a median age of 58y were screened for HLA abs class I (CLI) and II (CLII) with ELISA and were stratified according to diagnose in 6 major disease groups (61 AA: Aplastic Anemia, 415 NHL: Non-Hodgkin Lymphoma, 33 HL: Hodgkin Lymphoma, 228 MM: Multiple Myeloma, 222 CL: Chronic Leukemia and 446 MDS/MPN: Myelodysplastic Syndrome/Myeloproliferative Neoplasms). Acute leukemia patients were not included in the study considering that patients with longer course of disease are more likely to be HLA-alloimmunized due to higher frequency of transfusions. Statistical significance was assessed via chi-square test and/or
Fisher’s exact test. The threshold for statistical significance was set to a p value of o0.05. R program v 3.1.0 was used for all statistical computations. Results: We found a statistically significant higher prevalence of HLA CLI and CLII abs in the AA (CLI:21.3%, Po0.001, CLII:16.4%, P ¼ 0.003) as well as in the MDS/MPN (CLI:15.7%, Po0.001, CLII:16.4%, Po0.001) group of patients in relation to patients in the other four groups which were subsequently classified as control group (CLI:7%, CLII:5.6%). It was also observed that female patients presented a higher rate of HLA alloimmunization (Po0.001). Subsequent analysis however, revealed no preferential distribution of patients according to sex in the various disease groups (P ¼ 0.21), and therefore female sex was excluded as possible confounding factor for our initial findings. Conclusion: Our data indicate that AA and MDS/MPN patients are more prone to develop HLA abs compared to those in other hematological disease groups. A plausible explanation could be the higher rate of blood and /or platelet transfusions among these patients. Past pregnancies can also explain why female patients had a significantly higher frequency of positive HLA-antibody results. Conclusively, based on our findings and on the existing data regarding the negative effect of patient’s HLA DSA on HSC engraftment, it would be reasonable to advise an initial screening for HLA abs in patients at higher risk for HLA-alloimmunization such as Aplastic Anemia or MDS/ MPN patients upon initiation of unrelated donor search. In cases when a fully matched donor is not an option, further identification of HLA abs and consideration of arising HLA antibody specificities upon donor selection would be strongly recommended. Disclosure of Interest: None declared. P183 Biosimilar G-CSF vs classical G-CSF in post allotransplant recovery. A case control study C. Targhetta1,*, D. Baronciani1, C. Depau1, F. Pilo1, M. Capasso2, E. Angelucci1 1 - Ospedale Oncologico di Riferimento Regionale ‘‘A. Businco’’, Cagliari, Italy., U.O. Ematologia-CTMO, Cagliari, 2Dept. of Molecular Medicine and Medical Biotechnologies CEINGE Biotecnologie Avanzate , University of Naples Federico II, Napoli, Italy Introduction: The use of biosimilarG-CSF was licensed by the European Medicines Agency (EMA) for all the registered indications of the originator G-CSF on the basis of extrapolation of efficacy and safety data without a specific clinical trial. Moreover the setting of allogeneic transplantation is unexplored. Because we believe that clinical data is the only cornerstone for clinical practice since February 2013 we started a case-control clinical study in patients undergoing allotransplant to verify biosimilars safety and clinical appropriateness in the post-transplantation hematopoietic recovery. Materials (or patients) and methods: We analyzed two cohorts comprising a total number of 86 patients suffering from various hematological diseases treated in our HSCT Centre. The first cohort included 43 consecutive patients undergoing allogeneic HCST between February 2013 and October 2014 who were treated with biosimilars of G-CSF (Tevagrastims or Ratiograstims or Zarzios). The second cohort (control group) included 43 consecutive patients who underwent allogeneic HSCT between January 2011 and December 2012 and received originator G-CSF (Granulokines). The injection of G-CSF began at day 7 after HLA identical sibling or MUD and at day 5 after haploidentical stem cell infusion. To compare the efficacy of the two G-CSF formulas we evaluated the day of engraftment and the incidence of graft failure in the two cohorts. Neutrophil engraftment was defined as a sustained (for more than 3 consecutive days) absolute neutrophil count of 500/ml without the use of growth factors. Primary graft failure was defined as failure to maintain absolute neutrophil count Z500/ml for 3 consecutive days following transplantation. Categorical variables have been
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evaluated by X2 test, continuous variables have been evaluated by Mann-Whitney test. Data are expressed as median with a range. Results: Study and control groups were matched for sex, type of disease, Karnofsky score, HCT-Comorbidity Index, status of disease at transplant, conditioning regimen, type of donor, HLA-matching, stem cell source, number of stem cells injected (P not significant). There were 41 and 40 evaluable patients in the study and control group respectively. White blood cell engraftment took place in median time of 13 (10-28) days in the study group and in 14 (10-22) days in the control group (P ¼ 0.205). Primary graft failure developed in 1 out of 41 and in 1 out of 40 evaluable patients in the two groups, respectively (P ¼ 0.4). To further enhance clinical relevance we evaluated on intention to treat basis the incidence of early infections that developed in 13/43 (30%) patients in the first cohort, in 18/43 (41%) in the second cohort (P ¼ 0.37). Conclusion: This study shows that biosimilars of G-CSF are equivalent to classical products in terms of safety and efficacy when used for white blood cell recovery after allogeneic hemopoietic stem cell transplantation. Disclosure of Interest: None declared. P184 Implementing International HCT Late Effects Screening Guidelines in Clinical Practice: Outcomes D. Greenfield1,*, V. Shanklin2, J. Snowden3 1 Cancer Services, Sheffield Teaching Hospitals NHS FT, 2Medical School, University of Sheffield, 3Haematology, Sheffield Teaching Hospitals NHS FT, Sheffield, United Kingdom Introduction: HCT survivors are at risk for developing late complications secondary to pre-, peri-, and post-transplantation exposures and risk factors. Updated recommendations for late effects screening for survivors of autologous and allogeneic HCT were published in 20121. The aim of this study was to assess the clinical efficacy of a screening clinic to identify late complications of HCT. Materials (or patients) and methods: Our centre implemented the internationally agreed guidelines in 2013 using a locally developed Standardised Operating Policy (SOP) through a nurse-led late effects screening clinic. 59 consecutive patients who attended the screening clinic for the first time during 1st Sept 2013 to 31st Aug 2014 were included in the audit. Data was extracted from the clinic letter annotated from the consultation and from results of the requested investigations as per the SOP. Presence or absence of each variable and investigation was coded and current results and actions were recorded in a spreadsheet. Audit approval confirmation was given by the Clinical Effectiveness Unit of our hospital trust. Results: 30 female and 29 male HCT survivors with a mean age of 49 years and a mean 6 years post-transplant attended the nurse-led late effects clinic as new patients in the time period. 49/65 were allogeneic HCTs. 54 had one transplant procedure, 4 had 2 and one had 3 procedures.13 patients received TBI radiotherapy. 43/59 primary indications for HCT were lymphoid diseases, 12/59 were myeloid diseases with 3/59 were solid tumour and 1/59 had an auto-immune disease. 134 late complications were identified in 59 patients with 114 requiring further action or referral, mean complication per patient ¼ 2. The most frequent late complications were pain 18/59 (31%), sleep disturbance 11/59(19%), fatigue 14/59(24%) and sexual function concerns 14/59(24%). Conclusion: To our knowledge this is the first time new international late effect screening guidelines have been audited. This new nurse-led screening clinics demonstrates clinical efficacy in identifying late complications using holistic screening. This will inevitably result in earlier detection of problems and may prevent and/or reduce morbidity therefore playing a central role in optimising quality of life for HCT survivors. Future research should look at complications and their link to certain treatments and whether these
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complications can be reduced using different transplantation exposures. References: 1. Majhail NS, Rizzo JD, Lee SJ, Aljurf M, Atsuta Y, Bonfim C, et al. Recommended screening and preventive practices for long-term survivors after haematopoietic cell transplantation. Biol Blood Marrow Transplant 2012 Mar 3; 18(3): 348–371. Disclosure of Interest: None declared. P185 Ruxolitinib for symptomatic patients with relapse of myelofibrosis after allogeneic hematopoietic transplantation D. Janson1,*, T. Stu¨big1, H. Alchalby1, F. Ayuk1, C. Wolschke1, M. Christopeit1, N. Kro¨ger1 1 University Medical Center, Hamburg, Germany, Department of Stem Cell Transplantation, Hamburg, Germany Introduction: The janus-activated kinase (JAK) 1/2 -inhibitor ruxolitinib is approved for the treatment of symptomatic myelofibrosis. Ruxolitinib can reduce constitutional symptoms and spleen size resulting in an improvement of the performance status and also in a potential prolonged survival. No data on ruxolitinib for relapsed patients after allogeneic hematopoietic transplantation have been reported so far. Materials (or patients) and methods: Five patients of our department with relapse or progressive disease of primary (n ¼ 3) or post ET/PV- (n ¼ 2) myelofibrosis were treated with ruxolitinib after allogeneic transplantation. The median age of the patients was 63 years (r: 50-72). Stem cell source had been PBSC (MRD n ¼ 2, MUD n ¼ 3) in all patients. The median time from allogeneic hematopoietic transplantation until the start of ruxolitinib was 8.8 months (r: 3.5-85.7). The median treatment duration was 268 days (r: 12-312). Ruxolitinib doses differed from 5mg to 20mg bid according to platelet counts. Two patients received additional DLI. One patient started ruxolitinib after non-responding to DLI and another patient received DLI due to decreasing chimerism during ruxolitinib treatment. All patients had mixed chimerism. Before starting ruxolitinib three patients had palpable splenomegaly and all had constitutional symptoms. Three patients had cGvHD (skin, liver or oral mucosa) at the time of treatment-start. Results: Constitutional symptoms improved in all patients (100%). The palpable spleen size was reduced in all three patients with splenomegaly (450% reduction n ¼ 2, 45% reduction n ¼ 1). In transfusion-dependent patients (n ¼ 3) a reduction of the transfusion frequency could be seen. No reduction of the JAK2-alllele-burden was seen in those patients with a JAK2V617F-mutation (n ¼ 2). During ruxolitinib treatment a further decline of donor chimerism was seen in all patients. In three out of four patients with an available bone marrow histology before and within the last days of ruxolitinib treatment the grade of bone marrow fibrosis aggravated. Two patients transformed to AML despite of response to spleen size and constitutional symptoms during ruxolitinib. Two patients with cGVHD had an improvement and even complete resolution of their GvHD. In one patient cutaneous GvHD (grade II) occurred during ruxolitinib treatment but immunosuppressive treatment had been stopped simultaneously. Within the time of ruxolitinib treatment no severe infectious complications were seen. Conclusion: Ruxolitinib is well tolerated, ameliorates MFspecific symptoms and reduces the spleen size in patients with relapse after allogeneic transplantation. Ruxolitinib does neither reduce the JAK2-allele burden nor does it lead to an increase of donor chimerism. Bone marrow fibrosis seems to aggravate whilst treatment with ruxolitinib. Transformation to AML was seen in two patients despite of clinical response to ruxolitinib. It is of note that in two patients an improvement of the cGvHD was seen. Disclosure of Interest: None declared.
P186 Central Venous Catheter Related Complications in a Pediatrıc Stem Cell Transplantation Unit F. Azik1, D. Gurlek Gokcebay1,*, P. Isik1, Z. Avci1, N. Ozbek1, B. Tavil1, D. Uc¸kan1, B. Tunc1 1 Pediatric Hematology, Ankara Children’s Hematology and Oncology Hospital, Ankara, Turkey
P187 CD4 þ cell count on day þ 30 predict overall survival and transplant related mortality in acute leukemia patients after alloPBSCT D. Pastore1,*, M. Delia1, A. Mestice1, P. Carluccio1, T. Perrone1, F. Gaudio1, A. Ricco1, P. Curci1, C. Pasciolla1, G. Specchia1 1 Hematology-Policlinico-Bari Italy, BARI, Italy
Introduction: Children undergoing hematopoietic stem cell transplantation (HSCT) require long term central venous catheters (CVC) for administrating of medications, blood products and blood sampling. The aim of the study is to assess infectious and mechanical complications of CVCs during HSCT. Materials (or patients) and methods: We retrospectively evaluated 95 consecutive patients between April 2010 and January 2013 who underwent HSCT at our Bone Marrow Transplantation Unit. Data included demographic information, CVC type and location of insertion, duration of insertion, occurrence of complications, antimicrobial therapy and causes of removal collected from medical records. Results: A total of 95 patients (42% girls, 58% boys) underwent 100 HSCT with median age of 8.9 years (range; 0.5-16.8). Ninety-two of the transplants were allogeneic, and 8 of them were autologous. Forty-five patients had malignant, 40 had non-malignant, and 10 had immunodeficiency disorders. Ninety-nine Hickman double lumen catheters were inserted in 91 (95.8%) of the patients for a total 6369 catheter days, with a median catheter life of 56 days (range; 23-201). Hickman catheters could not be inserted in 4 (4.2%) of the patients because of repeated CVC insertions or radiation therapy scars; they had nontunneled jugular or femoral catheters during HSCT. Forty CVC-related infections were recorded with a rate of 6.28/1000 catheter days. Coagulase negative staphylococci were the predominant microorganisms in 42.5% (17/40). Twenty-nine of the CVC’s (27%) were removed during the study period as 22 of them became infected, 5 had mechanical complications (3 ruptures, 1 malposition, 1 leakage), and 1 was revised because of blocking. One of the CVC which was blocked opened using tissue-plasminogen activator. Overall mechanical complication rate was 1.09/1000 catheter days. Enoxaparin at a dose of 0.8 mg/kg/day by subcutaneous injection was administrated as prophylaxis of veno-occlusive disease for median 34 days (total 3016 days, range; 11-54), and overall 5.2% (5 out of 95) of the patients developed CVC-associated thrombosis. Type of the CVC did not influence the frequency of mechanical or infectious complications. Children o8 years of age were more likely to develop infectious and mechanical complications leading to CVC removal (P ¼ 0.01). Conclusion: The rate of CVC-related infections were varying from 0.87 to 8.24, and mechanical complications were varying from 0.45 to 4.5 per 1000 catheter days in children with hemato-oncological diseases; which were in accordance with our study. Prior studies have suggested that thrombosis occurs in 4.4%49% of pediatric HSCT recipients. Although enoxaparin was used for prevention of VOD in this study, the thrombosis rate did not differ from another reports. In younger children with smaller size CVCs more frequently developed a complication leading to removal which shown in previous studies, it is important that the proper care of the CVC by nurses and parents. References: 1. Fratino G, Molinari AC, Parodi S, et al. Central venous catheter-related complications in children with oncological/hematological diseases: an observational study of 418 devices. Ann Oncol. 2005; 4: 648–654 2. Lagro SW, Verdonck LF, Borel Rinkes IH, Dekker AW. No effect of nadroparin prophylaxis in the prevention of central venous catheter (CVC)-associated thrombosis in bone marrow transplant recipients. Bone Marrow Transplant 2000; 26: 1103– 1106. Disclosure of Interest: None declared.
Introduction: Allogeneic peripheral stem cell transplantation (alloPBSCT) from a related or unrelated donor is a wellestablished strategy for patients with acute leukaemia. However, this procedure is associated with increased morbidity and mortality because of graft versus host disease (GvHD), immune reconstitution impairment and a high risk of infections. In our study we evaluated the lymphocyte subset recovery after alloPBSCT and its impact on transplant-related mortality (TRM) and overall survival (OS) in acute leukemia patients. Materials (or patients) and methods: We evaluated the immune reconstitution of CD3 þ /CD4 þ , CD3 þ /CD8 þ and NK cells performed at 30, 100, 180 and 360 days after alloPBSCT in 115 patients with acute leukaemia. Patients were transplanted with unmanipulated PBSC from an HLA matched related donor (MRD) (n ¼ 80) or an HLA (8/8) matched unrelated donor(MUD) (n ¼ 35). Median age was 38 years (range 18-61); diagnoses were acute myeloid leukaemia (n ¼ 90) and acute lymphoblastic leukaemia (n ¼ 25); 80% of patients underwent myeloablative conditioning (busulphan, cyclophosphamide in MRD and busulphan, cyclophosphamide and ATG in MUD) and 20% underwent reduced intensity conditioning (busulphan, fludarabine, ATG) Results: The median counts of CD3 þ /CD4 þ were 98, 160, 200, 262 ml at 30, 100,180 and 360 days, respectively. The median counts of CD3 þ /CD8 þ were 180, 350, 500 and 670 ml at 30, 100, 180 and 360 days, respectively. The median counts of NK cells were 110, 260, 270 and 260 mL at 30, 100, 180 and 360 days, respectively. Considering the entire patient population the median cell count rose above 200 ml after day 180 for CD3 þ /CD4 þ and after day 45 for CD8 þ cells; the median NK cells count rose above 100 ml after day 30. The median CD3 þ /CD4 þ cell count on day þ 30 for the entire patient population was 98 ml( range 20-190) and TRM at 2 years was significantly higher in patients not achieving this CD4 cell count (38% vs 15%, Po0.001); patients with a low CD3 þ / CD4 þ count (o 98 ml) on day þ 30 had a higher risk of dying of infections (30% vs 11%, P40.003). Median OS in patients not achieving CD4 þ of 98 ml at 30 days was 40 months while median OS in patients with more than 98 CD4 þ at 30 days was not reached. Univariate analysis at 100 days showed a significant association between CD4 cell recovery and transplant from a related donor (P ¼ 0.003), myeloablative conditioning (P ¼ 0.04) and absence of aGvHD (grade II-IV). In multivariate analysis, transplant from a related donor (P ¼ 0.05) and absence of aGvHD( grade II-IV) (P ¼ 0.002) were significantly associated with a better CD4 cell recovery. CD3 þ / CD8 þ and NK cells recovery did not correlate with a different TRM risk or OS. Conclusion: Our results support the relationship between immune reconstitution of CD4 þ , OS and TRM. The CD4 þ cell count on day þ 30 is able to predict OS and TRM after myeloablative alloPBSCT in acute leukemia patients. Disclosure of Interest: None declared.
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P188 Restrospective comparative analysis of clinical outcomes between elderly and young patients in allogeneic hematopoietic stem cell transplantation E. Chapchap1,*, L. N. Kerbauy1, F. Santos1, M. Rodrigues1, I. Esteves1, J. Sobrinho1, F. Kerbauy1, A. Ribeiro1, N. Hamerschlak1 1 Haematology and Oncology, Albert Einstein Hospital, Sao Paulo, Brazil Introduction: The number of allogeneic hematopoietic stem cell transplantations (Allo-SCT) has been rising for older patients in the past decade. This is mostly due to advances in treatment and supportive care, as the use of reduced intensity and non myloablative conditioning regimens together with post-transplant immunomodulatory strategies. In this retrospective study, we analyzed the outcomes of two groups: elderly group (EG) versus young group (YG) that underwent Allo-SCT, from January 2007 to June 2014, at Israelita Albert Einstein Hospital in Brazil, in the light of prognostic factors and toxicity. Materials (or patients) and methods: The two groups, YG (r55 years old) and EG (455y), were analyzed with the following endpoints: overall survival (OS); therapy-related mortality (TRM) in the first 100 days and after 1 year post transplant; admission to intensive care unit (ICU), need for renal replacement therapy (RRT) and total days as inpatient in the first year. STATA software (Kaplan-Meier curve and Cox regression) were used for statistical analysis. We also analyzed potential risk factors for mortality in the elderly group. Results: A total of 111 patients were analyzed, 75 in the young group (YG) and 36 in the elderly (EG). MDS and AML were the most common indications in both groups: 47.2% (YG) and 40.7% (EG). Half of patients in the EG underwent Allo-SCT with MRD and 60% in the YG. Haploidentical donors were used in the young group: 18 transplants (24%) and in 5 (13.9%) in the elderly. In both groups, the most frequent conditioning regimen was IV Busulfan and Fludarabine±ATG with adjusted busulfan dose per pharmacokinetics/AUC analysis. The OS rates in the YG and EG were 84% versus 75% (P ¼ 0.01) at D þ 100 and 71% vs 53% (P ¼ 0.01) at one year. TRM were higher in the EG at D þ 100: 17% vs 14% (P ¼ 0.04) and 29 % vs 17% (P ¼ 0.04) at one year, respectively. Admission to ICU was more common in the elderly 44% vs 23% (P ¼ 0.019). The requirement of RRT was higher in elderly: 15% vs 25% (P ¼ 0.18). The median days as inpatient in the first year for elderly versus young was 73 vs 60 (P ¼ 0.67). The cumulative GVHD incidence was similar in both groups: 52% vs 50% (P ¼ 0.88), while grade III-IV GVHD were 14% vs 11%. The relapse rate in the first year was: 23% vs 20% (P ¼ 0. 67). None of these variables: age older than 65y; conditioning regimen; graft source and the primary disease (AML/MDS versus others), had impact in OS or TRM in the elderly group. Conclusion: In our population, despite the mortality was higher in elderly group, this was not found to be related to relapse or GHVD. Probably, the co-morbidities and loss of functionality in the elderly group are important factors that impacted in the OS, since the TRM increase over time from 100 days to one year. Comorbidity index and functional assessment should be used in the elderly patients in order to define the best transplant candidate and treatment strategy choice before and after transplant. References: 1. Alatrash G et. al (2011) Myeloablativereducedtoxicity iv busulfan and allogeneic hematopoietic Fludarabinestem cell transplant for acute myeloid leukemia with patientes or myelodysplastic syndrome in the sixth through eighth decades of life. Biol Blood MarrowTransplant. 10,1490-1496. 2. Appelbaum F. (2011) Preparative regimens and ageism. Biol Blood Marrow Transplant 17(10), 1419–1420 Disclosure of Interest: None declared.
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P189 Veno occlusive disease of the liver after hematopoietic stem cell transplantation (HSCT) in pediatric patients: Padova experience E. Calore1,*, S. Rossin1, M. Pillon1, M. Tumino1, C. Mainardi1, T. Toffolutti2, S. Varotto1, R. Destro1, M. V. Gazzola1, G. Basso1, C. Messina1 1 Department of Pediatrics, Clinic of Pediatric Hematology Oncology, 2Institute of Radiology, Unit of Pediatric Radiology, University of Padova, Italy Introduction: Hepatic veno-occlusive disease (VOD) is one of the most severe complication due to HSCT. The incidence of VOD has been described between 10-60% in children, depending to many factors. Materials (or patients) and methods: We performed a prospective study analyzing 117 pediatric patients that underwent HSCT from 2011 to 2014 in the Bone Marrow Unit of Pediatric Onco-Hematology in Padova. Thirty nine patients underwent autologous HSCT, 88 allogeneic HSCT. Seven patients received more than one HSCT. The conditioning regimen was TBI based in 38 patients, busulfan based in 32. Ninety two patients were Caucasian, 6 Black, 5 Asiatic e 14 south American. Patients with one or more risk factors for VOD (58) were enrolled to receive intravenous Defibrotide (DF) prophylaxis, while the other patients (58) were enrolled to receive Heparin; 1 patient didn’t receive any prophylaxis. Results: VOD has been diagnosed in 4/117 patients (2 received autologous HSCT, 2 allogeneic HSCT) with an incidence of 3.4% using Seattle criteria, while using Baltimora criteria in 2/117 patients (1.7%). Inversion of portal flow has been found in all 4 patient at a median time of 5.5 days (range 0-7) after clinical diagnosis of VOD. Two children have been affected by severe VOD while the others 2 by a moderate VOD. All patients have been treated with rt-PA and Heparin after the occurrence of inversion of portal flow and it has been reverted in all patients with resolution of VOD in a median time of 15 days (range 9-22). No one had bleeding due to the therapy with rt-PA and no one died for VOD. Taking into account our statistical analysis, gender, primary disease, conditioning regimens, type of transplant (auto or allo), stem cell source, pre-existing hepatic disease, abdominal irradiation in the year before HSCT and ferritin level 42000 ng/ml are not risk factors for VOD. Non caucasian ethnicity is the risk factor for VOD (p-value ¼ 0,03). The 4 patients with VOD were in prophylaxis with DF. Comparing the two groups of patients in prophylaxis with Heparin and DF we saw that patients in prophylaxis with DF had lower incidence of hepatotoxicity (lower levels of AST/ ALT) than patients in prophylaxis with Eparina; we didn’t observe any difference about incidence and severity of acute Graft versus Host Disease (aGHVD) and Transplant related mortality (TRM). Conclusion: Our study shows that the incidence of VOD is lower than that reported in literature. Moreover the incidence of VOD is different depending on diagnostic criteria used (Seattle or Baltimore). All 4 patients developed inversion of portal flow and doppler US may be helpful in identification of the patients with VOD. All 4 patients with VOD were in prophylaxis with DF but they also had risk factors for VOD. From our analysis, non Caucasian ethnicity is the only risk factor for VOD. This data hasn’t been reported in literature and it requires a larger series to be confirmed. Prophylaxis type doesn’t affect on incidence and severity of aGVHD in patient with allogeneic HSCT. Disclosure of Interest: None declared.
P190 Peripherally Inserted Central Catheter (PICC) Power Groshong 5 Fr device for management of peripheral blood stem cell collection and transplantation in haematological patients: a single centre study from the Rome Transplant Network E. Conte1,*, A. Ferrari1, M. P. Bianchi1, M. Piedimonte1, C. Guglielmi1, V. Naso1, R. Palmieri1, D. Grigore1, F. Sollazzo1, M. Shafii Bafti2, D. Meo2, A. Tafuri1, W. Arcese3 on behalf of Rome Transplant Network 1 Haematology, Sant’Andrea Hospital, "Sapienza" University of Rome, 2Immunohematology and Transfusion Medicine, "Sapienza" University of Rome, 3Stem Cell Transplant Unit, Haematology, Tor Vergata University of Rome, Rome, Italy Introduction: PICC is inserted bedside, via peripheral vein such as cephalic, basilic or brachial veins, avoiding general anesthesia, sedation and surgical procedures. However, only few data are reported on PICC management in peripheral blood stem cell (PBSC) collection and transplantation. Herein, we are reporting our experience in using PICCs during these procedures. Materials (or patients) and methods: From October 2012 to November 2014 a single lumen silicone 5 FR Power Groshongs PICC (Bard Access, Usa), a new device recently available, was inserted in 48 haematological patients (28 F and 20 M; median age 54, range 24-68), affected by Acute Myeloid Leukemia (n ¼ 9), Non Hodgkin Lymphoma (n ¼ 18), Multiple Myeloma (n ¼ 20), Hodgkin Disease (n ¼ 1). Power Groshongs PICC was used to collect PBSC in 27 patients, 18 of whom later underwent an autologous stem cell transplantation (AuSCT). In the remaining 17 and 4 patients, PICC was used only in AuSCT or an allogeneic stem cell transplantation (AlloSCT), respectively. In all patients PICC was used for PBSC reinfusion. All PICCs were positioned and managed by a team of specifically trained physicians and nurses, at our Haematology Unit. Results: The 48 PICCs remained in place for an overall period of 7,099 days (median time 137 days, range 21-391). The infective complications observed were represented by 1 Central Line Associated Bloodstream Infection (CLABSI) by Staphylococcus Epidermidis and 9 Bloodstream Infections (BSI): 3 by Staphylococcus Epidermidis, 1 by Staphylococcus Haemolyticus and 5 by Escherichia Coli. The incidence of CLABSI and BSI were 2% (0.14 per 1000 PICCs days) and 18% (1.26 per 1000 PICCs days), respectively. PICCs were removed in 38/48 patients: 35 for completion of therapy (72%, 4.9 per 1000 PICCs days), 1 for BSI (2%, 0.14 per 1000 PICCs days), 1 for breakage (2%, 0.14 per 1000 PICCs days), 1 lumen occlusion (2%, 0.14 per 1000 PICCs days). The remaining 10 PICCs are still in place. Conclusion: In summary, Power Groshongs PICC 5 Fr seems a feasible, safe and effective alternative to tunnelled central venous catheters (CVCs) for PBSC harvesting (using this device to return blood components) and reinfusion. PICC resulted useful for the complete management of transplanted patients (administration of high dose chemotherapy, antibiotic agents, blood cell product transfusion and parenteral nutrition). The results obtained by the use of Power Groshongs PICC should be therefore compared with those of tunnelled CVCs, on a large number of patients and in a prospective randomized trial. The above proposed study may evaluate, in addition, quality of life and will be conducted by the same team composed by specifically trained physician and nurses. Disclosure of Interest: None declared.
P191 Clinical Impact And Prognosis About Multiple Transfusions Prior To Allogeneic Transplantation Of Haematopoietic Progenitors E. Alonso1,*, T. Botin2, J. R. Grifols1, M. Morgades2, C. Ferra`2, I. Rodrı´guez3, B. Xicoy2, J. M. Ribera2 1 Banc de Sang i Teixits, Hospital Germans Trias i Pujol, 2 Hematologic Clinic-Institut Catala` d’Oncologia, 3Red Cell Pathology Unit-Hematologic Clinic-Institut Catala` d’Oncologia, Hospital Germans Trias i Pujol-Institut de Recerca contra la Leucemia Josep Carreras, Badalona, Spain Introduction: Hyperferritinemia in patients who undergo allogeneic progenitor cell transplantation (alloHSCT) has been related to lower overall survival (OS) and higher non-relapsed mortality (NRM), probably due to iron overload toxicity in hepatic and cardiac cells, and to an increased risk of fungal and bacterial infections. The aim of this study was to analyse if the number of red blood cells (RBC) units received prior to alloHSCT could impact on the probability to develop infections (bacterial or fungal), hepatic sinusoidal obstruction syndrome (SOS), graft-versus-host disease (GVHD) and OS. Materials (or patients) and methods: Retrospective analysis of 88 patients submitted to alloHSCT from January 2008 to December 2013. We compared the patients who had been transfused more than 20 RBC units before alloHSCT (n ¼ 46) with the patients who had received less than 20 RBC units (n ¼ 42). We collected demographic and clinical data, transfusional history, value of serum ferritin and hepatic function previous and after alloHSCT (at 3, 6 and 12 months later), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) pre-alloHSCT, chelating treatment pre- and postalloHSCT, history of bacterial sepsis (two blood culture positives), invasive fungal infections (IFI) and the incidence of SOS and III-IV mucositis grade, GVHD type (acute and chronic), OS, as well as, NRM and relapse incidence (RI). Results: There were no statistically significant differences between two groups as to age, sex, diagnosis, ABO and HLA compatibility, progenitors source, donor type, pre-alloHSCT risk, type of conditioning, CD34 cells infused, Left Ventricular Ejection Fraction (LVEF) pre-alloHSCT, median of neutrophils recovery days neither chelation treatment pre-alloHSCT. There were statistically significant differences in ferritinemia prealloHSCT (higher in the most transfused group [3639 ng/mL vs. 1094 ng/mL, P ¼ 0.001]). A greater tendency to present SOS was observed in the group of patients who received more than 20 RBC units (9/46 vs. 3/42, P ¼ 0.090); without significant differences in III-IV mucositis grade, GVHD type and infections incidence between the two groups (but higher incidence of infections by multiresistant pathogens was observed in the more transfused group, without significant differences). The OS at 5 years was lower in patients who had received more than 20 RBC units (27% [95% CI, 14% - 40%] vs. 43% [25% 61%] P ¼ 0.04), without significant differences in RI (26% [12% -40%] vs. 17% [6%; 28%], P ¼ 0.427) or NRM (48% [30% -66%] vs. 47% [31% -63%], P ¼ 0.578). Conclusion: In this study, the patients who received more than 20 RBC units before alloHSCT, presented higher serum ferritin values, major SOS incidence and worst OS than the patients who received less than 20 RBC units before HSCT. So, it is important to insist on an adequate chelation treatment before submit a patient to alloHSCT, whenever possible. References: Sivgin et al. Ann Hematol 2013; 92: 577–586. Sivgin et al. Transfusion and Apheresis Science 2013 48: 103–108. Sakamoto et al. Int J Hematol 2013; 97: 109–116. Disclosure of Interest: None declared.
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P192 Analysis of early life-threatening complications in children and adolescents undergoing allogeneic hematopoietic stem cell transplantation Z. Szmit1, E. Gorczynska1,* on behalf of Kalwak K., Owoc-Lampach J , Mielcarek M., Ussowicz M., Kulej D., Musial J, Chybicka A. 1 Medical University, Wroclaw, Poland, Department of Pediatric Hematology, Oncology and Bone Marrow Transplantation, Wroclaw, Poland Introduction: Allogeneic hematopoietic stem cell transplant (allo-SCT) is a treatment method of cancer, bone marrow failure resistant to standard therapy, and immune or metabolic disorders. This procedure has high risk of life-threatening complications. The aim of the study was analysis of severe complications and treatment related mortality (TRM) in early period post allo-SCT. Materials (or patients) and methods: Medical records of 526 patients aged from 1month to 21 ys (median 9.4ys), who underwent first procedure of allo-SCT in Department of Pediatric STC, Hematology and Oncology in Wroc"aw were analyzed. Since 2007 range of diagnostic methods (regular determination of EBV, ADV, BKV by quantitative method of PCR, and fungal antigens) and infection prophylaxis (antifungal drugs including Aspergillus spp. and Ciprofloxacin for BKV-induced cystitis) were expanded, therefore the patients cohort was divided into two groups. Group I:231 patients transplanted between Jan.2002-Dec.2006 (59 matched sibling donor MSD ,135 matched donor MD, 37 mismatched donor MMD). Group II: 294 patients (74 MSD, 188 MD, 32 MMD) transplanted in the period Jan. 2007-Mar.2014. Frequency of early severe complications ( until day 180 ) such as: organ damage, infections, acute graft versus host disease (GvHD) and TRM were evaluated depending on type of stem cell donor (MSD vs alternative donor, including MD and MMD). Results: Early life-threatening complications in group I and group II were as follow: Fever of unknown origin (FUO) 64.9% vs 63% (P ¼ 0.59), severe toxic mucositis 47.2% vs 47% (P ¼ 0.95), bacterial infection 32.5% vs 21.8% (P ¼ 0.013), fungal infections 21% vs 14.5% (P ¼ 0.06), BKV induced haemorhagic cystitis (HC) 25.5% vs 15.3% (P ¼ 0.0046), CMV infection 28% vs 36.1% (P ¼ 0.0428), VOD 9.5% vs 7.1% (P ¼ 0.3234), neurological disorders 17.8% vs 15.3% (P ¼ 0.45), severe aGvHD 22% vs 24.1% (P ¼ 0.334), respiratory failure 23.8% vs 12.7% (P ¼ 0.0016), EBV-PTLD 6.1% vs 6.5% (P ¼ 0.88). TRM 19.9% vs 17.7% (P ¼ 0.67). After MSD transplantation major TRM causes in in group I and II were: sepsis 37.5% vs 18.2%, aGvHD 12.5% vs 18.2%, viral infections 12.5% vs 9% neurological disorders 12.5% vs 9%, VOD 0% vs 27.3%, MOF 0% vs 18.2% HC 12.5% vs 0% (P ¼ 0.2). Post transplantation from alternative donor, major causes of TRM in group I and II were as follow: aGvHD 7.8% vs 39% (P ¼ 0.0012), sepsis 26.4% vs 21.9%, MOF 13.5% vs 19.5%, viral infections 18.4% vs 7.3% (P ¼ 0.05), VOD 5.2% vs 2.4%, rejection 7.8% vs 0% (P ¼ 0.06), fungal infections 0% vs 4.9%. Conclusion: 1. Extended prophylaxis significantly decreased frequency of HC and systemic fungal infections. 2. Regular EBV diagnostics had no influence on occuring of EBV-PTLD. 3. Despite decrease in frequency of life-threatening complications no TRM decrease was noticed. Only the causes of death structure has changed. 4. As aGvHD is actually major cause of TRM in early period after allo-SCT it would be expedient to extend early diagnostic including monitoring of plasma GvHD biomarkers such as ST2. Disclosure of Interest: None declared.
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P193 Sistemic Inflamatory Response Syndrome (SIRS) associated to stem cell infusion in haploidentical transplantation recipients F. Lopez1,*, S. Alonso2, M. Manzanares3, O. Lopez-Godino1, O. Ferre´1, L. Garcia2, E. Perez-Lopez2, A. Redondo-Guijo2, F. Sanchez-Guijo2, L. Vazquez2, M. C. del Can˜izo2, D. Caballero2, L. Lopez-Corral2 1 HOSPITAL UNIVERSITARIO DE SALAMANCA, salamanca, Spain, 2 hematologia, HOSPITAL UNIVERSITARIO DE SALAMANCA, Salamanca, 3Hospital of Jerez, Jerez de la Frontera, Spain Introduction: Haploidentical stem cell transplantation has been used as an option for patients needing rapid allogeneic stem cell transplantation (SCT) in the absence of a sibling donor. Some specific side effects have been reported, some of them being considered a probable consequence of a cytokine release associated with activation of donr T cells after graft infusion Materials (or patients) and methods: This is a retrospective descriptive study in which we included all consecutive patients receiving haploidentical stem cell transplantation in our Hospital Results: 26 patients were included in this study. Median age was 58 years (17-68).Gender M/F ratio was 14/12. Underlying diseases were lymphoblastic (n ¼ 2, 8%) and myeloblastic leukemia/myelodysplasia (n ¼ 14, 54%), Hodgkin (n ¼ 7, 27%) and non-Hodgkin (n ¼ 1, 4%) lymphomas, myelofibrosis (N ¼ 2, 8%). Disease status at transplant was:complete response in 12 patients (46.2%), minor residual disease in 2 patients (7.6%), partial response in another patient (3.8%), aplasia without blasts in 3 patients (11.5%), active disease in 3 patients (11.5%), untreated relapse in 3 (11.5%). Some patients had received a prior transplant: ASCT 30%, and allogeneic 20%. 1 patient had graft failure after a previous cord transplant. Conditioning regimen consisted on busulfan (BUS), fludarabine (FLU) and cyclophosphamide (CTX) in 17 patients, and in BUS, FLU & thiothepa in 6. Three (11.5%) received total body irradiation. Graft versus host disease prophylaxis (GVHD) was tacrolimus (TCR) plus mycophenolate (MMF) starting on day þ 5, & CTX at days þ 3 & þ 4 in 23 patients (88.5%), those receiving non-myeloablative conditioning and days þ 3 & þ 5 in the remaining 3 (11.5%) who received myeloablative conditioning. MMF was dropped out at day þ 35 while TCR was maintained until day þ 80/100 if disease was in complete remission. 25 patients (96%) had fever around SC infusion day. All received antibiotics when fever was onset. In 7, fever started before infusion with an infectious origin; 18 (78%) developed fever during the first 24 hours after SC infusion. Only in one there was microbiological documentation and fever persisted until engraftment. From the remaining 17, in 14 (82%) fever resolved at day þ 4 or þ 5, while in the other 3, fever spontaneously disappeared within the first 24 hours. In all of these 17 patients, median PCR value at infusion day was 1 mg/dL (0-2) (upper limit 0,5), and it rose up to 7.5 mg/dL (2-19) at day þ 2 and went down to 4.0 mg/dL (0-9.1) at days þ 4 or þ 5. Conclusion: An important part of our haplo patients developed fever closed to stem cell infusion. Although all of them received antibiotics, the low PCR at fever onset suggest a noninfectious origin. Cytokine storm around stem cell infusion has been already reported previously, however our patients did not have other associated symptoms suggesting that several degrees of this cytokine release is possible in the haploidentical transplant setting. Biological studies are required for understanding these phenomena. References: 1. Luznik L, O’Donnell P, Ephraim JF: Post-transplantation cyclophosphamide for tolerance induction in HLA-haploidentical BMT. Semin Oncol. 2012; 39(6): 10
2. Colvin GA, Berz D, Ramanathan M, Winer ES, Fast L, Elfenbein GJ, Quesenberry PJ. Nonengraftment haploidentical cellular immunotherapy for refractory malignancies: tumor responses without chimerism. Biol Blood Marrow Transplant 2009; 15(4): 421–431. Disclosure of Interest: None declared. P194 Superior efficacy of piperacillin-tazobactam vs. ceftriaxone for the prophylaxis of neutropenic fever in lymphoma patients managed at-home after autologous stem cell transplantation conditioned with BEAM F. Fernandez Aviles1,*, M. Rovira1, C. Martı´nez1, M. Sua´rez-Lledo´1, N. Martı´nez1, A. Gaya1, C. Gallego1, A. Hernando1, S. Segura1, J. Gu¨ell1, A´. Urbano-Ispizua1 1 Haematology, HOSPITAL CLI´NIC I PROVINCIAL, Barcelona, Spain Introduction: Bacterial infections remain a major cause of morbidity in patients undergoing autologous stem cell transplantation (ASCT). In this study we analyze the efficacy of primary prophylaxis with either ceftriaxone (Ct) or piperacillin-tazobactam (P-T), in two consecutive cohorts of patients managed at-home receiving BEAM as preparative regimen. Materials (or patients) and methods: Between March 2007 and September 2014, 75 consecutive patients received an ASCT for Hodgkin and Non-Hodgkin lymphomas and all were managed at-home since day þ 1. All patients received the same conditioning regimen with BEAM (mg/m2): BCNU 300, etoposide 800, cytarabine 800 and melphalan 140. The first group of patients received prophylaxis with Ct 1 g/day i.v. (n ¼ 28; group A) and the remaining patients with P-T 4.5 g/8h i.v. (n ¼ 47; group B). Both started these antibiotics on day þ 1 and were maintained until febrile neutropenia or granulocyte count over 1x109/l. First-line therapy of neutropenic fever was P-T 4.5 g/6h i.v. in group A, and meropenem 1 g/8h i.v. in group B. The nurse visited the patient at-home once or twice daily, while the physician in the hospital visited the patient only in case of clinical complications. Indications for readmission to the hospital were: willingness of the patient or caregiver; uncontrolled nausea, vomiting or diarrhoea; mucositis requiring total parenteral nutrition or i.v. morphics; fever with focal infection or signs of severe sepsis. Results: There were no differences between the two groups with respect to the age of the patients, sex, performance status, diagnosis, stage of disease, and source of stem cells. The only difference was the median quantity of CD34 þ cell dose (x106/kg) infused: 3.8 (1.5-21) in group A and 5.9 (1.617.6) in group B; P ¼ 0.0001. After the transplant, there were no differences in the duration of neutropenia r0.5 and o0.1x109/l between groups. Whereas the incidence and intensity of oral and gastrointestinal toxicity (NCI-CTC-score) were similar in both groups, neutropenic fever occurred in 82% of patients in group A and 53% in B (P ¼ 0.02). Patients of group A required two daily visits by nurses at-home more frequently (64% vs. 30%; P ¼ 0.007) but not more physician visits in the hospital (14% vs. 16%). Bacterial infection was documented in 2 febrile episodes in group A and 5 in group B. Re-admission was required for persistent fever in 6 (21%) patients in group A, and 1 (2%) in group B (P ¼ 0.009). Conclusion: This study suggests that the use of prophylaxis with piperacillin-tazobactam reduces significantly the incidence of febrile neutropenia and the need for hospital readmission in patients receiving BEAM, optimizing both domiciliary and hospital resources. Disclosure of Interest: None declared.
P195 Mega-dose double conditioning chemotherapy of thiotepa and melphalan is feasible about renal function for the treatment of pediatric solid tumor F. Yamazaki1,*, C. Kiyotani1, Y. Shioda1, T. Osumi1, K. Terashima1, D. Tomizawa1, K. Matsumoto1 1 Children’s Cancer Center, National Center of Child Health and Development, Tokyo, Japan Introduction: Thiotepa (TT) and melphalan (LPAM) are alkylating anti-cancer drugs which are often used as conditioning regimen of high dose chemotherapy (HDC) for treating pediatric solid tumors. Hara et al. reported feasibility and efficacy of double conditioning regimen including TT and LPAM with one-week interval. Due to excessive dosage of LPAM, patients undergoing this therapy are at risk of renal insufficiency. Here, we retrospectively analyzed our institutional experience of the double-conditioning regimen for patients with solid tumors, focusing on renal toxicity and its efficacy to control tumors. Materials (or patients) and methods: Thirty-eight patients with solid tumors (neuroblastoma 18; brain tumor 13; other 7) were treated by the double-conditioning regimen with TT and LPAM followed by peripheral blood stem cells or bone marrow rescue between March 2003 and January 2010. The double conditioning regimen consisted of two cycles of TT and LPAM with a 1-week interval. TT (patients aged 42 years; 200 mg/ m2/day, o2 years; 8 mg/kg/day) and LPAM (patients aged 42 years; 70 mg/m2/day, o2 years; 1.5 mg/kg/day) were administered on days -12, -11, -5, and -4. When creatinine clearance (Ccr) waso100 ml/min/1.73m2 in patients aged 42 years, dosage was adjusted according to the following formula: given dose (mg/m2) ¼ (Ccr/100) 200 mg/m2/day (TT) or 70 mg/m2/day (LPAM). We analyzed renal function before and after HDC of the patients who had sufficient medical records and who did not received additional treatment for one year after HDC. Results: Four out of 27 patients experienced 1.5 times more elevation of serum creatinine level at one year post-HDC compared with one at pre-HDC, but only one patient had creatinine level higher than institutional normal range at one year post-HDC. The ratio of serum creatinine at one year after HDC to pre-HDC was 0.75-3.20 (median 1.19). This ratio was significantly higher in neuroblastoma patients (1.55; 95% CI, 1.18 to 1.92) than in other solid tumors (1.13; 95% CI, 1.05 to 1.21) (P ¼ 0.034). Ccr before HDC was associated with the elevation of serum creatinine level after HDC (P ¼ 0.041). We analyzed progression free survival (PFS) in 18 patients with neuroblastoma. Median follow-up time of the 9 patients who remain alive without progression is 5.3-12.0 years (median 7.8 years). PFS and overall survival (OS) at 5 years from diagnosis were 56% (95% CI, 33% to 79%) and 72% (95% CI, 52% to 93%), respectively. One patient died from transplant-related toxicity. Relapse occurred in 8 (44%) of 18 patients. Conclusion: HDC using the double conditioning regimen comprised of two cycles of TT and LPAM with a one-week interval is acceptable in renal toxicity and effective for treating high-risk neuroblastoma. Renal insufficiency after the double conditioning regimen could be predicted by the Ccr before HDC even in patients who received renal adjusted dose of TT and LPAM. For the patients with potential risk of renal insufficiency, such as patients with low Ccr, this mega-dose HDC should be used with meticulous care. References: Hara J et al. Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors. Bone Marrow Transplant. 1998; 22: 7-12 Disclosure of Interest: None declared.
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P196 Safety and feasibility of romiplostim treatment for patients with persistent thrombocytopenia after allogeneic stem cell transplantation G. Battipaglia1,2,*, A. Ruggeri1, A.-C. Mamez1, L. Drevon1, F. Malard1, A. Gomez1, E. Brissot1, R. Belhocine1, A. Vekhoff1, S. Lapusan1, F. Isnard1, O. Legrand1, T. Ledraa1, M. Labopin1, M.-T. Rubio1, M. Mohty1 1 Hoˆpital Saint-Antoine, Hoˆpital Saint-Antoine, Paris, France, 2 Federico II University of Naples, Federico II University of Naples, Naples, Italy Introduction: Prolonged thrombocytopenia after allogeneic stem-cell transplantation (SCT) is a common complication, with a negative impact on survival. It can occur as secondary failure platelet recovery (SFPR) defined as platelet (PLT) counts o20,000/mL or requiring transfusion support after achieving sustained counts Z50,000/mL without transfusions for 7 consecutive days after SCT. Furthermore, thrombocytopenia could lead to potentially lethal bleeding, thus needing the use of iterative PLT transfusions. There is a lack of effective and reliable methods to promote PLT recovery and to prevent hemorrhagic complications and PLT transfusion needs in SCT recipients. The use of the weekly subcutaneous recombinant thrombopoietin agonist romiplostim (Rm) could be an attractive option, but many concerns still exist about its safety and feasibility after SCT. Materials (or patients) and methods: We report the use of Rm in 7 thrombocytopenic patients (pts) who experienced thrombocytopenia after SCT. All pts were transplanted for high risk hematological malignancies (4 acute myeloid leukemia, 1 myelodysplastic syndrome, 1 non Hodgkin lymphoma, 1 T-cell leukemia/lymphoma) in complete remission. Results: Two pts had received a previous allo-SCT, and one an auto-SCT. Median follow up after SCT is 249 days (range 115-520). Median age at SCT was 58 (range 16-67) years. All pts received peripheral blood stem cell as stem cell source, 5 from an haploidentical donor, 1 a sibling donor and 1 a matched unrelated donor. All pts achieved neutrophil engraftment with full donor chimerism. Five pts had PLT engraftment in a median of 11 days after SCT (range 11-41),while 2 were on therapeutic PLT transfusions for concomitant hemorrhagic cystitis (HC). In the engrafted pts SFPR was observed at a median of 140 days after SCT (range 121-313) in 3 patients, while 2 pts developed HC with transfusions when PLTr50000/mL. Thrombocytopenia was of central origin in 4 pts, peripheral in 2 pts, mixed in 1 patient. Reason for starting Rm was therapeutic transfusion dependence in 4 pts with HC and SFPR in 3 pts. Median time between SFPR or therapeutic transfusion dependence and start of Rm was 23 and 37 days, respectively. Starting dose was 3 mg/kg with weekly escalation of 1 mg/kg till a maximal dose of 10 mg/kg (reached in 3 pts). Median number of injections was 7 (range 5-29). All SFPR pts achieved PLT 450000/mL without transfusion after a median of 2 injections (range 1-5), while HC and transfusion needs weren’t significantly improved by Rm. Three pts had concomitant aGVHD, 2 presented thrombotic thrombocytopenic purpura signs, 6 experienced CMV reactivation, 3 infectious complications, and 3 received concomitant myelotoxic therapies. Three non-responders pts received a CD34 þ boost due to overall poor graft function. PLT recovery was achieved in all but one, who needed to restart Rm. Conclusion: Overall Rm was well tolerated and no patient needed to discontinue drug treatment because of adverse events, making this agent a feasible therapeutic option after allo-SCT. As to its efficacy, we can speculate in SFPR that Rm improved PLT counts but in hemorrhagic patients it didn’t either reduce PLT transfusion needs or hemorrhagic syndrome. However, these preliminary results need to be confirmed in larger studies with longer follow-up to improve assessment of efficacy and safety profiles in this setting. Disclosure of Interest: None declared.
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P197 Erythrocytosis following allogeneic hematopoietic stem cell transplantation in six cases G. Pekcan1,*, E. Atilla1, P. Ataca1, P. Topcuoglu1, A. Uslu2, S. Civriz Bozdag1, M. Kurt Yuksel1, O. Arslan1, M. Ozcan1, O. Ilhan1, H. Akan1, M. Beksac1, G. Gurman1, S. K. Toprak1 1 ANKARA UNIVERSITY DEPARTMENT OF HEMATOLOGY, 2ANKARA UNIVERSITY DEPARTMENT OF INTERNAL MEDICINE, ANKARA, Turkey Introduction: Post transplant erythrocytosis (PTE) is not an expected complication after allogeneic hematopoietic stem cell transplantation (HSCT). Only Mahmood et al reported PTE in 3 aplastic anemia cases in literature (1). We present PTE in 3 acute myeloblastic leukemia (AML), 2 paroxysmal nocturnal hemoglobinuria (PNH) and 1 myelodysplastic syndrome (MDS) from 945 allogeneic HSCT in our center. Materials (or patients) and methods: Patient 1: AML, 38 years old, male, second complete remission, HLA fullmatch related donor, Cyclosporin (CsA) þ methotrexate (MTX) for graft versus host disease (GVHD) prophylaxis, myeloablative conditioning regimen. Grade 2 acute skin GVHD, liver chronic GVHD. In remission, no complications. Patient 2: PNH/bone marrow failure, 29 years old, male, HLA fullmatch related donor, CsA þ MTX for GVHD prophylaxis, reduced intensity conditioning regimen. Grade 1 acute skin GVHD. In remission, no complications. Patient 3: AML, 49 years old, male, second complete remission, HLA fullmatch related donor, CsA þ MTX for GVHD prophylaxis, myeloablative conditioning regimen. Grade 1 chronic skin, oral mucosa and liver GVHD. In remission, followed by skin, liver, eye and lung (bronchiolitis obliterans (BO)) GVHD. Patient 4: MDS, 17 years old, male, 1 antigen mismatch unrelated donor, CsA þ metilprednisolon for GVHD prophylaxis, myeloablative conditioning regimen. Grade 3 acute skin, liver and gastrointestinal GVHD. In remission, no complications. Patient 5: PNH/bone marrow failure, 26 years old, male, HLA fullmatch related donor, CsA þ mycofenolatemofetil (MMF) for GVHD prophylaxis, reduced intensity conditioning regimen. In remission, no complications. Patient 6: AML, 33 years old, male, second complete remission, HLA fullmatch related donor, CsA þ MTX for GVHD prophylaxis, myeloablative conditioning regimen. Grade 2 acute liver GVHD. In remission, no complications. Results: During follow-up, patients’ hemoglobin (gr/dL) and hematocrit (%) values are as listed: 17,9 and 52 in 1st patient at 5th year; 17,6 and 51 in 2nd patient at 3rd month; 17,5 and 54,5 in 3rd patient at 4th year ; 17,7 and 51,7 in 4th patient at 4th year, 17,5 and 55 in 5th patient at 3rd year; 17,6 and 51 in 6th patient at 6th year. None of them had either leukocytosis or thrombocytosis. All of the patients had hyperviscosity symptoms with no organomegaly. All the patient had erythropoietin levels in the upper limit of normal. No JAK2V617F mutation positivity was detected. None of the patients had pulmonary symptoms or oxygen requirement. Conclusion: Liver GVHD, BO and cyclosporine could lead to secondary erythrocytosis however there is no evidence for causality. All of our patients were male which is a risk factor for erythrocytosis similar to hypertension and smoking.Remarkably, all of the patient except one had erythrocytosis three years after allogeneic HSCT. Further surveillance of the cases will improve our knowledge over erythrocytosis. References: 1. Bone Marrow Transplantation 2011; 46: 1163– 1165. Disclosure of Interest: None declared. P198 Improving the re-vaccination programme for patients post allograft H. Leonard1,*, C. Anthias1 1 THE ROYAL MARSDEN , Sutton, United Kingdom Introduction: International consensus guidelines recommend commencing re-vaccinations at 3-6 months post-transplant,
even in patients on immune suppression. We carried out an audit to determine compliance with international revaccination guidelines at the Royal Marsden Hospital. Materials (or patients) and methods: Electronic patient records of 100 consecutive patients who received allogeneic transplants between Nov 2012 and March 2014 were retrospectively examined. Information collected included whether revaccinations had been commenced at 21st October 2014, number of months post transplant that revaccinations were commenced, and whether revaccinations schedules had been sent to general practitioners. Patient clinical status (remission, relapse and presence or absence of GVHD) was also recorded. Results: 22 patients had died or relapsed within 6 months of allograft and were therefore excluded from further analyses. Of the remaining 78 patients, 47 (60%) had commenced the revaccination programme. 7 (14%) of these were international patients who had received vaccinations in their home country. Of the 47 that had received vaccinations, practice was inconsistent with respect to timing of when the vaccinations commenced. The reasons for failure to initiate revaccinations in the remaining 31 patients (40%) were examined. These patients tended to be more complex, with recurrent hospital admissions. There was an inconsistent approach by the medical team to commencing vaccinations for patients with chronic GVH on immunosuppression, with some team members recommending delaying the start of the vaccination programme. Documentation of both discussion of the vaccination programme with patients and GPs and of when/if vaccinations had commenced was also variable and difficult to identify from patient records. Conclusion: This audit raises several issues with our approach to revaccination. There is currently no formal method of ensuring and documenting that vaccinations for post transplant patients are carried out. We have therefore proposed the following changes: Prior to transplantation, all patients will have a clinic appointment set up for 6 months post transplant with the post transplant CNS. At this appointment the rationale for revaccinations will be discussed with patients, a standard letter with the revaccination schedule will be posted to the GP with a request to arrange vaccinations. This will be then clearly documented. The post transplant CNS will follow up all patients by telephone 1 month after this 6-month appointment to ensure vaccinations have commenced and to answer any queries. For the international patients a reminder will be sent by e-mail to the referring consultant with a vaccination schedule. Lastly, to address the varied practice by the medical team in recommending timing of revaccination, the vaccination SOP will be reviewed; contraindications to vaccination agreed with the transplant lead and subsequent training of the medical team will be arranged, ensuring consistency in practice. We will re audit in 6 months to evaluate the effectiveness of these changes. Disclosure of Interest: None declared. P199 Successful treatment with dexamethasone palmitate for patients with post-transplant cytokine storm syndrome H. Sakaguchi1,*, K. Matsumoto2, K. Narita1, M. Hamada1, S. Kataoka1, N. Yoshida1, M. Ito3, K. Kato1 1 Department of Hematology and Oncology, Children’s Medical Center, Japanese Red Cross Nagoya First Hospital, Nagoya, 2 Children’s Cancer Center, National Center for Child Health and Development, Tokyo, 3Department of Pathology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan Introduction: Post-transplant cytokine storm syndrome (PTCSS) is an early complication after hematopoietic stem cell transplantation (HSCT) caused by a massive release of proinflammatory cytokines, macrophage colony-stimulating
factor, erythropoietin, products of degranulation and oxidative metabolism, in which activated macrophage play a key role, and consists of hyper acute graft versus host disease (GVHD), pre-engraftment immune reaction, engraftment syndrome, idiopathic pneumonia syndrome, and hemophagocytosis. Standard therapy for PTCSS is not established yet. Liposomeincorporated dexamethasone, dexamethasone palmitate (DP), is known to suppress activated macrophages by readily taken up via phagocytosis and strongly retained in the cytoplasm. In this retrospective study, we aimed to identify the incidence of PTCSS, evaluate the efficacy, feasibility, and clinical impact of DP treatment for PTCSS in HSCT. Materials (or patients) and methods: We reviewed retrospectively 139 consecutive HSCT (autograft, n ¼ 17; allograft, n ¼ 122) for children (median age at HSCT 8.6 years; range, 0.5–18.2) with hematological malignancies (n ¼ 93), solid tumors (n ¼ 15), and non-malignant disorders (n ¼ 31), performed in our institution between April 2006 and September 2014. Allografts were donated from family members (n ¼ 35), unrelated volunteer donors (n ¼ 39), or cord blood (n ¼ 48). Among patients with malignant disease, 44 patients received HSCT at high risked stage; third remission or non-remission. The diagnosis of PTCSS consists of non-infectious cause of fever more than 3days with skin rash, pulmonary infiltration, or bone marrow aspiration findings; proliferation of activated macrophage or phagocytosis at peri-engraftment period. DP treatment (2.5 mg/m2/day i.v. for 3 consecutive days) indicated for all patients diagnosed as PTCSS, and on demand DP treatment for refractory cases. Results: Forty-five patients (33%) were diagnosed as PTCSS and treated with DP for 3 days at median day 14 (range, 7–22) after HSCT. PTCSS was resolved in 39 of 45 cases; 6 patients needed additional methylprednisolone treatment. Of 45 patients with PTCSS, graft failure was concomitantly observed in 3 patients donated from sibling (n ¼ 1) and unrelated cord blood (n ¼ 2). The interval from PTCSS to engraftment: median, 5 days (range, 0–27 days). The incidence of acute GVHD for patients with PTCSS was as comparable as that of patients without PTCSS (12% versus 12%, P ¼ 0.99), however, the incidence of chronic GVHD of the former was higher than that of the latter (18% versus 8% in 5 years, P ¼ 0.11). The incidence of transplant related mortality for patients with PTCSS was as comparable as that of patients without PTCSS, on the other hand, relapse rate in malignant disease of the former was lower than that of the latter (32% versus 46% in 5 years, P ¼ 0.06). Finally, the overall survival of patients with PTCSS was superior to that of patients without PTCSS (76% versus 55% in 5 years, P ¼ 0.10), even in high risked patients’ cohort (57% versus 36% in 5 years, P ¼ 0.10). Conclusion: Our results suggested that DP could control the PTCSS, at the same time, could save the immune reaction which was important for graft versus leukemia effect; DP could control acute allo-reaction effectively, but not for later immune reaction. Prospective multi-institutional study is warranted to confirm our findings. Disclosure of Interest: None declared. P200 Evaluation of The Long-Term Effects of Hematopoietic Stem Cell Transplantation on Oral and Dental Health of Children After Transplantation for Benign and Malign Hematologic Diseases ¨ . M. Akgu¨n1, C. Yıldırım1, I. Eker2,*, O. Gu¨rsel2, O. Babacan2, O F. Bas¸ak1, A. E. Ku¨rekc¸i2 1 Pediatric Dentistry, 2Pediatric Hematology, Gulhane Military Medical Faculty, Ankara, Turkey Introduction: Because of the severe immunosuppression in chilren after hematopoietic stem cell transplantation (HSCT), any problem in the oral cavity may become life-threatening and may increase the complications, treatment costs and duration of hospitalization. In this study, pre-transplant and post-transplant dental health evaluation results of pediatric
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patients with benign and malign hematological diseases, who underwent HSCT at Gulhane Military Medical Faculty, Pediatric Hematopoietic Stem Cell Transplantation Center are presented. Materials (or patients) and methods: DMFT index (decayed, missing, filled teeth index), plaque index (PI), gingival index (GI) and bleeding on probing (BOP) scores, which were obtained from oral and dental health assessment of 37 HSCT patients were analyzed retrospectively. In our center, HSCT patients are evaluated for oral and dental health at pretransplant period and on post-transplant 100th day and 6th, 12th and 24 th months. Pedodontist, who made the evaluation of the patient at pretransplantation period, continued the all other evaluations on the time points above. Assessment was made with the reflector light by using mirror and periodontal probe. Results: The median age of the patients was 10 (1-18) years, 10 were female and 27 were male. Oral and dental health assessments of all patients were completed on the pretransplant period and on post-transplant 100 th day. But on post-transplant 6th month 94,6%, on 12 th month 81% and on 24 month only 33% of the patients’ oral and dental health assessments could be completed. The remaining of the asessments could not be done because of the incompliance of the other patients. The patients’ pre-transplant and posttransplant 100 th day and 6 th month oral and dental health evaluation results of DMFT index GI, PI and BOP were very similar. But patients’ decay teeth number, formation of plaques, gingival inflammation and bleeding scores were increasing, especially starting at 12th month evaluation and become more prominent at 24th. In other words, patients’ adherence to oral and dental health care were decreasing and proportional with these, oral and dental problems were increasing especially starting at 12th month after HSCT and become more prominent at 24th. Conclusion: Our findings showed an increase in oral, periodontal and gingival problems after HSCT, especially in the longterm period, which was consistent with other literatures on the issue. This is because of the decreasing importance, given to oral health at post-HSCT period. It is very important to take measures to ensure regular assesments of patients’ pedodontic monitoring and treatment of the problems in post transplant long term period, like as in the pre-transplant and post-transplant early periods. Preparation of guidelines and patient education programs about oral and dental health in HSCT, together by pedodontists and pediatricians; and regularly pedodontic evaluations on the early and late postHSCT periods are of extremely important for the success of transplantations. Disclosure of Interest: None declared.
[P200]
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P201 Comprehensive Pulmonary Rehabilitation in patients with pulmonary chronic graft versus host disease I. C. Song1,*, S. J. Jee2, S. W. Baek1, Y. S. Choi1, H. W. Ryu1, J. Y. Moon1, H. J. Lee1, H. J. Yun1, S. Kim1, K. H. Cho2, D. Y. Jo1 1 Internal Medicine, 2Rehabilitation Medicine, Chungnam National University Hospital, Daejeon, Korea, Republic Of Introduction: Pulmonary chronic graft versus host disease (cGVHD) is a serious late complication of allogeneic hematopoietic stem cell transplantation (HSCT), which hampers quality of life and increases mortality of survivors. Medical treatments have limited efficacy at improving pulmonary function. We investigated the therapeutic effects of comprehensive pulmonary rehabilitation (CPR) in patients with deteriorated lung function due to pulmonary cGVHD. Materials (or patients) and methods: We retrospectively analyzed the medical records of patients who had pulmonary cGVHD and participated in the CPR program, which consisted of aerobic exercise, inspiratory muscle strengthening, accessory respiratory muscle strengthening and mechanically assisted coughing. The subjects were planned to undergo maximal cardiopulmonary exercise test and a body composition test with bioelectrical impedance analysis at baseline and 3 months. Exercise testing parameters, skeletal muscle mass of lower extremities and body mass index (BMI) were analyzed. Results: Nine patients were assessed (two males, seven females; median age 40 (range 24–64) years). Donor lymphocyte infusion was performed in two patients. All patients had skin, mucosal and visceral manifestations, in addition to pulmonary cGVHD. When starting CPR, the median FEV1 was 25% (range 18% – 51%). Eight patients received concomitant corticosteroid and three received imatinib. At a median followup of 11 (range 2–16) months, the patients had participated in the CPR program for a median of 40.5 (range 10–148) days. Eight patients (88.9%) had completed more than 20 sessions of the CPR program. The rehabilitation programs were readily tolerable and had no adverse effects. The median change in the maximum oxygen consumption (VO2max) was 28.5% (range –31.8% to 60.1%) and the muscle mass of the lower extremities changed by 3.0% (range –0.2% to 24.1%). All four patients who had an increased muscle mass in the lower extremities had an increase in VO2max. There were no marked changes in BMI. Conclusion: Our comprehensive pulmonary rehabilitation program for patients with pulmonary cGVHD with limited lung function was feasible and improved the exercise capacity in a population of patients. Disclosure of Interest: None declared.
P202 Prospective, randomized trial of physical function in patients before and after haematopoietic stem cell transplantation H. Schumacher1, S. Stru¨we1, N. Greger1, P. Blaschke1, S. Freitag1, C. Junghanss1, I. Hilgendorf1,2,* 1 University Hospital Rostock, Rostock, 2Jena University Hospital, Jena, Germany Introduction: Physical and sports therapy during stationary treatment and aftercare operations, in patients receiving hematopoietic stem cell transplantation (HSCT), is becoming more and more important. Positive effects are known for strain dependent physical practice after HSCT. Nevertheless, there is lack of capacities and problem orientated therapies. Materials (or patients) and methods: Methods: Here we report on a prospective, randomized study comparing a multimedia sensor-based practice with a classical physiotherapeutic treatment in 30 HSCT recipients. Both groups performed the exercises under the supervision of a physical therapist. Physical function was evaluated at the date of hospital admission (T1), 14 days after HSCT (T2) and 30 days after HSCT (T3). The conditional status was assessed by Treadmill and a two minute walk test. Furthermore, a grip strength test was performed with a hydraulic hand dynamo meter and balance was evaluated using the Berg Balance Scale. Results: Patients after HSCT for hematologic malignancies were randomized into the control group receiving physiotherapeutic treatment (n ¼ 17) or the experimental group with exercising on the Nintendo Wii (n ¼ 13). The median age of patients in the control group was 60 (46–69) years and 55 (22– 69) years in the experimental group. Treadmill intervention showed a median performance of 100 W (25–125) (T1), 75 W (25–100) (T2) and 25 W (25 - 75) (T3) in the control group and 100 W (75 - 125), 50 W (50 - 125), 50 W (50 - 125) in the Nintendo Wii-group, respectively. In the two minute walk test the median distances in the control group were 166 m (105 211) (T1), 154 m (73 - 208) (T2), 147 m (105 - 204) (T3), while the experimental group walked an average distance of 166 m (134 - 210), 143 m (60 - 196) and 139 m (124 - 156). The grip strength test resulted in mean values of 35 kg (13 - 51) (T1), 34 kg (11 - 53) (T2), 27 kg (11 - 50) (T3) in the control group and 37 kg (19 - 61), 34 kg (13 - 60), 32 kg (21 - 53) in the experimental group, respectively. The control group scored a mean value of 55 (52 - 56) (T1), 53 (34 - 56) (T2), 53 (52 - 56) (T3) in Berg Balance Scale (max. 56) compared with 56 (54 56), 54 (47 - 56), 56 (55 - 56) in the experimental arm. Conclusion: Discussion: The functional status of patients decreased after HSCT in both groups and impairment remained at least until one month (T3) after HSCT. However, compared to classical physiotherapeutic treatment the group with sensor-based practice showed a slightly lower endurance, while none differences were observed in balance testing. Exergaming is feasible as well as enjoyable in the stationary setting of HSCT and may be used in addition to physiotherapeutic treatment. Disclosure of Interest: None declared. P203 Reduced Incidence of CMV Reactivation with Prophylactic Use of Ganciclovir in Allogeneic MUD Transplants Using Alemtuzumab as Part of Conditioning J. Vogelaar1,*, V. Rodriguez1, A. Nageswara Rao 1, J. Gourde1, S. Khan1 1 Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, United States Introduction: Cytomegalovirus (CMV) disease is a cause for morbidity and mortality in the allogeneic transplant setting. Ganciclovir is an effective treatment for CMV infection. However, given concern for myelosuppression and other side effects, there continues to be a debate between prophylactic
versus preemptive therapy in allogeneic transplant patients. At our institution, alemtuzumab (Campath) is part of the conditioning regimen for matched unrelated donor (MUD) transplants. Due to concern of increased risk of early and late CMV disease secondary to alemtuzumab-induced T-cell depletion, it is our standard practice to use ganciclovir prophylactically in these patients. In contrast, in patients undergoing matched related donor (MRD) transplantation, we use preemptive ganciclovir therapy upon CMV reactivation. Materials (or patients) and methods: We reviewed data in our allogeneic transplant patients (MUD transplants: n ¼ 42; MRD transplants: n ¼ 37) from 2003-2014. All patients with matched unrelated donors who were CMV seropositive, or CMV seronegative (-ve) with a seropositive ( þ ve) donor, were started on ganciclovir once their absolute neutrophil count (ANC) was greater than 1000 and continued until day þ 100. Patients with matched related donors and similar CMV status received preemptive ganciclovir upon CMV reactivation. Weekly CMV PCR was obtained for close followup. Results: Of the 42 patients with unrelated donors who received Campath, 66.6 % were at risk for reactivation. They included recipients CMV þ ve (n ¼ 14), recipients CMV-ve and donors CMV þ ve (n ¼ 6), recipients and donors CMV þ ve (n ¼ 8). Only four (9.5%) had CMV reactivation (days þ 9 through þ 60). Only one patient was on ganciclovir prophylaxis at the time of reactivation (day þ 60, started ganciclovir day þ 24). One patient was on ganciclovir prophylaxis for only two days prior to reactivation. The two remaining patients were not on ganciclovir prophylaxis due to an ANC of less than 1000. Among patients receiving prophylaxis, only one patient had discontinuation of ganciclovir due to myelosuppression. However, this patient’s cause of myelosuppression remains disputed as the patient had also been on cidofovir due to urine and plasma BK virus activity. This patient was started on ganciclovir on day þ 22 and both cidofovir and ganciclovir were discontinued on day þ 40 due to continued myelosuppression. Of the 37 patients who underwent MRD transplants and were followed closely with CMV PCR, two had CMV reactivation (5.6%), at day þ 26 and day þ 31. Both patients were started on ganciclovir. Unfortunately, one patient died seven days later due to veno-occlusive disease, multi-organ failure, and CMV viremia. The other patient completed a 14 day course of twice daily ganciclovir with resolution of CMV infection. Conclusion: Based on our review, ganciclovir prophylaxis reduced the incidence of CMV reactivation despite using Campath in MUD transplants. No patient had active CMV disease and there were no deaths due to CMV infection. The incidence of reactivation in these patients is comparable to our matched sibling transplants that do not receive Campath, and only receive preemptive therapy on CMV reactivation. Disclosure of Interest: None declared. P204 Poor outcome following ciclosporin-related neurotoxicity in paediatric allogeneic haematopoietic stem cell transplantation K. Straathof1,*, P. Anoop1, Z. Allwood1, J. Silva1, O. Nikolajeva1, R. Chiesa1, P. Veys1, P. Amrolia1, K. Rao1 1 Department of Blood and Marrow Transplantation, Great Ormond Street Hospital, London, United Kingdom Introduction: Ciclosporin A (CSA)-related neurotoxicity poses a significant clinical problem in allogeneic haematopoietic stem cell transplantation (HSCT) as it necessitates withdrawal of one of the most effective agents in the prevention and treatment of graft-versus-host disease (GVHD). The spectrum of CSA-related neurological complications has been well characterised in several case series of paediatric HSCT recipients, but long term follow up data are lacking.
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P205 Abstract Withdrawn
P206 Intravesical Sodium Hyaluronate and Sodium Chondroitin Sulfate As A Prophylaxis For Hemorrhagic Cystitis In Pediatric Hematopoietic Stem Cell Transplantation Patients K. Yalc¸ın1,*, F. Tayfun2, N. Eker1, S. Kaya1, S. Yu¨cel3, E. Gu¨ler1, A. Ku¨pesiz1 1 Pediatric Hematopoietic Stem Cell Transplantation Unit, Akdeniz University School of Medicine, Antalya, 2Pediatric HematologyOncology, Diyarbakır Children Hospital, Diyarbakır, 3Pediatric Urology, Acıbadem Atakent Hospital, ˙Istanbul, Turkey
Here, we report the outcome of a cohort of paediatric HSCT recipients who experience CSA-related neurotoxicity. Materials (or patients) and methods: From a cohort of 569 consecutive paediatric allogeneic HSCT recipients between 1st January 2003 and 31st December 2011 at our institution we identified those children who developed neurological complications at any time after start of conditioning whilst receiving CSA. Children with obvious alternate causes for neurotoxicity such as infectious meningoencephalitis, prior history of seizure disorder, structural brain lesions, CNS relapse of underlying disease, busulfan-induced seizures and intracranial haemorrhage were excluded. Results: During this 9-year period, 26 of 569 (4.6%) children developed CSA-related neurotoxicity. The median time from HSCT to neurotoxicity was 47.5 days (range day -1 to þ 545). Clinical symptoms included seizures (n ¼ 22), headaches (n ¼ 8), visual disturbances (n ¼ 3) and reduced sensorium (n ¼ 3). 24/26 patients had cranial imaging with MRI or CT or both; 19 had classical findings of PRES, 2 showed non-specific changes consistent with encephalopathy and 3 had normal appearances of brain. CSA and MMF was the most commonly employed GVHD prophylaxis (n ¼ 19), followed by CSA and MTX (n ¼ 4) and CSA alone (n ¼ 3). At the time of neurotoxicity, 18 had prior acute GVHD (8 grade I/II, 5 grade III, 5 grade IV). Five patients had chronic GVHD prior to developing neurotoxicity. Initial management consisted of discontinuation of CSA and supportive care. After resolution of neurological symptoms, 18 patients were re-challenged with CSA and/or FK506. In 8/18 this resulted in recurrence of neurotoxicity symptoms. Additional GVHD prophylaxis/treatment consisted of continuation or (re) introduction of MMF (n ¼ 15), corticosteroids (n ¼ 25), sirolimus (n ¼ 4), immunomodulatory antibodies (n ¼ 12), methotrexate (n ¼ 1), anti-thymocyte globulin (n ¼ 1) and/or mesenchymal stem cells (n ¼ 3). Prognosis in this cohort was poor: 14/26 (54%) died due to severe refractory GVHD and its associated complications. In contrast, during the same study period in our institution, the overall survival at 5 years following allogeneic HCST was 67.7% for haematological malignancy, 82.1% for immunodeficiency and 87.2% for metabolic disorders. Of those surviving 5/12 (42%) live with extensive chronic GVHD. Prognosis was poorest in those who did not tolerate re-introduction of a calcineurin inhibitor (1/8 (12%) survival). Conclusion: Our results suggest that the development of CSArelated neurotoxicity is a poor prognostic factor associated with high mortality and morbidity due to severe GVHD. Survival is enhanced in patients who tolerate the re-introduction of an immunosuppressive agent. Disclosure of Interest: None declared.
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Introduction: Hemorrhagic cystitis (HC) is a relatively common and potentially severe complication observed after hematopoietic stem cell transplantation (HSCT). Many prospective and retrospective studies addressed the etiology and mechanisms of HC in HSCT and a variety of risk factors have been reported in pediatric population but especially cyclophosphamide (Cy) was identified as a cause of HC several decades ago. According to literature, none of the therapeutic modalities which have been tested in the treatment of HC, proved to be effective to date. Also, hyaluronic acid and chondroitin sulfate have been used in the treatment of HC but in this study, we used intravesical hyaluronic acid and chondroitin sulfate as a prophylaxis for HC. Materials (or patients) and methods: Eighty-six children affected by malignant or non-malignant hematological disorders undergoing HSCT from January 2012 to September 2014, in Akdeniz University Pediatric Hematopoetic Stem Cell Transplantation Unit, are enrolled in this study. We focused on 38 children who have Cy in the conditioning regimen. 38 patients had been divided in two groups, 17 patients received sodium hyaluronate and sodium chondroitin sulfate (Ialurils) as a prophylaxis for hemorrhagic cystitis and 21 patients had no prophylaxis. We analyzed the possible risk factors for post-hematopoietic stem cell transplantation hemorrhagic cystitis and focused on the effects of intravesical sodium hyaluronate and sodium chondroitin sulfate prophylaxis. Results: The median age of 38 children who have Cy in the conditioning regimen was 8,2 years old. In all, 24 patients (%63) developed HC a median 23,8 days after HSCT. 16 patients had unrelated, 4 patients had haploidentic HSCT and the incidence of HC was not different according to the type of donor. The incidence of HC was significantly lower in prophylaxis group than non-prophylaxis patients (P ¼ 0.002) In all prophylaxis group only 2 patients had HC (%11), but in non-prophylaxis group 13 patients had HC (%61). Also HC degree was lower in prophylaxis group. Median degree was 1 in the prophylaxis group while it was 3 in non-prophylaxis group. Conclusion: HC is a common cause of morbidity after HSCT and may give rise to serious complications. The reported incidence varies from o10% to more than 70%. High-dose Cy is a well-known risk factor associated with the development of HC. In this study, we administrate intravesical sodium hyaluronate and sodium chondroitin sulfate as a prophylaxis for the patients who have Cy in the conditioning regimen. Lower HC and HC degree were found in patients on prophylaxis group. We consider that the results of our study are promising and the efficacy of sodium hyaluronate and sodium chondroitin sulfate in the prophylaxis of post-transplant HC should be further investigated in larger cohorts of patients. Disclosure of Interest: None declared.
Conditioning regimen I P207 Therapeutic drug monitoring (TDM) of iv busulfan (Bu) in adults undergoing allogeneic hematopoietic stem-cell transplantation (allo-HSCT) to better control the targeted exposure after a 3-day Bu-based myeloablative conditioning (MAC) A. AMIN1,*, P. BOURGET1, A. GOMEZ2, F. ADER1, A.-C. JOLY3, A.-C. MAMEZ2, E. BRISSOT2, M. T. BAYLATRY3, M. MOHTY2, M.-T. RUBIO2 1 Department of Clinical Pharmacy, University Hospital NeckerEnfants Malades, 2Department of Hematology, 3Department of Clinical Pharmacy, University Hospital Saint-Antoine, Paris, France, Paris, France Introduction: High-dose IV Bu is the backbone of different preparative regimens prior to allo-HSCT. However, Bu has a narrow therapeutic window, exhibits large pharmacokinetic (PK) variability, and its clearance is non-linearly related to body weight (BW). To optimize Bu duration/intensity exposure, we studied the contribution of reiterated TDM in patients (Pts) receiving 3-day Bu-based MAC (experimental cohort [EC]). We compared the experimental vs expected values of Bu exposure after performing 3 daily PK. Selected parameters of the EC were compared to a control cohort (CC) of non-monitored Pts. Materials (or patients) and methods: In this observational study (07/2013-10/2014), Pts with hematologic malignancies in CR receiving a 3-day Bu-based MAC in combination with Flu±Thiotepa were prospectively included. The regular Bu dosing schedule of 3 daily 3-hr infusions (72-hr exposure) was used. Bu PK was performed using non-compartmental method with area under the concentration-time curve (AUC) calculation from the 1st (PK1) to the 3rd (PK3) dose; 12 blood samples (4/PK) were assayed by LC-MS2. In the EC (n ¼ 20), dose 1 was calculated from BW (3.2 mg/kg) and corrected in 8 obese Pts using ideal BW; doses 2 and 3 were adjusted based on PK1 (TDM1) and PK2 (TDM2), respectively, with an expected AUC of 4,400-6,000 mM*min/day. AUC values calculated after each dose (AUC1 þ 2 þ 3) were compared to the expected AUC and to the theoretical AUC sum without TDM (AUC1x3) using a Wilcoxon signed-rank test. In the CC (n ¼ 20, 03/2009 09/2014), Pts received 3.2 mg/kg/day of Bu x 3 d. Parameters from the EC and CC were compared using w2, Student t, and log-rank tests. Results: EC and CC Pts had similar sex ratio, median age (56 vs 60 yrs, P ¼ 0.7), BW (68 vs 72 kg, P ¼ 0.9), follow up (263 vs 269 days, P ¼ 0.2), and were transplanted for high-risk diseases (16/ 20 vs 17/20, P ¼ 1.0). In the EC: (a) reiterated TDM showed no statistical difference between the expected AUC [13,20018,000 mM*min] vs AUC1 þ 2 þ 3 (P ¼ 0.3053), (b) AUC1 þ 2 þ 3 and total dose of Bu were significantly different from the AUC1x3 and Dose1x3 without TDM (P ¼ 0.0094 and P ¼ 0.02829, respectively), (c) EC Pts received higher Bu doses compared to CC Pts (mean total dose 717 vs 666 mg [P ¼ 0.028] and 10.2 vs 9.2 mg/kg [P ¼ 0.0014], respectively). Median time to engrafment for neutrophil and platelets were 14 vs 17 days (P ¼ 0.1) and 14 vs 12 days (P ¼ 0.6) in EC and CC Pts, respectively. Grade (Gr) 2-4 liver toxicities were observed in 20 vs 5% of EC and CC Pts (P ¼ 0.3), and renal Gr 2-4 toxicity in 25 vs 10%, respectively (P ¼ 0.4). One death in the EC was attributed to toxicity (SOS after a 2nd allo-HSCT). Grade 2-4 acute GVHD at day 100 was 30% in both groups (P ¼ 0.8). Chronic GVHD at 1 yr in the EC and CC were 31% vs 17% (P ¼ 0.4), while NRM were 20.1 and 5.8% (P ¼ 0.4), respectively. 1-yr relapse incidence, DFS, and OS were 29, 68, and 82.5% in the EC vs 65, 31, and 42.5% in the CC (P ¼ 0.1, 0.13, and 0.18). Conclusion: Reiterated TDM of Bu in a 3-day Bu-based MAC for allo-HSCT is relevant to achieve optimal Bu exposure. It led to administer higher total Bu dose without significant increased toxicity. Disclosure of Interest: A. AMIN: None declared, P. BOURGET: None declared, A. GOMEZ: None declared, F. ADER: None
declared, A.-C. JOLY: None declared, A.-C. MAMEZ: None declared, E. BRISSOT: None declared, M. T. BAYLATRY: None declared, M. MOHTY Funding from: Pierre-Fabre (lecture honoraria and research support)., M.-T. RUBIO: None declared. P208 Allogeneic bone marrow transplantation in patients with hematological malignancy using busulfan, thiotepa, fludarabine as conditioning regimen A. M. Raiola1, A. Dominietto1, R. Varaldo1, F. Gualandi1, C. Di Grazia1, T. Lamparelli1, S. Bregante1, M. T. van Lint1, F. Galaverna1, A. Ghiso1, C. Marani1, A. Bacigalupo1,* 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: In 2012 Sanz and coworkers reported a conditioning regimen containing thiotepa (5mg/kgx2), intravenous busulfan (3,2 mg/kgx3), and fludarabine (50 mg/ m^2x3) (TBF) in patients receiving transplants from single unit cord blood (Sanz et al BMT 2012). Materials (or patients) and methods: We have used the same conditioning regimen for patients with hematologic malignancies : in the present study we analyzed the outcome of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Patients: This is a unicentric retrospective analysis of 97 patients undergoing an allogeneic bone marrow transplantat for AML (n.50) or MDS (n ¼ 47) following the TBF regimen . Patients over the age of 60 or with comorbidities were given TBF with 2 days of bulsulfan (BU2) instead of the full 3 days (BU3). Fifty one patients were conditioning with BU3 and 46 with BU2 regimen. More patients in the BU2 group had advanced disease (57% vs 35%, P ¼ 0.03). BU2 patients were older: median age 50 years (range 18-65) versus 62 years (range 44 – 74) (P ¼ 0,001). Donor type was equally distributed between the two groups with a prevalence for transplant from haploidentical donor (70%). Results: The cumulative incidence of rejection was 0% for BU3 and 8% for BU2 (P ¼ 0.04 Fisher test). The CI for II-IV grade of acute GVHD and of moderate- severe chronic GVHD was 17% and 15% with no difference between the two groups. The 1-year CI of transplant-related mortality was 14% (13% for BU3 and 15% for BU2; P ¼ ns). The CI of relapse related death (RRD) was 12% (BU3) and 15% (BU2) (P ¼ 0.7). After a median followup of 328 days (100-1515) 39 of 51 patients for BU3 (77%) and 33 of 46 (70%) patients for BU2, are surviving. The 5-year actuarial overall survival for patient in CR1/CR2 and advanced disease patients was 83% and 45% in the BU3 group , versus 81% and 36% in BU2 group respectively (P ¼ ns). Also DFS was comparable. Conclusion: This analysis shows that thiotepa-busulfanfludara is an effective regimen for patients with AML and MDS, with encouraging outcomes. Patients receiving the BU2 regimen had outcome in terms of TRM, RRD, and OS , comparable to BU3, despite older age and more advanced disease. A TRM of 15% for patients with a median age of 62 is noteworthy. Disclosure of Interest: None declared. P209 TEPADINAs (thiotepa) plus Busulphan i.v as conditioning regimen before Autologous Stem Cells Transplantation (ASCT) in patients with acute myeloid leukaemia (AML) A. M. Carella1,*, E. Merla1, M. M. Greco1, G. Pisapia2, G. Palazzo2, N. Cascavilla1 1 Hematology, IRCCS CASA SOLLIEVO DELLA SOFFERENZA HOSPITAL, San Giovanni Rotondo, 2Hematology, Taranto, Taranto, Italy Introduction: From June 2010, 24 acute myeloid leukemia (AML) patients (pts) in first Complete Remission (CR) were
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included in a prospective multicenter trial to evaluate the efficacy and toxicity of a new preparative conditioning regimen based on TEPADINA and intravenous (iv) Busulphan for autologous stem cell transplantation (ASCT). Materials (or patients) and methods: This new conditioning regimen consisted of Tepadina 10 mg/kg (5 mg twice every 12 hours) with an intravenous infusion of 4 hours on day -7 and Busulfan (i.v.) 3.2 mg/kg given intravenously on day -6 to-3 (cumulative dosage: 12,8 mg/kg). Patients older than 65 years (n ¼ 6) received Tepadine at the dose of 8 mg/kg (4 mg twice every 12 hour) on day -6 and Busulfan on day-5 to 3 at cumulative dosage of 9 mg/kg. Most patients were female (n ¼ 15, 63%), and the median age at transplantation was 58 years. Results: All patients received peripheral blood stem cells (PBSC), the median CD34 þ cells doses were 5 x106/kg (range: 2,6-9,0). From day þ 2 all pts received granulocyte colony stimulating factor every day until the neutrophil count was at least 41000x109/L for 3 consecutive days. Twenty pts showed normal kariotype, the other 4 pts showed unfavourable cytogenetics. Two pts had mutation of NPM1 gene, none pts had mutation of FLT3 gene. All pts achieved leukocyte and platelets recovery after a median time of 9 (range: 9-14) and 12 (range: 9-30) days, respectively. Grade III-IV mucositis occurred in 6 (25%) pts. Only one patient died due to transplant-related causes after transplantation; in particular this patient died at day þ 26 because of a gram-negative sepsis worsened by kidney and liver failure. The relapse rate was 33% (8 pts relapsed between 3 and 31 months after autoSCT) and the cumulative treatment-related mortality was less than 1%. The average number of days of hospital stay was 20 (range: 19-35). Febrile neutropenia occurred in 14 patients (59%) with a median duration of 2 days (range: 0-10); out of these patients,11 experienced a grade I-II fever of unknown origin (FUO). Two patients developed a fever caused by grampositive infection (n ¼ 1), while 1 had gram-negative fever. Only 1 patient developed a cytomegalovirus infection. To date 15 pts (62%) are in CCR with a median follow-up of 25 months (range 3-49). Two patients (9%) died of progressive disease between 16 and 53 months after autografting. Twenty-one (87%) pts are alive. Conclusion: In conclusion our experience with this new conditioning regimen has shown a significant efficacy and a low toxicity profile. Only minor grades of treatment related nausea and vomiting were observed, also gastro intestinal and liver dysfunction were minor events in our study. Disclosure of Interest: None declared. P210 Comparison of two fludarabine-based reduced-intensity conditioning regimens in elderly or infirm patients with myeloid malignancies treated with allogeneic hematopoietic stem cell transplantation A. Czyz1,*, B. Nasilowska-Adamska2, A. Lojko-Dankowska1, M. Matuszak1, E. Bembnista1, M. Kozlowska-Skrzypczak1, K. Halaburda2, K. Warzocha2, M. Komarnicki1 1 Poznan University of Medical Sciences, Poznan, 2Institute of Hematology and Blood Transfusion, Warsaw, Poland Introduction: An optimal pre-transplant reduced-intensity conditioning (RIC) regimen for elderly or infirm patients with myeloid malignancies has not been established. Materials (or patients) and methods: We compared fludarabine (Flu) plus intravenous busulfan (Bu) regimen with Flu plus melphalan (Mel) therapy to determine long-term outcomes after allogeneic hematopoietic stem cell transplantation (alloSCT) in myeloid malignancies. A total of 90 patients (median age 59 years, range 19-70) with acute myeloid leukemia (n ¼ 61), myelodysplastic syndrome (n ¼ 6), chronic myeloid leukemia (n ¼ 17) and other myeloid neoplasms (n ¼ 6) were given peripheral blood stem cells from HLA– identical siblings (n ¼ 22) or matched unrelated donor (n ¼ 68) after conditioning with Flu combined with a total Bu dose
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ranging from 6.4 to 8.0 mg/kg (n ¼ 30) or Flu combined with a total melphalan dose of 140 mg/m2 (n ¼ 60) between 2009 and 2014. Allotransplantation was performed in advanced phase of the disease (beyond first complete remission in AML or first chronic phase in CML) in 33% of patients. Patients younger than 50 years old (n ¼ 24) were qualified to treatment with RIC regimen due to clinically significant comorbidities or prior alloSCT. Graft versus host disease (GvHD) prophylaxis consisted of CSA and methotrexate in standard dose. In addition, ATG was used in alloSCT from unrelated donors. Patients conditioned with FluBu were older (median age 59 years, range 49-70) than those treated with FluMel (median age 51 years, range 19-59) (Po0.001). The other characteristics such as type of the disease, advanced phase of the disease or type of the donor were similar between the groups. Results: Engraftment was observed in all but four patients who died within 28 days of alloHSCT from infection (n ¼ 3) or multiorgan failure (n ¼ 1). The median time to neutrophil count (0.5 G/L) and platelet count (20 G/L) recovery was shorter after FluBu in comparison with FluMel regimen (18 days vs 22 days; P ¼ 0.045 and 14 days vs 23 days; Po 0.001). Acute GvHD grade II-IV occurs in 17% of FluBu and 33% of FluMel patients (P ¼ 0.13). The respective frequency of moderate/severe chronic GvHD was 12% and 32% (P ¼ 0.078). The median follow-up of surviving patients is 23 months (range 2-70). The 3-year overall survival for patients treated with FluBu and for those conditioned with FluMel was 51% (95%CI 27-74) and 61% (95%CI 47-74) (P ¼ 0.82), respectively (Figure 1). The respective 3-year leukemia-free survival was 56% (95%CI 52-71) and 54% (95%CI 35-75) (P ¼ 0.72). Similarly, two-year non-relapse mortality [21% (95%CI 10-44) vs 21% (95%CI 12-35)] and relapse incidence [18% (95%CI 8-40) vs 20% (95%CI 11-35)] did not differ between two groups. Conclusion: In conclusion, our results confirm that FluBu and FluMel as RIC regimens for alloSCT provide similar survival for the elderly or infirm patients with myeloid malignancies. Nonrelapse mortality is acceptable after both type of RIC alloSCT. Disclosure of Interest: None declared.
P211 Reduced risk of oral mucositis among autoHSCT recipients treated with total marrow irradiation compared to either total body irradiation or chemotherapy-based conditioning. Results of a prospective, observatory study A. M. Polakiewicz-Gilowska1,*, T. Czerw1, M. Sadus´-Wojciechowska1, J. Najda1, W. Mendrek1, L. Miszczyk2, K. S´losarek2, J. Ho"owiecki1, S. Giebel1 1 Department of Bone Marrow Transplantation and OncoHematology, 2Department of Radiotherapy, Maria SklodowskaCurie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland Introduction: Oral mucositis (OM) is one of the most frequent complications of high-dose therapy and considered as a risk factor for infections, prolonged hospitalization, and the need for parenteral nutrition and analgesia. High incidence of OM has been reported after both total body irradiation (TBI) and chemotherapy-based conditioning regimens. In recent years, a new protocol including total marrow irradiation (TMI) has been introduced in clinical practice. However, its impact of the occurrence of OM has not been documented so far. The goal of this study was to prospectively evaluate the incidence of OM according to the type of conditioning, including TMI. Materials (or patients) and methods: 155 consecutive patients (median age 55, range 17-70 years) with multiple myeloma (n ¼ 84), non-Hodgkin lymphoma (n ¼ 45) and Hodgkin’s lymphoma (n ¼ 26) referred for autologous HSCT were included in the analysis. Conditioning regimen consisted of high dose melphalan or BEAM chemotherapy (n ¼ 67), TBI (12 Gy in 3 fractions) alone (n ¼ 11), TBI þ chemotherapy (n ¼ 21) or TMI (12 Gy in 3 fractions) alone (n ¼ 56). All patients had intra-oral assessment before conditioning and were thoroughly instructed in everyday oral hygiene. Oral mouth washes with saline, natrium bicarbonicum, and chlorhexidine were provided during hospitalization at least 3 times daily. In the melphlan setting cryotherapy with crushed ice was additionally used. Standard systemic anti-infectious prophylaxis (quinolones, fluconazole, acyclovir) was administered. Filgrastim 5 mg/kg/day was used to enhance neutrophil engraftment. OM assessment was made daily until discharge. Results: In the whole group, OM occurred in 34% of patients with the median duration of 7.5 days (range 2-19). Its incidence was 40% in the chemotherapy group, 64% after TBI alone, 57% after TBI þ chemotherapy and 11% in the TMI group (P ¼ 0.0001). The median time from start of conditioning to peak OM score was 14 days. Severe OM (WHO score G3 and G4) occurred in 5% of all patients without significant differences between groups. In a multivariate analysis, adjusted for other risk factors, the incidence of OM (grade 1-4) was significantly reduced for TMI (HR ¼ 0.36, 95%CI, 0.220.58, P ¼ 0.0001) together with increasing values of BMI. In contrast to some previous reports, the risk of OM was not related to age, performance status, oral hygiene status and gender. Conclusion: The use of TMI as conditioning prior to autoHSCT for patients with lymphoproliferative disorders is associated with reduced risk of OM compared to both TBI and high-dose chemotherapy. Disclosure of Interest: None declared. P212 LACE-conditioned allogeneic transplantation for high-risk haematological malignancies: treatment outcomes in 62 patients from a single centre B. Sevillano1,*, J. Pavlu1, R. Szydlo1, A. Rotolo1, A. Khoder1, M. Sever1, M. Lasa1, R. Palinacawandar1, C. Giles1, A. Chaidos1, D. MacDonald1, A. Karadimitris1, I. Gabriel1, A. Marks1, D. Milojkovik1, E. Olavarria1, A. Rahemtulla1, J. Apperley1, E. Kanfer1 1 Haematology, Hammesmith Hospital, London, United Kingdom
potential for cure in high-risk haematological malignancies, but with a high risk of non-relapse mortality (NRM), particularly in the elderly or those with significant co-morbidities. The concept of reduced intensity conditioning (RIC) SCT attempts to address this risk. 25 years ago we developed the RIC conditioning protocol (LACE) consisting of oral lomustine 200 mg/m2, (day -7), iv etoposide 1000 mg/m2 (day -7) iv cytarabine 2000 mg/m2 (day -6 and -5) and iv cyclophosphamide 1800 mg/m2 ( days -4, -3 and -2). Here we summarise outcomes of SCT with this regimen. Materials (or patients) and methods: From 1989 to June 2014, 62 patients (median age 45 years; range 13-68) underwent LACE SCT. Recipients of matched sibling allografts (n ¼ 46) received GvHD prophylaxis with Cyclosporine and Methotrexate. T-cell depletion with Campath was used in recipients of matched unrelated (n ¼ 14), haploidentical (n ¼ 1) and mismatch sibling (n ¼ 1) transplants. Patients not eligible for myeloablative conditioning due to advanced age or associated comorbidities were selected with the following diagnoses: AML (n ¼ 23, 37%), non-Hodgkin lymphoma (NHL, n ¼ 16, 26%), MDS (n ¼ 12, 19%), and CML in accelerated (n ¼ 2, 3%) and second chronic phase (n ¼ 1, 2%). All malignancies were classified as high risk for relapse. 7 patients (11%) were not in remission at the time of transplantation. Results: NRM at day 100 was 27%, with the following causes: infection (n ¼ 9), hepatic veno-occlusive disease (n ¼ 1), multi organ failure (n ¼ 1) and unknown (n ¼ 3). After day 100, mortality was mainly related to disease relapse. Acute GvHD occurred in 20 patients (33%). These were scored as grade I in 8 (13%), grade II in 5 (8%) and grade III in 7 patients (11%). None of the patients developed grade IV acute GvHD. Chronic GvHD occurred in 12 (19%) patients. It was graded as limited (localized skin involvement and/or hepatic dysfunction) in 8 and extensive in 4 patients. The probability of overall survival and progression free survival at 5 years was 40% and 37% respectively, with a median follow up of 3.6 years. Kaplan Meyer curves indicating probability of survival according to disease are shown below. Conclusion: LACE RIC showed acceptable toxicity, with a relatively low incidence of acute and chronic GvHD. A significant proportion of patients with high-risk haematological malignancies unfit for MAC achieved good long-term results with this conditioning protocol. Disclosure of Interest: None declared.
Introduction: Haematopoietic stem cell transplantation (SCT), preceded by a myeloablative-conditioning regimen provides a
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P213 Research of Iron chelator treatment in allogeneic hematopoietic stem cell transplantation C. Li1,2,*, J. Ma1, L. Ye1, X. Ye1, C. Ma1, C. Gu1, T. Wang1, Q. Zhou1, X. Chen1, D. Wu1 1 The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, 2Immunology, School of Biology & Basic Medical Science, Medical College of Soochow University, Suzhou, China Introduction: To investigate the efficacy and safety of ironchelator treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Materials (or patients) and methods: a total of 50 allo - HSCT recipients with IO were retrospectively analyzed. Patients were divided into two groups. The experimental group consisted of 23patients who did not receive any chelator treatment. The control group consisted of 27 patients who received ironchelator treatment. Results: The iron chelating and allo-HSCT treatment significantly reduced serum ferritin levels (1262, 824, min 501–max 1305 vs min 1000–max 2352) (Po0.01). In the Treatment group after HSCT the median time of grain giant nuclear reconstruction is 12±2 days and 20±8 days, 13±3 days and 25±14 days in the control group. Nuclear hematopoietic reconstruction time was significantly shortened with iron chelation therapy (P ¼ 0.049). In the treatment group , 39.1% (9/23) cases occurred infection, while 74.1% (20/27) cases occurred infection in control group. The difference was found statistically signicant (P ¼ 0. 014). 17.4% (4/23) cases were found GVHD in the treatment group and 55.6% (15/27) cases were found GVHD in control group. There was a significant reduction in the risk of GVHD (P ¼ 0. 008). The median OS in control group was 12 (min:0.3 B max:50) and 18 (min:3.5max:78) months in Treatment group. The difference was found statistically signicant (P ¼ 0.050). Conclusion: Iron chelation therapy can safely and effectively to reduce iron load of allo - HSCT patients with iron overload, and promote hematopoietic reconstruction, reduce the infection and the incidence of GVHD, prolong survival time. Disclosure of Interest: None declared. P214 Comparison between BEAM and Yttrium-90 Ibritumomab Tiuxetan (Zevalin)-BEAM as conditioning regimen before autologous stem cell transplantation in patients with nonHodgkin B-Cell Lymphoma C. CHIC ACEVEDO1,*, E. GARCIA TORRES1, M. J. LLAMAS POYATO1, G. RODRIGUEZ1, C. MARTIN1, R. ROJAS1, J. SERRANO1, S. TABARES1, J. A. VALLEJO1, J. SANCHEZ1 1 HEMATOLOGY, HOSPITAL UNIVERSITARIO REINA SOFIA, CORDOBA, Spain Introduction: Patients with chemosensitive relapsed nonHodgkin B-Cell lymphomas are candidates to intensification with autologous stem cell transplantation (ASCT). The addition of Ibritumomab Tiuxetan (Zevalin) in combination with highdose chemotherapy as BEAM (Z-BEAM) has proven to be safe and effective in patients with poor-risk features. However the precise role of Z-BEAM as conditioning regimen for ASCT is not well established. The aim of this study is to analyze the safety and efficacy of Z-BEAM as conditioning for ASCT in nonHodgkin B-lymphoma. Materials (or patients) and methods: In this retrospective study we analyze 54 patients diagnosed with non-Hodgkin B-lymphoma candidates to ASCT who received conditioning regimen with BEAM or Z-BEAM in our institution, from 2004 to 2014. 31 (57.4%) were males and 23 (42.6%) females. Median age was 50years (30-68). Zevalin was administered 7days previous to standard BEAM at dose of 0.4mCi/Kg. Patients were assigned to each arm according to lymphoma-risk features. Toxicity was measured according to CTCAEv4.1. Overall Survival (OS) and Progression free survival (PFS) were
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compared. Statistical analysis was performed using SPSS.v17 software package. Results: 27 patients received BEAM and 27 Z-BEAM. We did not find statistical differences in age, sex, IPI, FLIPI, histological grade and diagnosis between both groups. However we found significant difference in disease-status at ASCT: 70.4% (n ¼ 19) in BEAM group were in complete remission (CR) vs. only 33.3% (n ¼ 9) in Z-BEAM group (P ¼ 0.006). Significant difference was also found in the number of prior chemotherapy (CT) lines (42 lines): 14.8% (n ¼ 4) vs 40.7%(n ¼ 11); P ¼ 0.033 for BEAM and Z-BEAM respectively. No statistical differences were found in neutrophil engraftment (4500/mm3): 11.59 days (11-12.7) in BEAM group vs 11.38 (10.6-12) in Z-BEAM (P ¼ 0.747) nor in platelet engraftment (420,000/mm3): 13 days (12-14) vs 15.7 (13.7-18); (P ¼ 0.073). Significant differences were neither found in transplant-related toxicity except for the mucositis which was significantly higher in Z-BEAM: 92.6% (n ¼ 25) than in BEAM: 62.9% (n ¼ 17); P ¼ 0.009. With a median follow-up of 34months (0-104), there was no statistically significant difference either in OS or PFS between both groups. OS was 76.5% (60.7-92) in BEAM group vs 67.1% (50.6-84) in Z-BEAM; P ¼ 0.65. PFS was 71% (53.4-88.6) vs 54.2% (36.1-72.2) respectively; P ¼ 0.387. Other clinical variables such as age, FLIPI and IPI did not impact on outcomes(OS or PFS) in our series. The number of previous CT lines negatively impact PFS: 74.8% (60.4-89.3) in d 2 lines vs 39.6% (15.1-64.1) for 42 CTlines (P ¼ 0.013). Disease-status at ASCT had a significant statistical impact on outcomes:OS was 87.3% in those in CR (74.2-100) vs 51.6% (35.3-67.9) in partial response (P ¼ 0.014) and PFS was 82.6% (67.6-98) vs 33.1% (1947); P ¼ 0.002 respectively. This variable remained as the only significant variable in multivariate analysis: OR: 3.45 (1.1-11.1) P ¼ 0.027 in SG; OR: 2.96 (1.1-8.7) P ¼ 0.05 in PFS. Conclusion: In our series, patients receiving Z-BEAM for ASCT harbored significantly higher risk features than those with BEAM. However we did not find statistical differences in outcomes comparing both groups. This finding suggests that patients who did not achieve CR before ASCT could benefit of the use of Z-BEAM as conditioning regimen. Disclosure of Interest: None declared. P215 Effects of Busulfan dose and administration route on transplantation outcome in adult patients with AML or MDS - a retrospective analysis of the German transplant registry DRST C. Scheid1,*, U. Holtick2, M. Bornha¨user3, N. Kro¨ger4, A. Ganser5, R. Arnold6, P. Dreger7, G. Stuhler8, M. Theobald9, C. Peschel10, M. Hallek11, D. Niederwieser12, J. Finke13, B. Hertenstein14, D. Beelen15 on behalf of Deutsches Register Stammzelltransplantation (DRST) 1 Stem cell transplantation program, University of Cologne, Ko¨ln, 2 Stem cell transplantation program, University of Cologne, Cologne, 3BMT, University of Dresden, Dresden, 4BMT, University Hospital Eppendorf, Hamburg, 5Hematology, Hannover Medical School, Hannover, 6BMT, Charite University Hospital, Berlin, 7BMT, University of Heidelberg, Heidelberg, 8BMT, Helios Kliniken, Wiesbaden, 9Hematology, University Hospital Mainz, Mainz, 10 Hematology, Klinikum rechts der Isar, Mu¨nchen, 11Dept. I of Internal Medicine, University of Cologne, Cologne, 12Hematology, University of Leipzig, Leipzig, 13BMT, University of Freiburg, Freiburg, 14Hematology, Kliniken Bremen-Mitte, Bremen, 15BMT, University Hospital Essen, Essen, Germany Introduction: Busulfan is frequently used as part of the conditioning for allogeneic stem cell transplantation. It can be given orally or intravenously in a variety of dosages. While the advantages of the intravenous administration compared to the oral formulation is well established, the optimal dose for each situation is less clear. Materials (or patients) and methods: To assess the current use of Busulfan in terms of application form and dosage in
Germany, we collected registry data of 1844 patients with AML (n ¼ 986), MDS (n ¼ 649) or secondary AML (n ¼ 209) receiving an allogeneic stem cell transplantation after a Busulfancontaining conditioning regimen. The median age was 53 years (range 18-80), 55% were male, 45% female. Karnofsky performance status was 100 in 24.7%, 90 in 44.1%, 80 in 18.8%, 70 or less in 6.1% and missing in 6.5%. 49.8% were in complete remission at transplantation, 11.7% were transplanted upfront and 48.5% had active disease after chemotherapy. Stem cell source was PB in 94%, BM in 5.3% and others in 0.7%. Results: Busulfan was given orally in 607 patients and intravenously in 1237 patients. The dose was up to 4mg/kg in 212 patients, 4.1- 8 mg/kg in 1084 patients, 8.1 -12 mg in 82 patients and more than 12 mg/kg in 466 patients. In the age group o40 yrs most patients received 412 mg/kg (61%), while in patients between 41 and 50 yrs 50% had 412 mg/kg and 32% had 4.1 -8 mg/kg. In contrast patients between 51 and 60yrs a reduced dose of 4.1-8 mg/kg was used in 57%, increasing to 78% and 82% in age groups 61-65yrs and 465 yrs respectively. Multivariate Cox model analysis revealed age and status at transplant as significant parameters for overall survival and non-relapse mortality (NRM). To exclude status at transplant as confounding variable, a subgroup analysis of AML patients in CR1 was performed. In this subgroup cumulative incidence of NRM at 24 months was 15.7% in patients o40 yrs, 17.6% in 41-50 yrs, 23.7% in 51-60 yrs, 31.8% in 61-65 yrs and 35.7% in patients 4 65 yrs. Overall survival at 24 months was 72%, 68%, 57%, 53% and 43% respectively. Conclusion: Busulfan can be applied in a wide dose range and adapted to age and clinical situation. Our data from the German stem cell transplantation registry indicate that most patients 450 yrs receive a reduced dose of up to 8mg/kg. With this age-adjusted dosing there was only a moderate increase of NRM between the different age groups 450 yrs, amounting to 35.7% in patients 465 yrs with AML in CR1 leading to an overall survival of 43% at 2 years. Disclosure of Interest: C. Scheid Funding from: Pierre Fabre, Amgen, Novartis, Janssen, Celgene, U. Holtick: None declared, M. Bornha¨user: None declared, N. Kro¨ger: None declared, A. Ganser: None declared, R. Arnold: None declared, P. Dreger: None declared, G. Stuhler: None declared, M. Theobald: None declared, C. Peschel: None declared, M. Hallek: None declared, D. Niederwieser: None declared, J. Finke: None declared, B. Hertenstein: None declared, D. Beelen: None declared P216 Haploidentical hematopoietic stem cell transplantation using a reduced-intensity conditioning regimen which consist rituximab and mesencymal stem cell infusion in children; preliminary results D. Atay1,*, F. Erbey1, A. Akcay1, M. Akbiyik1, O. Harman1, E. Ovali2, G. Ozturk1 1 PEDI˙ATRI˙C HEMATOLOGY ONCOLOGY BONE MARROW TRANSPLANTATI˙ON UNIT, 2Acibadem Labcell, ACIBADEM UNIVERSITY MEDICINE FACULTY, Istanbul, Turkey Introduction: Haploidentical stem cell transplantation (haploHSCT) has been developed as an alternative transplant strategy for children with hematological disorders without a HLA matched donor. Reduced-intensity conditioning (RIC) regimens allow haplo-HSCT with less toxicity, but graft versus host disease (GVHD) remains still an actual problem. T cell receptor (TCR) a and b/CD3 depletion for the prophylaxis of GVHD achieved a significant success in haplo-HSCT. The aim of our study is to investigate the efficacy of rituximab (RTX) and mesencymal stem cell (MSC) administration for GVHD prevention in haplo-HSCT. Materials (or patients) and methods: We analyzed the outcome of eleven pediatric patients (median:8.6 years) with various hematological diseases who underwent haplo-HSCT using a RIC regimen [ATG (days –13 to –9), fludarabine (days – 8 to –5) thiotepa (day –4) and melphalan (days –3 and –2)] and TCRab /CD3 depletion at our institution from January, 2014 to
October, 2014. To prevent EBV related posttransplant lymphoproliferative disease (PTLD) and GVHD, RTX 375mg/m2 was added into conditioning regimen on day -2. MSC was infused at day -1 to suppress alloreactive donor anti-host T-cell responses and to use their ability to promote angiogenesis and support micro-environment, which facilitate engraftment. Mycophenolat mofetil was used for GVHD prophylaxis. PCR screening for BK, Adeno, EBV and CMV viruses were performed routinely weekly. Analysis on immune reconstitution was performed at day 30, 60, 90, 120 and 180. Results: Reducing a/b of the graft was 99.9%. The grafts contained a median of 36.08x106 (range: 14.27-281.13) CD34 þ cells. Engraftment of neutrophils and platelets was achieved at a median of 12.5 (8-17) days and 14.5 (9-22) days, respectively. Lymphocyte count 41000/mm3 was seen at a median of 24 days (13-186). Seven patients engrafted with full donor chimerism. Two patients were mix chimeric. Graft rejection occurred in two patients, one of them was successfully rescued by CD34 infusion from same donor. The regimen was generally well tolerated. Two patients developed grade 3-4 acute GVHD. One patient died due to massive gastrointestinal system bleeding. A number of 2/11 patients suffered from CMV-reactivation at day 13 and 35 without clinical or radiological involvement, with prompt response to specific antiviral therapy. Adeno virus reactivation was seen in three patients at day 33, 66, 82; they didn’t need any therapy. BK virus reactivation was seen in four patients at day 30,12,10,11; they were only treated with hydration. None of the patients developed EBV infection or severe bacterial infection during or after HSCT. Transplant related mortality (TRM) at day 30 and at day 100 was 0%. Median discharge day was þ 27. Our data at þ 30, þ 60 and þ 90 from engraftment showed delayed immune reconstitution. Conclusion: These data indicate that a selective graft manipulation and MSC results into effective prevention of acute GVHD, rapid recovery of neutrophil and platelet counts without severe complication at acute phase of HSCT and low TRM. Rituximab is less expensive then hardware-based depletion and more applicable. But the median observation time is limited. Following investigation of advantages and risks of Rituximab and MSC application are required. Disclosure of Interest: None declared. P217 Clinical Comparison of Weight and Age Based Strategy of Dose Administration in Children Receiving Intravenous Busulfan for Hematopoietic Stem Cell Transplantation D. Gurlek Gokcebay1,*, F. Azik1, N. Ozbek1, P. Isik1, Z. Avci1, B. Tavil1, B. Tunc1 1 Pediatric Hematology, Ankara Children’s Hematology and Oncology hospital, Ankara, Turkey Introduction: Busulfan (Bu), combined with therapeutic drug monitoring-guided (TDM) dosing, is associated with fewer graft failure/relapse and lower toxicity in children who had hematopoietic stem cell transplantation (HSCT). Because TDM of Bu cannot be performed in Turkey formerly, based on previous pharmacokinetic analyses, we planned this retrospective study for comparison of weight and age based dosing in terms of clinical outcomes. Materials (or patients) and methods: Patients underwent HSCT from April 2010 to February 2013 and completed 100 days after transplantation in our Hospital were enrolled in this study. Intravenous Bu was administrated according to age in Group 1 that includes 37 patients. The dose of Bu was 1 mg/kg foro4 years and 0.8 mg/kg for Z4 years. In Group 2 that includes 24 patients Bu doses were adjusted according to actual body weight; patients o9 kg received 1 mg/kg, 9-16 kg 1.2 mg/kg, 1623 kg 1.1 mg/kg, 23-34 kg 0.95 mg/kg, and 434 kg 0.8 mg/kg. Results: A total of 61 patients (median age 8.2 years, range 0.54-16.8) received allogeneic HSCT. The weight based dosing group received higher amount of Bu, and this group was significantly prone to develop sinusoidal obstruction
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syndrome (SOS) (P ¼ 0.001). A statistically significant but mild correlation was found between Bu dose and the incidence of SOS (r:0.26, P ¼ 0.04). There was no relation between high levels of serum aspartate aminotransferase or ferritin and SOS. Although univariate analysis revealed association between SOS and conditioning regimen, weight or age based Bu dosing strategy, total amount of Bu and cyclophosphamide, and use of Pesaro protocol 26 as conditioning regimen (Po0.05); multivariate analysis revealed that only weight based dosing of Bu was a significant predictor of SOS (HR: 9.46; P ¼ 0.009). Febrile neutropenia, hypoxia and dyspnea occurred more frequently in the weight based dosing group, and this group also had higher bilirubin concentration, and longer duration of hospitalization (Po0.05). However, no relation was found between two groups in terms of hemorrhagic cystitis, engraftment syndrome, acute or chronic graft-versus-host disease (GvHD), time to engraftment, chimerism, transplant related mortality, survival rates. Conclusion: Therapeutic drug monitoring of Bu is essential for improvement of efficacy and reduction of toxicity. We suggest use of age based dosing strategy for Bu administration in countries where TDM cannot be performed. References: 1. Tran HT, Madden T, Petropoulos D, et al. Individualizing high-dose oral busulfan: prospective dose adjustment in a pediatric population undergoing allogeneic stem cell transplantation for advanced hematologic malignancies. Bone Marrow Transplant 2000; 26: 463–470. 2. Nguyen L, Fuller D, Lennon s, et al. Iv Busulfan in pediatrics: a novel dosing to improve safety/efficacy for hematopoietic progenitor cell transplantation recipients. Bone Marrow Transplant 2004; 33: 979–987. 3. Zwaveling J, Bredius RGM, Cremers SC, et al. Intravenous busulfan in children prior to stem cell transplantation: study of pharmacokinetics in association with early clinical outcome and toxicity. Bone Marrow Transplant 2005; 35: 17–23. Disclosure of Interest: None declared. P218 Once daily intravenous infusion Busulphan plus Fludarabine as conditioning for Acute Myeloid Leukemia and MDS allogeneic transplantation. A single Centre experience D. Baronciani1,*, C. Depau1, C. Targhetta1, A. A. DiTucci1, E. Angelucci1 1 Ospedale Oncologico di Riferimento Regionale ‘‘A. Businco’’, Cagliari, Italy., U.O. Ematologia-CTMO, Cagliari, Italy Introduction: Allogeneic HSCT is the treatment of choice for higher risk AML and MDS patients. The traditional preparative regimens TBI-CY and BU-CY have been widely used but extra hematologic toxicity remains a significant concern. Even if the combination of intravenous formulation Busulphan/CY has shown lower toxicity and suggests favorable safety and efficacy new drugs associations are continuously explored. Recently the association Busulphan –Fludarabine has replaced the Bu-Cy regimen with the aim to reduce toxicity. Materials (or patients) and methods: In our Institution we started to use this regimen as preparation for allogeneic HSCT in patients with AML and MDS in October 2008. Primary objectives were evaluation of overall survival, disease free survival, transplant related mortality and relapse. Since October 2008 up to October 2014, 28 consecutive (21 males, 7 females) patients entered this study. Median age was 55 years (range 3563). Underlying diseases were: AML (23 patients: CR1 ¼ 18, advanced disease ¼ 5) and MDS (5 IPSS Intetermediate II patients). Conditioning consisted of iv Busulphan (Busilvexs 9.6 -12.8 mg/kg) given once daily in a 4 hours infusion together with Fludarabine (160 mg/m2) for 4 consecutive days. The association CSA þ short MTX was used as GVHD prophylaxis and anti-lymphocyte globulins (Thymoglobulins or ATG Freseniuss) were used in case of MUD transplants. Sixteen patients received HSCs from an HLA identical siblings, 1 from a mismatched family donor, 11from unrelated donors. Source of stem cells was bone
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marrow in 14 patients, peripheral blood stem cells in 13 patients and both in 1 patient. Results: All patients regularly engrafted (median time of ANC 4500 was 13 days (range 11-15 ). One patient presented late rejection in aplasia 2 months after a unrelated transplant. She received rescue with an haploidentical family donor transplant. Five patients presented a pattern of mixed chimerism in a state of complete remission. None of them received donor lymphocyte infusion; all recovered to full donor status with immunosuppression tapering. Nine patients experienced gastro-intestinal toxicity; three patients had grade II cystitis. No other extra-hematologic toxicity occurred. Acute grade III gastro-intestinal GVHD occurred in one patient; 13 patients presented grade I skin GVHD, associated with grade II gastrointestinal in one. Cr.GVHD occurred in 6 over 20 evaluable patients (it was mild in 5 and moderate in 1). One patient presented early infection (pneumonia), four late infections (3 fungal infections, 1 Pneumocystis Jirovecii pneumonia); Cytomegalovirus reactivation occurred in 18 over 28 evaluable patients (64%). Nineteen patients are alive (68%), 18 in complete remission with a median follow-up of 12 months (range 1-41). Nine patients died (32%): eight by recurrent disease and one by acute GVHD. Among the 18 patients transplanted in first complete remission, 14 are alive and well. Four patients have died for high risk leukemia recurrence. Conclusion: This single Centre report, even if with limited number of patients, underlines the association iv Busulphan– Fludarabine is a well-tolerated and effective conditioning regimen in AML/MDS patients. Intravenous Busulphan administered once daily is convenient, effective and well tolerated. Disclosure of Interest: None declared. P219 Therapeutic drug monitoring of oral and intravenous busulfan in paediatric patients E. Fernandez De Gamarra1,*, M. Torrent2, L. Sisinni2, E. Zapico3, T. Olive4, C. Dı´az de Heredia4, I. Badell2 1 PHARMACY DEPARTMENT, 2PEDIATRIC BONE MARROW TRANSPLANTATION UNIT, 3BIOCHEMISTRY DEPARTMENT, HOSPITAL SANTA CREU I SANT PAU, 4PEDIATRIC BONE MARROW TRANSPLANTATION UNIT, HOSPITAL VALL D’HEBRON, BARCELONA, Spain Introduction: Busulfan is commonly used in preparative regimens for hematopoietic stem-cell transplantation in adults and children for a variety of malignant and non-malignant diseases. It has a narrow therapeutic range and a large pharmacokinetic variability that could have consequences in treatment outcomes and toxicity. It is available for oral and intravenous administration. The aim of this study is to assess the benefits of therapeutic drug monitoring (TDM) to guide busulfan dosing and to review efficacy and safety of busulfan. Materials (or patients) and methods: Data from 36 paediatric patients treated with busulfan between 2010 and 2014 in two bone marrow transplantation units was collected. Protocols were chosen according to the underlying disease. We included children treated with oral or intravenous busulfan who underwent TDM. We applied a non-linear regression method and used ID3 software for pharmacokinetic studies. Area under the curve target was 55000-95000 ng/ml.h, depending on the conditioning protocol (reduced intensity or myeloablative). Graft failure and incidence of veno-occlusive disease (VOD) were recorded to assess efficacy and safety. Results: Thirty-six patients with ages ranging from three months to 18 years were included in the study. Twenty-five of them received IV busulfan and 11 patients received oral busulfan. Allogeneic or autologous transplantations were performed in 33 and three patients respectively. Baseline diseases in the allogeneic group were: 27 malignant and 16 non-malignant haematological diseases; and in the autologous group: 2 neuroblastoma and 1 Ewing’s sarcoma. Regarding the conditioning regimen, 26/36 were myeloablative and 10/36 non-myeloablative. Busulfan initial doses ranged from 2.8 and
5.1 mg/kg/day (related to adjusted body weight), according to the protocol and the weight band. Oral busulfan was administered in four divided doses (every six hours) and daily IV busulfan dose was either administered in one or four doses. All patients received seizures prophylaxis with phenytoin. TDM and follow-up results for oral treatments (N ¼ 11): all treatments were myeloablative. Ten patients needed a dose adjustment (four reductions and six increases) to guarantee a drug exposure within the desired range. Any of them experienced graft failure or VOD. TDM and follow-up results for intravenous treatments (N ¼ 25): - Myeloablative regimens (N ¼ 15): 13 patients needed a dose adjustment (nine reductions and four increases) to guarantee a drug exposure within the desired range. Any of them experienced graft failure. - Non-myeloablative regimens (N ¼ 10): all patients needed a dose adjustment (four reductions and six increases) to guarantee a drug exposure within the desired range. Secondary graft failure was observed in four patients (in three of them it was recovered). These four patients had a nonmalignant baseline disease. Any VOD was reported for this group of intravenous treatments. Globally, considering all 36 patients, 33 needed a dose variation: 17 reductions (average -18.6%) and 16 increases (average þ 21.4%). Conclusion: TDM of oral and IV busulfan in paediatric patients is a valuable tool to guide drug dosing. It should be strongly recommended even for intravenous treatments, in spite of its theoretical advantage of less variability compared to the oral form. TDM might provide improvements in efficacy and safety. Disclosure of Interest: None declared. P220 Final results of a prospective multicenter phase II study using Busulfan AUC intensification in AML patients undergoing allogeneic HSCT in first complete remission F. Suarez1,*, P. Bourget2, E. P. Alessandrino3, A. Bartoli4, G. Socie´5, M. Michallet6, J. Bourhis7, A. Paci8, D. Blaise9, A. Berceanu10, V. Kemmel11, J. Sierra12, M. E. Moreno Martinez13, A. Amin2, A. A. Colombo14, A. Xhaard5, F.-E. Nicolini6, X. Thomas6, S. Fu¨rst15, B. Lioure16, R. Martino17, C. Levrault18, A. Pe´tain19, C. Ta Thanh Minh20 1 Hematology, 2Pharmacy, Hoˆpital Necker-Enfants Malades, Paris, France, 3Hematology and Oncology, Fondazione IRCCS Policlinico San Matteo and University of Pavia, 4Clinical Pharmacokinetic Laboratory, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 5Hematology and Bone marrow Transplantation, Hoˆpital Saint-Louis, Paris, 6Hematology and Bone Marrow Transplantation, CHU Lyon Sud, Lyon, 7Hematology, 8Service de Pharmacologie & d’Analyse du Me´dicament (SIPAM), Institut Gustave Roussy, Villejuif, 9Hematology and Bone Marrow Transplantation department, Institut Paoli-Calmettes, Marseille, 10 Hematology, CHU Besanc¸on, Besanc¸on, 11Service de Biochimie, CHRU Strasbourg, Strasbourg, France, 12Hematology and Bone Marrow Transplantation, 13Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 14Hematology and Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 15Hematology and Bone Marrow Transplantation, Institut Paoli-Calm16 Hematology and Bone Marrow ettes, Marseille, Transplantation, CHRU Strasbourg, Strasbourg, France, 17Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 18Institut de Recherche Pierre Fabre, Boulogne, 19De´partement de Pharmacocine´tique, Pierre Fabre Me´dicament, Toulouse, 20PIERRE FABRE MEDICAMENT, PIERRE FABRE MEDICAMENT, Boulogne Billancourt, France Introduction: Despite major advances in the risk-stratification and management of acute myelogenous leukaemia (AML) based on cytogenetics and molecular biology, the relapse rate remains as high as 25%. Therefore, a prospective phase II study aimed to assess a strategy based on Busulfan (Bu) AUC (area under the curve) intensification in AML patients was designed. The
administration of IV Bu in once-daily infusion over 3 hours combined with Fludarabine prior to haematopoietic stem cell transplantation (HSCT) was approved recently in adult patients who are candidates for a reduced-intensity conditioning regimen. Since limited experiences were available with the combination IV BuCy2, in this protocol, IV Bu was infused in a single daily infusion during 4 days, followed by Cyclophosphamide. Materials (or patients) and methods: The primary objective was to target the upper range of the Bu TW (therapeutic window) [4400 - 6000 mmol/L.min]. A daily pharmacokinetic (PK) guided adjustment from Dose 2 to Dose 4 of Bu treatment was expected to enable the same targeting performance (Z70 to 80% of patients within the TW) as that achieved for the common large TW[3600-6000 mmol/L.min] without adjustment. The secondary objective was to determine the clinical outcomes at 2 years. The eligible patients were required to have AML in first complete remission (CR1) and eligible for an allogeneic HSCT following a myeloablative regimen. The graftversus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitor and IV methotrexate. The addition of antithymoglobulin for unrelated donors was decided by each centre based on their institutional guidelines. Results: Thirty adult patients with a median age of 43.5 years were treated. Ninety percent of patients reached the cumulated therapeutic window of AUC [17600-24400 mmol.L/ min]. Clearance after once daily administration was similar to clearance with a quarter in die regimen demonstrating the linearity of Bu pharmacokinetic from 0.8 mg/kg to 3.2 mg/kg. Overall, the cumulative incidence of acute graft-versus-host disease (aGVHD) grade II-IV at 100 days was 26.7% [23.7-29.6]. The cumulative incidence of aGVHD grade III-IV was 6.7% [5.0; 8.3]. None of the patients developed grade IV aGVHD. Five patients died of late non relapse mortality (NRM) between day þ 197 and day þ 391 (2-year cumulative incidence of NRM was 16.7%). No patient died before day þ 100. The causes of late NRM were GVHD (2 patients), hemorrhage (1 patient), non-infectious interstitial pneumonitis (1 patient), vasculitis (1 patient). No death due to VOD was observed. The cumulative incidence of relapse at 2 years was 20% [17.3 – 22.7]. The 2year overall survival (OS) was 63.2% [43.4-77.6]. This study proved the feasibility of a daily adjustment according to PK measurement. The 2-year OS was satisfactory with a low relapse rate; these values are comparable with previous publications reporting experiences with IV BuCy2 without AUC intensification. The 2-year NRM was comparable with the previously published safety profile of a myeloablative BuCy2. Conclusion: The clinical outcomes (OS, Relapse rate) were good in this homogeneous cohort of high-risk AML patients allografted in CR1. However, compared to the literature, no additional benefit could be demonstrated using Bu AUC intensification. Disclosure of Interest: F. Suarez: None declared, P. Bourget Funding from: Pierre Fabre Medicament, Conflict with: Travel grants –Pierre Fabre Medicament, E. P. Alessandrino: None declared, A. Bartoli: None declared, G. Socie´: None declared, M. Michallet Conflict with: Fundings and Personal Financial interest : Advisory boards and consultancy BMS, Novartis, Genzyme, MSD, Hospira, GILEAD, J. Bourhis: None declared, A. Paci Funding from: Pierre Fabre Me´dicament, Personal Interest: Pierre Fabre Me´dicament, D. Blaise Funding from: Pierre Fabre Medicament, Personal Interest: Pierre Fabre Medicament, A. Berceanu: None declared, V. Kemmel: None declared, J. Sierra: None declared, M. E. Moreno Martinez: None declared, A. Amin: None declared, A. A. Colombo: None declared, A. Xhaard: None declared, F.-E. Nicolini Funding from: Novartis Pharma, Personal Interest: Novartis Pharma, Conflict with: Advisory boards; consultancy, speakers bureau; BMS: consultancy, advisory boards, speakers bureau; ARIAD: Advisory board, speakers bureau, X. Thomas: None declared, S. Fu¨rst: None declared, B. Lioure: None declared, R. Martino: None declared, C. Levrault Employee of: Pierre Fabre Me´dicament, A. Pe´tain Employee of: Pierre Fabre Me´dicament, C. Ta Thanh Minh Employee of: PIERRE FABRE MEDICAMENT.
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P221 Comparison of two conditioning regimen for autologous stem-cell transplantation for lymphoma. FEAM vs TEAM A. Gravetti1, R. Della Pepa1, S. Avilia1, N. Pugliese1, F. Pane1, A. M. Risitano1, G. De Rosa1,* 1 Hematology, Federico II University, Naples, Italy Introduction: The treatment of choice for relapsed/refractory non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL) consists of high-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT). Several HDC regimens with different drug combinations have been in use, however, no randomized data is available to demonstrate superiority of any one regimen and retrospective comparative reports are sparse. BEAM (BCNU, etoposide, cytarabine, melphalan) is a widely adopted conditioning regimen for autologous hemopoietic SCT for lymphoma transplants, and has an acceptable toxicity and high efficacy. Recently, there was a shortage of BCNU globally, to overcome this situation and the increased pulmonary toxicity associated with BCNU we instead used two modified regimens, FEAM or TEAM, where BCNU was replaced with Fotemustine or Thiotepa, respectively. Materials (or patients) and methods: In this study we analyzed these alternative drug schedules and compared them, focusing on early toxicities, infectious complications and hemopoietic engraftment. We studied 35 patients with relapsed or refractory lymphoma (18 NHL-17 HL) who were treated with HDC followed by ASCT in the Centro Trapianti Midollo Osseo–Federico II between October 2010 and April 2014. HDC regimen administered was FEAM in 17 patients and TEAM in 18 patients. FEAM consisted of Fotemustine 150 mg/m2 on days –8, –7, etoposide 200 mg/m2 and aracytin 400 mg/m2 on days –6 to –3 and melphalan 140 mg/m2 on day –2. TEAM consisted of Thiotepa 10 mg/kg on day -8, -7, etoposide 200 mg/m2 and aracytin 200 mg/m2 on days –6 to –3 and melphalan 140 mg/m2 on day –2. The median age of the study group was 46 years (range 17–66 years). The evaluation of the toxicity and tolerability profile were performed by comparing the two groups on the following variables: time to haemopoietic recovery (plt420.000/109 dL, neutrophils41500/109 dL), transfusion support therapy, presence of fever, days on intravenous antibiotic therapy, appearance and degree of mucositis, onset of hepatic and renal abnormalities. Results: We observed no significant difference in the peritransplant outcomes in terms of engraftment, febrile complications, regimen related organ toxicities. The time to neutrophil engraftment and to platelet recovery are the same in the two groups (10 and 14 days respectively). 8 patients (47%) in the FEAM group and 9 patients (50%) in the TEAM group need transfusion support therapy with RBC units; 16 patients (94.1%) in the FEAM group and 18/18 in the TEAM group need transfusion support therapy with platelet unit. 13 patients (76%) in the FEAM group and 16 patients (88%) in the TEAM group developed neutropenic fever. The median duration of intravenous antibiotic use are 7 and 6 days for FEAM and TEAM group respectively. The most common grade 3 or 4 organ toxicity is oral mucositis and is observed in 6 patients (35%) in FEAM group and in 4 patients (22%) in TEAM group, while G2 diarrhea is observed in 3 (17%) and 7 (38%) patients, respectively. No severe hepatic or renal toxicity is detected. Conclusion: In conclusion, we would like to document that Fotemustine and Thiotepa could be a readily available alternative to BCNU. According to preliminary comparative results we can conclude that the two regimens, FEAM and TEAM are equivalent for safe and feasible, but longer follow-up and a wider sample are needed to evaluate fully their efficacy and safety. Disclosure of Interest: A. Gravetti: None declared, R. Della Pepa: None declared, S. Avilia: None declared, N. Pugliese: None declared, F. Pane: None declared, A. M. Risitano: None declared, G. De Rosa Funding from: no, Employee of: no, Personal Interest: no, Conflict with: no.
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P222 Different Doses of Anti-Thymocyte Globulin (ATG) in the Conditioning Regimen for Allogeneic Hematopoetic Stem Cell Transplantation G. Pekcan1,*, B. Atesagaoglu1, M. Merter1, P. Topcuoglu1, S. Civriz Bozdag1, S. K. Toprak1, M. Kurt Yuksel1, O. Ates1, M. Ozcan1, T. Demirer1, O. Ilhan1, H. Akan1, M. Beksac1, N. Konuk1, G. Gurman1 1 HEMATOLOGY, ANKARA UNIVERSITY DEPARTMENT OF HEMATOLOGY, ankara, Turkey Introduction: ATG as a part of conditioning regimens has been known to reduce the incidence and severity of acute and chronic graft versus host disease (GvHD). We retrospectively evaluated the impact of ATG at two different doses (r45mg/kg-low vs 445 mg/ kg-high) on the early and late complications and the probability of progression free- or overall survival. Materials (or patients) and methods: Between July 2003 and October 2014, 102 adult patients (61M;41F) with hematological malignancies underwent allo-HSCT from an HLA matched or mismatched donor. Median age was 39 years (18-62 ys). Their diagnoses were acute leukemia (n ¼ 71), chronic myeloprolipherative neoplasia (n ¼ 14), MDS/CMML (n ¼ 10), lymphoma, (n ¼ 6) and myeloma (n ¼ 1). Stem cell sources were peripheral blood (PB, n ¼ 94) and bone marrow (BM, n ¼ 8). Most patients were transplanted from an unrelated donor (83.3%). The ratio of HLA matched:mismatched donor was 55:47. Eighty-one patients (79.4%) received a myeloablative conditioning regimen. While most of the patients (n ¼ 78) received less than 45 mg/kg of ATG-Fresenius (F) in totally (7-45 mg/kg), more than a total of 45 mg/kg was given to 24 patients. (Max: 60 mg/kg/day). ATG-F was infused to the patients in equal divided doses from day minus 3 to 1. Results: The patients’ age and gender was similar between two groups (P40.05). Most of the patients in two groups had acute leukemia (69.2% in low-dose ATG vs 78.8% in high-dose ATG). High dose ATG-F was frequently used in patients with HLA mismatched and/or unrelated donors (Table). The intensity of conditioning regimen and stem cell source were similar distributions between two groups. Transplant outcomes including acute or chronic GvHD, transplant-related mortality and the probability progression-free or overall survival were not affected by ATG-F doses (Table). When we repeated the similar analysis in patients with an HLA-mismatched donor or in those with an unrelated donor, the statistical results did not show any differences.
Table.
Patients’ features and transplant outcome
Variables /ATG Fresenius HLA mismatched donor, present (n ¼ 55) Unrelated donor, present (n ¼ 85) Engraftment kinetics Graft failure Neutrophil (0.5x10e9/L) Platelet (20x10e9/ L) CMV reactivation or infection Acute GvHD, present Gr0-I vs GrII-IV cGvHD TRM Relapse 2-year PFS 2-year OS
r 45 mg/ kg (n ¼ 78)
445 mg/kg (n ¼ 24)
P
41.0% (n ¼ 32)
95.8% (n ¼ 23)
o.0001*
78.2 % (n ¼ 61)
100 % (n ¼ 24)
0.01*
7.0% 17.5 (1028) 19 (7-150)
0.5% 17 (10-26)
1.0 0.67
17.5 (0-40)
0.60
94.7 %
0.60
45.0 % 70% vs 30%
0.78 0.64
35.6% 38.5% 33.3% 39.7±11.9% 27.9±1.51%
0.96 0.23 0.12 0.89 0.71
91.0 % 48.6 % 64.3% vs 35.7% 35.0% 25% 16.7% 31.0±5.4% 40.7±5.8%
Conclusion: In conclusion, based on these result lower doses of ATG might be as effective as higher doses. Therefore we need to find the lowest efficient doses of ATG Fresenius by randomized prospective trials. Disclosure of Interest: None declared. P223 EBMT metaanalysis of treosulfan conditioning before allogeneic hematopoietic stem cell transplantation in paediatric patients with haematological malignancies H. Boztug1,*, E. Glogova1, U. Po¨tschger1, M. Zecca2, P. Veys3, A. Lankester4, A. Cant5, R. Skinner6, M. Slatter7, J. Wachowiak8, K.-W. Sykora9, C. Peters1 on behalf of on behalf of the EBMT Pediatric Diseases Working Party 1 St. Anna Kinderspital and Children´s Cancer Research Institute, Department of Paediatrics , Medical University of Vienna, Vienna, Austria, 2Paediatric Haematology / Oncology, Fondazione IRCCS, Policlinico San Matteo Foundation, Pavia, Italy, 3Great Ormond Street Hospital for Children National Health Service Trust, London, United Kingdom, 4Department of Paediatrics, Leiden University Medical Center, Leiden, Netherlands, 5Department of Pediatric Immunology, Great North Children’s Hospital, 6Department of Paediatric and Adolescent Haematology and Oncology, and Children’s HSCT Unit, Great North Children’s Hospital, Royal Victoria Infirmary, 7Paediatric Immunology Department, Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom, 8 Department of Paediatric Haematology, Oncology, and Haematopoietic Stem Cell Transplantation, University of Medical Sciences, Poznan´, Poland, 9Department of Paediatric Hematology/Oncology, Hannover Medical School, Hannover, Germany Introduction: Standard myeloablative conditioning regimens for allogeneic haematopoietic stem cell transplantation (HSCT) in children with haematological malignancies are mainly based on total body irradiation or busulfan. Their profound short and longterm side effects have necessitated the exploration of less toxic alternatives. Treosulfan is increasingly used in view of its potent immunosuppressive and cytotoxic effects and low toxicity. Materials (or patients) and methods: To further investigate the role of treosulfan in children, the EBMT paediatric diseases working party performed a retrospective analyses of 193 children with haematological malignancies (ALL n ¼ 71, AML n ¼ 47, MDS/ MPS n ¼ 40, other leukaemia/lymphoma n ¼ 25) undergoing allogeneic HSCT following treosulfan conditioning.
Results: Early regimen-related toxicity was low and mainly gastrointestinal. There was no association of toxicity with type of disease or higher treosulfan dose (43x13 g/sqm). High grade early toxicity was not higher in infants (n ¼ 11) or patients undergoing second or later transplantation (n ¼ 51). Treatment-related mortality was low at 14%. Three-year event-free survival was 45±4% and not significantly influenced by number of HSCT (first HSCT 46±4%, 4first HSCT 41±7%, P ¼ 0.32), however it was significantly better in infants (P ¼ 0.022). When compared to treosulfan plus fludarabine, the combination of treosulfan, fludarabine and an alkylator (either thiotepa or melphalan) resulted in significantly better overall survival (OS, P ¼ 0.048) and a trend towards better EFS. In multivariate analysis, OS and EFS were significantly worse for patients transplanted not in remission (P ¼ 0.033 and 0.003). Conclusion: In conclusion, treosulfan based conditioning is a safe and efficacious approach for paediatric haematological malignancies, and especially well tolerated by patients undergoing second or later transplantation and infants. Disclosure of Interest: H. Boztug Funding from: HB received a travel grant from Pierre Fabre, E. Glogova: None declared, U. Po¨tschger: None declared, M. Zecca: None declared, P. Veys: None declared, A. Lankester: None declared, A. Cant: None declared, R. Skinner: None declared, M. Slatter: None declared, J. Wachowiak: None declared, K.-W. Sykora Funding from: KWS received travel grants and study support from medac., C. Peters Funding from: CP received travel grants and study support from medac, Pierre Fabre, Sanofi and Novartis. P224 Allogenic Hematopoietic Stemcell Transplantation Results of Treosulphan Based Conditioning Regimens in Hematological Malignancies: Seven Case Reports From a Single Center ¨ zgu¨r2, O. Gu¨rsel1, M. Yıldırım2, O. Babacan1, I. Eker1,*, G. O 2 S. Sayın , F. Avcu2, A. E. Ku¨rekc¸i1 1 Pediatric Hematology, 2Adult Hematology, Gulhane Military Medical Faculty, Ankara, Turkey Introduction: Treosulphan (Treo), which is structural analog of Busulphan (Bu) is an alkylating agent that widely used in the treatment of hematologic malignancies and solid tumors. Recent years, Treo has become an important component of the conditioning regimens due to its predictable pharmacokinetics,
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sufficient immunosuppressive activity for engraftment and high antileukemic effect. Here we report allogenic hematopoietic stemcell transplantation results (HSCT) of adults and children with hematological malignancies, who had undergone HSCT in Gulhane Military Medical Faculty HSCT Center. Materials (or patients) and methods: Between September 2012 and January 2014, HSCT with treosulphan based conditioning regimen, performed for 7 high-risk leukemia (6 with AML, 1 with ALL) patients between the ages of 4-40 years. The characteristics of the patients and HSCT are summarized in Table 1. Three patients were in first complete remission, while other patients were in Z second complete remission. Two cases were treated with HSCT before, with a TBI based regimen and the other two cases received intensive antileukemic treatment, while the other three cases had no risk factors. Three of the transplantations were performed with peripheral derived stem cells, while the others were performed with bone marrow derived stem cells. In five of the transplantations, donors were full matched siblings (one syngenic), while haploidentical HSCT was performed in one patient. Conditioning regimen of all patients were including 36g/m2 Treo. Results: Full engraftment was achieved in all patients. The mean discharge day of the patients was þ 38 (35-42). While hemorrhagic cystitis, sepsis and HBV reactivation developed in two cases, no complications were observed in other five patients. Primary disease relapsed after HSCT in three cases, within the time periods ranging from 4-9 months. Other four cases didn’t relapsed during follow up. Three of the seven cases (all of them were pediatric cases) are in complete remission without complications for 10, 15 and 20 months follow up period. Two cases died due to primary disease relapse and two cases died due to complicaitons, though they were in complete remission. Conclusion: In the literature it is reported that especially in pediatric myeloid malignancies and in second HSCT, Treo has same myeloablative, immunosuppressive and cytotoxic activity with Bu, but it is reported that it is better than Bu in splenic B and T cell depletion. Treosulphan is associated with much more lower risk of seizures, severe mucositis or hepatotoxicity than Bu, our experiences also supported this. Treo is therefore increasingly replacing Busulphan in radiation free conditioning regimens, especially in pediatric cases. Further safety and efficacy studies are needed especially, for the useage of Treo in HSCTs for adult hematologic malignancies. Disclosure of Interest: None declared. P225 Low dose of ATG-Fresenius is effective to reduce severe Graft versus host disease with low non relapse mortality in high risk allogenic hematopoietic stem cell transplantation I. Ormazabal Velez1,*, A. Bermu´dez1, L. Ya´n˜ez1, B. Lo´pez-Pereira1, G. Martin2, E. Conde1 1 Hospital Universitario Marques de Valdecilla, Hospital Universitario Marques de Valdecilla, 2Banco de Sangre y Tejidos de Cantabria, Banco de Sangre y Tejidos de Cantabria , Santander, Spain Introduction: Graft versus host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). ATG-Fresenius (ATG-F) has demonstrated its efficacy in reducing the risk of acute GvHD
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(aGvHD)1 and chronic GvHD (cGvHD)2 at a dose of 60mg/kg, and at a dose of 30mg/kg3. We analysed the incidence of aGvHD grade I-IV, limited/extense cGvHD, early and late infections including CMV and EBV-PTLD, overall survival (OS), disease free survival (DSF) and non relapse mortality (NRM) in a cohort of patients who received low dose of ATG-F (2132mg/kg). We compare these data with the literature (Ju¨rgen Finke1, Gerard Socie´2). Materials (or patients) and methods: Between 2012 and 2014, 20 patients received ATG-F in our hospital. We used ATGF at a dose of 21 mg/kg (7 mg/Kg days –3,-2,-1) in unrelated peripheral blood donor and in mismatched donor or ATG-F at a dose of 28-32 mg/kg (7-8mg/kg days -4 to –1) in aplasia. Median age was 53 years (9-68) and sixteen patients (80%) received myeloablative conditioning regimen. The diagnosis were 5 acute myeloid leukaemia (25%), 2 acute lymphoid leukaemia (10%), 3 myelodisplastic syndrome, 2 non Hodgkin lymphoma (10%), 1 chronic myeloid leukaemia (5%), 3 aplasia (15%), 1 myelofibrosis (5%) and 3 other (15%) (chronic myelomonocitic leukaemia, blastic plasmacytoid dendritic cell neoplasm and secondary engraftment failure). There were nine patients (45%) in first complete remission at the moment of undergoing HSCT. Results: With a median follow up of 11 months (1-24), the cumulative incidence of aGvHD grade I-IV and grade III-IV were similar (13.6% and 45%) to described by Ju¨rgen Finke (11.7% and 56,3%1). The incidence of CMV infection was higher (70% vs. 54%1 ) which might be due to a higher rate of seropositive patients (85% vs. 62%1) being of high risk (receptor þ /donor -) the 25% of them. Four patients (3 receptor þ /donor -) developed recurrent CMV Three patients (15%) had EBV viremia and one patient developed EBV-PTLD (5% vs. 4,8%1). Five patients suffered JC/BK cystitis o100 days. The percentage of patients who experienced at least one early (o100 days) bacterial and fungal infection were 60% and 5% (vs. 62,3%1 and 33%1), respectively. Fourteen patients were valuable for cGvHD. Eight patients developed cGvHD (57%) and three were extensive (21,4% vs. 12,2%2 ). Late infection (4 100 days) appear in 8/14 patients so that the percentage of bacterial, viral (excluding CMV and EBV) and fungal infection were 21%, 36% and 0%, respectively (viral and fungal infection were not mentioned in the referenced article2). The OS and DFS rates were 75% and 70%. Relapse occurred in 3 patients. The incidence of NRM in the first 100 days was lower (15% vs. 19,6%1,2) being the death causes EBV-PTLD, aGVHD grade IV and multiresistant pseudomonas pneumonia. Conclusion: In our cohort that includes older patients than other published low dose of ATG-F is effective to reduce the incidence of severe GvHD with low early NRM and high OS rate, though a longer follow up is necessary to confirm this results. References: 1. Ju¨rgen Finke, Lancet Oncology, 2009. 2. Ge`rard Socie`, Blood, 2011. 3. Ayuk F, Biol bone marrow transplant, 2008. Disclosure of Interest: None declared.
P226 Comparable outcomes of hematopoietic stem cell transplantation with myeloablative and reduced intensity conditioning in patients with acute myeloid leukemia – a single centre study J. Vydra1,*, V. Valkova1, M. Markova1, L. Novakova1, J. Schwarz1, C. Salek1, P. Cetkovsky1, A. Vitek1 1 Institute of Hematology and Blood Transfusion, Prague, Czech Republic Introduction: We analyzed retrospectively outcomes of allogeneic hematopoietic cell transplantation for acute myeloid leukemia performed at our centre with respect to the type of conditioning regimen. Materials (or patients) and methods: Patients treated with allogeneic hematopoietic cell transplantation (HCT) for acute myeloid leukemia at Institute of Hematology and Blood Transfusion in Prague between 1987 and 2014 were included in the study. Results: 225 patients were identified, 109 males and 116 females. Median age was 46 years (range; 19 – 67). 184 patients received myeloablative conditioning (MA group) and 41 reduced intensity conditioning (RIC group). Myeloablative conditiong regimens used were TBI/Cy (n ¼ 52), Bu/Cy (n ¼ 22), Bu/Flu(n ¼ 27), TBI/Flu/Ida (n ¼ 24) and TBI/Flu/AraC/ Ida(n ¼ 59). Reduced intensity conditioning regimen was Flu/Mel (n ¼ 32) or Bu8/Flu (n ¼ 9). In the MA group, 71 (39%) of patients received graft from matched sibling donor, 58 (32%) from 10/10 matched unrelated donor and 55 (29%) from mismatched unrelated donor (8/10 or 9/10). In the RIC group, 10 (24%) of patients received graft from matched sibling donor, 21 (51%) from 10/10 matched unrelated donor and 10 (25%) from mismatched unrelated donor (8/10 or 9/10), the difference in donor type was statistically significant between the groups (P ¼ 0.025). Prophylaxis of GvHD was cyclosporine and mycophenolate mofetil in RIC patients and in 56% of MA patients, other MA patients received cyclosporine with methothrexate. ATG-Fressenius 20 – 40mg/kg was given to patients with unrelated donors. Patients in the RIC group were older, with median age 60 years (range 23 – 67) compared to 43 (19 – 64) in the MA group (P ¼ 1e-9). Cytogenetic risk group, disease status, CMV serology and AB0 matching were not significantly different between the groups Median OS in the MA group was 2.31 years and 2.08 years in the RIC group (P ¼ 0.281). Cumulative incidence of relapse was 23% and 24% in the MA and RIC groups, respectively. In multivariate analysis of the effect of pretransplant and transplant variables on overall survival, only HLA mismatch, CMV status of the donor and graft type had statistically significant effect on overall survival. HLA mismatch led to decreased OS with HR of mortality of 4.33 and 3.16 for 8/10 and 9/10 mismatch, respectively. Age, gender match, cytogenetic risk group, AB0 match and type of conditioning used were not statistically significant. The overall survival was not different between MA and RIC groups even after adjustment for other transplant characteristics using the multivariate Cox proportional hazards model. Conclusion: In our retrospective single centre analysis, outcome of RIC based allogeneic transplantation for AML was comparable to myeloablative transplantation despite older patient population. Disclosure of Interest: None declared.
P227 Reduced-toxicity conditioning with 4 days of once-daily 100mg/m2 intravenous Busulfan associated with fludarabine and antithymocyte globulins prior to allogeneic stem cell transplantation in patients with high risk hematological malignancies J. Elcheikh1,*, A. wanquet1, R. crocchiolo1, S. furst1, A. granata1, C. faucher1, R. devillier1, S. harbi1, C. lemarie1, B. calmels1, R. bouabdallah2, N. vey2, P. J. weiller2, C. chabannon2, L. castagna1, D. blaise1 1 transplantation, 2hematology, institut paoli calmettes, marseille, France Introduction: The optimal intensity of myeloablation delivered as part of a reduced-intensity/toxicity conditioning (RIC/ RTC) regimen to decrease the recurrence rate, without increasing non relapse mortality (NRM), remains to be established and the disease control remains a major challenge. The introduction of RTC regimens has allowed allogeneic hematopoietic cell transplantation to be performed in patients who were previously considered too old or otherwise unfit. When busulfan Pharmacokinetic is not available the optimal dose is difficult to determine. In this perspective we made the hypothesis that decreasing the daily dose can be safer and efficient in high risk patients. Materials (or patients) and methods: We studied the outcome of 27 patients (median age, 50 years; range, 21-65 years) with hematological malignancies were included. The conditioning regimen based on busulfan at a dose of 100 mg/m2/day intravenously for 4 days, fludarabine at a dose of 30 mg/m2/day for 5 days, and antithymocyte globulins at a dose of 2.5 mg/kg/day for 2 days. Patient, disease and transplant characteristics are shown in Table 1. Results: No patients experienced graft rejection. The median HCT comorbidity index score was 2 (range, 0 to 5). With a median follow-up of 13 months (range, 3-16months), the cumulative incidences of grade 2 to 4 acute graft-versus-host disease (GVHD) and chronic GVHD (all grades) were 43% (95% CI, 26%460%) and 44% (95% CI, 20%468%), respectively. The Kaplan-Meier estimates of overall and disease-free survival at 1 year were 63% (95% confidence interval [95% CI], 42%484%) and 49% (95% CI, 27%471%), respectively. At 1 year, the cumulative incidence of recurrence/disease progression was 32% (95% CI, 12%452%). Non relapse mortality (NRM) was 4% and 19% at day 100 and at 1 year respectively. Patient age, diagnosis, donor type, sex, presence of comorbidities, and the Hematopoietic cell transplantation-specific comorbidities index did not appear to have any statistically significant impact on NRM, recurrence/disease progression, disease-free survival, or overall survival. Conclusion: This well-tolerated conditioning platform can lead to long-term disease control The RTC regimen used in the current study appeared to be safe, with a low NRM rate at 1 year noted among high-risk patients, and efficient disease control, warranting prospective phase 3 trials. Disclosure of Interest: None declared. P228 Application of global metabolomics to predict busulfan pharmacokinetics J. W. Lee1,*, B. Kim2, H. J. Kang1, C. R. Hong1, H. Y. Ju1, J. Y. Choi1, H. Kim1, K.-S. Yu2, I.-J. Jang2, J.-Y. Cho2, K. D. Park1, H. Y. Shin1, H. S. Ahn1 1 Department of pediatrics, Cancer Research Institute, 2Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine, Seoul, Korea, Republic Of Introduction: Busulfan shows narrow therapeutic range. High exposure is associated with systemic toxicity such as venoocclusive disease (VOD) and underexposure is associated with graft failure or relapse. In children, inter-patient pharmacokinetic variability was found to be high even after the use of intravenous (IV) busulfan. In this study, we identified
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biomarkers to predict busulfan pharmacokinetics using global metabolomics. Materials (or patients) and methods: Therapeutic drug monitoring (TDM) was done for pediatric patients who underwent busulfan-based hematopoietic stem cell transplantation (HSCT). The 6-hours interval urine samples were collected before busulfan administration in 59 patients. Patients were divided into 2 groups according to the area under the curve (AUC) of busulfan at the 1st day of busulfan administration; group 1 (n ¼ 50), AUC o25,000 mg*h/L and group 2 (n ¼ 9), AUC Z25,000 mg*h/L. The urinary metabolite profiling was performed by ultra performance liquid chromatography time of flight mass spectrometry (UPLC-TOF-MS) in conjugation with multivariate statistical analysis. Results: Multivariate analysis was performed between group 1 and group 2. Principle components analysis (PCA) of urinary profiling was not separated between group 1 and group 2 clearly, but group 2 tended to cluster together. The urinary metabolites associated with high level of busulfan AUC were identified as deferoxamine metabolites. Because deferoxamine was used for patients with high ferritin level before HSCT, we analyzed the association between ferritin level and the 1st day AUC in 136 pediatric patients. The 1st day AUC/dose (mg/m2) was higher in patients whose ferritin level Z1,000 ng/mL compared to that of the other patients (192.5±56.5 mg*h/L vs 164.4±40.0 mg*h/L, P ¼ 0.006). The optimal busulfan dose to meet the target AUC of 18,750 mg*h/L/day was 120.7±28.1 mg/m2 in patients with ferritin o1,000 mg/mL and 107.6±28.0 mg/m2 in patients with ferritin Z1,000 ng/mL (P ¼ 0.025). Conclusion: This study suggested the possibility of decreased busulfan metabolism in patients with iron overload or a possibility of drug interaction between busulfan and deferoxamine. Further study is needed to identify the exact mechanism of these results. Disclosure of Interest: None declared. P229 Impact of Anti-Thymocyte Globulin (ATG) on overall survival depends on HLA-C group KIR ligand status – Significant benefit in C1 homozygous HSCT recipients J. Clausen1,*, A. Bo¨hm1, V. Buxhofer-Ausch1, S. MachherndlSpandl1, D. Nachbaur2 1 Elisabethinen Hospital Linz, Linz, 2Internal Medicine V, University Hospital Innsbruck, Innsbruck, Austria Introduction: Anti-thymocyte globuline (ATG) is commonly used to decrease the risk for severe acute and chronic GVHD following allogeneic hematopoietic stem cell transplantation (HSCT). However, ATG has not been shown to significantly improve overall survival in randomized studies. Natural killer cell development after SCT is affected by T-cells, and both graft versus host (GVH) and graft versus leukemia (GVL) effects are influenced by killer immunoglobuline-like receptor (KIR) genotype and KIR ligands. Thus, in-vivo T-cell depletion by ATG may have different effects in subgroups defined by the recipient’s HLA-C KIR ligand status (C1/1, C1/2, or C2/2). Materials (or patients) and methods: We analyze retrospectively a two-center cohort of 553 consecutive, HLAmatched, related (n ¼ 361) or unrelated (n ¼ 192) HSCT. Graft source was peripheral blood (n ¼ 434), or bone marrow (n ¼ 110). HSCT were performed between 11/1988 and 1/ 2014. Anti-T-cell serotherapy was applied during conditioning in 178 transplants (mainly ATG-Fresenius (n ¼ 128), median dose 25mg/kg, range 10-90; or Thymoglobuline (n ¼ 44), median dose 5mg/kg, range 1.75-7.5; or Campath (n ¼ 6), 50100mg). Serotherapy was used for an unrelated donor in most instances (143), whereas some matched related transplants (35) included ATG (e.g., per protocol). The predominant indication for SCT was a hematologic malignancy, in an advanced phase in 313 cases.
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Results: In C1/1 homozygous patients (n ¼ 215), cumulative incidence of non-relapse mortality (NRM) at 4y was significantly lower following ATG- versus non-ATG based transplants (18% vs 39%; P ¼ 0.003), resulting in a trend towards better 4y overall survival (OS; 50% vs 39%, P ¼ 0.05). This effect of ATG was not found in C1/2 patients (n ¼ 249) or in C2/2 patients (n ¼ 89). By multivariate analysis considering major confounding factors (recipient age, donor age, disease phase, donor relationship, conditioning type, sex mismatch female-to-male), C1/1, but not C1/2 or C2/2 recipients significantly benefited from ATG in terms of overall mortality (RR, 0.50; P ¼ 0.01), treatment failure (progression or death) (RR, 0.67; P ¼ 0.04) and NRM (RR, 0.31; P ¼ 0.003). In contrast, survival of C2/2 recipients was inferior after ATG-based transplants (RR, 2.4; P ¼ 0.009). Conclusion: We show for the first time that the impact of ATG on HSCT outcomes depends on the recipient’s HLA-C KIR ligand status. In C1/1 homozygous recipients, ATG significantly reduced overall and non-relapse mortality, while in C2/2 recipients, ATG had the opposite effect. In the era of high-resolution HLA-typing, immunological determinants as the KIR ligand status may become more important for the decision for or against ATG, than the donor-recipient family relationship. Disclosure of Interest: None declared. P230 Low incidence of veno-occlusive disease after using intravenous busulfan in allogenic hematpoietic stem cell transplantation for acute lymphoblastic leukemia M. Ramzi1,*, M. Dehghani1, R. Vojdani1, M. Karimi1 1 department of hematology and oncology&stem cell transplantation, Shiraz university of medical sciences, Shiraz, Iran, Islamic Republic Of Introduction: BU and CY is a common conditioning regimen for allogeneic hematopoietic stem cell transplantation (HSCT).The use of i.v. busulfan instead of the oral busulfan can improve outcomes in patients undergoing HSCT by reducing toxicity and transplantation related mortality. Materials (or patients) and methods: We prospectively treated 42 Acute Lymphoblastic Leukemia( ALL) patients who underwent allogeneic HSCT at our center with BU/CY using i.v. BU without dose adjustment. IV BU was administered at a dosage of 0.8 mg/kg through a central venous catheter for 2 hours every 6 hours at a total of 16 doses for 4 days (On days -8 to -5). CY 40 my/kg /day was administered through a central venous catheter for 3 days on days -4 and -2. After one day rest (-1), GCSF-mobilized peripheral blood stem cell (PBSC) without T-cell depletion was infused on day 0. Results: All 42 patients achieved hematopoietic engraftment at a median 14days (range 9-23). All adverse events were those that are commonly observed in HSCT and no specific events related to i.v. BU were observed. The transplant related mortality for were 14.2%. The 1- and 2-year disease free survival (DFS) for i.v. BU group was 59.5%and 54.7% respectively. Disease relapse was the most common cause of death. We found also incidence of veno occulusive disease was very low with i.v. BU (4.8%). Conclusion: Our study demonstrates that i.v. BU conditioning regimen was associated with lower toxicity and promising survival. We found also lower incidence of veno occulusive disease after using i.v. BU in comparison with previous reported data and concluded i.v. BU/CY regimen is less toxic, safe, well tolerated and effective in our ALL patients. Disclosure of Interest: None declared.
Minimal residual disease, tolerance, chimerism and immune reconstitution I P231 Mixed chimerism after allogeneic hematopoietic stem cell transplantation in sickle cell disease: preliminary results after chimerism analysis on peripheral blood sorted cells and erythroid progenitors A. Magnani1,2,*, J. Magalon1,2, F. Touzot1,2, C. Cuzin1, J.-A. Ribeil1,2, F. Bernaudin3, D. Bories4, M. Cavazzana1,2 1 De´partement de biothe´rapie, Hopital Necker-Enfants malades, 2 Institut Imagine, Paris, 3Centre de re´fe´rence de dre´panocytose, CHIC Centre Hospitalier Intercommunal de Cre´teil, 4He´matologie Mole´culaire, Hoˆpital Henri Mondor, UPEC, Cre´teil, France Introduction: A small proportion of patients following hematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD) develop a persistent mixed chimerism (MC) over time associated with clinical control of the disease despite a seemingly low donor engraftment. In order to investigate this condition we analyzed the chimerism in sorted myeloid and lymphoid subpopulations, and erythroid progenitors. Materials (or patients) and methods: Between May 1990 and November 2012, 108 SCD patients followed in the pediatric Cre´teil center underwent allogeneic HSCT in different centers. Myeloablative conditioning consisted of busulfan (16 mg/Kg), cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (20 mg/kg). Among the 104 patients with available chimerism at 2 years, 3 had total autologous reconstitution, 54 (51.9%) had a MC, i.e. o95% of cells from donor origin on total white blood cells (WBCs), the majority ((n ¼ 50) of whom with MC 450%. These patients displayed the same hemoglobin (Hb) electrophoresis than their donors and were free of any SCD symptoms. We analyzed the chimerism in CD14 þ , CD15 þ , CD3 þ , CD19 þ and erythroid burst-forming units (BFU-E) in patients presenting with: (a) donor MCo70% on WBCs, (b) follow-up Z1 year after HSCT and (c) the same Hb electrophoresis as their donors. Results: Preliminary results obtained from 9 patients reveals heterogeneous pattern of subpopulation chimerism (Table 1). The mean follow-up duration is 88.6 months (range 21-155). Patients present a stable Hb comparable to the donor’s. A discrepancy in donor chimerism measured in total WBCs compared to the subpopulations or the erythroid precursors is observed. Patients were divided into two groups according to the donor chimerism in the CD3 þ and in the myeloid compartment. The first group includes patients with donor chimerism in CD3 þ cells lower than in the myeloid compartment. This could be explained by the expansion of recipient’s memory T cells after transplantation and/or a lower T-cell progenitor engraftment in these patients. The second group includes patients with a donor chimerism higher in the CD3 þ than in the myeloid compartment. Interestingly, there is a trend to an higher percentage of donor chimerism in the BFU-E as compared to the myeloid compartment suggesting a selective advantage of donors cells during erythropoiesis.
Donorpheno type
Hb (g/dl)
HbA (%)
HbS Donor chimerism (%) (%) TotalCD3 þ CD14 þ CD15 þ CD19 þ BFUE
AA AA AS AS AS AA AS AS AA
14.2 11.8 13.5 10.6 8.4 10.5 9.7 9.5 10.5
84.5 79.7 50.2 54 24 78.3 31.7 43.4 79.8
0 5.6 37.9 33.3 68.1 7.6 60.6 47.5 4.3
66 19 58 53 21 44 16 18 30
40.5 12 34 44 70 46 31 30 63
98.5 23 71 44 9.4 41 12 26 17
495 31 na 46 5.4 na 10 16 18
90 13 49 58 31 50 13 11 23
Conclusion: These data show that the analysis of chimerism on total WBCs is a poor indicator of the myeloid and erythroid engraftment in SCD patients. Analysis of chimerism on sorted subsets and erythroid progenitors allows a more detailed investigation of this population. Final analysis will include about twenty cases of MC and further investigation on memory/naive T cells compartment as well as on granulocyte monocyte colony forming units (CFU-GM) are necessary to complete these informations. This study may have remarkable implications not only in the setting of HSCT but also for gene therapy trials based on the autologous transplantation of genetically modified CD34 þ cells. Disclosure of Interest: None declared. P232 Delayed CD4 þ T Lymphocyte reconstitution in Haploidentical T Cell Repleted Transplantation with Postransplant Cyclophosphamide compared with HLA Identical Sibling Donor Transplantation in the setting of reduced intensity conditioning regimen A. M. Pe´rez-Corral1,*, D. Champ1, A. C. Franco1, J. Gayoso1, C. Pascual1, J. Anguita1, M. Bastos1, M. Kwon1, I. Gonzalez1, C. Mun˜oz1, D. Serrano1, J. L. Dı´ez-Martı´n1 1 Hematology, Gregorio Maran˜on Hospital, Madrid, Spain Introduction: Haploidentical T Cell Repleted Transplantation(Haplo HSCT) with Postransplant Cyclophosphamide (PTCy) has become a therapeutic option for high risk patients without HLA identical sibiling donor. As previously described, immune reconstitution seems to be appropriate in these patients but few publications comparing with conventional HLA identical sibling transplantation (HLAid HSCT) have been reported to date. Materials (or patients) and methods: We retrospectively recorded 28 pts who received RIC Haplo HSCT with PTCy and 13 pts who received RIC HLAid HSCT between 2007-2014 in our institution. Conditioning included fludarabine (Flu), cyclophosphamide (Cy) and busulfan (Bux) for Haplo and Flu/Bux/melphalan for HLAid. Prophylaxis for GVHD consisted of high dose Cy on þ 3/ þ 4, cyclosporineA (CsA) and
na 52 76 25 12.5 61 17 34 31
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mycophenolate for Haploid and CsA/methotrexate for HLAid. Stem cell source was mobilized peripheral blood in all cases. Immune reconstitution was assessed in terms of lymphoid subpopulations quantification by 5 colour flow-cytometry on FC500 or Navios (Beckman Coulters) on months þ 1, þ 3, þ 6, þ 9, þ 12. X2 and Mann–Whitney test were used for comparison. Results: No statistical differences between patients characteristics in the two groups were found regarding sex, disease status at transplantation, CD34 þ cells infused and acute GVHD incidence. Some differences were shown in diagnosis with more non Hodgkin Lymphoma in HLAid group and more Hodgkin Lymphoma in Haploid group (p0.04), and in median age (38 for Haploid and 52 for HLAid, p 0.006). Table 1 shows the median lymphocyte counts in both groups. In general terms Haploid group reconstitution was good. No differences were recorded in NK cell total counts on any of the studied time points. Total T cell counts, CD4 þ cells , CD8 þ cells were significantly lower in the first three months (time points þ 1 month and þ 3 months) in the Haploid group (Po0.01). On þ 6, þ 9 and þ 12 months CD4 þ cells remained at slightly lower levels in Haploid group without statistically significant differences, while total T cells and CD8 þ lymphocytes were similar in both groups. B cells were practically absent in the first month in Haploid group, but reconstituted from the third month on, with no significantly differences on þ 3, þ 6, þ 9 and þ 12 between both groups. Conclusion: As previously shown, immune reconstitution in Haploid HSCT pts is good. Despite the heterogeneity in initial diagnosis, we observe worse immune reconstitution in our Haploid HSCT group in the first month and a tendency for more delayed CD4 þ cells reconstitution compared with this HLA id HSCT patients. More studies are needed to confirm these findings and determine its clinical implications. Disclosure of Interest: None declared. P233 Immune reconstitution after allogeneic stem cell transplantation: Impact of reduced conditioning regimen, alemtuzumab, anti-thymoglobulin and CMV infection A.-S. Hartjen1, A. Krumbholz2, F. Thieme3, M. Bulduk1, N. Schub1, A. Humpe1, M. Gramatzki1, A. Guenther1,* 1 Division of Stem Cell Transplantation and Immunotherapy, 2nd Medical Department, Christian-Albrecht University of Kiel, 2 Institute for Infection Medicine, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein Campus Kiel, 3Central laboratory, University Medical Center SchleswigHolstein Campus Kiel, Kiel, Germany Introduction: The renewal of the immune system after allogeneic stem cell transplantation is a complex process and not fully understood. Recently, the use of reduced intensity conditioning (RIC) regimens, new anti-viral drugs and intensified GvHD treatment changed the landscape of allogeneic transplantation. However, establishment of a functional immune response against the underlying disease as well as control of infections is still crucial for the long-term outcome. Here, the influence of several parameters on the development of the cellular and humoral immunity of 136 patients consecutively transplanted in a single center was analyzed. Materials (or patients) and methods: All adult patients (81 male, 55 female, median age 52 years) transplanted within three years starting in September 2010 were analyzed. Retrospectively immunoglobulin levels (turbidimetric method) and lymphocyte subset counts by FACS staining were evaluated. In 91 % of the patients a reduced or intermediate conditioning regimen was used; nearly all patients received either anti-thymoglobulin (ATG) or alemtuzumab (25/136 matched related donors) leading to a relatively low rate of chronic GvHD (40%). The immune parameters achieved one year after stem cell transplantation were correlated with the conditioning regimen, diagnosis, relapse, age, sex and history
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of CMV infection, acute and chronic GvHD using Pearson correlation, t-test and regression analysis (SPSS). Results: A normal CD4/CD8 ratio at day þ 360 was only seen in 14% of available patients (3% elevated, 83% reduced) mostly caused by impaired CD4 cell count (90%onormal) while CD8 cell count was increased in 42%. The IgG level was normal in 51%, reduced in 31% and increased in 17 % of tested patients. The CD8 cell count was significantly higher in patients after CMV infection (Po 0.001) and without alemtuzumab (P ¼ 0.017) in the conditioning regimen; the IgG level correlated with history of CMV infection (P ¼ 0.002), CD4, NK (both P ¼ 0.002) and CD8 (P ¼ 0.025) cell count; age, sex, diagnosis, relapse, use of reduced intensity conditioning, acute and chronic GvHD had no significant impact on immune system one year after stem cell transplantation Conclusion: Despite using frequently RIC regimens, one year after allogeneic stem cell transplantation nearly all patients showed a compromised immune system mainly characterized by a reduced CD4 cell count. In contrast, the CD8 cell count is often elevated, in part probably reflecting response to viral infections. Interestingly, the immunoglobulin levels are highly variable ranging from antibody deficiency to hyper-immunoglobulinemia not fully explained by viral triggers. Surprisingly, GvHD had only a minor impact on the immune reconstitution what may be explained by the frequent use of alemtuzumab and ATG in the investigated patient cohort. Disclosure of Interest: None declared. P234 Integrative Profiling of Natural Killer Cell Repertoires Reveal a Role for Less Differentiated NK Cells in Protection from Leukemia Relapse A. T. Bjo¨rklund1,2,*, T. Clancy3, M. Schaffer4, P. Ljungman1, K.-J. Malmberg3 1 Hematology Center Karolinska University Hospital, 2Center for Infectious Medicine, Karolinska Institute, Stockholm, Sweden, 3 Dep of immunology, Radiumhospitalet, Oslo, Norway, 4Dep of Immunology, Karolinska University Hospital, Stockholm, Sweden Introduction: Reactivation of cytomegalovirus (CMV) following transplantation leads to profound changes in the recipient natural killer (NK) cell repertoire with expansion and differentiation of educated NK cells expressing self-specific inhibitory killer cell immunoglobulin-like receptors (KIRs). It has been postulated that such adaptive-like NK cell responses may contribute to anti-leukemic activity thereby protecting from relapse. Materials (or patients) and methods: To address this possibility, we examined donor NK cell repertoires in 106 peripheral stem cell donors and the dynamic reconstitution in 65 donor-recipient pairs. Hierarchical clustering of key parameters (frequencies of CD56bright, CD57 þ , NKG2A þ , NKG2C þ , educated NK cells) that define the heterogenic NK cell compartment, linked clusters with distinct NK cell repertoires, to clinical outcomes. Results: Clusters defining naı¨ve NK cell repertoires in the donor were associated with decreased risk for relapse; HR 0.15, 95% CI (0.14-0.93), P ¼ .04. Corroborating this finding, recipients with naı¨ve NK cell repertoires at 9-12 months and those that displayed resetting of donor repertoires to less differentiated repertoires had lower risk for leukemic relapse; HR 0.18, 95% CI (0.05-0.71), P ¼ .01, and better overall survival; HR 0.11, 95% CI (0.03-0.65), P ¼ .01. Conclusion: In contrast to current beliefs our data suggests that maintenance of a naı¨ve NK cell repertoire rather than an expansion and differentiation of memory-like NK cells is associated with protection against leukemia relapse after allogeneic stem cell transplantation. Disclosure of Interest: None declared.
P235 Donor KIR A haplotype is associated with an increased in T cell recovery after DLI and better overall survival after HLA match sibling pediatric stem cell transplantation A. Pe´rez Martı´nez1,*, I. Romera2,3, J. Valentı´n4, L. Fernandez5,
J. L. Vicario6, M. A´. Dı´az7 1
Servicio de Hemato-Oncologı´a Infantil. Hospital La Paz., Universidad Auto´noma de Madrid, 3Servicio de Hematooncologia infantil, Hospital Monteprı´ncipe, 4IdiPAZ, 5Centro nacional de investigaciones oncolo´gicas (CNIO), 6Servicio de tipaje y HLA, Centro regional de transfusiones de la Comunidad de Madrid, 7Servicio de Hemato-Oncologı´a Infantil., Hospital Nin˜o Jesu´s, Madrid, Spain
P236 CD4 þ FoxP3 þ Regulatory T Cells Induce Tolerance to Bone Marrow Grafts and Promote B Cell Differentiation A. Pierini1,*, H. Nishikii1, Y. Pan1, J. Baker1, D. Schneidawind1, M. Alvarez-Rodriguez1, D. Leveson-Gower1, M. Florek1, R. Negrin1 1 Department of Medicine, Division of Blood and Marrow Transplantation, Stanford University, Stanford, United States
2
Introduction: Graft versus leukemia (GvL) effect after hemotopoietic stem cell transplantation (HSCT) is mediated by donor immune cells recovery. Natural Killer (NK) cell alloreactivity is controlled by the interaction of activatory receptors and inhibitory killer-immunoglobulin-like receptors (KIRs) with major histocompatibility locus class I antigens on the leukemia cells. Haploidentical setting is a plattform for NK cell alloreactivity however HLA identical setting remains unclear. Materials (or patients) and methods: We performed KIRgenotyping of HLA-identical sibling donors in 35 pediatric CD34 selection peripheral blood stem cell transplantations with donor lymphocyte infusion (DLI) at day þ 30 to identify genetic factors affecting leukemia relapse and overall survival. Univariate analysis of leukemia relapse and KIR genotyping was performed in order to identify independent variables predictive of outocome for pediatric acute lymphoblastic leukemia (ALL), (n ¼ 20) and acute myeloblastic leukemia (AML), (n ¼ 15). Results: Donor B haplotype was observed in 21 cases (60%). Statistical analysis shown that donor B haplotype was associated with significantly more relapse in leukemia pediatric patients (38% vs. 0%) and worse overall survival (40% vs. 7%). Further analysis revealed that 2DL5a, 2DS3 and 2DS5 were associated with an increased rate of leukemia relapse (47% vs. 6%, 54% vs. 10% and 58% vs. 13%, respectively) and 2DL5a, 2DS1 and 3DS1 were associated with a worse overall survival (48% vs. 6%, 64% vs. 10% and 58% vs. 13%, respectively). No difference was observed in patients KIR haplotype or KIR donor-recipient KIR haplotype mismatch. We also observed a positive impact of DLI on immune reconstitution, specifically in KIR A donors. T-cell reconstitution was increased on KIR A donors with a median of 1282 vs 477 CD3 þ cells/ml, 205 vs 146 CD3 þ CD4 þ cells/ml and 1002 vs 294 CD3 þ CD8 þ cells/ml on day þ 60, respectively. NK cell reconstitution was impired on KIR A donors with a median of 182 vs 242 CD56 þ cells/ml. Conclusion: In our study, which included only reduced intensity conditioning without antithymocyte globulin followed by related peripheral blood stem cell transplantation and DLI, we found a significant better impact of donor KIR A haplotype in stem cell transplantation outcome. Our results suggest that donor KIR A haplotype had a better T cell expansion after DLI and could decrease leukemia relapse in HLA-identical sibling HSCT. In this setting, T cell alloreactivity dominates NK alloreactivity. References: Kro¨ger N1, Binder T, Zabelina T, Wolschke C, Schieder H, Renges H, Ayuk F, Dahlke J, Eiermann T, Zander A. Low number of donor activating killer immunoglobulin-like receptors (KIR) genes but not KIR-ligand mismatch prevents relapse and improves disease-free survival in leukemia patients after in vivo T-cell depleted unrelated stem cell transplantation. Transplantation. 2006 Oct 27; 82(8): 10241030. Disclosure of Interest: None declared.
Introduction: CD4 þ FoxP3 þ regulatory T cell (Treg) adoptive transfer has proved to prevent GVHD while Treg impact on immune reconstitution and engraftment has been less well studied. Materials (or patients) and methods: We tested the impact of Diphtheria Toxin (DT) induced Treg ablation in C57BL/6 FoxP3DTR mice in different transplantation models. We used mass cytometry (Cytof) and FACS analysis for studying bone marrow populations. Cytokines were quantified by ELISA assays. Bone marrow cell localization has been explored with confocal microscopy. Sorted purified CD4 þ CD25 þ FoxP3 þ Treg from lymph nodes and spleen were adoptively transferred for several chimerism and reconstitution studies as reported. Results: Transplantation of lethally irradiated (TBI 10 Gy) Treg depleted mice with allogeneic (BALB/C or FVB/N) T-cell depleted bone marrow (TCD BM) or purified hematopoietic stem cells (HSCs) resulted in donor rejection or mixed chimerism (Po0.01). Treg depletion favored host CD4 þ (Po0.001), CD8 þ (Po0.01) and GR1 þ cell (Po0.01) persistence and delayed B cell reconstitution (Po0.001). Treg transfer to DT treated mice rescued engraftment (Po0.01) and boosted B cell reconstitution (Po0.001). Syngeneic transplanted Treg depleted mice engrafted but had markedly delayed B cell reconstitution (Po0.01) thus Treg promote donor B cell differentiation in manner not dependent upon alloreactivity. These mice had higher numbers of donor Lin-Sca1 þ cKit þ HSCs (Po0.05) and Lin-Sca1 þ cKit þ Flt3 þ lymphoid progenitors (Po0.05) while B220 þ IgM-CD19 þ cKit þ Pro-B cells (Po0.05) and total CD19 þ cells (Po0.01) were reduced showing a block of maturation in the early phases of B cell differentiation. IL-7 production was reduced after Treg depletion (Po0.05) and IL-7 administration rescued B cell reconstitution (Po0.05) in syngeneic transplanted Treg depleted mice demonstrating that Treg control B cell differentiation through an IL-7 dependent mechanism. We found that Treg localize near the endosteum (Po0.0001) co-located with donor B220 þ B cell clusters and gather in the epiphyseal areas where donor HSCs were mainly detectable suggesting that Treg act as an immunological barrier for HSCs and B cell progenitors, providing a protective niche. Treg transfer induced B cell reconstitution (Po0.01) in non irradiated immune deficient BALB/C rag2-/-gc-/- mice that received allogeneic TCD BM. These mice had higher numbers of Pro-B (Po0.05) and total CD19 þ cells (Po0.05) with reduced levels of inflammatory cytokines such as INFg and IL12 and increased IL-7 and VEGF. In a non myeloablative (TBI 5.5 Gy) allogeneic (C57BL/6 -4 BALB/C) model of rejection, host Treg adoptive transfer induced persistent full donor chimerism if Treg were injected together with a sublethal dose of donor T cells (P ¼ 0.016) and enhanced donor chimerism if Treg were injected with low dose IL-2 (Po0.001) without impacting on GvHD. Conclusion: Our findings indicate that Treg act as a key regulator of B cell differentiation promoting production of mature B cells from lymphoid precursors through an IL-7 mediated mechanism not dependent on alloantigen recognition. Our data suggest that Treg play a role in building the donor HSC and B cell precursor niche. Finally, Treg adoptive transfer induces immune reconstitution and tolerance to bone marrow grafts even in the absence of conditioning providing a new tool for clinical translation especially in children with SCID or hemoglobinopathies. Disclosure of Interest: None declared.
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P237 Immunogenetic Cross-Talk in Patients Transplanted for AML: CMV Reactivation Is Not a Strong Stimulus for Immune Response Against Leukemia A. Papalexandri1,* on behalf of Hematology Department and BMT unit, George Papanicolaou Hospital, Z. Boussiou1, C. Apostolou1, S. Charalampidou1, P. Zerva1, T. Touloumenidou1, V. Konstantinou1, D. Mallouri1, I. Batsis1, A. Anagnostopoulos1, I. Sakellari1 1 Hematology Department and BMT Unit, G.PAPANIKOLAOU HOSPITAL, THESSALONIKI, Greece Introduction: Early cytomegalovirus (CMV) re-activation after allogeneic hematopoetic stem cell transplantation (allo-HCT) for AML has been correlated with enhanced graft versus leukemia (GvL) effect, thus implying an immunogenetic cross- talk between donor-derived cytotoxic response against both CMV and leukemia. Data on this are conflicting and correlation could not be confirmed for special group populations. Materials (or patients) and methods: In this study we explore whether CMV re-activation favors the outcome in a group of 49 AML patients consecutively allo-transplanted between 2008-2012. Forty-nine patients (29 male, 20 female), aged 14-63 years (median 42) underwent allo-HCT with myeloablative (42/49) or reduced-intensity conditioning (7/49), receiving graft from an HLA- identical sibling (25/49), matched volunteer (23/49) or haplo-identical (1) donor. Most patients were transplanted in complete remmision (CR), 22/49 in CR1 and 11/49 in CR2 and 16/49 patients had refractory disease. Monitoring of CMV viral load was performed weekly for 3 months with real-time PCR, or more if patients were treated for GvHD. Upon clinical suspicion, other samples were tested. Results: Overall, 19/49 patients (39%) presented with CMV reactivation {viremia (4 ¼ 1000 copies/ml) in 16/49, gastritis in 2/49, cystitis in 1) at a median of 46 days post-tranplant (13402). Leukemia relapse occured in 7/19 patients with CMV at a median of 189 days (2-512), versus 7/30 CMV-free patients (37% vs 23%, P ¼ ns). Disease free survival (DFS) was 591 days for CMV patients (95% CI: 417-939) and 609 for CMV-free (95% CI: 474-871, P ¼ ns). Overal survival (OS) was 746 (95% CI:472963) and 625 (95% CI:504-899) days, respectively, P ¼ ns. Only acute and chronic GvHD were correlated with longer DFS and OS. Sixteen out of 19 CMV (84%) patients presented with increasing/full donor chimerism 30 days after reactivation, due to expansion of donor cytotoxic lymphocytes; however, this did not protect from imminent relapse. No particular clinical correlations could be found even for 14/19 patients with early (o100 days) CMV reactivation (RR: 28% vs 28% for all other patients, DFS: 714 vs 503 days, OS: 746 vs 625 days, P ¼ ns for all comparisons). Conclusion: In conclusion, CMV reactivation in AML transplanted patients is not related to graft allo-reactivity. Late relapses in this cohort of AML patients, transplanted often in remission, are not influenced by immunogenetic dynamics at the first semester, when CMV occurs. Regardless CMV reactivation, OS was comparable in all patients, providing evidence on a succesfull pre-emptive treatment of CMV. Larger prospective studies are strongly warranted in order to explore possible immunogenetic interactions. Disclosure of Interest: None declared.
P238 Long-term stable mixed chimerism in patients undergoing HSCT for non-malignant disorders A. Stikvoort1,2,*, J. Mattsson1,2, M. Sundin3,4, M. Uhlin1,2, B. Sundberg1, M. Uzunel5 1 Oncology-Pathology, Karolinska Institutet, Stockholm, 2Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Huddinge, 3Clinical sciences, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, 4 Hematology/Immunology section, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Huddinge, 5Therapeutic Immunology, Karolinska Institutet, Stockholm, Sweden Introduction: Long-term stable mixed chimerism (MC) is rare after allogeneic hematopoietic stem cell transplantation (HSCT). MC is defined as 5-95% residual recipient hematopoietic cells beyond one-year post-HSCT. The mechanisms directing hematopoietic recovery to MC in some patients and to full donor chimerism (DC) in others are poorly understood. Materials (or patients) and methods: We compared patients with DC and long-term stable MC for a median of 9.5 years postHSCT. Both the clinical situation and the immune system (functional and phenotypic properties) were investigated by questionnaires, western blot, multiplex bead-based immunoassay, ELISA and flow cytometry. Blood samples were drawn at 2 weeks and 45 years post-HSCT. Additionally, lineage-specific chimerism status in the MC patients was determined. The results were analyzed by the Mann-Whitney U test, Fisher’s exact test, Spearman’s rank correlation coefficient, ordinary fit least squares linear regression and robust fit linear regression. Results: At 2 weeks post-HSCT cytokine levels did not differ between the groups. Neither did the groups differ in long-term outcome in terms of general well being, immunoglobulin response to vaccinations and in the most central phenotypic features of the immune system (e.g., differentiation status, CD4/ CD8 ratio, B and NK cell frequency). More than 5 years post-HSCT, MC patients had significantly higher NKT cell levels (CD94 þ CD8 þ and CD56 þ CD8 þ T cells) than DC patients. In depth analysis of the MC patients revealed that recipient chimerism could be observed in a large variety of immune cell lineages (i.e., total, CD4 þ , CD8 þ and gd þ T cells; B cells; NK cells; myeloid cells; and cytokine producing cells). Furthermore, the percentages of recipient chimerism in different cell lineages were positively correlated with each other, e.g., an increase in CD3 chimerism was associated to an increase in CD56 chimerism. Both recipient and donor derived cells in MC patients produced cytokines to the same degree in response to mitogen stimulation. No difference in mitogen response was observed between the groups. Conclusion: Long-term MC does not appear to negatively affect the well being of the patients. It could even be argued that the recipient system has a more differentiated phenotype than the donor system, indicating that MC might be of benefit, protecting against infections. The recipient immune system may still be functional in MC patients, as both recipient and donor derived cells responded to mitogens. Two scenarios could possibly explain such a symbiotic relationship between donor and recipient system: a superfluous duplicity of the entire immune system or an adjuvant role by the recipient system to the donor system. Disclosure of Interest: None declared. P239 Early post-allogeneic transplantation WT1 transcript positivity predicts AML relapse C. Messina1,*, E. Sala1, M. Carrabba1, F. Pavesi1, L. Vago1, B. Gentner1, C. Tresoldi1, M. Tassara1, E. Xue1, C. Corti1, J. Peccatori1, F. Ciceri1, M. Bernardi1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Patients (pts) with refractory or relapsed acute myeloid leukemia (AML) or high risk myelodysplastic syndrome (MDS) receiving allogeneic hematopoietic stem cell transplantation (HSCT) with active disease have a poor outcome. Thus,
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HSCT and post transplant immunotherapy need to be further optimized to treat pts with persistent disease at HSCT. When complete remission (CR) is achieved after myeloablative transplant conditioning, reliable approaches are essential to detect minimal residual disease (MRD) and predict the risk of relapse. The Wilms’ tumor gene 1 (WT1) is over-expressed in the bone marrow (BM) of 480% of AML and advanced MDS pts, and is considered a promising marker for MRD detection. We retrospectively evaluated data from BM of pts with active AML or MDS transplanted at our Institute and in CR after HSCT, to evaluate the sensitivity and specificity of quantitative WT1 monitoring to predict disease relapse. Aim of the study was to evaluate if early post-HSCT WT1 positivity in BM of AML/MDS pts in CR correlates with subsequent overt relapse. Materials (or patients) and methods: We reviewed our institutional database in order to select AML/MDS pts receiving HSCT for active WT1 overexpressing disease. Between 1/2009 and 11/2013, 38 consecutive pts: 22 AML, 4 sAML, 3 tAML, 1 AML-GS and 8 MDS; source of HSCT: 7 MRD, 2 MUD, 27 MMRD, 2 CB. During post-transplant follow-up (FU), BM WT1 levels were quantified using the ELN WT1 assay with a cut-off level of positivity of 250 copy numbers [1], from 34 evaluable pts. We analysed BM data from the three earliest time points of FU after HSCT (day þ 30, þ 60 and þ 90) and latest FU available for each patient. Results: At day þ 30 after HSCT 33 out of 34 pts (97%) achieved CR. One patient experienced a WT1-negative relapse and 4 pts were excluded because they lacked complete molecular data for the time points considered in the present analysis. Thus, 28 pts were evaluable for early WT1 monitoring after HSCT. Overall, at a median follow-up (FU) of 472 days (range 55-2110), 16 pts (60%) had relapsed while 12 pts (40%) were still in CR. Of the relapsed pts, 12 (75%) showed at least one early BM WT1 positivity while in haematological CR and 4 (25%) had WT1 levels4250 during all time points. On the contrary, only 3/12 (33%) of pts who did not relapse had a positive WT1 at Z1 timepoint while 9/12 (67%) had WT1 levels below the threshold of 250 during all time points. We observed a significant correlation between positive early BM WT1 levels and subsequent haematological relapse and between negative early BM WT1 levels and persistence of CR (Fisher’s test P ¼ 0.02). BM WT1 positivity anticipated the haematological relapse of 158 days (range, 20-360). Conclusion: Early post HSCT BM WT1 positivity is a promising tool to predict relapse in AML/MDS pts transplanted with active disease that achieved haematological CR after HSCT. A single quantitative level above the threshold of MRD positivity in the early post HSCT phase is predictive of subsequent overt disease relapse, with a median time to relapse of 158 days. Our results suggest that quantitative monthly monitoring of BM WT1 in the first 3 months after HSCT is useful to predict relapse and to drive proper post transplant immunotherapy (e.g. immunosuppression withdrawal and/or donor lymphocyte infusion) to induce an anti-leukemia effect at an early stage. References: 1. Cilloni et al. J Clin Oncol. 2009 Nov 1;27(31):5195-201. Disclosure of Interest: None declared. P240 Droplet Digital PCR for DNMT3A and IDH1/2 Mutations to Improve Early Diagnosis of Acute Myeloid Leukemia Relapse after Allogeneic HSCT C. Brambati1,*, C. Toffalori1, E. Xue1,2, L. Crucitti2, R. Greco2, A. Crippa3, L. Chiesa1, B. Mazzi1, C. Tresoldi3, S. Galbiati4, N. Soriani4, J. Peccatori2, M. G. Carrabba2, M. Bernardi2, M. Ferrari4, V. Lampasona4, L. Vago1,2, F. Ciceri2 1 Unit of Molecular and Functional Immunogenetics, 2Unit of Hematology and Bone Marrow Transplantation, 3Molecular Hematology Laboratory, 4Genomics for the Diagnosis of Human Pathologies Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Leukemia relapse after allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) represents a major
unsolved issue, and efforts are aimed to anticipate relapse detection and treatment to the Minimal Residual Disease (MRD) stage. Mutations in DNMT3A and IDH1/2 occur early during the step-wise process of Acute Myeloid Leukemia (AML) tumorigenesis, possibly representing disease founder mutations (Shlush et al., 2014) and thus putatively optimal markers for MRD monitoring. Materials (or patients) and methods: By conventional Sanger sequencing, we screened for presence of the mutations of interest (DNMT3A R882H, IDH1 R132C, IDH1 R132H, IDH2 R140Q and IDH2 R172K) 67 AML samples collected at diagnosis from patients who underwent allo-HSCT at our center. We selected 19/67 patients who carried at least one of the mutations and tested a total of 79 bone marrow samples collected longitudinally over time by ultra-sensitive droplet digital PCR (ddPCR) assays. As controls, we tested diagnosis bone marrow samples from 10 patients typing negative for the mutations, and peripheral blood samples from 15 healthy individuals. ddPCR assays were performed using the Bio-Rad QX100 system: each sample was tested in duplicates, employing 25 ng of genomic DNA in each reaction well. Samples with a mutant-to-wild-type ratio above 0.1% were considered positive. ddPCR results were compared to those obtained testing the same samples by quantitative PCR (qPCR) assessment of the WT1 gene transcript and of hematopoietic chimerism. Results: All the samples which resulted positive by conventional sequencing were confirmed by ddPCR, and in all of them the population carrying the mutant allele, quantified by ddPCR, consistently exceeded the morphological count of leukemic blasts, suggesting the presence of the mutation also in apparently normal hemopoietic cells. All the samples tested at post-transplantation relapse resulted positive for the mutations present at diagnosis, except for one case, originally carrying both DNMT3A and IDH2 mutations and typing negative for the latter at relapse. When post-transplantation remission samples were tested, 41/42 (98%) of those harvested from 5 patients who remained long-term leukemia-free (median follow-up after allo-HSCT: 20 months) resulted negative for the mutations of interest. 4/5 patients who subsequently relapsed presented at least one sample harvested during apparent disease remission which resulted positive by ddPCR, 3 of which immediately preceeding relapse. Of notice, only 2 of those 5 patients displayed WT1 transcript overexpression before relapse and only one displayed host chimerism above the 1% threshold. Conclusion: Although the very small number of patients included in this preliminary analysis warrants for caution, ddPCR for DNMT3A and IDH1/2 mutations appears extremely promising, displaying very high specificity and sensitivity in relapse prediction, and comparing favorably with our present and historical results obtained by qPCR-based post-transplantation monitoring techniques. References: Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia (Shlush et al., Nature, 2014). The origin and evolution of mutations in acute myeloid leukemia (Welch et al., Cell, 2012). Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia (Kro¨nke et al., Blood, 2013). DNMT3A Mutations in Acute Myeloid Leukemia (Ley et al., NEJM, 2010). Disclosure of Interest: None declared. P241 Automated workflow for microchimerism analyses A. Wijngaard1, B. Luiken1, E. Torun2, N. Westerink1, J. Geerligs1, E. Rozemuller1,3, D. Bost3,* 1 Research and Development, GenDx, Utrecht, Netherlands, 2ATQ Biyoteknoloji, Ankara, Turkey, 3Research and Development, KimerDx BV, Utrecht, Netherlands Introduction: In an effort to enhance qPCR-based microchimerism analysis, we are investigating the ability to
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automate microchimerism analysis workflow using robotic pipetting stations and the Rotor-Genes qPCR platform. The use of robotic systems with our proprietary reagents and software will likely automate the process of post-transplant monitoring almost completely. The robotic station prepares and dispenses the reaction mixes, while our software provides reaction set up and analysis tools. With its rapid software analysis and not needing post-PCR sample processing, our microchimerism analysis workflow may become the most automated to date. Materials (or patients) and methods: We currently utilize analysis software and a panel of 30 qPCR assays to comparatively genotype and quantify DNA samples. The assays are designed and validated to detect INDEL and copy number variants between individuals on 20 different chromosomes. These differences are then used to track an individual’s genomic material in a mixed sample. Results: The genotyping is performed using 10 mixtures, each containing 3 qPCR assays multiplexed in a single well. We program the robot to set up the genotyping experiment and load the samples into the 72-well ring. Our software, combined with the RGQ 72-well ring configuration, enables simultaneous comparative genotyping of up to six samples. By tracking the relationships between samples, the software automatically selects informative markers between 2 or more individuals. The results from the genotyping are stored by the software for recall during subsequent monitoring. For monitoring an individual in a mixture, informative markers are used in a singleplex configuration, with each marker validated to a sensitivity of 0.05% of a genomic mixture. Reports from a monitoring test may be generated from a single time point or longitudinally, using the data collected from the individual over time. Longitudinal reports provide a temporal view for better understanding of rejection or relapse kinetics. Conclusion: With the development of our software, reagents and automated platform approach, our workflow reduces manual calculations, increases throughput and helps to minimize errors in test execution, potentially making it a fully-automated solution for microchimerism analysis. Disclosure of Interest: A. Wijngaard Employee of: Employee, B. Luiken Employee of: Employee, E. Torun Employee of: Employee, N. Westerink Employee of: Employee, J. Geerligs: None declared, E. Rozemuller Employee of: Employee, Conflict with: Shareholder, D. Bost Employee of: Employee, Conflict with: Shareholder. P242 Nucleophosmin 1 Mutation Monitoring after Allogeneic Hematopoietic Stem Cell Transplantation to Predict Relapse and Guide Preemptive Therapy E. Xue1,2,*, E. Sala1, C. Tresoldi3, L. Crucitti1,2, R. Greco1, C. Messina1, M. Carrabba1, L. Vago1,2, M. Bernardi1, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, 2Unit of Molecular and Functional Immunogenetics, 3Molecular Hematology Laboratory, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: Nucleophosmin 1 (NPM1) mutations represent the most frequent genetic alterations in adult patients with acute myeloid leukemia (AML). In addition, NPM1 mutations are considered early events in leukemogenesis, shared by the majority of leukemic subclones in a given patient and remaining stable throughout the disease course. For these reasons, they represent promising minimal residual disease (MRD) markers for the longitudinal monitoring of AML patients. In the present study we investigated the potential value of NPM1 mutation monitoring in the context of allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT). Materials (or patients) and methods: Between January 2011 and June 2014, we performed 135 alloHSCTs for AML, and in 25/135 (18.5%) we could monitor NPM1 mutations during the
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post-transplantation follow-up. Among these 25 patients, 7 were transplanted in active disease (AD), while 18 had achieved complete remission (CR) before the start of conditioning; 20 patients were treated with full dose treosulfan-based conditioning, while 5 patients were treated with busulfan-based myeloablative conditioning. To monitor NPM1 mutations we employed Real Time quantitative PCR (RTqPCR), using as reference the ABL gene and as template genomic DNA extracted from bone marrow (BM) samples harvested monthly during the post-transplantation follow up, starting from day þ 30 after alloHSCT. Results: The median follow up in this cohort was 255 days (range 68-1332); 9/25 patients experienced leukemic relapse after alloHSCT with a median time to relapse of 136 days (range 68-384), whereas median follow-up for the 16 nonrelapsed patients was 556 days (range 126-1332). Out of the 9 patients who experienced relapse, 8 remained positive for NPM1 mutation at time of relapse, whereas 1 was persistently negative for NPM1 during all his follow up and ultimately experienced a NPM1-negative relapse. Among the 8 patients with NPM1-positive relapses, each of them showed at least one detectable NPM1 value during follow up, 6/8 showed two consecutive positive values and 5/8 showed an increasing value of NPM1 quantification. 4/8 patients never displayed negative values. The average time from molecular conversion to morphological and clinical relapse was 127 days. Among the 16 patients who remained in LT CR, 8/16 of them showed persistent negativity for NPM1 during all the follow up, whereas 8/16 patients showed at least one sample with detectable NPM1. Notably in 4 out of these eight patients the positive samples were those early after alloHSCT, and NPM1 became subsequently negative. Only 2/16 non-relapsed patients showed two consecutive positive values, and only one displayed increasing values. Conclusion: In line with the biological notion that NPM1 mutation is an early and stable event in AML, in our analysis its monitoring could predict relapse in 8/9 patients with a useful time of advance, confirming its usefulness as MRD marker also in the transplantation setting. In particular, increasing values in consecutive BM samples are highly suggestive of impending relapse. Although this study was performed on a small patient cohort, it suggest that NPM1 is a useful tool to identify those patients that will experience relapse and to timely carry out a preemptive therapy. Disclosure of Interest: None declared. P243 Impact of pretransplant minimal residual disease detection by flow cytometry in acute myeloid leukemia E. Perez1,*, O. Lo´pez-Godino1, M. Cabrero1, J. Labrador1, S. Alonso1, J. J. Perez1, L. Lo´pez-Corral1, L. Vazquez2, M. Gonzalez1, J. M. Hernadez1, N. Puig1, M. Diez-Campelo1, A. Martin1, M. V. Mateos1, B. Paiva2, M. C. del Can˜izo1, J. San Miguel1, M. D. Caballero1, M. B. Vidriales1 1 HOSPITAL UNIVERSITY OF SALAMANCA, 2HOSPITAL UNIVERSITARIO DE SALAMANCA, Salamanca, Spain Introduction: The value of minimal residual disease (MRD) analysis by flow cytometry (FC) in acute myeloid leukemia (AML) patients early during treatment have been reported by several groups, including our own. However, few data is available about the impact of MRD detection by FC before stem cell transplantation (SCT). The purpose of the present study is evaluate the impact of pre-transplant MRD detection by FC in terms of disease free survival (DFS) in AML patients. Materials (or patients) and methods: We included 122 ‘‘de novo’’ non APL AML patients, who achieved complete remission (CR) after induction chemotherapy and who further underwent an allogeneic (n ¼ 62) or autologous (n ¼ 60) SCT, all of them having immunophenotypic aberrancies detected in blast cells at diagnosis, suitable for MRD monitoring during follow-up. Bone marrow samples were analysed by four-colour multiparametric FC.
Results: With a median follow-up of 61 months, we demonstrated that pre-transplant MRD allowed to stratify relapse risk when considering both the whole group of 122 patients (5 years DFS: 51% vs 71% for patients with MRDo10-4 (n ¼ 43) or MRD4 ¼ 10-4 (n ¼ 79) respectively; P ¼ 0.004) or the autologous stem cell transplant group (5 years DFS: 66% vs 40% for patients with MRDo10-4 (n ¼ 18) or MRD4 ¼ 10-4 (n ¼ 42) respectively; P ¼ 0.05). However, in the subanalysis performed in patients who underwent allogeneic SCT, no statistical differences in DFS were detected according with the MRD level (MRDo10-4 or MRD4 ¼ 10-4 76% vs 66%; P ¼ 0.37),that could suggests that allogeneic SCT can overcome the adverse impact of high MRD level. In addition, considering those patients with higher MRD levels (410-4), allogeneic SCT as a consolidation treatment (n ¼ 37) was associated with a better outcome compared to autologous SCT (n ¼ 42), with a 5y-DFS of 66% vs 40% respectively; P ¼ 0.10), while no differences were detected according with the type od SCT in low MRD level (o10-4) patients. In the multivariate analysis performed in the whole group of patients, consolidation with allogeneic SCT (P ¼ 0.04) and MRD levels o10-4 (P ¼ 0.01) were independent factors associated with DFS, together with cytogenetic at the diagnosis (P ¼ 0.02). When considering only the allogeneic SCT group, development of chronic graft versus host disease (P ¼ 0.006) and cytogenetic at the diagnosis (P ¼ 0.02) were the only factors with independent impact on DFS, while pre-transplant MRD lost its significant impact in this group. Conclusion: These results support the role of pre-transplant MRD detection by FC in AML patients, which could be useful to decide therapeutic strategy early after transplant. In addition, according to our results, allogeneic SCT could overcome adverse prognosis associated with higher MRD levels confirming the existence of a graft versus leukemia effect. Disclosure of Interest: None declared. P244 Time-points for minimal residual disease monitoring in patients with acute myeloid leukemia who underwent allogeneic stem cells transplantation: a comparison between multiparameter flow cytometry and Wilm’s tumor 1 expression G. Rossi1,*, A. M. Carella1, M. M. Minervini1, F. di Nardo2, M. M. Greco1, E. Merla1, L. Savino1, G. P. de Cillis1, N. Di Renzo3, A. Melpignano4, S. Capalbo5, G. Pisapia6, N. Cascavilla1 1 Department of Hematology, IRCCS "Casa Sollievo della Sofferenza", San Giovanni Rotondo, 2Department of Hygiene, Catholic University of Sacred Heart, Rome, 3Department of Hematology, "Vito Fazzi’’ Hospital, Lecce, 4Department of Hematology, ‘‘Perrino’’ Hospital, Brindisi, 5Department of Hematology, ‘‘Riuniti’’ Hospital, Foggia, 6Department of Hematology, ‘‘San Giuseppe Moscati’’ Hospital, Taranto, Italy Introduction: Allogeneic stem cell transplantation (allo-SCT) represents the only effective therapy for high risk patients with acute myeloid leukemia (AML). Nevertheless, relapse remains a crucial issue in this setting and new methods able to prevent it are needed. The detection of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) and WT1 expression showed to be a powerful predictor of prognosis Anyway, the most sensitive method in predicting the relapse as well as the optimal time point to evaluate MRD in allografts remain to be determined yet. Thus, with this purpose, we studied technical performance and prognostic impact of MRD assessed at the optimal cut-off by MFC and WT1 expression, before and after transplant Materials (or patients) and methods: Between June 2010 and January 2014, 30 adult AML patients achieving a complete remission (CR) underwent allo-SCT at the Hematology Department of IRCCS ‘‘Casa Sollievo della Sofferenza’’ Hospital. Samples for MRD analysis were collected before (-10 days), at day þ 30 and þ 90 after transplant. Samples were investigated by 6-color MFC and quantitative analysis of WT1. All patients received a myeloablative conditioning regimen.
Results: When MFC and WT1 were compared at the optimal cut offs before transplant MRD identified by MFC showed better values of sensitivity (71.4% vs 57.1%), specificity (87.0% vs 72.7%), positive predictive value (PPV) (62.5% vs 40.0%) and negative predictive value (NPV) (90.9% vs 84.2%) than WT1. At day þ 30 post-transplant, MFC displayed a higher sensitivity (85.7% vs 60.0%) but a lower specificity (68.2% vs 90.9%), PPV (46.7% vs 60.0%) and similar NPV (93.8% vs 90.9%) compared to WT1. Finally, at day þ 90 diagnostic performance of WT1 was better than MFC. Combining the methods by considering as positive the patients who were positive by both MFC and WT1, pre and post-transplant specificity improved (91.3%, 95.8% and 87.0%, respectively at each check-point). Patients with pre-transplant MRD values greater than 0.1% by MFC showed a shorter DFS compared to others with lower values (P ¼ 0.006). Only a trend to significance was found between ptients having more and less than 64 copies WT1/104 ABL (P ¼ 0.081). One month after transplant, patients having MRD higher than 0.05% by MFC and 138.0 copies WT1/104 ABL by WT1 showed a poorer DFS (P ¼ 0.005 and P ¼ 0.010, respectively). Multivariate analysis confirmed results obtained from univariate analysis. At day þ 90, only MRD by WT1 higher than 103 copies WT1/104 ABL seems to identify pts with high risk to experience a recurrence (P ¼ 0.085). Conclusion: MFC may be used indifferently before and thirty days after the transplant to detect MRD, while WT1 expression should be preferentially measured after the transplant, based on different diagnostic performance and prognostic impact reported in our series. At day þ 30 post- transplant we recommend that minimal residual disease should be investigated by either or both methods, as it has a strongly predictive role. MRD measurements appear to be less predictive at later time points. The combination of MFC and WT1 may be preferred to a single one when further treatments should be administered to prevent the relapse. In fact, only in this case patients have a high probability of experiencing a recurrence. Disclosure of Interest: None declared. P245 Immune Reconstitution after HLA-Haploidentical and HLA-Matched Allogeneic Transplantation (BMT) Utilizing Posttransplantation Cyclophosphamide G. Cengiz-Seval1,*, A. Vulic1, S. McCurdy1, C. Kanakry1, R. Varadhan2, H. Symons1, L. Luznik1 1 Oncology, SKCCC at Johns Hopkins, Baltimore, MD, 2Oncology and Biostatistics, SKCCC at Johns Hopkins, Baltimore, United States Introduction: Posttransplantation cyclophosphamide (PTCy) limits severe acute and chronic graft-versus-host disease (GVHD) and facilitates engraftment, permitting HLA-matched donor (MD) BMT without additional GVHD prophylaxis and the safe performance of HLA-haploidentical (haplo) BMT. Low rates of transplant related mortality, infection, and posttransplant lymphoproliferative disorders (PTLD) support that PTCy selectively depletes proliferating alloreactive T-cells, while preserving resting memory T-cells essential for immunologic recovery. However there is limited data regarding immune reconstitution after haplo or MD BMT utilizing PTCy. Materials (or patients) and methods: We assessed immune reconstitution in 71 patients undergoing myeloablative (MA) haplo BMT with PTCy (50mg/kg on days þ 3 and þ 4), mycophenolate mofetil (MMF) (days þ 5-35), and tacrolimus (days þ 5-180) as GVHD prophylaxis and 73 patients undergoing MA MD BMT with PTCy (50mg/kg on days þ 3 and þ 4) as sole GVHD prophylaxis. Flow cytometric immunophenotyping was performed on peripheral blood samples collected serially at predetermined time points. Results: Immune Reconstitution was not statistically different after HLA-matched related and HLA-matched unrelated BMT; therefore these groups are reported together. In all groups NK cells recovered to normal donor counts by 6 months. In patients without GVHD, NK recovery was more rapid with no
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significant difference by 2-3 months. By 1 year following haplo and MD BMT, median ALCs were in the normal range (11004800 cells/ml) and B-cell counts were higher than those in normal donors. Results were similar after MD and haplo BMT. However, CD4 þ and CD8 þ T-cell counts at 1 month were statistically significantly lower after haplo BMT (both p’so0.0001). Median CD4 þ T-cell counts at 1 year were significantly lower after both MD and haplo BMT compared with normal donors, but higher after haplo compared with MD BMT. At 6 months and 1 year there was no significant difference in CD8 þ T-cells after haplo or MD BMT compared with normal donors. There was a trend towards lower total CD8 þ T-cell counts at all time-points after haplo compared with MD BMT. Phenotypic effector memory (EM) and terminally differentiated effector memory T-cells (TEMRAs) recovered rapidly after BMT, particularly within the CD8 þ fraction after both haplo and MD BMT. However, phenotypically naı¨ve cells remained low throughout the first post-BMT year. Consistent with our prior reports, Tregs comprised a larger portion of CD4 þ T-cells after MD or haplo BMT compared with donors (Po0.0001). The statisticaly significant difference in expression of PD-1 and TIM3 but not CD160, 2B4 and BTLA on CD4 þ and CD8 þ T was noted beetwen patients that received MD vs haplo BMT. Conclusion: Haplo and MD BMT lead to comparable reconstitution of NK and B-cells, however CD4 þ and CD8 þ T-cell early recovery lags after haplo BMT. This delay is likely attributable to the addition of MMF and tacrolimus, which may be mitigated by discontinuation of MMF at Day 35, resulting in equivalent CD4 þ T-cell and CD8 þ T-cell numbers by 3 and 6 months respectively. Further deciphering of co-inhibitory molecules expression is needed. Excellent immune reconstitution by 1 year supports the low rates of PTLD and infectious deaths after haplo or MD transplantation with PTCy. Disclosure of Interest: None declared. P246 Evaluating of posttransplant relapse risk in patients with acute leukemia using the gene expression markers A. Shakirova1, I. Barkhatov1,*, O. Shakirova2, V. Teplyashina1, A. Evdokimov1, Y. Gudozhnikova1, S. Bondarenko1, L. Zubarovskaya1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, First Pavlov Saint Petersburg State Medical University, 2St.Petersburg State Polytechnic University, St.Petersburg, Russian Federation Introduction: It is known that up to 50 percent of cases of acute myeloid leukemia (AML) does not have informative genetic markers. At the same time, the studies of donor chimerism do not fully indicate the degree of tumor cells elimination and not always allows to estimate the risk of posttransplant relapse. Thus, finding of universal markers allowing for adequate therapy in post-transplant period, is quite important. WT1, BAALC, EVI1 and PRAME gene expression analysis is one of the possible approaches is this field. Materials (or patients) and methods: Our study included 63 patients with AML (1 to 60 years old) (M0-2pts, M1-11pts, M213pts, M3-2pts, M4-21pts, M5-11pts, M6-1pt, M7-2pts ) who underwent allogeneic transplantation of hematopoietic stem cells (allo-HSCT). In 24 patients myeloablative conditioning regimen were used, 39 – received reduced toxicity protocols. Assessing the levels of WT1, BAALC, EVI1, PRAME gene expression and the level of chimeric transcripts was performed by means of RQ-PCR with normalization for ABL gene expression. For the donor chimerism monitoring a panel of STR-markers was used. Results: As based on gene expression in healthy donors, we have established cut-off overexpression (gene exp. / ABL exp. X100) values for the genes: WT1 – 250, EVI1-10, BAALC – 20, PRAME - 200. For the present patient setting, we found no statistically significant differences in WT1, BAALC, EVI1, PRAME genes expression between the patients with different FAB variants of AML. However we were able to identify a trend to
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higher values of PRAME and BAALC gene expression in patients with M1 variant, and WT1 gene values among patients with M4 variant of AML. In patients who underwent transplantation in relapse state, we have noted a significant overexpression of EVI1 (P ¼ 0.006), WT1 (Po0.001), BAALC (Po0.001). A similar trend was observed for PRAME gene (P ¼ 0.08). EVI1 gene overexpression was revealed for 6 patients (33%), WT1 in 13 cases (72%), BAALC in 10 patients (55%) and PRAME in 4 patients (22%). When comparing the data on chimeric transcripts expression, we detected a correlation between expression levels of the studied genes and chimeric transcripts (PML-RARa and RUNX1-RUNX1T1, Po0.05) as well as with donor chimerism levels. At the same time, we did not find this relationship, when comparing with data about of expression of a CBFB-MYH11 chimeric gene. When evaluating the test sensitivity and specificity analysis we revealed a bellow-cutoff value of the expressed genes in presence of the chimeric transcript less than 2%. Conclusion: Evaluation of universal gene marker expression is an attractive approach for assessing efficiency of therapy in patients with AML. Thus, their use in early diagnostics of relapse in post-transplant period is quite promising. However, due to low specificity caused by basal expression in normal cells, futrher application of these markers is limited, when detecting minimal residual disease levels. Disclosure of Interest: None declared. P247 Analysis of donor chimerism kinetics to assess relapse in allogeneic hematopoietic stem cell transplantation in patients with hematologic malignancies I. Barkhatov1,*, A. Evdokimov1, A. Shakirova1, V. Teplyashina1, S. Bondarenko1, E. Semenova1, A. Chukhlovin1, L. Zubarovskaya1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, First Pavlov Saint Petersburg State Medical University, St.Petersburg, Russian Federation Introduction: Allogeneic hematopoietic stem cell transplantation is an effective treatment for different hematological malignances. Efficiency of donor cell engraftment is determined by donor chimerism evaluation, i.e. determining of genetically different cell populations coexistence in patient. We assume that the kinetics of chimerism in patients with hematologic malignancies, in turn, may be defined in terms of relapse probability, and it could be used as a marker of minimal residual disease (MRD) after allogeneic transplantation of HSCs (allo-HSCT). Materials (or patients) and methods: We have analyzed 440 allo-HSCT’s in patients with various hematologic malignancies. For the donor chimerism monitoring we used standard panels of human DNA markers based on STR variability (short tandem repeats, microsatellites). The ratios of donor and recipient cells were calculated according to the data derived from fragment analysis, using the Genemarker program. The study was performed on days þ 15, þ 30, þ 45, þ 60 and þ 100 after transplantation. Results: Achieving of full donor chimerism (95%) significantly increases the 5-year survival (P ¼ 0.001) and reduces the probability of disease recurrence (P ¼ 0.01). Among patients with mixed chimerism the stability of donor chimerism level is most important, being associated with lower risk of relapse (P ¼ 0.013). Thus, in patients with stable mixed chimerism, the markers molecular markers of MRD had no significant effect either on overall survival, or the likelihood of relapse (P ¼ 0.55 and P ¼ 0.34 respectively). Meanwhile, we had found an inverse correlation between the level of donor chimerism and expression of relapse markers, i.e. WT1 (R ¼ -0.4, P ¼ 0.0001) and EVI1 (R ¼ -0.16, P ¼ 0.03). Similar data were obtained when analyzing expression of the chimeric transcripts: BCR-ABL (R ¼ -0.26, P ¼ 0.0001), MLL-AF4 (R ¼ -0.36, P ¼ 0.003), TEL-AML (R ¼ -0.37, P ¼ 0.003). Hovewer, this dependence with other markers (CBFB-MYH11, PML-RARa, RUNX1-RUNX1T1) could be also obtained.
Conclusion: Monitoring of donor chimerism after allo-HSCT adequately reflects the dynamics of donor cells engraftment and may be used as an adequate method for predicting recurrence of the disease, but it can not be used as a single method in MRD analysis. Disclosure of Interest: None declared. P248 Fetal microchimerism in development of acute GVHD after haploidentical BMT I. Barkhatov1,*, A. Shakirova1, O. Smykova1, V. Teplyashina1, A. Evdokimov1, A. Chukhlovin1, L. Zubarovskaya1, B. Afanasyev1 1 R.M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, First Pavlov Saint Petersburg State Medical University, St.Petersburg, Russian Federation Introduction: Haploidentical transplantation of hematopoietic stem cells (haplo-HSCT) is a feasible therapeutic approach in patients lacking an HLA-identical donor. However, high risk of graft-versus-host disease (GVHD) is among major complications in haplo-BMT. Currently, there exist some data concerning associations between maternal microchimerism levels (detection of maternal cells in recipient’s blood) and increased risk for GVHD development. Meanwhile, the effects of microchimerism upon GVHD probability are still controversial. The aim of our study was to adapt a technique for quantitative evaluation of fetal (recipient-cell) and mathernal (donor-cell) microchimerism and investigation of its effects upon actual risk of common complications after HSCT. Materials (or patients) and methods: Determination of microchimerism was carried out by real-time alle-specific polymerase chain reaction (AS RQ-PCR). The analytic panel included 20 SNP markers located at different chromosomes. Evaluation of PCR sensitivity was carried out using DNA mixtures from several established cell lines (HL60, K562, 293T, MOLT3, A549) at serial dilutions. The samples from 20 donorrecipient pairs were analyzed. The patients’ age ranged from 2 to 27 years. Results: The sensitivity thresholds for the SNP-based test panel ranged from 10^-4 to 10^-5. We have found out that detection of fetal microchimerism in the donor organism is associated with lower risk, or lower degree of acute GVHD (P ¼ 0.01). Moreover, we observed a trend to higher timeframe for transplant engraftment, and lower probability of full donor chimerism achievement, if the donor exhibited fetal microchimerism (P ¼ 0.12). The patients in our setting transplanted from donors with detectable fetal microchimerism had a tendency to a higher overall survival (P ¼ 0.14). Hovewer, we did not reveal any significant association between microchimerism levels (fetal and maternal), and higher probability of chronic GVHD development, as well as any differences in other posttransplant complications for recipients with maternal microchimerism. Conclusion: Evaluation of fetal microchimerism may be considered a useful and informative approach to selection of potential donors, or a method of GVHD prediction in haploidentical HSCT. Disclosure of Interest: None declared. P249 B-cell intrinsic and extrinsic defects in apoptosis, migration and T-cell communication impede the germinalcenter formation of memory B cells after allogeneic-HSCT A. Mensen1, Y. Oh1, S. Demski1, P. Hemmati2, C. Jehn2, J. Westermann2, G. L. Vuong2, B. Do¨rken2,3, C. Scheibenbogen1, R. Arnold2, I.-K. Na1,2,4,* 1 Institute for Medical Immunology, Charite´ Berlin, 2Department of Hematology, Oncology and Tumorimmunology, Charite´ Berlin, 3 Max-Delbru¨ck Center (MDC) for Molecular Medicine, 4Experimental and Clinical Research Center (ECRC), Berlin, Germany Introduction: Sustained CD27 þ memory B-cell deficiency despite normalized lymphocyte counts is associated with
prolonged susceptibility to infections and poor vaccination responses within one year after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Little is known about mechanisms contributing to defects in the germinal-center (GC) formation of memory B cells in allo-HSCT patients. Materials (or patients) and methods: We have analyzed the peripheral B and T-cell subset reconstitution and their expression of functional markers for 37 adult acute leukemic patients at day (D) 90, D180 and D360 after allo-HSCT. In addition, efflux capacity of naı¨ve B cells as an intrinsic contributor to B-cell survival as well as B-cell apoptosis after T-cell dependent B-cell activation have been investigated. Quantification of cytokines and chemokines in plasma at D180 was performed with the luminex multiplex technology. Results: We could confirm a sustained CD27 þ memory B and CD4 þ T-cell deficiency at one year after allo-HSCT. Additionally, we found significantly decreased percentages of CXCR5expressing memory T follicular helper (TFH) cells in patients when compared to age-matched healthy controls (Mean %±SEM: HC 7±1, alloHSCT 2±1; pr0.05). Moreover, CD27-IgD- double negative B cells of putative GC-independent origin, were fully recovered. This suggested defects in the GC reaction, which was further supported by significant lower CXCR5 expression on naı¨ve B cells (MFI±SEM: HC 5899±975, alloHSCT 3494±554; pr0.001), important for the entry into B-cell follicles. CCR7, mediating the migration into the T-cell zone, was only slighty decreased. Lower HLA-DR expression on naı¨ve B cells further could impair antigen-presentation and thus maintenance of TFH cells in patients (HC 20446±4742, alloHSCT 9150±2386). B and CD4 þ T cells exhibited an increased Fas expression ex vivo, indicating an apoptoticprone/activated state (Mean %±SEM: naı¨ve B, HC 4±1, alloHSCT 13±2, pr0.001; CD4 þ T, HC 19±5, alloHSCT 63±8; pr0.05). This might result from an inflammatory milieu in patients as expressed by increased plasma level of G-CSF, GMCSF, IFNa, TNFa, IP-10, MCP-1, MIP-1b and Eotaxin and increased B-cell subset expression of CD86 and CXCR3. We also observed a significantly reduced capacity of naı¨ve B cells from Cyclosporin A-treated patients to efflux the dye rhodamine 123, which further could increase apoptosis susceptibility (Mean % effluxing naı¨ve B cells±SEM: HC 51±6, alloHSCT 28±7, pr0.05). Upon T-cell dependent stimulation with CpG/CD40L/PWM/ionomycin, patient B cells up-regulated Fas, which was paralleled by significantly increased Fas-L expression on CD4 þ and CD8 þ T-cell subsets and associated with significantly increased B-cell apoptosis for patients than for HC (Mean % cells±SEM: HC 15±2; alloHSCT D360 60±6, pr0.001). Conclusion: We conclude that B-cell intrinsic and extrinsic survival defects in the T-cell dependent B-cell activation impede the GC formation of memory B cells up to one year after allo-HSCT. This emphasizes the importance to consider prolonged infectious prophylaxis, to control cellular vaccination responses and to evaluate the adoptive transfer of memory B cells as a new treatment option to improve B-cell immune function after alloHSCT. Disclosure of Interest: None declared. P250 Minimal residual disease analysis by flow cytometry patients with acute myeloid leukemia before and after allogeneic hematopoietic stem cell transplantation I. Kozanoglu1,*, G. Unver1, M. Yeral2, C. Gereklioglu2, A. P. Korur2, N. Buyukkurt2, S. Solmaz2, M. Kasar2, C. Boga2, H. Ozdogu2 1 Cell Processing Unit, 2Clinical Unit, Baskent University Adana Adult Bone Marrow Transplantation Center, Adana, Turkey Introduction: Allogeneic stem cell transplantation has become a treatment choice in patients with acute myeloid leukemia (AML). Monitoring of minimal residual disease (MRD) by multiparametric flow cytometry (MFC) has the advantage of being applicable in most patients. The aim of the present
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study was to evaluate the efficacy of monitoring of MRD using 8-color MFC both before and after transplantation to predict relapse in allo-SCT patients with AML. Materials (or patients) and methods: We identified 24 consecutive AML patients who had undergone allo-SCT between March 2011 and December 2014. To be included in the study, patients had to have at least one leukemiaassociated immunophenotypes (LAIPs) at diagnosis. The study was performed on erythrocyte-lysed whole bone marrow samples, after staining with directly conjugated monoclonal antibodies. Samples were analyzed using 8-color MFC (FACS Canto II; BD, Bioscience), and FACS DIVA was used for data analysis. When possible, the same monoclonal antibody and fluorochrome were used at diagnosis and for subsequent monitoring of MRD. Positive MRD was defined as the observation of a cell population with 40.1% LAIPs at diagnosis in bone marrow samples. Minimal residual disease testing was performed before transplantation (immediately before initiation of the conditioning regimen) and at months 1, 3, 6, and 12 after allo-SCT. Results: Sixteen patients who were shown to be MRDnegative by MFC at transplantation and underwent allo-SCT had lower rates of relapse (18.75% vs. 33.4%, P ¼ 0.04), better overall 1-yr survival (85% vs. 34%, P ¼ 0.01) and a lower cumulative incidence of relapse (P ¼ 0.032) than patients who were MRD-positive (40.1%). All post-transplant MRDpositive patients underwent a therapeutic intervention after transplant (tapering of immunosuppression, donor lymphocyte infusion, or re-transplant) with the intention of preventing relapse. Disease was controlled and MRD disappeared in four of these patients. Disease recurred in the other four patients Conclusion: We can conclude that follow-up with MFC for the detection of MRD in AML before and after SCT is useful for predicting relapse. In the post-transplant setting, monitoring of MRD by MFC could be a key preemptive intervention. References: 1.Walter RB, Gyurkocza B, Storer BE, Godwin CD, Pagel JM, Buckley SA, Sorror ML, Wood BL, Storb R, Appelbaum FR, Sandmaier BM. Comparison of minimal residual disease as outcome predictor for AML patients in first complete remission undergoing myeloablative or nonmyeloablative allogeneic hematopoietic cell transplantation. Leukemia. 2014 Jun 3. 2. Bastos-Oreiro M, Perez-Corral A, Martı´nez-Laperche Prognostic impact of minimal residual disease analysis by flow cytometry in patients with acute myeloid leukemia before and after allogeneic hemopoietic stem cell transplantation. Eur J Haematol. 2014 Sep;93(3):239-46. Disclosure of Interest: None declared.
Paediatric issues I P251 The outcomes of haploidentical hematopoetic stem cell transplantation performed with unmanipulated donor stem cells and post-transplant cyclophosphamide in high risk children: a single center experience A. Yes¸ilipek1,*, V. Uygun1, G. Karasu1, H. Dalog˘lu1 1 Pediatric Bone Marrow Transplantation Unit, Bahc¸es¸ehir University, School Of Medicine, Istanbul, Turkey Introduction: In recent years, some of haploidentical transplantations are performed with unmanipulated bone marrow or peripheral blood stem cells which the GVHD prophylaxis includes a more intense chemoterapy approach. This approach is now more popular by post-transplant high dose cyclophosphamide. In this study, we present the children who transplanted haploidentically in our center, with unmanipulated bone marrow or peripheral blood stem cells
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and GVHD prophylaxis with post-transplant high dose cyclophosphamide. Materials (or patients) and methods: We assessed 16 HSCT in 15 patients retrospectively, who underwent haploidentical related HSCT with unmanipulated bone marrow or peripheral blood stem cells and used post-transplant high dose cyclophosphamide for GVHD prophylaxis. Five of patients were male and 10 were female between 3.5-17 years old, median 9 years old. One patient was AML developed after Ewing Sarcoma and one was ALL progressed from CML. Another patient was transplanted for aplasia developed after a haploidentical transplantation from his mother which had been performed with CD34 positive selection. One patient underwent second haploidentical transplantation because of the relapse. Four patients were in the first complete remission, five were in the second and three were in the third in the transplantation date. Donors were their mother in ten transplantation, the father in five, and the sibling in one patient. For the conditioning regimen BU þ FLU þ Etoposid was used for seven patients and two patients with BU þ FLU þ ATG/, BU þ CY þ Mel, /BU þ FLU þ ATG and FLU þ CY þ Mel, and one patient with BCNU þ Etoposid þ ARA-C þ Mel. Fifty mg/kg CY on the 3rd and 5th day and CsA or tacrolimus with MMF or MP were also used for GVHD profilaxis. The source of stem cells was bone marrow in six transplantation while bone marrow and peripheral blood stem cell were used in ten transplantation. The median count of TNC, MNC, CD34 þ and CD3 þ was 12,9x108 (4,1-39), 8,8x108 (4,8-14,3), 5,9 x106 (2,5-16) and 2,9 x108 (1,1-7,8), respectively. Results: All patients engrafted with a median of 16 days (1623) and 18 days (11-37) for neutrophil and thrombocyte recovery, respectively. Grade 1 acute GVHD developed in one patient, and grade 2 in 4 and 3 in 3 patients, while limited chronic GVHD developed in two patients. Three patients had hemorrhagic cystitis and four developed VOD. Two patients were lost in the first 100 days because of sepsis (TRM 12, 5%). After 100 days, a patient died of progression of the primary disease and one patient was lost because of pneumonia. One patient with diagnosis of Hurler showed graft rejection. The rest ten patients (66.6%) are disease-free (OS is 73.3%) and on the follow up of median 10 months (between 4-20 months). Conclusion: The advantages of using unmanipulated hematopoetic stem cells are low laboratory costs, no worry for waiting for a suitable donor, and less risk for the graft failure or T cell deficiency. Our results are consistent with these circumstances in our high risk patients and encouraging for haploidentical HSCTs. However our results should be accepted as a preliminary report with these few patients. Disclosure of Interest: None declared. P252 Chronic Granulomatous Disease (GCD): diagnosis and therapy A. Beghin1,*, M. Zucchi1, R. Baffelli1, F. Bolda1, F. Porta2, A. Lanfranchi1 1 Lab Cell Stam,Children’s Hospital, Spedali Civili di Brescia, 2 Pediatric Oncohematology and BMT Unit, Brescia, Italy Introduction: Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by early onset of recurrent and severe infections. The underlying pathophysiological mechanism in CGD is a defective respiratory burst associated with an impaired microbicidal function of phagocytes. The molecular defects causing CGD are generally because of the absence, low expression or malfunctioning of one of the NADPH oxidase components. The X-linked form of the disease is caused by defects in the heavy chain of the cytochrome b588 component (gp91phox coding by CYBB gene) and accounts approximately 60% of the cases. The autosomal recessive forms (AR-CGD) are caused by defects in one of the cytosolic components of the NADPH oxidase: p47phox coding by NCF1 gene (approximately 30% of the cases), p67phox, p40phox, or
even the cytochrome b588 light chain component p22phox coding respectively by NCF2, NCF4 and CYBA genes (all together the remaining 10% of the cases).Screening for CGD is usually performed by functional assays such as the neutrophil respiratory burst assay, performed by flow cytometry (DHR123). Genetic analysis is critical for diagnostic confirmation, genetic counseling, prenatal diagnosis and gene therapy. Materials (or patients) and methods: All patients (pts) were diagnosed by dihydrorhodamine 123 (DHR123).We indentified 13 pts with impaired phagocytic functions by DHR123 assays: 10 males and 3 females. We perfomed molecular analysis in 12 of 13 pts. 4 males were related members: 2 brothers and an uncle and his grandson. Results: We investigated 7 males for CYBB mutationIn 2 Pts, no alteration in the coding region were found, in 2 pts splice site mutation was identified. For 3 a frameshift was detected and for 1 pts it was identified a missense mutation while it was found a nonsense mutations in 1 pt. For one patient no DNA was available for analysis.Moreover we analyzed genes for ARCGD in 3 females.Only 1 pt shows mutation in NCF1 gene, while the other 2 have only polymorphisms.8 pts, all males, underwent transplantation: 5 MUD (pt 8 undergoing two HSCT) and 3MRD (Matched Unrelated Donor), the mean number of CD 34 þ infused was 9.58x106/kg and the mean number of CD 3 þ infused was 267x105/kg. 30 days later HSCT, they showed 100% donor chimerism for 4 patients and a detection of mixed chimerism for 4 patients, while pt 8 underwent a second transplantation. Conclusion: CYBB was the most frequently mutated gene in our cohort of pts with an heterogeneity of mutations. For the 2 brothers in which the coding region results wild type, we hypothesized that it will be mutation in promoter region or will be a deletion in intronic region not yet analyzed. Moreover these preliminary data show a characteristic cluster of snp in AR-CGD patients. Particularly all share the same Arg90His snp in heterozigousis, that Olsonn et al. dimostrated significantly reduced reactive oxygen species production. 2 patients have no mutation but share the same snp in CYBA and NCF1 gene.In our setting of pts (5/8) have a good outcome. No severe or chronic was observed: GvHD I grade in 2 of 8. Disclosure of Interest: None declared. P253 Outcome of hematopoietic stem cell transplantation using full matched grandparents in pediatric patients lacking an HLA identical sibling A. A. Hamidieh1,*, M. Behfar1, M. Ostadali1, S. Alizadeh1, A. Kasaeian1, A. Ghavamzadeh1 1 Hematology, Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic Of Introduction: The extended family search is a more practical method to find full matched other relative donors in regions where consanguineous marriage is more frequently practiced. However, few studies showed that searching extended families such as parents, grandparents, aunts and uncles for an appropriate donor is useful. The purpose of this retrospective study was to investigate the outcome of HSCT using grandparents as hematopoietic stem cells donors. Materials (or patients) and methods: Twenty one patients (11 female, 10 male) aged r15 years old who had received HSCT from their grandparents since 2007 to 2014 were enrolled. We compared the result obtain in 12 patients who had less than or equal to 55-year age difference with their donor (group 1) with 9 patients who had greater than 55-year age difference with their donor (group 2). Results: Indications for HSCT were Fanconi Anemia (n ¼ 6), Thalassemia Major (n ¼ 5), Acute Leukemia (n ¼ 4), Primary immunodeficiency (n ¼ 4) and Mucopolysaccharidosis (n ¼ 2).
The median age of patients was 6 years (range: 0.25-11 years) and it was 60 years for donors (range: 45-75 years). The mean age difference was 54.5 years (range: 36.5-72.9 years). All the donors were completely full matched. Regarding graft versus host disease (GvHD) occurrence, Although Acute GvHD was significantly higher in group1 (P-value ¼ 0.008), there was no significant difference in chronic GvHD between two groups. With the median fallow up 3.17 years (range: 1.4- 4.6 years), overall survival (OS) and disease free survival (DFS) was 80.9% and 76.15, respectively. Also there were no significant differences in OS and DFS in two group (P-value o0.66 & P-value o0.33). Conclusion: Although searching the extended family for HLAmatched donors especially among grandparents seems very uncommon, this can increase the chance of well-tolerated and success transplantation especially in regions where consanguinity is common. Disclosure of Interest: None declared. P254 Allogeneic HSCT for HR-ALL in I CR in children treated according ALL IC-BFM 2002 program - the Polish Pediatric Group for Hematopoietic Stem Cell Transplantation report A. Pieczonka1,*, O. Zajac-Spychala1, A. Chybicka2, J. Owoc-Lempach2, M. Wysocki3, R. Debski3, J. R. Kowalczyk4, K. Drabko4, J. Gozdzik5, J. Wachowiak1 1 Department of Oncology, Hematology and Pediatric Transplantology, University of Medical Sciences, Poznan, 2Department of Pediatric BMT Hematology and Oncology, Wroclaw Medical University, Wroclaw, 3Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, 4Department of Pediatric Hematology, Oncology and Transplantology, Medical University of Lublin, Lublin, 5Jagiellonian University Colegium Medicum, Krakow, Poland Introduction: We present results of allo-HSCT in pediatric patients with high risk group acute lymphoblastic leukemia in first complete remission (HR ALL I CR) who were prospectively qualified and uniformly treated according the same therapeutic protocol ALL IC-BFM 2002. Materials (or patients) and methods: Between 2003-2013, 112 children (41 girls, 71 boys), median age 8.5 years (1-17.8 years) were transplanted from matched sibling donor (MSD) (n ¼ 49/112; 43.7%) or matched unrelated donor (MUD) (n ¼ 63/112; 56.3%). Stem cells source were: bone marrow 61.6% (69/112) or peripheral blood in 37.5% (42/112) or cord blood in 0.9% (1/112). The majority of patients (75.9%; 85/112) received conditioning with FTBI, remaining Busulfan-based 20.5% (23/112) or Treosulfan-based 3.6% (4/112). Cyclosporine A was used as a basic drug of graft versus host disease prophylaxis (GvHD) 100% (112/112), children prepared for MUD transplantation received additionally anti-thymocyte globulin and ‘‘short’’ MTX. Results: Engraftment was achieved in 111 patients (99.1%). Two boys were transplanted twice from MUD (1- no graft take; 1- secondary graft insufficiency). Acute GvHD grade II-IV or cGvHD was observed in 46.9% (23/49) and 16.3% (8/49) children after MSD and in 54% (34/63) and 20.6% (13/63) patients who received transplantation from MUD. Cumulative incidence of transplant related mortality after 1 year was 0.063±0.035 and 0.179±0.049 for children transplanted from MSD (3/49; 1- aGvHD; 2 infection in patients with cGvHD) or MUD (11/63; 9- GvHD-related; 1-lack information; 1- sepsis) respectively. Relapse rate was 6.1% (3/49) and 9.5% (6/63) after MSD or MUD transplantation respectively. After 5 years the probability of leukemia-free survival for MSD or MUD transplantation were 0.929±0.040 and 0.872±0.050 respectively, while probability of event-free survival 0.813±0.056 and 0.720±0.059, respectively. Forty children transplanted from MSD and 46 from MUD are alive in I CR. Overall survival at 5 years for MSD was 0,832±0.054, while for MUD 0,752±0,057 for MUD. Forty one (83.6%) patients transplanted
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from MSD alive 12-138 (median 86) months whereas 48 (76.2%) children after MUD alive 13-132 (median 68) months. Conclusion: In children with ALL in ICR treated according ALL IC BFM 2002 - 1. Allo-HSCT demonstrated excellent antileukemic effect. 2 TRM after transplantation from MSD was lower than after MUD. 3. The GvHD was the most common cause of TRM, indicating the need of further optimization of donor selection, appropriate cells source choice and GvHD prophylaxis. Disclosure of Interest: None declared. P255 Successful allogeneic HSCT after treosulfan-based conditioning in two children with PNH: single-center experience A. Pieczonka1,*, J. Skalska-Sadowska1, A. Sobkowiak-Sobierajska1, K. Wachowiak-Szajdak1, R. Tomaszewska2, T. Szczepanski2, J. Wachowiak1 1 Department of Oncology, Hematology and Pediatric Transplantology, University of Medical Sciences, Poznan, 2Department of Pediatric Hematology and Oncology, Zabrze, Medical University of Silesia, Katowice, Poland Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a complex hematologic disorder of the hematopoietic stem cells. Patients present with recurrent intravascular haemolysis, venous thrombosis and hematopoietic failure. In children, PNH is very rare, clinical course is similar to that in adults, but the majority of children present with bone marrow failure. Nowadays, apart from symptomatic treatment two options of treatment are used: eculizumab (humanized monoclonal antibody against complement protein C5) or allogeneic HSCT (allo-HSCT). However, little information is available in children regarding efficacy and safety of either eculizumab or allo-HSCT and the risk of VOD is very high. Materials (or patients) and methods: We report two children with documented PNH and treated with allo-HSCT after treosulfan-based preparative regimen. Results: Patient 1. Twelve year-old boy with leucopenia, anemia, recurrent infections and hemolysis, hemoglobinuria, hepatopathy, thrombosis and with proven presence of GPInegative neutrophils and erythrocytes in peripheral blood was transplanted from MUD (10/10) in 2010. Preparative regimen consisted of treosulfan (3 x 14g/m2), fludarabine (5 x 30 mg/ m2) and thymoglobulin (7.5 mg/kg) was used. He received 6.0 x 106/kg CD34 þ cells from peripheral blood. For GvHDprophylaxis CsA and ‘‘short’’ MTX course 10 mg/m2 were given. Reticulocytes appeared on day þ 7, granulocytes 40.5x109G/L on þ 14, platelets 4 20 G/L on þ 13. Full donor chimerism was proven þ 28. On day þ 14 engraftment syndrome was diagnosed and treated with steroids. Since this time patients suffered from acute (II) and chronic extensive skin GvHD. He was treated with corticosteroids (CS) and ECP. Currently GvHD seems to be resolved, CS were withdrawn in July 2014. Patient general condition is good, without any PNH symptoms with full donor chimerism, however, he suffered vertebral fracture. No liver toxicity or thrombosis occurred. Patient 2. Eleven-year-old girl with pancytopenia, chronic bacterial eyelid infection, recurrent hemolysis, red blood cells and platelet transfusion-dependent and PNH granulocytes and erythrocytes clones proved in 2013 was transplanted from UD (9/10) in June 2014. Treosulfan-based conditioning was the same as in Patient 1. She received 2.55x106/kg bone marrow derived CD34 þ cells. GvHD prophylaxis consisted of CsA with MFF (-1 to þ 28 day). Reticulocytes appeared on þ 18 day, granulocytes 40.5 x 109 G/L on þ 28, platelets 420 G/L on þ 20 was present. Full donor chimerism was proven on day þ 28. Liver toxicity and thrombosis were not observed. Nowadays, 6 months after transplantation her general condition is excellent, with normal peripheral blood and bone marrow counts and full donor chimerism.
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Conclusion: Questions about the most effective treatment of PNH remain open not only in children. Eculizumab seems to be the best option for patients with hemolysis, but it’s expensive, ineffective in about one-third of PNH patient and induce chronic inhibition of C3 activation, which increases susceptibility to infection especially with Neisseria meningitidis. Allo-HSCT is the only curative treatment for patients with PNH and bone marrow failure but information about optimal conditioning regimen are scarce. According to our experience, in children with PNH the treosulfan-based conditioning demonstrates low organ toxicity and excellent long-lasting engraftment. Disclosure of Interest: None declared. P256 Safety of peritoneal and pleural drain placement in pediatric stem cell transplant recipients with severe veno-occlusive disease A. L. Madenci1,2,*, A. Stetson3, C. Weldon1,3, L. Lehmann3,4 1 Department of Surgery, Boston Children’s Hospital, 2Department of Surgery, Brigham and Women’s Hospital, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, 4Division of Hematology/Oncology, Department of Medicine, Boston Children’s Hospital, Boston, United States Introduction: Veno-occlusive disease (VOD) of the liver is a serious complication of hematopoietic stem cell transplantation (HSCT) that has historically been associated with high mortality. The mechanism of VOD involves hepatic endothelial injury resulting in a clinical syndrome of ascites, hyperbilirubinemia, and painful hepatomegaly. Severe cases have lead to hepatorenal syndrome, multiorgan failure, and death. Defibrotide, a polydeoxyribonucleotide-derived medication that enhances the fibrinolytic pathway, is one of limited medical treatment options although still an investigational agent in the United States. Drainage of ascites via peritoneal catheters can improve respiratory excursion and mitigate renal injury from intra-abdominal hypertension. Drainage of pleural fluid can improve both ventilation and oxygenation. However, there is often concern regarding surgical complications or infections occurring as a consequence of peritoneal or pleural drain placement in critically ill, immunocompromised children. Materials (or patients) and methods: We conducted an IRB-approved retrospective review of all HSCT recipients at a single pediatric center who developed VOD (defined using Bearman criteria) of sufficient severity to be eligible for one of two clinical research protocols using defibrotide between 2000 and 2012. The primary outcome was complication from peritoneal or pleural drain placement. Secondary outcomes included mortality and infectious complications. Results: Thirty-two patients were treated with defibrotide for severe VOD. Median age at transplantation was 2.9 years (interquartile range [IQR], 1.3–5.8 years). Defibrotide treatment began at a median (IQR) 15.5 (12–21) days after transplant and lasted for median (IQR) 22 (17–30) days. Overall mortality was 47% (n ¼ 15). Six (19%) patients required renal replacement therapy, which was associated with mortality (n ¼ 6, 100% vs. n ¼ 9, 35%, P ¼ 0.004). Twenty-four patients (75%) had peritoneal drains inserted (median [IQR] ¼ 16.5 [13-25] drain days). Interventional radiologists placed 92% of drains (13% at bedside), while critical care physicians placed the remainder at bedside. Median (IQR) absolute neutrophil count at the time of peritoneal drain placement was 0.6 (0.01–4.9) K/uL. The procedure was safe and not associated with increased mortality (P40.99). Eight (25%) peritoneal drains were exchanged due to poor functioning. Seven (23%) patients had undergone prior abdominal surgery, but there were no instances of visceral perforation or hemorrhage with drain placement. Among the 24 patients with peritoneal drains, only one (4%) patient had a positive culture from ascitic fluid (Staphylococcus non-aureus
species). At the time peritoneal drains were removed, median creatinine and total bilirubin were decreased by 0.1 (0–0.3) and 1.9 (0.3–6.8) mg/dL, respectively, compared to the time of placement. Eight (25%) patients underwent pleural drain placement (median [IQR] ¼ 9 [6–35] drain days). No pleural drain-related surgical complications or infections occurred. Four (50%) patients with pleural drains had de-escalation in oxygen requirement at the time of drain removal, compared to the time of placement. Conclusion: VOD remains a devastating complication of HSCT, with a 47% mortality rate among patients with severe disease. Drain placement in the peritoneal or pleural space is a safe treatment modality for children with severe VOD. Disclosure of Interest: None declared. P257 First Report of Pediatric Hematopoietic Stem Cell Transplantation Activities in the Eastern Mediterranean Region, 1984-2012: On Behalf of Pediatric Cancer Working Committee of the Eastern Mediterranean Blood and Marrow Transplantation (EMBMT) Group A. A. Hussein1,*, A. Hamidieh2, A. Elhaddad3, M. Ramzi4, T. Ben Othman5, F. Hussain6, D. Dennison7, P. Ahmed8, M. Abood9, A. Al-Ahmari6, A. Wahadneh10, J. Fathy3, M.-A. Bekadja11, S. Al-Kindi7, S. Benchekroun12, A. Ibrahim13, M. Behfar2, M. Samra3, S. Ladeb5, S. Adil14, H. El-Solh6, M. Ayas6, A. Ghavamzadeh2, A. Al-Seraihy6 on behalf of Pediatric Cancer Working Committee of the Eastern Mediterranean Blood and Marrow Transplantation (EMBMT) Group 1 Bone Marrow and Stem Cell Transplantation Program, King Hussein Cancer Center, Amman, Jordan, 2Tehran University of Medical Sciences, Hematology, Oncology and SCT Research Center, Tahran, Iran, Islamic Republic Of, 3National Cancer Institute, Cairo University, Cario, Egypt, 4Shiraz University of Medical Science, Shiraz, Iran, Shiraz, Iran, Islamic Republic Of, 5Center National de Greffe de Moelle Osseuse de Tunis, Tunis, Tunisia, 6King Faisal Specialist Hospital and Research Centre, Riyadh, Riyadh, Saudi Arabia, 7Sultan Qaboos University Hospital, Muscat, Oman, 8Armed Forces Bone Marrow Transplant Center, Rawalpindi, Pakistan, 9 American University Beirut Medical Center, Beirut, Lebanon, 10Queen Rania Al-Abdulla Hospital, Amman, Jordan, 11Oran University-1st November Hospital, Oran, Algeria, 12 Service d’Hematologie et Oncologie Pediatrique, Casablanca, Morocco, 13Makassed General Hospital, Beirut, Lebanon, 14 Agha Khan University Hospital, Karachi, Pakistan Introduction: There is no available data in the literatures describing the state of hematopoietic stem cell transplantation (HSCT) activities for children in the Eastern Mediterranean (EM) region. Materials (or patients) and methods: Data on transplants performed for children less than 18 year of age between 1984 and 2012 in eight EM countries (Saudi Arabia, Jordan, Tunisia, Iran, Oman, Pakistan, Egypt, and Lebanon) were collected using survey sheets. The data were collected according to primary disease categories, indication, donor type, stem cell source, intensity of conditioning regimen (myeloablative versus reduced intensity), and year of transplantation. Results: The first HSCT for children in the EM region was performed in 1984. Between 1984 and 2012, 5187 transplants were performed, 4513 (87 %) allogeneic, and 674 (13%) autologous. In all countries, the number of allogeneic HSCT was significantly larger than autologous. The largest number of transplants were performed in Saudi Arabia with a total of 1977 transplants (38.2%), followed by 1197 in Iran (23.1%), 811 in Egypt (15.6%), 361 in Jordan (7%), 325 in Pakistan (6.2%), 249 in Tunisia (4.8%), 162 in Oman (3.1%), and 105 in Lebanon (2%). Lymphoma was the main indication for autologous HSCT (43.2%), followed by solid tumors (36.8%), and leukemia (20%). In the allogeneic HSCT group, leukemia was the main indication (37.5%), followed by hemoglobinopathy (24%), bone marrow failure (21%), primary immunodeficiency
disorders (13.3%) and inherited metabolic diseases (2.9%). Overall, the indication for transplantation was malignant diseases in 1736 patients (38.5%) and non-malignant in 2777 cases (61.5%). A myeloablative conditioning regimens were used in 88% of the allogeneic HSCT. Bone marrow (BM) was the most common stem cell source (56.2%), though an increasing use of peripheral blood stem cell (PBSC) was observed in the last decade. The stem cell source of autologous HSCT has shifted from BM to PBSC, and 80.9% of autologous transplants were from PBSC. Among the recipients of allogeneic transplants, 94.5% were from related donors, while 5.5% were from an unrelated source, mainly cord blood. There was a significant increase in the number of HSCT over the study period. The number of transplants increased from 132 transplants in the first decade (1984-1993) to 1300 transplants between 1994-2002 to 3755 transplants between 2003-2012. Conclusion: This is the first report on the state of HSC activities for children in the EM region. There is an overall increased rate of HSCT for children in the last 2 decades. Unlike western countries; the non-malignant disorders are the main indications for allogeneic transplantation. Unrelated transplants are still markedly underutilized. Development of regional cord blood donor banks and registries might improve the access to unrelated donor sources. Further studies on detailed clinical outcome data are planned. Disclosure of Interest: None declared. P258 Hematopoietic stem cell transplantation in Fanconi anemia and acquired aplastic anemia in children: one center experience B. Kuskonmaz1,*, T. Bayhan2, S. Aytac¸2, S. Unal2, B. Tavil2, F. Gumruk2, M. Cetin2, D. Uckan Çetinkaya1 1 Department of Pediatrics, Divison of Pediatric Bone Marrow Transplantation Unit, 2Department of Pediatrics, Divison of Hematology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) from HLA matched sibling donor is a curative therapy for patients with severe aplastic anemia either acquired (AAA) or inherited. Fludarabine based conditioning regimen is preferred in patients with FA in recent years. Here we report the results of HSCT in two groups of patients; patients with FA and patients with AA. Materials (or patients) and methods: Thirty six patients with FA and 21 patients with AA underwent HSCT between February 1995 and May 2014. The median age of the patients was 10.5±3.4 y (5.6-17.6 y) and 26 of them (72.2%) were males in FA group; median age was 9.5±3.2 y (4.3-14.3 y) 15 of them (66.7%) were male in AAA group. One patient in FA group and one patient in AA group underwent second HSCT due to engraftment failure. Fludarabine based conditioning regimen had been used in 27 of the 28 patients with FA who underwent HSCT since April 2004 as shown on Table 1. Two of the FA patients developed AML before transplantation. One of them received fludarabine based conditioning regimen. Results: Engraftment was achieved in 35 of 36 patients in FA group (97.2%) and 20 of 21 patients in AAA group (95.2%). Transplant related events were shown on Table 1. Survival rates were 82.9% (29/35) in patients with FA and 95.2% (20/21) in patients with AAA. When patients with FA who received fludarabine based regimen were evaluated separately, survival rate was found to be 92.9% (25/27). One patient with FA underwent second HSCT due to poor graft function and died with sepsis. The two patients with AML among the FA group died after HSCT due to AML relapse and acute GVHD, respectively. Conclusion: In FA patients survival rate is reported to be about 75% after HSCT from HLA identical sibling in experienced centers. In our study, survival rate is found as 82.9% in FA patients after HSCT. The survival rates increased to
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Table 1.
Transplant charactersitics and results of trasplantation Fanconi anemia
Donor characteristics HLA matched sibling HLA matched other relatives HLA 1 ag mismatched relatives Conditioning regimen
GVHD prophylaxis
Stem cell source BM PB BM þ CB Engraftment Neutrophil engraftment day Acute GVHD (Zgrade 2) Chronic GVHD VOD1 Survival/median follow-up
1
Acquired aplastic anemia
26 (72.2%)
16 (76.2%)
7 (19.4)
2 (9.5%)
3 (8.3%)
3 (14.3%) Cy þ ATG: 18 (85.7%) Bu þ Cy þ ATG: 2 (9.5%) Flu þ Cy þ ATG: 1 (4.8%)
27 (75.0%) 8 (22.2%) 1 (2.8%) 35 (97.2%) 14.2±2.8 d (9-19 d)
20 (95.2%) 1 (4.2%) 20 (95.2%) 16.7±3.4 d (1022 d) 2 (9.5%)
2/33 (6.1%) 8 (22.2%) Total patients: 29/36 (80.6%) (53 mo) Flu based regimen: 25/ 27 (92.6%) (49 mo)
Conditioning regimen Bu þ Cy1 Bu þ Flu þ ATG2 Bu þ Cy þ ATG1
Flu þ Cy þ ATG: 27 (75.0%) Cy þ TBI±ATG: 4 (11.1%) Bu þ Cy: 3 (8.3%) Cy þ ATG: 1 (2.8%) Cy þ TBI þ ATG þ Bu: 1 (2.8%) CsA þ MTX: 27 (75.0%) CsA/CsA þ MPZ: 9 (25.0%)
7/35 (20.0%)
Table 1. Characteristics of patients and results of transplantation
CsA þ MTX: 21 (100%)
1 (4.5%) (-) 20 (95.2%) (97mo)
Seven of them mild-moderate and one is severe VOD.
92.9% in FA patients who received fludarabine based regimen as expected. Allogeneic HSCT is a preferred therapy for young patients with severe AAA who have a HLA matched sibling donor with long-term survival rates reaching 80–95%. In this study, although 23.8% donors were not HLA identical siblings, disease free survival was achieved in 95.2% of the patients. Disclosure of Interest: None declared. P259 Hematopoietic stem cell transplantation in children with chronic myeloid leukemia B. Kuskonmaz1,*, T. Bayhan2, S. Aytac¸2, S. Unal2, B. Tavil2, F. Gumruk2, M. Cetin2, D. Uckan Çetinkaya1 1 Department of Pediatrics, Divison of Pediatric Bone Marrow Transplantation Unit, 2Department of Pediatrics, Divison of Hematology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Introduction: Chronic myeloid leukemia (CML) is composed of 3% of pediatric leukemias. This makes it difficult to perform clinical trials that can define the best therapeutic option, when considering the impact of tyrosine kinase inhibitors (TKIs) versus the established approach of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report the results of the 17 CML patients who underwent HSCT. Materials (or patients) and methods: Seventeen patients with CML who underwent HSCT between October 1997 and December 2011 were included in the study. The mean age of the patients was 11.5±3.7 y (5.0-16.1 y) and 11 of them were males (64.7%). Three of the 17 patients underwent second HSCT due to relapse of CML. Before HSCT 12 patients had chronic phase CML and 5 patients had accelerated phase CML. Donor characteristics were as follows: HLA identical sibling 14 (82.4%), HLA 1 ag mismatched family donor 2 (11.8) HLA identical father 1 (5.9%). Sources of stem cells was bone
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GVHD prophylaxis CsA þ MTX/CsA Engraftment/Neutrophil engraftment day Acute GVHD (Z grade 2) Chronic GVHD VOD (moderate/severe) Survival/median follow up
14 (82.4%) 2 (11.8%) 1 (5.9%) 16 (94.1%)/1 (5.9%) 17 (100%)/17.1±2.6 day (1222 day) 5 (29.4%) 3 (17.6%) 1 (5.9%) 13 (76.5%)/135 months
1
Myeloablative conditioning regimen, 2Reduced intensity conditioning regimen.
marrow (BM) in 13 (76.5%), peripheral blood (PB) in three (17.6%) and BM þ PB in one (5.9%) of the patients. Before use of imatinib in pediatric CML, patients were treated with various chemotherapeutic agents, hydroxyurea, interferon alpha before HSCT. Imatinib was used in 5 patients; solely in two patients and in combination with other agents in three patients. Imatinib was switched to dasatinib in one patient due to failure of molecular remission. Results: The results of HSCT were shown on Table 1. Engraftment was achieved in all patients. Relapse of CML developed in 5 patients (29.4%). Relapse was observed in only one of the 12 patients (8.3%) who underwent HSCT at chronic phase and four of the five patients (80.0%) at accelerated phase. Engraftment and survival were achieved in all three patients who underwent second HSCT due to disease relapse. One of them received imatinib and the other received imatinib and donor lymphocyte infusion due to molecular t(9;22) positivity. Survival rate was 91.7% (11/12) in patients who underwent HSCT at chronic phase and 40.0% (2/5) in patients who underwent HSCT at accelerated phase. Conclusion: Because there have been no randomized controlled trials comparing transplant and imatinib in pediatrics, the decision on how to treat has been individualized. Consistent with these recommendations the number of HSCT has decreased in recent years at our center as well. In a registry report from the EBMT, the overall survival (OS) at 3 years for children transplanted in first chronic phase from sibling donors or unrelated donors was 75%, and 65%, respectively. Outcome for patients who received transplants in advanced stage was significantly worse. In our study, the OS was 76.5% for all patients, this ratio was found as 91.7% in patients who underwent HSCT at chronic phase of CML. Disclosure of Interest: None declared. P260 Hematopoeitic stem cell transplantation in patients with genetic hemophagocytic lymphohistiocytosis B. Kuskonmaz1,*, S. Aytac¸2, D. Ayvaz3, T. Bayhan2, B. Tavil2, S. Unal2, M. Cetin2, F. Gumruk2, I. Tezcan3, D. Uckan Çertinkaya1 1 Department of Pediatrics, Divison of Pediatric Bone Marrow Transplantation Unit, 2Department of Pediatrics, Divison of Pediatric Hematology, 3Department of Pediatrics, Divison of Pediatric Immunology, Hacettepe University, Faculty of Medicine, Ankara, Turkey Introduction: The genetic form of hemophagocytic lymphohistiocytosis (HLH) can be divided into 2 subgroups: familial HLH (FHL) and those associated with primary immunodeficiency syndrome including Griscelli syndrome 2 (GS2), Chediak-Higashi syndrome (CHS), X linked lymphoproliferative disorder and other rare immunodeficiency syndromes. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment modality in genetic HLH.
Table 1. Characteristics of patients and results of transplantation Donor characteristics HLA identical sibling HLA identical other relatives HLA 1 ag mismatched relatives Conditioning regimen Engraftment Acute GVHD (4grade 2) Chronic GVHD VOD (moderate-severe) Survival rate for all patients Survival rate at CR1
16 (64.0%) 5 (20.8%) 4 (16.0%) Bu þ Cy±vepesid/ATG: 20 (80%) Bu þ Flu: 4 (16.0%) Flu þ Mel þ alemtuzumab: 1 (4.0%) 24 (92.3%) 2/23 (8.7%) 3/22 (13.6%) 6 (24.0%) 18 (72%) (median follow up 67 months) 16/19 (84.2%) (median follow up 60 months)
Materials (or patients) and methods: Twenty five children with HLH who underwent HSCT between October 1997 and September 2014 are included in the study. The mean age of the patients was 4.8±5.3 (5 mo-16 y), 15 of them (60.0%) were females. Fourteen patients had familial HLH and 10 had GS2 associated HLH, one had CHS associated HLH. At the time of HSCT, 19 patients had complete remission 1 (CR), three patients had CR2, two patients had active disease, one had CR3. Source of stem cells was bone marrow (BM) in 15 patients (60%), peripheral blood in 9 patients (36%) and BM þ cord blood in one (4%) patient. The other characteristics of the patients and transplantation were shown on Table 1. Three patients underwent second HSCT and two of them underwent third HSCT due to primary or secondary engraftment failure. Results: Engraftment was achieved in 24 patients (92.3%) but one of them developed secondary graft failure. The mean engraftment day was 13.5±2.6 day (10-21 day). The survival rate for all 25 patients was 72% (16/26); when the patients who underwent HSCT at CR1 were evaluated separately, survival rate was found as 84.2% (16/19) (Table 1). In four patients in whom bu and flu were used as conditioning regimen survival was achieved. All patients who survived are disease free at present. Conclusion: The survival rate of HSCT with myeloablative conditioning was reported as 53–71%. The advent of reducedintensity conditioning with lower toxicity improved survival of HSCT. In this study survival rate for all patients was 72%, in patients transplanted at CR1 survival rate increased to 84.2%. Disclosure of Interest: None declared. P261 Infectious profile and infection-related mortality of allogeneic stem cell transplantation in children: A single center experience C. De Miguel Sanchez1,*, M. Lopez Duarte1, A. Bermudez1, M. Colorado1, B. Gonzalez-Mesones1, P. Ibarrondo1, E. Conde1 1 Hematology, HOSPITAL UNIVERSITARIO MARQUES DE VALDECILLA, Santander, Spain Introduction: Infectious diseases are important causes of morbidity and mortality in paediatric patients who undergo an allogeneic haemopoietic stem-cell transplantation (allo-HSCT). We analyze the infectious complications occurring during three different posttransplantation phases (days 0-30, 30-100, and 100-365 after allo-HSCT) and their effect on mortality in a high risk allo-HSCT pediatric cohort during the first year after allo-HSCT. Materials (or patients) and methods: Thirty one patients younger than 18 years underwent allo-HSCT in our center from 2004 to 2014. We retrospectively analyzed all documented
infections in 35 allo-HSCT(4 patients had a previous allo-HSCT) reviewing medical and microbiological records in the first year after allo-HSCT. No primary antibacterial or antifungal prophylaxis was employed. Acyclovir was used until immunosuppression was stopped and TMP-SMX until CD4 counts were greater than 200/mm3. Twenty patients were male. Twenty patients had a malignant disease. Median age at time of transplantation was 10 years (1-18). There were 15 mismatched donors (13 unrelated). The stem cell source was bone marrow (19), peripheral blood (12) and cord blood (4). Mieloablative conditioning regimen was used in 27 and timoglobuline was used in 16 allo-HSCT. Sixteen patients had acute graft versus host disease (aGVHD), but 8 had greater than grade 2 GVHD. Seven patients developed moderate-severe chronic GVHD during the study period. Results: One hundred forty-one infections were documented in 31 patients. Seventy-seven (55%) bacterial infections were registered, being more frequent in the first period (46 vs 26 vs 5 cases respectively). We found 46 Blood Stream Infections (BSI) which were the most frequent bacterial infection in first two periods (28 and 16 BSI respectively). Staphylococcus Epidermidis was the main pathogen of BSI (22 cases). Cumulative incidence for BSI in the 3 periods was 80%, 47% and 7% respectively. There were 7 cases of C. Difficile Infection (6 in first period). Forty-four viral Infections (31%) were documented, being equally prevalent in all 3 periods (15 vs 15 vs 14 cases) but with different etiologies at each period. Two patients developed Posttransplantation Lymphoproliferative Disease (PTLD) by Epstein Barr (EBV)(first and fourth month). Nineteen Fungal Infections (13%) were documented, being more frequent in the 2nd and 3rd period (1 vs 11 vs 7 cases). Candida Albicans was the major etiology (12 cases, all with oral candidiasis) and 7 invasive fungal infections (IFI) were detected. One patient (0,7%) was diagnosed of Cerebral Toxoplasmosis at 8th month. 365 days after allo-HSCT, 19 patients were alive. Infection was a direct cause of death in 2 patients with cord blood transplant (Early PTLD-EBV in Fanconi Anemia and IFI by Rhizopus sp in a patient with primary graft failure) during the first year after allo-HSCT. Seventeen patients are currently alive (54%) with a median follow up of 48 months (0-127). One death due to Pneumococcal sepsis on sixth year after allo-HSCT was registered in this period. Conclusion: Bacteria and viruses were the most prevalent infections documented in a high risk paediatric group of patients. However direct infection-related mortality was less than 10% with no bacterial-related mortality one year after allo-HSCT even though primary antibacterial or antifungal prophylaxis was not used. Disclosure of Interest: None declared. P262 In vivo B-cell depletion with Rituximab following transplantation with T-cell depleted haploidentical donor hematopoietic cells efficiently prevents EBV reactivation and EBV-PTLD without the need of ex vivo B-cell depletion of the garft C. Jepsen1,*, C. Pronk1, J. Toporski1, J. Dykes2, D. Turkiewicz1 1 Department of Pediatric Oncology and Hematology, 2Department of Immunology and Transfusion Medicin, University Hospital in Scania, Lund, Sweden Introduction: Haploidentical stem cell transplantation (haploSCT) is an established treatment for patients that require an allogeneic SCT but lack a HLA matched donor. Ex vivo T-cell depletion (TCD) efficiently prevents GvHD, but the consequential delayed immunologic recovery increases the risk of EBV reactivation. Herein we aim to investigate the efficacy of ex vivo B-cell depletion using one dose of Rituximab on day þ 1, in the context of TCD haploSCT, to preventing EBV reactivation and EBV-PTLD. Materials (or patients) and methods: We evaluated a total of 50 haploSCT performed in 42 patients, 29 males and 15 females, at the Dep of Paediatric Oncology and Haematology in Lund, Sweden between 2005 and 2014. Median age of
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patients was 7 years and 8 months (from 4 months to 18 years). Six children received more than one haploSCT due to graft failure or relapse. Twenty patients were diagnosed with a malignant disease (ALL 13, AML 4, MDS/JMML 2), 17 with solid tumours, (NBL 14, RMS 1, Ewing Sa 1, Wilms 1) and 8 with nonmalignant diseases (3 with SCID, 2 with MPS VI, 1 Fanconi Anemia, 1 Osteopetrosis and 1 SAA). In all cases, either donor or recipient or both were EBV serologically positive before haploSCT. Conditioning consisted of either Fludarabine or Clofarabine combined with Thiotepa and Melphane. One patient with SCIDXl did not receive any conditioning. For rejection prophylaxis either OKT3 (0.1 mg/kg until day þ 21) or ATG-Fresenius 10mg/kg/day (day -12 to -10) was used. To prevent GvHD, immunomagnetic TCD of either CD3 þ or TCRab þ T-cells was performed (CliniMACS, Miltenyi Biotec). One dose of Rituximab 375mg/m2 on day þ 1, without ex vivo CD19-depletion of the graft, was administered to all patients to prevent EBV associated complications. All patients were followed weekly with EBV PCR in plasma until recovered normal immunologic function and monitored clinically for the symptoms of EBV-PTLD. Results: Median follow-up was 428 days ranging from 9 days to 9 years. No case of EBV-PTLD was observed. Only 4 patients had at least one positive result of EBV-PCR resulting in cumulative incidence of EBV positivity of 0.08 (95% confidence interval 0.02 – 0.18). Time from haplosSCT to the first positive EBV-PCR result ranged from 88 to 159 days. The highest observed number of EBV copies ranged from 270 to 4600 copies/mL. In 3 patients, EBV-PCR positivity converted spontaneously within 2 weeks without intervention. One patient with a DLI-induced acute and chronic GvHD had repeated episodes of transient EBV positivity during 338 days but finally became negative. Three out of 4 patients were serologically EBV neg prior to haploSCT while the donors were positive. All long-term survivors recovered with normal B-cell function and do not require IVIG substitution. Conclusion: Prevention of EBV-PTLD after TCD haploSCT is achieved by either or both in vivo and/or in vitro B-cell depletion. Post-haploSCT in vivo B-cell depletion given one day after infusion of non-B cell depleted graft have an advantage of eliminating both residual autologous and donor B-cells. Our results strongly suggest that in vivo B-cell depletion with one dose of Rituximab is sufficient to prevent EBV reactivation and EBV-PTLD without the risk of long-term B-cell dysfunction. Disclosure of Interest: None declared. P263 Impact of donor-recipient ABO mismatch on allogeneic hematopoietic stem cell transplantation in pediatric thalassemic patients D. Atay1,*, F. Erbey1, A. Akcay1, G. Ozturk1 1 PEDI˙ATRI˙C HEMATOLOGY ONCOLOGY BONE MARROW TRANSPLANTATI˙ON UNIT, ACIBADEM UNIVERSITY MEDICINE FACULTY, Istanbul, Turkey Introduction: The ABO incompatibility between donor and recipient is not considered a barrier to successful allogeneic hematopoietic stem cell transplantation (HSCT), even if it can be associated with several immunohematologic complications. Nevertheless, conflicting data still exist about the influence of major, minor, bidirectional ABO incompatibility on graftversus-host disease (GVHD), venoocclusive disease (VOD) incidence, hematologic recovery and engraftment in pediatric thalassemic patients. Materials (or patients) and methods: From May 2011 to August 2014, 52 children with beta thalassemia major who underwent allogeneic HSCT and followed for a minimum of hundred days post-transplantation were enrolled this study. Results: The median age of patients at the time of transplant was 6 years (range ¼ 11 months-18 years). The distribution of
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Pesaro risk class I and II categories was 29 and 23 children. The median serum ferritin level was 1700 ng/mL. All patients received bone marrow (BM) stem cells from matched sibling (n ¼ 43), related (n ¼ 5) or unrelated (n ¼ 3) donors after myeloablative conditioning regimens, which include ATG in 14 patients. Except 6 patients (major:4, none:2), all patients achieved initial donor cell engraftment. Thalassemic reconstitution was observed in 6 of the transplant patients (major:1, minor:1, none:4). Post-transplant pure red cell aplasia occurred in two patients, both were major ABO mismatched patients. Among BM recipients, overall incidence of acute and chronic GVHD, VOD, neutrophil and platelet engraftment were similar in ABO-matched and ABO mismatched transplants (P40.05). However, especially in major and bidirectional ABO mismatched patients, a delayed erythroid recovery was recorded as compared to the group receiving an ABO compatible graft (p:0.02 and p:0.03). Median time to red cell transfusion independence was significantly longer in major ABO-incompatible BM recipients (median time, 95 days vs 32 days; p:0.001). This difference appears to be independent of the amount of cells infused during transplantation, with a median number of CD34 cells of 4.89x 106/kg in ABO-mismatched patients and 5.40x106/kg in control ABO-matched patients (P40.05). Conclusion: In our experience, major and bidirectional ABO incompatibility leads mainly to delayed RBC recovery after HSCT. Therefore, whenever feasible, major ABO-mismatched donors should be avoided in HSCT recipients, to prevent delayed erythroid recovery with prolonged RBC transfusion needs and impaired quality of life. Disclosure of Interest: None declared. P264 CD45RA depleted donor lymphocyte infusions for antiviral boost following ex-vivo T-cell depleted haploidentical hematopoietic stem cell transplantation D. Turkiewicz1,*, J. Dykes2, C. Pronk1, J. Toporski1 1 Department of Pediatric Oncology and Hematology, 2Department of Immunology and Transfusion Medicin, University Hospital in Scania, Lund, Sweden Introduction: Extensive ex-vivo T cell depletion is a very effective method of graft versus host disease prevention. It allows performing even a haploidentical stem cell transplantation (haploSCT) without any pharmacological post-transplantation immunosuppression. However, to be so effective T-cell depletion must be nearly total. This, in turn, leads to elimination of the early phase of T-cell recovery thus increasing the risk of viral infections. One possible way to manage the problem of T-cell gap and provide the patient with the donor antiviral immunity is by donor lymphocyte infusion. However, infusion of unselected lymphocytes poses a risk of severe graft versus host disease. Since the alloreactive T-cells are predominantly contained in the naı¨ve T-cell compartment, depletion of CD45RA positive cells from DLI decreases the frequency of GvHD initiating cells while preserving the pathogen specific memory lymphocytes. Infusion of CD45RA depleted donor lymphocytes might therefore allow for safe transfer of donor-derived immunity with a limited GvHD risk. Materials (or patients) and methods: Infusion of CD45RA depleted lymphocytes has been performed in 4 children after haploidentical SCT. The clinical data is summarized in a table below. All patients had an active viral infection at time of infusion, all were in complete remission of the underlying disease and had a profound T-cell lymphopenia. Two patients had clinical symptoms of viral diseases: HHV6 encephalitis (one patient) and concomitant active varicella, colitis and rotavirus infection (one patient). None of the children had any GvHD. Three patients received one infusion of 2,5x104/kg, one patient received two infusions: 2,5x104/kg and four weeks later 5x104/kg.
Results: UPN Disgnosis
1
2
3
4
Virus ifection
Clinical symptoms
CD45RA depleted DLI - cell dose
Outcome
Recovery VZV, CMV gan- varicella, colitis 2.5x104/ with loss of kg ciclovir resis- keratitisretinitis sight in one Day tant, Rotavirus eye no GvHD þ 80 CMV neg from day þ 73 HSCT from DLI VZV neg day þ 22 from DLI Rota neg day þ 22 from DLI ALL 2CR CMV - increasnone DLI#1: Full recovery ing number of 2.5x104/ no GvHD copies despite kg HAdV neg cidofovir treatday day þ 31 ment, HAdV þ 74 from DLI#2 from CMV neg HSCT day þ 84 DLI#2: from DLI#2 5x105/kg day þ 93 from HSCT Recovery ALL 3CR HHV6, HAdV encephalitis 2.5x104/ kg with residual day neurological þ 63 symptoms, from no GvHD HSCT HAdV neg day þ 26 from DLI 4 SAA HAdV - increasnone 2.5x10 / Full recovery kg ing copy numno GvHD day ber despite HAdV neg þ 68 cidofovir day þ 75 from treatment from DLI HSCT SCID IL7Ra
Conclusion: All patients survived, eliminated all viruses and none developed graft versus host disease. The depletion of CD45RA þ cells with CliniMACS system was reliable, effective and affordable. Since the depletion step does not alter the biological properties of the cells, the regulatory issues are simpler to comply. However, a clinical study is needed to establish the role of this method in decreasing morbidity and mortality of haploSCT. Disclosure of Interest: None declared. P265 Outcome following haploidentical haematopoietic stem cell transplantation using ex-vivo T-cell depletion and reduced intensity conditioning in children with acute leukemia D. Turkiewicz1,*, M. Ifversen2, J. Dykes3, C. Heilmann2, C. Pronk1, K. Mueller2, A. Fischer-Nielsen4, J. Toporski1 1 Department of Pediatric Oncology and Hematology, University Hospital in Scania, Lund, Sweden, 2Pediatric Clinic II, 4074, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 3Immunology and Transfusion Medicin, University Hospital in Scania, Lund, Sweden, 4Rigshospitalets Blodbank, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark Introduction: Haploidentical stem cell transplantation (HaploSCT) is an established treatment modality for children who require an allogeneic stem cell transplantation (alloSCT) but lack a matched (sibling or unrelated) donor. The reduced intensity conditioning permits performing alloSCT even in patients with a high risk of transplant related mortality (TRM). We present the results of TCD haploSCT in children with acute
leukemia performed between 2005 and 2014 in 2 Nordic HSCT centers in Lund and Copenhagen; a result of an established cooperation in the Oresund region. Materials (or patients) and methods: HaploSCT was performed in 23 children with acute leukemias, 15 with ALL and 8 with de novo AML. Four patients were transplanted with Z5% blasts in bone marrow and 19 were in complete remission (CR; CR1: 3, CR2: 9, CR42: 7). Eight patients (35%) had previously received an alloSCT, median time from the 1st alloSCT to haploSCT was 375 days (50 days-2.3 years). Conditioning regimen consisted of a combination of either clofarabine 200mg/m2 or fludarabine 125mg/m2 with 10mg/ kg Thiotepa and 120mg/m2 Melphalane. For rejection prophylaxis, either OKT3 (0.1mg/kg/day until day þ 21) or ATG-Fresenius (30mg/kg day -12 to -9) was used. GvHD prevention consisted of ex-vivo immunomagnetic depletion of either CD3 þ (n ¼ 9) or TCR ab þ (n ¼ 14) cells (CliniMACS, Miltenyi Biotech). Median number of transplanted CD34 þ cells/kg was 11.4x106. Median number of co-infused ab þ T-cells/kg was 2.7x104. One dose of Rituximab 375mg/m2 was given on day þ 1 to prevent EBV-PTLD. Sixteen patients received a short course of MMF (n ¼ 16) until day þ 28. Fourteen patients received low-dose donor lymphocyte infusions (DLI) either as a relapse prophylaxis or to clear viral infections. Results: 22/23 patients engrafted. One patient with primary graft failure was successfully regrafted with another haploidentical donor. Median time to ANC and PLT engraftment was 12 and 15 days, respectively. Cumulative incidence (CI) of primary grade I to II aGvHD was 0.21 (95% Confidence Interval 0.07-0.39). No primary grade III-IV aGvHD was observed. No cGvHD occurred in children who did not receive DLI. Post-DLI aGvHD was observed in 6/14 patients (43%), of which 5 had grade III/IV. Two patients developed extensive cGvHD following DLI. Thirteen patients are alive; 12 in CR, with median follow up of 2 years and 2 months (from 147 days to 9 years). Ten patients died: 6 of relapse (including 4 patients transplanted without remission) and 4 of TRM (2 post-DLI GvHD, 1 AdV infection and 1 secondary graft failure). For the whole cohort, 1 year EFS, CI of relapse and TRM were 0.5 (95% CI 0.32 – 0.76), 0.32 and 0.18 respectively. For patients transplanted in remission, 1-year EFS reached 0.6 (95%CI: 0.32 – 0.76), CI of relapse 0.17 and TRM 0.22. EFS for children with previous alloSCT was 0.47 (95% CI: 0.2 – 1) at 1 year. Conclusion: Our results show that TCD HaploSCT is a valid therapeutic alternative for children with high risk acute leukemia that achieve CR. Despite reduced conditioning, cumulative incidence of relapse is acceptable in this high risk population. The role of post-haploSCT DLI remains to be determined as it may cause life threatening GvHD despite the relatively low doses of lymphocytes infused. Disclosure of Interest: None declared. P266 Fertility Preservation in Female Pediatric HSCT Patients: A Multidisciplinary Approach for the French Speaking Part of Switzerland F. Bernard1,2,*, L. Crosazzo3, C. Girardin1, M. Yaron4, I. Andrieu-Vidal5, F. Phan-Hug3, D. Wunder6, M.-P. Primi6, J.-P. Simon7, V. Schwitzgebel1, I. Streuli4, Y. Chalandon8, M. Ansari1,2, M. Beck-Popovic3, F. Gumy-Pause1,2 1 Pediatrics, University Hospital of Geneva, 2CANSEARCH Research Center, Geneva, 3Pediatrics, University Hospital of Canton de Vaud, Lausanne, 4Gynecology-Obstetrics, 5Pediatric Surgery, University Hospital of Geneva, Geneva, 6Gynecology-Obstetrics, 7 Legal Affairs Unit, University Hospital of Canton de Vaud, Lausanne, 8Hematology, University Hospital of Geneva, Geneva, Switzerland Introduction: Conditioning regimens prior to hematopoietic cell transplantation (HSCT) are known to be highly gonadotoxic (especially total body irradiation and alkylating agents such as Busulfan (Bu), Cyclophosphamide (Cy) and Melphalan
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(Mel)). Unfortunately, impaired reproductive function and infertility are frequently seen in long term survivors. Ovarian failure rate post HSCT ranges from 72 to 100% and may negatively impact the survivor’s quality of life. Different fertility preservation options (FPO) are available for pubertal patients (sperm banking for males and cryopreservation of oocytes for females). Recently, ovarian tissue cryopreservation (OTC) has emerged as a new experimental option. This technique can be proposed to pubertal and prepubertal females. Theoretically, two different options are available once the patient is ready to conceive: ovarian auto-transplantation (experimental method, feasible today) or in vitro maturation of primordial follicles followed by in vitro fertilization (still in research, nowadays not feasible). Materials (or patients) and methods: In 2010, two multidisciplinary swiss pediatric teams in Lausanne (CHUV) and in Geneva (HUG) were formed under the auspices of the ‘‘Re´seau Romand de Cancer et Fertilite´’’. Each team consists of pediatricians specialized in hemato-oncology, gynecology, endocrinology, surgery, psychiatry, reproductive medicine, radiation oncology, adolescent medicine and genetics. Regular video-conference meetings are organized during which new cases are presented, with the objectives of determining 1) indications for fertility preservation and 2) the best FPO for the individual patient. Meetings also provide an opportunity to discuss guidelines, literature and difficult cases. Results: To date, ovarian fragments were collected and cryopreserved for 12 (7 autologous and 5 allogenic) HSCT prepubertal girls, with a median age of 5 years (range 6 months to 11 years). In all cases, an informed consent form was signed. Except one patient with hemophagocytic lymphohistiocytosis (HLH), all patients suffered from malignant diseases: metastatic neuroblastoma (n ¼ 6), acute lymphoblastic leukemia (n ¼ 4), and metastatic Ewing sarcoma (n ¼ 1). OTCs were realized before HSCT conditioning regimens (5 BuMel, 4 Bu-Cy, 1 Treosulfan-Mel, 1 CEM (Carboplatin-EtoposideMel) and 1 TBI-Etoposide). All patients except the one with HLH, underwent several courses of chemotherapy before OTC and conditioning phase, the number of courses was dictated by their disease. These chemotherapy courses are not known to have a sterilizing effect. Post surgery follow up was uneventful in all 12 patients. OTC was also proposed to 2 other HSCT patients (1 acute lymphoblastic leukemia and 1 juvenile myelomonocytic leukemia) but refused by the parents. Conclusion: Fertility preservation techniques are now also available for prepubertal females and continue to develop. The opportunity to provide accurate information and hope for future fertility in young patients is invaluable. In view of the paucity of cases, only unification of medical forces will enable us to offer for each of our patients the best possible care. Disclosure of Interest: None declared. P267 Low transplant-related mortality in children with acute leukemia given hla-haploidentical hematopoietic stem cell transplantation after removal of Alpha/Beta þ t cells and of CD19 þ b cells from the graft F. Locatelli1,*, A. Bertaina1, P. Merli1, B. Lucarelli1, L. P. Brescia1,
coupled with B-cell depletion accomplished through the use of an anti-CD19 monoclonal antibody to prevent the occurrence of Epstein-Barr Virus (EBV)-related post-transplant lymphoproliferative disorders (PTLD). Materials (or patients) and methods: Enrolled in the study were 70 patients (pts, 48 M, 22 F), with acute leukemia and a median age at HSCT of 11.3 yrs (range 0.6-17.9). Fifty-one pts had ALL and 19 AML. All children were transplanted in morphological complete remission (CR): 26 pts were transplanted in CR1, 41 in CR2 and 3 in more advanced CR. All pts transplanted in CR1 had either poor cytogenetic/molecular characteristics or high levels of minimal residual disease (MRD) at the end of induction therapy. The donor was either the mother (n ¼ 42) or the father (n ¼ 28); according to the model of KIR/KIR ligand disparity, 31 pts were transplanted from an NK-alloreactive donor. The median number of CD34 þ cells, NK cells, gd þ T cells, and ab þ T cells were 13.6, 34.7, 8.4, 0.08 and 0.04x106/kg, respectively. A myeloablative regimen, containing TBI in 52 cases, was given to all children, who also received rabbit ATG (12 mg/kg over 3 days, from -5 to -3). Rituximab (200 mg/m2) was administered on day -1 to further prevent EBV-related PTLD. Results: Primary engraftment occurred in 69/70 pts, the remaining child being successfully re-transplanted from the other parent. The median time to reach neutrophil and platelet recovery was 13 days (9-19) and 11 days (8-20). No child developed gut or liver acute GVHD. Twenty pts experienced skin-only grade I-II GVHD, this leading to a cumulative incidence (CI) of 28.6% (Standard Error, SE, 5.4). Only 3 pts developed skin limited chronic GVHD, the CI of this complication being 4.7% (SE 2.7). Three pts died for causes other than disease relapse (all in the first 100 days after HSCT), the CI of transplantation-related mortality (TRM) being 4.3% (SE 2.4). Fourteen pts relapsed, the CI of disease recurrence being 21.2% (SE 5.1). With a median follow-up of 27 months (range 6-48), the 2-year Kaplan Meier estimate of leukemiafree survival (LFS) was 74.5% (SE 5.4). The 2-year LFS probability of children with ALL and AML was 75.8% (SE 6.1) and 70% (SE 11.5), respectively. The 2-year LFS of pts who did or did not experience skin-only acute GVHD was 90% (SE 6.7) and 68.7% (SE 6.7, P ¼ 0.11), respectively, while that of children given or not TBI as part of the conditioning regimen was 82% (SE 5.5) and 51.5% (SE 12.7, P ¼ 0.009.) Conclusion: These data obtained in one of the largest single center cohorts of HLA haploidentical-HSCT in childhood indicate that a selective graft manipulation results into effective prevention of both acute and chronic GVHD, rapid recovery of neutrophil and platelet counts and low TRM. The 2-year LFS probability observed in these children favourably compares with that reported in studies where the donor was either an HLA-identical sibling or an unrelated donor of adult stem cells or cord blood cells. Disclosure of Interest: None declared.
D. Pagliara1, R. Pinto1, R. Masetti2, D. Pende3, S. Ceccarelli1, M. Falco4, A. Moretta5, L. Moretta4, G. Li Pira1
P268 Invasive Fungal Infection in Pediatric Hematopoietic Stem Cell Transplantation H. D. Rodriguez Vega1,*, C. Fuentes Socorro1, B. Torres Guerola1, J. M. Fernandez Navarro1 1 Pediatric Oncology, Hospital Universitari i Politecnic La Fe, Valencia, Spain
Introduction: We conducted a prospective, single center study (NCT01810120) in children with acute leukemia aimed at evaluating the safety and efficacy of HLA-haploidentical HSCT after a graft manipulation based on depletion of T lymphocytes carrying the ab chains of the T-cell receptor (TCR),
Introduction: Invasive fungal infections (IFI) are a major cause of mortality and morbidity among pediatric patients receiving hematopoietic stem cell transplantation (HSCT). Diagnosis and treatment of IFI are complex, so that it becomes necessary to know those features that allow to identify patients at high risk. Publications reporting data about IFI risk factors in pediatric population are scanty. Materials (or patients) and methods: We retrospectively analyzed medical records from children with oncohematological disorders who underwent HSCT in a reference Pediatric Oncology Service of a tertiary hospital between January 1991
1
BAMBINO GESU HOSPITAL, Rome, 2University of Bologna, Bologna, 3Istituto di Ricovero e Cura a Carattere Scientifico, Azienda Ospedaliera Universitaria San Martino , 4Istituto Giannina Gaslini, 5Dipartimento di Medicina Sperimentale and Centro di Eccellenza per la Ricerca Biomedica, Universita` di Genova, Genova, Italy
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and December 2013. We defined IFI cases based on current EORTC/MSG criteria. In our centre, we use fluconazol prophylaxis in autologous HSCT from the beginning of mucositis until neutropenia resolves. In allogeneic HSCT, we use voriconazol prophylaxis from the end of the conditioning to the immunosuppressants withdrawal. Since 2009, we twiceweekly do serum galactomanan leves in all HSCT patients during the more risky period. We initially described the incidence and epidemiological features of the different kinds of IFI in our centre. We also carried out an univariate analysis and subsequently a binary logistic regression in an attempt of identify risk factors for IFI. Results: The study population includes 403 pacients who underwent 445 HSCT. The median age was 7,26 years (±4,42). Acute Leukemia was the most frequent diagnosis (55,7% of HSCT). Autologous HSCT was the most common (66%). Sixteen percent of patients suffered from acute graft versus host disease (aGVHD) whereas 4,3% did it from the chronic form (cGVHD). We observed an overall incidence of IFI of 3,4%. Invasive Aspergillosis was the more frequent kind of IFI with 12 cases (incidence of 2,7%). Pulmonary Aspergillosis was the most frequent presentation form (75% of cases). Overall mortality was 44,9% among patients who underwent HSCT but 68% in the IFI group. Patients with a prior diagnosis of lymphoma showed the higher risk for IFI with an Odds Ratio (OR) 25 (2,18-306,98). Conclusion: In our centre, we found an incidence and mortality of IFI similar to that communicated in the literature. As in other centers, after using azole prophylaxis, number of Candida infections has decreased and Aspergillosis is the most frequent IFI. In the univariate analysis, prior diagnosis of lymphoma, allogeneic HSCT, and aGVHD were significantly related to the presence of IFI. After binary logistic regression only the diagnosis of lymphoma was risk factor for IFI with an Odds Ratio (OR) 25 (2,18-306,98). This could be explained by the presence of two cases of candidiasis among only 16 patients with lymphoma. Allogeneic HSCT, specially blood cord and haploidentical transplantation, showed trends of being a risk factor for IFI though it didn’t reach statistical significance. References: 1. Srinivasan A, Wang C, Srivastava DK, et al. Timeline, epidemiology, and risk factors for bacterial, fungal, and viral infections in children and adolescents after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 94–101. 2. Castagnola E, Faraci M, Moroni C, et al. Invasive mycoses in children receiving hemopoietic SCT. Bone Marrow Transplant 2008;41 Suppl 2:S107-11. Disclosure of Interest: None declared. P269 Allogeneic hematopoietic stem cell transplantation for pediatric T lineage acute lymphoblastic leukemia H. Sakaguchi1,*, A. Narita2, K. Narita1, S. Kataoka1, M. Hamada1, N. Murakami2, Y. Sekiya2, K. Suzuki2, N. Kawashima2, Y. Okuno2, S. Doisaki2, H. Muramatsu2, N. Yoshida1, N. Nishio2, A. Hama2, Y. Takahashi2, S. Kojima2, K. Kato1 1 Department of Hematology and Oncology, Children’s Medical Center, Japanese Red Cross Nagoya First Hospital, 2Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan Introduction: The historically unfavorable outcome of patients with T lineage acute lymphoblastic leukemia (T-ALL) has recently improved through the use of highly effective treatment protocols with risk classification. With appropriately intensive therapy, children with T-ALL have an outcome similar to that of children with B-precursor acute lymphoblastic leukemia. However, patients with T-ALL remain at higher risk for remission induction failure, early relapse, and isolated CNS relapse. These high-risk-patients indicate hematopoietic stem cell transplantation (HSCT). In this study, our purpose was to evaluate clinical importance of HSCT in treatment for pediatric T-ALL.
Materials (or patients) and methods: This retrospective study included 22 consecutive pediatric patients diagnosed with T-ALL who underwent HSCT at our institutes between Jan 1990 and Mar 2013. Both institute maintain a comprehensive and prospective database on all patients who undergo transplantation. We reviewed all available medical records for this study. The median age at diagnosis was 7.6 years old (range, 1.8-13.0); that of at transplantation was 8.3 years old (range, 3.3-16.2 years). The clinical status at HSCT was composed of first complete remission (1CR) for 9 patients (including 5 patients with delayed primary remission), second complete remission (2CR) for 7 patients, and advanced stage (AS) including third remission and non-remission for 6 patients. Conditioning regimens consisted of total body irradiation (TBI) based therapy (n ¼ 17), busulfan based myeloablative therapy (n ¼ 2), and reduced intensity therapy (n ¼ 3). Hematopoietic cells were donated from autologous bone marrow (n ¼ 1), human leukocyte antigen (HLA)-identical sibling or parent (n ¼ 11), HLA-haploidentical parent (n ¼ 3), volunteered unrelated donor (n ¼ 5), and unrelated cord blood (n ¼ 2). Overall survival (OS) was defined as the time from HSCT to death or last follow-up date. Disease free survival (DFS) was defined as the time from HSCT to relapse, death, or last follow-up date. Results: The median period of observation was 38 months (range, 4-195 months). Primary graft failures occurred in 1 patient (4%), who received cord blood graft and underwent second cord blood transplantation resulting primaly graft failure again, finally were rescued by HLA-haploidentical transplantation with sustained engraftment and complete remission. The probabilities of 3years OS/DFS were 65%/63% for total cohort, 100%/100% for patients at 1CR, 34%/43% for patients with 2CR, and 38%/38% for patients at AS, respectively. Among 13 patients at 2CR or AS, 6 patients survive with remission, including 3 patients who received HLAhaploidentical HSCT, 2 patients who received HLA-identical sibling HSCT with TBI-based myeloablative conditioning, and the other patient conditioned with nelarabine combined chemotherapy following cord blood transplantation. While, 7 patinets at 2CR or AS died during observation; cause of death consisted of progressive diaseas (n ¼ 4), respiratory distress (n ¼ 2), and hepatic veno-occlusive disease (n ¼ 1). Conclusion: Our findings suggested that HSCT yielded long term disease free survival to the patients at 1CR even with delayed primary remission, on the other hand, the prognosis of patients at 2CR or AS treated with conventional HSCT was poor; these patients could be rescued by more novel approach such as HLA-haploidentical HSCT or nelarabine combined conditioning. Disclosure of Interest: None declared. P270 Haploidentical hematopoietic stem cell transplantation using ex vivo T cell-depleted grafts in pediatric patients H. J. Im1,*, K. N. Koh1, J. K. Suh1, S. W. Lee1, E. S. Choi1, S. Jang2, J. J. Seo1 1 Pediatrics, 2Laboratory Medicine, Asan Medical Center, Seoul, Korea, Republic Of Introduction: Currently, haploidentical hematopoietic cell transplantation (HHCT) is considered a valid option for patients who have diseases curable with hematopoietic cell transplantation (HCT) but who lack a matched related or unrelated donor. The purpose of this study was to assess the efficacy and feasibility of transplantation using ex vivo T cell-depleted grafts from haploidentical family donors in children and adolescents with malignant or non-malignant diseases. Materials (or patients) and methods: Between July 2008 and November 2014, 57 pediatric patients underwent ex vivo T cell-depleted HHCT after reduced intensity conditioning (RIC). Twenty-eight received CD3-depleted stem cells (CD3-HHCT) and 29 received TCRab-depleted grafts (TCRab-HHCT). Among 57 patients, 22 had non-malignant disease (NM; 1 with Fanconi
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anemia, 19 with acquired SAA, 1 with CDA and 1 with WiskottAldrich syndrome), 31 had hematologic malignancy [6 with ALL (2 CR1, 2 CR2, 1 CR3, 1 non-CR), 16 with AML (6 CR1, 6 CR2, 4 non-CR), 1 with mixed lineage leukemia not in CR, 4 with MDS (2 RCMD and 2 RCC), 2 with JMML and 2 with NHL (1CR2, 1 CR3], and 4 had solid tumors [2 with rhabdomyosarcoma (1 CR2, 1 refractory), 1 with refractory neuroblastoma, and 1 with Ewing sarcoma in CR3]. Eight of 35 patients with malignant disease had refractory disease at the time of HHCT. RIC regimen consisted of fludarabine, cyclophosphamide and r-ATG. Low-dose total body irradiation was incorporated into the conditioning regimen in 45 patients. Results: Of a total of 57 patients, two patients failed to achieve primary engraftment, and five patients experienced graft rejection (GR) within 28 days post-transplant. All graft failure (GF) including GR occurred in CD3-HHCT. No patients with TCRab-HHCT experienced GF. The cumulative incidence (CI) of acute GVHD grade II-IV and III-IV were 31% and 14%, respectively. Five of 53 evaluable patients developed extensive chronic GVHD with a CI of 10%. Four patients from CD3-HHCT died of non-relapse causes [2 of CMV disease, 1 of encephalopathy, and 1 of autoimmune hemolytic anemia)], whereas none from TCRab-HHCT died of non-relapse cause. The non-relapse mortality (NRM) was 6.5% at 1 year and 9.2% at 2 years. Eleven patients relapsed and nine of them died of disease. Of 22 patients with NM, only one patient with CD3HHCT died, resulting in OS of 95% at 2 years. As for 35 patients with malignant disease, the estimated EFS at 1 year was 54%. Patients with refractory disease at the time of transplant had a poor outcome (EFS: 0% for 8 patients with refractory disease versus 75% for the remaining 27 patients, P ¼ 0.000). Conclusion: In this study, HHCT using ex vivo T-cell depleted grafts showed a favorable outcome with low NRM for pediatric patients with NM and malignant disease in CR at transplant. However, further study is warranted to improve the outcome for patients with refractory malignant disease. Disclosure of Interest: None declared.
P271 Zoledronic acid is able to potentiate cd T-cell antileukemic activity in patients receiving ab þ T cell and CD19 þ B lymphocytes depleted grafts from haploidentical donor I. Airoldi1,*, A. Bertaina2, A. Petretto3, B. Lucarelli2, A. Zorzoli3, P. Merli2, C. Lavarello3, G. Barbarito3, L. P. Brescia2, V. Bertaina2, E. Inglese3, G. M. Milano2, F. Locatelli2 1 Laboratory of Oncology, Istituto Giannina Gaslini, Genova, 2 IRCCS Bambino Gesu` Children’s Hospital, Rome, 3Istituto Giannina Gaslini, Genova, Italy Introduction: A new method of graft manipulation based on physical removal of ab þ T cells and CD19 þ B cells, leaving mature NK cells and gd T cells in the graft, has been recently employed for HLA-haploidentical HSCT. We demonstrated that gd T cells collected from transplanted patients (pts) are endowed with limited killing capacity of leukemia cells; we hypothesized that in vivo treatment with zoledronic acid (ZOL) may expand these lymphocytes and increase their ability to lyse primary ALL and AML blasts. Materials (or patients) and methods: Thirty-three pts with acute leukemias were enrolled in a phase I/II trial of allogeneic HSCT from an HLA-partially matched family donor after TCRab þ T cells/CD19 þ B lymphocyte negative depletion. Pts were treated with ZOL within 1 month after HSCT and then every 28 days for at least 2 times whenever possible (according to a protocol approved by the Ethical Committee). gd T cells from pts before and after ZOL treatment were studied till at least 7 months after HSCT by flow-cytometry, degranulation assay and high-resolution mass spectrometry. Results: ZOL treatment did not affect the relative proportion of Vd1 and Vd2 subsets (A), while it significantly decreased the central memory (CM) population in both Vd1 and Vd2 cells. ZOL also increased the terminally differentiated (TD) subset in the Vd2 cell subset. Both Vd1 and Vd2 cells from pts treated once with ZOL showed increased cytotoxicity against primary
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leukemia cells, as assessed by the degranulation assay (B). Such an ability was further increased in Vd2 cells, but not in Vd1 lymphocytes, in those patients that received more than one treatment (B). Proteomic analysis, evaluated through the use of high-resolution mass spectrometry (HRMS), performed on gd T cells purified from pts, before (n ¼ 5) and after (n ¼ 5) the first treatment with ZOL, revealed a significant change (6789 proteins identified, T-Test FDRo0.01 S040.5, 377 significant proteins) in the proteome signature (C). Noteworthy, gd T cells from treated pts revealed an up-regulation of proteins involved in activation processes and immune responses, paralleled by a down-regulation of proteins involved in the proliferation processes (D). The gd T proteotype and the associated activation pathways were further modulated upon repeated infusion of ZOL treatment. Conclusion: Analyses of gd T cells from patients given TCRab þ T cells/CD19 þ B lymphocyte depleted HLA-haploidentical HSCT showed that ZOL treatment is able to: i) promote an increase of TD Vd2 cells; ii) modulate pathways involved in activation/proliferation of gd T cells; iii) potentiate the cytotoxic killing of gd T cells. Altogether, our results provide a robust biological rationale for implementing clinical trials with ZOL infusions in the post-transplant setting aimed at reducing the risk of leukemia recurrence and of life-threatening infections. Disclosure of Interest: None declared. P272 Outcome after Hematopoietic Stem Cell Transplantation for Pediatric MDS J. A. Park1,*, Y. J. Lim2, K.-N. Koh3, J. J. Seo3, H. J. Im3 1 Pediatrics, Inje University Haeundae-Paik hospital, Busan, 2 Pediatrics, Chung-Nam University Hospital, Daejeon, 3Pediatrics, Asan Medical Center Children’s Hospital, Seoul, Korea, Republic Of Introduction: Myelodysplastic syndrome (MDS) in childhood is extremely rare and accounts for less than 5% of all hematopoietic malignancies. Allogeneic hematopoietic stem cell transplantation (HCT) is the only curative treatment option for MDS. However, due to its rarity, safety and efficacy of HCT for pediatric MDS have not been fully determined. We
investigated outcome of HCT and impact of WHO classification 2008 on outcome after HCT for pediatric MDS. Materials (or patients) and methods: We retrospectively analyzed transplant outcomes for 29 pediatric MDS patients, median age 10.9 years, undergoing HCT between 1997 and 2014. Three were secondary MDS, and 26 were primary MDS. By WHO 2008 classification, 19 had refractory cytopenia of childhood (RCC), 8 had refractory anemia with excess blasts (RAEB), and the remaining two had refractory anemia with excess blasts in transformation (RAEB-t). Donor was matchedrelated in 12, matched-unrelated in 6, mismatched-unrelated in 2, umbilical cord blood (UCB) in 2, and haploidenticalrelated in 7. The conditioning regimen was busulfan and cyclophosphamide based myeloablative (MA) in 18 and fludarabine based nonmyeloablative (NMA) in 11 patients. Results: Cumulative incidence of neutrophil engraftment by day 28 was 71%, but all patients with graft failure were rescued with second HCT. Cumulative incidence of acute GVHD by day 100, and chronic GVHD by 1 year were 43% and 21%, respectively. After median follow-up of 3.2 years, TRM and relapse were 15% and 24.3%. EFS and OS after HCT were 64% (54%474%, 95% CI) and 68% (59%478% 95% CI), respectively. WHO classification significantly affected EFS after HCT for pediatric MDS patients (P ¼ 0.0089; Fig. 1a). Shorter interval between diagnosis and HCT (o 6 months) was associated with more favorable outcome (P ¼ 0.017, Fig. 1b). No significant difference in EFS was seen in patients with pre-HCT complete remission or o5% blasts versus in patients with 45% blasts (P ¼ 0.39). EFS in patients who had received active treatment including chemotherapy and immunosuppressive treatment before HCT was lower, compared to patients who had not (P ¼ 0.075). EFS rates were similar between MA and NMA conditioning (P ¼ 0.26) and did not differed by stem cell donors (P ¼ 0.27). Conclusion: We confirmed that WHO classification 2008 for pediatric MDS was significantly associated with patients’ outcome after HCT. Additionally, our data suggested that pediatric patients with MDS had more favorable outcome if they were transplanted early in the course of their disease, and marrow blasts o5% at HCT did not improve EFS, but rather, active treatment before HCT showed a trend of decreasing EFS
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rates. This study needs to be confirmed in larger, prospective studies including more pediatric MDS patients. Disclosure of Interest: None declared. P273 Encouraging results of reduced-intensity allogeneic haematopoietic stem cell transplantation for paediatric patients with acute lymphoblastic leukemia in second complete remission K. Higuchi1,*, M. Sato1, O. Kondo1, A. Mayumi1, M. Shimizu1, A. Sawada1, M. Yasui1, M. Inoue1 1 Haematology/Oncology, Osaka Medical Center And Research Institute For Maternal And Child Health, Izumi, Osaka, Japan Introduction: We have been performing reduced-intensity stem cell transplantation (RIST) to avoid preconditioningrelated complications. However, the effectiveness of RIST in paediatric patients with acute lymphoblastic leukemia (ALL) remains to be clarified. Materials (or patients) and methods: We retrospectively reviewed 18 paediatric patients with ALL in second complete remission (CR2) who underwent first allogeneic haematopoietic stem cell transplantation (allo-SCT) between 2000 and 2013 in our institute. We compared the outcomes of RIST with those of myeloablative stem cell transplantation (MAST). Results: The median age at allo-SCT was 9 years (range, 2 to 18 years). There were B-lineage ALL only, and none of Philadelphia chromosome-positive ALL. Four patients received HLA-matched bone marrow (2 related; 2 unrelated), 9 HLAmismatched bone marrow (8 unrelated; 1 HLA haploidentical related), 4 cord blood, and 1 CD34 positive peripheral blood stem cells (HLA haploidentical related). In all patients, the 5year overall survival (5y-OS) rate and the 5-year event free survival (5y-EFS) rate were 65.0% and 55.0%, respectively. Seven patients underwent RIST and 11 patients underwent MAST. The median follow-up durations of RIST and MAST groups were 3.3 years (range, 0.9 to 8.2 years) and 2.1 years (range, 0.2 to 13.7 years), respectively. The 5y-OS rates in RIST and MAST groups were 85.7% and 53.0% (P ¼ 0.19), and the 5y-EFS rates were 71.4% and 45.5% (P ¼ 0.28), respectively. The 5-year cumulative transplant-related mortality (TRM) rates in RIST and MAST groups were 0% and 31.8% (P ¼ 0.16), and the 5-year cumulative relapse rates were 28.6% and 31.4% (P ¼ 0.81), respectively.
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Conclusion: In our series, the cumulative relapse rate in RIST group was similar with that in MAST group, and the cumulative TRM rate in RIST group was lower than that of MAST group. Therefore, both of the 5y-OS and the 5y-EFS rates in RIST group seem to be better than those in MAST group. The outcomes of RIST in our series do not seem to be poorer. Although further studies are needed because of the small size of patients’ population and short follow-up duration, RIST can be considered as the transplantation for paediatric patients with ALL in CR2. Disclosure of Interest: None declared. P274 Donor/recepient CMV serological status is a key prognostic factor for occurence of health- and lifethreatenig complications after haematopoietic stem cell transplantation in children J. Gozdzik1,2, M. Wozniak1,*, W. Czogala1, A. KrasowskaKwiecien1,3, A. Dluzniewska1, O. Wiecha1, S. Skoczen1, M. Sawa1 1 Transplantation Center, University Children’s Hospital, 2Immunology, Chair of Clinical Immunology and Transplantation, Jagiellonian University Medical College, 3Department of Transplantation, Chair of Clinical Immunology and Transplantation, Krakow, Poland Introduction: Cytomegalovirus (CMV) infection is still a reason of life-threatening complications in hematopoietic stem cell transplant (HSCT) recipients. Donor/recipient CMV serological status is an important risk factor. In Poland almost 90% of adult population is CMV-positive while many donors come from countries where CMV-positive population is in minority (o40%). The aim of the study was to assess the incidence, clinical course and effectiveness of treatment CMV infections after HSCT taking into account donor/recipient CMV serological status. Materials (or patients) and methods: Between 2005-2014 in our center 113 children underwent 126 HSCT (100 patients received one transplant, 12 – two transplants from the same donor and 1 patient received three transplants from 2 donors). The HSCT were performed for malignancy and non-malignant disease in 67 and 46 patients, respectively; 41 HSCT were from related donor and 72 from unrelated donor. The conditioning regimen was ablative (85 transplants), reduced toxicity (39), or non conditioned (2). The study group comprised the following recipient/donor pairs: CMV þ /CMV þ (52), CMV þ /CMV- (36), CMV-/CMV þ (15) and CMV-/CMV- (11). Congenital CMV infection were found in 3 children, and 1 was infected in early infancy. CMV DNA real-time PCR testing was performed
once weekly during immunosuppression. Positive evidence of CMV replication was an indication for ganciclovir (GCV) in a dose of 10 mg/kg (I-line treatment). In case of ineffectiveness foscarnet (FCV) was given in a dose of 120mg/kg (II-line). In case of further lack of effectiveness upon Z14-day therapy the third-line treatment was introduced (GCV and FCV, 120 mg/ kg). Due to resistance to GCV and FCV, cidofovir was administered (5-week regimen, 5 mg/kg, once a week [IV-line treatment]). The preemptive therapy was used until a negative result and continued with reduced dose for next 14 days. Results: After the first HSCT, 34 patients (30%) with CMV replication were identified, on average day þ 38 (0-224). 28 patients experienced a single episode lasting from 7 days to 4 months. In 5 patients recurrence of CMV reactivation was observed. First reactivation ocurred average on 14 day (7-30) and the second on 42 (30-180) day after HSCT; 1 patient (CMV þ /CMV-) experienced three episodes. In spite of treatment (I-IV line), he died of CMV pneumonia. There was 1 case of fatal hepatitis in CMV þ infant with congenital CMV transplanted from a CMV- alternative donor. Among the 12 patients undergoing second HSCT CMV replication was confirmed in 2 patients, both were CMV þ , with CMV- donors. After the first transplantation from the same donors no reactivation was observed. After the second HSCT, 1 patient had three reactivation episodes, with no symptomatic disease. The second patient with CMV reactivation developed retinitis, meningitis and CNS involving. In this case the treatment with two agents was long and successful. The patient, who underwent 3 HSCT (from different donors) experienced no CMV replication. Both the patient and the donors were CMV þ .
Conclusion: 1. Recepient/donor serological status CMV þ /CMVis an extremaly high risk factor of life-threatenig complications of CMV reactivation in children. 2. Even very intensive and longterm treatment (triple HSCT) in case of CMV þ recipient and donor is not a risk factor of CMV reactivation. Disclosure of Interest: None declared. P275 Allogeneic stem cell transplantation in pediatric myelodysplastic syndromes and juvenile myelomonocytic leukemia- a retrospective study of Polish national group M. Ussowicz1,*, J. Musial1, K. Drabko2, A. Zaucha-Praz˙mo2, A. Pieczonka3, J. Wachowiak3, J. Styczynski4, E. Gorczyn´ska1, K. Kalwak1, L. Gil5, A. Chybicka1 on behalf of Polish Pediatric Group for Hematopoietic Stem Cell Transplantation (PPGHSCT) 1 Department of Pediatric BMT, Hematology and Oncology, Wroclaw Medical University, Wroclaw, 2Department of Pediatric Hematology, Oncology and Transplantology, Medical University of Lublin, Lublin, 3Department and Clinic of Pediatrics Oncology, Hematology and Transplantation, Medical University in Poznan, Poznan, 4Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, 5 Department of Hematology, Poznan University of Medical Sciences, Poznan, Poland Introduction: Myelodysplastic syndromes (MDS) and Juvenile Myelomonocytic Leukemia (JMML) in childhood are rare malignancies treated with megachemotherapy and allogeneic stem cell transplantation (SCT). The Polish Pediatric Group for
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Hematopoietic Stem Cell Transplantation analysed the results of 96 children and adolescents transplanted in years 19972013. Materials (or patients) and methods: The group consisted of 36 girls and 60 boys (M/F ratio 0.375). Patients median age was 5.9 years (range 0.2-19 years), median interval from diagnosis to SCT was 6.43 months (range 1.5-103 months), the median follow up time after SCT was 21 months (range 0.1-209). Of 96 patients, 17 were transplanted with refractory cytopenia (RC), 40 with Refractory Anemia with Excess of Blasts (RAEB/RAEBT), 12 with Myelodysplasia Related Acute Myeloid Leukemia (MDR-AML), 5 with undefined MDS, and 22 with JMML. 53 patients were transplanted from matched unrelated donors (MUD), 27 from matched sibling donors (MSD), and 15 from haploidentical (haplo) donors. Graft material was BM in 43, PBSC in 50, and cord blood in 2 patients. Conditioning regimen was myeloablative (busulfan-based) in 76 patients or non-myeloablative in 20 (RIC, mostly treosulfan-based). Results: The neutrophil engraftement was achieved in 91,6% of patients, and platelet 4 20 K/uL in 82% patients. Acute GVHD was seen in 30% (29 patients), and chronic GVHD in 15% (12/77 patients surviving beyond day 90). Relapse incidence was 24% (16 patients) with median incidence of 6 months. The long term overall survival was 32% in JMML and 48% in MDS. In univariate analysis MDS subtype, graft material, conditioning intensity, incidence of aGVHD or cGVHD had not affected the SCT outcome either in MDS or in JMML. The OS was superior in patients transplanted earlier than 6 months from diagnosis (64% vs 32%, P ¼ 0.005). The choice of haploidentical donor was a factor of adverse prognosis in MDS patients (P ¼ 0.001). The OS was superior in patients transplanted after year 2005 (57% vs 30%, P ¼ 0.01). Conclusion: Early allogeneic SCT is advantageous in children with MDS or JMML. The outcome in MSD and MUD transplantations is comparable. All relapses were observed within 24 months from SCT, what warrants close follow up during this posttransplant period. Similarly to results observed in adults, progress in supportive care resulted in improved transplantation outcomes in children. Disclosure of Interest: None declared. P276 Treosulfan, melphalan and thiotepa megachemotherapy backbone is well tolerated in heavily pretreated children and facilitates reliable engraftment M. Ussowicz1,*, E. Wawrzyniak1, J. Weclawek-Tompol1, M. Mielcarek1, J. Musial1, J. Peregud-Pogorzelski2, K. Kalwak1 1 Department of Pediatric BMT, Hematology and Oncology, Wroclaw Medical University, Wroclaw, 2Department of Pediatrics, Hematology and Oncology, Medical University, Szczecin, Poland Introduction: The conditioning regimen is an important prerequisite in the stem cell transplantation settings which is responsible for both the anticancer effect and establishing of environment suitable for stem cell engraftment. Here we report the results of megatherapy containing the treosulfan, melphalan and thiotepa backbone (Treo-Mel-TT) in heavily pretreated children with solid tumours. Materials (or patients) and methods: The group consisted of 9 patients (pts) with disseminated, relapsing malignancies, with median age 5.7 years (range 3-14.8). Diagnoses were neuroblastoma (NBL, 7 patients), primary neuroectodermal tumour (PNET, 1 pt), and nephroblastoma (WT, 1 pt). 6 patients with NBL underwent busulphan-melphalan megachemotherapy with autologous SCT, and relapsed afterwards. The megatherapy backbone consisted of i.v. treosulfan at 10 g/ m2 daily from day -8 to -6 (total dose 30 g/m2), i.v. thiotepa at 5 mg/kg daily from day -4 to -3 (total dose 10 mg/kg), and i.v. melphalan at 70 mg/m2 daily from day -2 to -1 (total dose 140 g/m2). For patients undergoing the haploidentical transplantation, i.v. fludarabine was added at 40 mg/m2 daily from day -8 to -5 (total dose 160 mg/m2), and ATG in total dose of 30 mg/ kg, usually from day -12 to -9. After Treo-Mel-TT megatherapy,
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8 patients underwent haploidentical transplantation from the parent (with CD3/19 depletion – 4 pts, or with TCR alpha-beta depletion – 4 pts), and 1 patient – second autologous SCT. The patients toxicities during the transplant procedure were assessed and graded according to Common Terminology Criteria for Adverse Events (CTCAE), Version 4.0. Results: Neutrophil engraftment was achieved by day þ 12 (range 10-24), and in all patients transplanted from haploidentical donors stable full donor chimerism was achieved. The median hospitalisation length was 37 days (range 28-49). WHO grades for body weight loss were 1 (5 pts) or 2 (2 pts), for body weight gain 1 (3 pts) or 2 (4 pts), diarrhea lasted for 1 to 12 days with grade 1 (2 pts), 2 (2 pts) or 3 (3 pts), oral mucositis grade was 1 (1 pt) or 3 (6 pts). All 9 patients developed neutropenia grade 4, with lymphopenia grade 3 (2 pts) or 4 (7 pts). Kidney function was not impaired (9/9 pts with normal creatinine levels), only mild liver toxicities were observed (AlAT43 x upper limit – 3 pts, AspAT43 x upper limit – 1 pt, no increase in alkaline phospatase or billirubin levels). 4 patients remain alive and well in remission with median follow up of 19 months (range 6.6-32.7 months). Conclusion: Although the reported group is small and heterogenous, the strikingly dismal prognosis was obvious in all patients. Despite long and intensive multiple lines of pretransplant chemotherapy, the patients tolerated the megachemotherapy very well, and some of them achieved remission and are disease-free. The treosulfan-melphalanthiotepa conditioning is a protocol that can be safely used in patients with solid tumours due to good tolerability and sustained engraftment. Disclosure of Interest: None declared. P277 Late effects in children after allogenic stem cell transplantation (Allo-SCT) M. J. LLAMAS POYATO1,*, J. R. MOLINA HURTADO1, C. CHIC ACEVEDO1, E. GARCIA TORRES1, G. RODRIGUEZ GARCIA1, C. MARTIN CALVO1, A. RODRIGUEZ VILLA1, R. ROJAS CONTRERAS1, F. MARTINEZ GUIBELARDE1, C. HERRERA ARROYO1, P. GOMEZ GARCIA1 1 HEMATOLOGY, REINA SOFIA UNIVERSITY HOSPITAL, CORDOBA, Spain Introduction: Late effects (LE) after Allo-SCT can appear in long-term survivors. LE expresses themselves as structural or functional impairment of organs or tissues secondary to AlloSCT or related to a new tumoral growth. The aim of this study was to determine the incidence of LE that contribute to longterm morbidity after Allo-SCT in pediatric patients. Materials (or patients) and methods: We analyse retrospectively 126 consecutive pediatric patients (r18 age)
undergoing Allo-SCT in our institution from May 1981 to November 2011 with a minimum overall survival more than two years after transplant. Data were derived from review of clinical histories in the last year. Results: Median age at transplantation was 11 years (range: 118). Seventy-one patients (56.3%) were male and 55 (47.7%) female. The most common underlying disease was acute lymphoblastic leukemia (45.2%) while 51 (40.5%) patients were in complete remission at Allo-SCT. Bone marrow was the most common stem cell source (82.5%) and conditioning regimen was myeloablative in 97.6% of patients. Median follow-up was 88 months (range: 24-341) with a progression free survival of 80.2% and an overall survival of 84.1%. Major death cause was relapse in 14 patients. A total of 40 (31.7%) patients developed chronic graft versus host disease (cGVHD). The most common long-term LE were: skin lesions in 38 (30.2%) patients like scleroderma (n ¼ 12) and lichen planus (n ¼ 4). Ocular involvement in 19 patients (15.1%), highlighting keratoconjunctivitis sicca (n ¼ 13). Oropharyngeal injury in 26 patients (20.6%) highlighting the lichen planus (n ¼ 14). Chronic lung involvement in 16 patients (12.7%): 12 of them developed bronchiolitis obliterans and it was related to previously cGVHD (Po0.001) and prolonged use of high-dose steroids (Po0.001). Liver complications in 11 (8.7%) patients (mostly increased liver enzymes). Uro-vaginal complications in 2 patients. Skeletal complications in 12 (9.5%) patients: avascular necrosis in 8 of them. Bone damage was related to Total Body Irradiation in conditioning regimen (P ¼ 0.003). Two patients developed cardiac insufficiency. Endocrine-metabolic complications developed in 24 patients (19%): hypothyroidism (n ¼ 4), hypogonadism (n ¼ 11), hypercholesterolemia (n ¼ 5), diabetes mellitus (n ¼ 3) and hypertension (n ¼ 1). Seven patients (4.8%) developed secondary tumors (six of them were aconditioned with TBI): they were more common in the group with cGVHD (P ¼ 0.01) and those receiving long term high-dose steroids (P ¼ 0.016). Regarding the growth, 23 (18.3%) and 22 (17.5%) patients have a height and weight less than the 25th percentile for their age (last measurement in the clinical history),respectively. Multivariate analysis showed that the risk of developing any LE in Allo-SCT survivors was associated with female sex of receptor (P ¼ 0.008),cGVHD (P ¼ 0.008) and high-dose steroids (P ¼ 0.004). If we divide the patients into two groups according to the date of Allo-SCT (before or after 2000) there were not significant differences in OS (P ¼ 0.63) or PFS (P ¼ 0.45). In the first group (before 2000 LE were more common compared to the other group (45.9% vs 29.2%;P ¼ 0.045). Conclusion: LE secondary to Allo-SCT in pediatric patients affect a high percentage of them,but not impede a normal life. Early diagnosis of these LE and possible treatment should be ensured by monitoring patients maintained over time. The prolonged use of steroids and cGVHD are associated with the occurrence of LE. Disclosure of Interest: None declared.
administered four-times-daily during four days. However, this administration modality is nursing-time consuming, responsible for organizational difficulties and discomfort for the patient. Even if once daily schedule is used in the adult population, only few data regarding this administration in pediatric population exist. Our study aims to compare the two administration modalities of Bu: four-times-daily versus once daily in a pediatric cohort. Materials (or patients) and methods: We performed a prospective study in 20 patients at the Strasbourg University Hospital during august 2012 and june 2014. The cohort was divided in two groups: in the first one (A), Bu was administered four times daily (12 patients), and in the second (B), Bu was administered once daily (8 patients). The posology of the first dose of Bu was determined according to the child’s weight, then the following doses were adapted to the measured AUC. The target AUC was between 900 to 1500 mM.min in fourtimes-daily schedule and 3600 to 6000 mM.min in once daily schedule. The plasmatic Bu concentrations were measured by gas chromatograph mass spectrometer method. AUC and accumulated dose received by every patient were calculated and finally efficiency, toxicity, organizational consequences and patient comfort were evaluated Results: In total, 78 AUC were determined (47 in group A, 31 in group B). The plasmatic concentration of Bu, 30 minutes after the end of the drip were measured between 268 and 1836 ng/ ml (mean, 1054 ng/ml) and 1919 to 6726 ng/ml (mean, 3005 ng/ml) in group A and B, respectively. These data confirm the large interpersonal variability. In the group A, 10/12 patients
P278 Comparative pediatric study of two modalities of administration of intravenous Busulfan (Bu) : four-timesdaily versus once daily schedule M. A. Puel1,*, V. Kemmel2, A. Spiegel1, P. Lutz1, J. M. Lessinger2, C. Paillard1 1 pediatrie, CHU Hautepierre, 2Laboratoire de Biochimie et Biologie Mole´culaire, CHU Hautepierre, CHU Strasbourg, strasbourg, France Introduction: Busulfan (Bu) is well known to have a large inter and intra-patient variability, especially during childhood. Exposure of high Bu concentrations have toxicity consequences due to overexposure, while no engraftment is observed with too low concentrations, due to underexposure. This is why Therefore therapeutic drug monitoring by following of the area under the curve (AUC) is essential, in pediatric population. Intra-venous Bu is classically
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had an average of AUC in the therapeutic window and 8/8 patient in the group B. As shown in Figure 1, the amplitude of dose adjustment was greater in group A. So the initial dose calculated for the protocol B was more effective to obtain the therapeutic target. On clinic view, no failure of engraftment was noticed. 3 deaths were reported but none related to Bu use. The only one patient, who presented a veino-occlusive disease was out range Bu concentration and had a favorable evolution after the return of the Bu concentrations in therapeutic range. In term of organization, the once daily administration permits to reduce 5 hours a day the perfusion time. This extratime was also responsible of an increase in patient’s comfort. Conclusion: Our study underlines that both schedule of Bu administration permits therapeutic drug monitoring with an equivalent patients care. Moreover, patient’s comfort and the nursing time optimization seems to be improved by once daily administration in pediatric patient. Disclosure of Interest: None declared.
Acute leukaemia I P279 Azacitidine maintenance after allogeneic stem cell transplantation is feasible in patients with acute myeloid leukemia and myelodysplasia A. Antar1,*, M. Kharfan-Dabaja2, H. Abou Ghaddara1, R. Mahfouz3, A. Bazarbachi1 1 Internal Medicine/Hematology-Oncology, American University of Beirut, Beirut, Lebanon, 2Blood and Marrow Transplantation, Moffitt Cancer Center, Tampa, FL, United States, 3Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon Introduction: 5-Azacidine (5-AZA) is a DNA hypomethylating agent with proven clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A recent non-randomized study reported promising results with the use of lower doses of 5-AZA as maintenance therapy after hematopoietic stem cell transplantation (HSCT). It is important to note that 5-AZA has an immunomodulatory effect and might enhance the graft-versus-leukemia (GVL) effect. Here, we report the successful use of 5-AZA maintenance following allogeneic HSCT in patients with high risk AML and MDS. Materials (or patients) and methods: Nine patients (M ¼ 6, F ¼ 3; median age ¼ 49 (36-65) years ) with high-risk AML (n ¼ 6 including 2 abnormal karyotypes) or MDS (n ¼ 3
[P279]
Table 1.
including 1 abnormal karyotype) received 5-AZA as posttransplant maintenance at a dose of 32mg/m2 daily for 5 days every 4 weeks starting at a median time of 100 (30-210) days post-transplant. All patients were in complete remission at initiation of 5-AZA. A median of 12 cycles (1-18) were delivered. Patients’ characteristics and response to treatment are summarized in Table I. Results: After a median follow-up of 19 months post HSCT and 15 months after starting 5-AZA treatment, five patients with normal karyotype are still in CR. Conversely, all three patients with abnormal karyotype rapidly developed disease recurrence while they were receiving 5-AZA after a median of 3 months. Overall, the actuarial 1-year progression free and overall survival rates were 65% and 90%, respectively. 5AZA was generally well tolerated with only mild thrombocytopenia observed in 2 patients. No clinically evident graftversus-host disease exacerbation was observed. Stem cell source for all patients : peripheral blood; all patients received cyclosporine as GVHD prophylaxis; CR: complete remission; PR: partial remission; MRD: matched related donor; MUD: matched unrelated donor; FB4: 5 days fludarabine plus 4 days busulfan(130 mg/m2/day); FB3: 5 days fludarabine plus 3 days busulfan (130 mg/m2/day); FB2: 5 days fludarabine plus 2 days busulfan (130 mg/m2/ day); ATG: anti-thymoglobuline; DLI: donor lymphocyte infusion. Conclusion: These results suggest that Low-dose 5-AZA is an effective maintenance therapy post- allogeneic SCT in high-risk AML and MDS particularly when a normal diploid karyotype is present. The relative lack of efficacy in the presence of an abnormal karyotype is intriguing and questions whether these subjects might benefit from higher doses of 5-AZA or other novel therapies within the context of a well-designed clinical trial. Prospective clinical trials and longer follow-up are needed to confirm these observations. Disclosure of Interest: None declared. P280 Mutated regions of nucleophosmin 1 elicit CD8 þ T-cell responses in patients with acute myeloid leukemia A. Sun1,*, Z. Cheng1, G. Chen1, X. Yang1, D. Wu1 1 The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou, China Introduction: Stimulating lymphocytes from healthy donors by DCs loaded with NPM1mut peptides in vitro in order to induce specific cellular immune responses against the peptides. And detecting the NPM1mut peptide specific T-cell in peripheral blood of patients with NPM1mut positive acute myeloid leukemia (AML), to provide a theoretical basis for immune therapy of AML.
Patients characteristics and Outcomes After Azacitidine maintenance
Subject # Disease status at HSCT Donor type Conditioning Disease recurrence Salvage therapy if recurrence Progression free survival, months Status at last follow up Survival, months
1
2
3
4
5
6
7
8
9
CR2
CR3
CR1
CR1
Refractory
CR1
PR
PR
CR1
MRD FB2 þ ATG
MRD FB3 þ ATG
MRD FB3 þ ATG
MRD FB4 þ ATG
MRD FB4 þ ATG
MRD FB4 þ ATG
MRD FB3 þ ATG
MRD FB2 þ ATG
no
no
yes
no
MUD FB3 þ ATG þ TBI (4Gy) yes
no
no
no
yes
N/A
N/A
Chemotherapy followed by DLI
N/A
None
N/A
N/A
N/A
Chemotherapy followed by DLI
13 þ
24 þ
1
24 þ
3
19 þ
21 þ
18 þ
10
CR
CR
CR
CR
died
CR
CR
CR
CR
13 þ
24 þ
18 þ
24 þ
5
19 þ
21 þ
18 þ
34 þ
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Materials (or patients) and methods: 1. newly diagnosed patients of AML underwent gene mutations screening routinely. 2. peripheral blood mononuclear cells (PBMCs) were isolatedfrom healthy donors with HLA-A* 0201 or A*1101 and NPM1mut positive AML patients in complete remission. 3. Inducing differentiation of PBMCs into DCs 4. Generation of NPM1-specific cytotoxic T cells 5. ELISPOT analysis and intracellular staining Results: 1. In the Han Chinese population, the most common alleles of HLA-A loci were A* 1101, A*2402, and A*0201 allele. 2. Through prediction of the aforementioned software, HLAA*0201 restricted wild type NPM1 amino acid sequences (DLWQWRKSL), mutated NPM1-A/D amino acid sequences (AIQDLCLAV), and HLA-A*1101 restricted mutated NPM1-A/D amino acid sequences (AVEEVSLRK) were synthesized. There were no proper epitopes for either HLA-A*1101 restricted wild type NPM1 protein or HLA-A*2402 restricted wild type and mutated NPM1 protein. 3. Expression of surface antigens of DCs on day 7 were as follows: CD14(2.6%), CD80(43.4%), CD83(7.8%), CD86(99.9%), HLA-DR(67.1%), which was consistent with DCs phenotype. 4. About 10 days after DCs were co-incubated with their own PBMCs, the number of lymphocytes increased significantly, especially in the NPM1mut peptide pulsed group. On day 7 of co-incubation, ELISPOT analysis results of all samples were negative. However, 3 cases’ ELISPOT results were positive for the mutated peptide holes on day 14, in contrast, the results of the wild type peptide holes were negative. The NPM1mut peptide-specific T lymphocytes positive rate [ ¼ (average number of spots in mutated peptide holes-the average number of spots in negative control holes)/total lymphocytes per hole] was about 1/2500. Intracellular staining showed that in the aforementioned 3 cases, the proportion of CD8 þ IFN-g þ cells of the mutant peptide group was higher than either the wild type peptide group or negative control group, but there was no difference between the later two groups. 5. 6 peripheral blood samples of patients with NPM1mut þ AML were performed ELISPOT analysis, with only 1 case (16.7%) showing a approaching positive result. Conclusion: NPM1mut peptide pulsed DCs can stimulate their own PBMCs from healthy donors in vitro to produce mutated NPM1 specific T lymphocytes, and it is expected to be used as immune therapy of AML. The mutated NPM1 specific CTL in NPM1mut þ AML patients are almost undetectable that indicates immune system have been comprised because of chemotherapy and disease, and can not responses to antigens efficiently. Perhaps AML patients can accept CTL transfusion from their relatives. Our study provide an experimental basis for cellular immunotherapy of AML. Disclosure of Interest: None declared. P281 Effect of in vivo T-cell depletion with ATG on cytomegalovirus (CMV) induced antileukemic effect in patients with acute myeloid leukemia (AML) receiving grafts from unrelated donors A. Busca1,*, C. Dellacasa1, C. Frairia1, S. Aydin1, R. Passera2, M. Pini3, F. Zallio3, E. Audisio1, L. Giaccone1, E. Maffini1, C. Costa4, R. Cavallo4, S. Manetta1, B. Bruno1 1 Onco Hematology, 2AOU Citta’ della Salute e della Scienza, Turin, 3Hematology, AO Nazionale Alessandria, Alessandria, 4 Microbiology, AOU Citta’ della Salute e della Scienza, Turin, Italy Introduction: Several studies provided evidence of a consistent antileukemic effect induced by CMV replication in AML patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). It is conceivable that the ‘‘virus-versus leukemia’’ effect promoted by CMV reactivation requires a robust T- or NK-cell immune response to elicit a graft-versus leukemia effect. In vivo T-cell depletion is typically associated with a delayed T-cell reconstitution, and may potentially abrogate the protective effect of CMV infection. Nevertheless, the influence of antithymocyte globulin (ATG, Thymoglobulin)
on the antitumor effect of CMV replication after HSCT is rather unexplored. To address this issue, we retrospectively analyzed a cohort of 101 patients with AML who received grafts from an unrelated donor in two italian institutions between 2004 and 2014, after a conditioning regimen including ATG. Materials (or patients) and methods: Overall, 66 patients (65%) had early disease phase at the time of HSCT and 83 patients (82%) received myeloablative regimens. Prophylaxis of GVHD consisted of cyclosporine and short course MTX combined with ATG at a dose of 5-7.5 mg/Kg over two or three days. Real-time qPCR testing in whole blood for CMV reactivation was routinely monitored twice weekly. CMV viral load 42000 copies/mL was considered as CMV reactivation. Results: The cumulative incidence of CMV reactivation was 59% at 12 months. The 5-year cumulative incidence of relapse in patients with CMV reactivation was 29% compared with 37% for patients without CMV reactivation (P ¼ 0.279). The only factor associated with a reduced 5-year cumulative incidence of relapse was an advanced disease status at HSCT. Similarly, CMV reactivation was not associated with different OS or NRM rates (P ¼ 0.850 and P ¼ 0.297 respectively). In the multivariable model adverse cytogenetics (HR 2.42, 95% CI 1.02-5.72; P ¼ 0.044) and acute GVHD (HR 3.36, 95% CI 1.328.54; P ¼ 0.011) were independent risk factors for reducing OS, while the presence of chronic GVHD was associated with a better OS (HR 0.37, 95% CI 0.15-0.89; P ¼ 0.027). Conclusion: The results of present study showed that patients with AML receiving T-cell depleted HSCT using ATG did not benefit from CMV reactivation. Larger studies are mandatory to confirm our preliminary data. Disclosure of Interest: None declared. P282 Allogeneic hematopoietic cell transplantation in AML with normal karyotype and isolated NPM1 mutation: a retrospective analysis from the Acute Leukemia Working Party of EBMT A. Bazarbachi1,*, M. Labopin2, M. Kharfan-Dabaja3, R. Schwerdtfeger4, L. Volin5, J. H. Bourhis6, G. Socie´7, E. Deconinck8, T. GeddeDahl9, A. Rambaldi10, P. Jindra11, G. Schlimok12, D. Blaise13, P. Chevallier14, A. Nagler15, C. Schmid16, J. Esteve17, M. Mohty18 1 Internal Medicine, Bone marrow transplantation program, American University of Beirut, Beirut, Lebanon, 2Acute Leukemia Working Party of EBMT, Paris, France, 3Department of Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center/University of South Florida College of Medicine, Tampa, Florida, United States, 4Deutsche Klinik fu¨r Diagnostik KMT Zentrum, Wiesbaden, Germany, 5Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland, 6Division of Hematology, Department of Medical Oncology, Gustave Roussy, institut de cance´rologie, BMT Service, Villejuif, 7Dept. of Hematology – BMT, Hopital St. Louis, Paris, 8Service d’He´matologie, Hopital Jean Minjoz, Besancon, France, 9Rikshospitalet Department of Medicine, The National Hospital, Oslo, Norway, 10Azienda Ospedaliera Papa Giovanni XXIII Hematology and Bone Marrow Transplant Unit, Bergamo, Italy, 11Dept. of Hematology/Oncology, Charles University Hospital, Pilsen, Czech Republic, 12Klinikum Augsburg II Medizinische Klinik, Augsburg, Germany, 13Programme de Transplantation&Therapie Cellulaire, Centre de Recherche en Cance´rologie de Marseille, Institut Paoli Calmettes, Marseille, 14 CHU Nantes Dept. D’He´matologie, Nantes, France, 15Chaim Sheba Medical Center, Tel Aviv University, Tel-Hashomer, Israel, 16 SCT Unit, Klinikum Augsburg, Germany, 17Department of Hematology, Hospital Clinic, IDIBAPS, Barcelona, Spain, 18De´partement d’He´matologie, Hopital Saint Antoine, Paris, France Introduction: NPM1 mutation, in the absence of FLT3-ITD, confers a survival advantage and lower risk of disease relapse in AML with normal diploid karyotype. The prognostic significance of mutated NPM1 in the setting of allogeneic HCT has not been described. Materials (or patients) and methods: we evaluated outcomes of 156 AML patients (females ¼ 83, 53.2%), median age
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of 54 (19.5-71) years, with normal diploid karyotype and mutated NPM1, who underwent an allogeneic HCT between 2006 and 2012. Donor source was limited to matched-related or unrelated donors, and cell source consisted of bone marrow or mobilized peripheral blood stem cells. Patient-, donor-, and disease-characteristics are summarized in Table 1. Results: The median time from diagnosis to allogeneic HCT was 310 (89-3713) days and the median follow-up from allografting was 32 (2-86) months. Overall, the 2-year cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were 27% (20-35) and 13% (8-19) respectively, and the 2-year cumulative incidence of chronic GVHD was 37% (29-46). Finally, the 2-year leukemia-free survival (LFS) and the 2-year overall survival (OS) were 60% (51-68) and 70% (62-77), respectively. In univariate analysis, transplantation in CR1 was associated with lower 2-year CIR [CR1 ¼ 14% (7-23), CR2 ¼ 37% (23-51), advanced/active ¼ 48% (26-67), P ¼ 0.0009], superior 2year LFS [CR1 ¼ 75% (64-86), CR2 ¼ 51% (36-65), advanced/ active ¼ 30% (11-49), Po0.0001] and superior 2-year OS [CR1 ¼ 81% (72-91), CR2 ¼ 67% (54-80), advanced/active ¼ 39% (19-59), Po0.0001]. Patients allografted from unrelated donors have higher 100-day cumulative incidence of 4 grade 2 acute GVHD [MUD ¼ 28% (19-39) vs. MRD ¼ 12% (5-22), P ¼ 0.02]. Patients older than 54.3 years had higher 2-year cumulative incidence of NRM [20% (11-31) vs. 7% [2-14], P ¼ 0.03] and inferior 2-year OS [61% (49-72) vs. 78% (68-88), P ¼ 0.02]. In multivariable analysis, transplantation in CR1 resulted in lower 2-year CIR and superior 2-year LFS and OS. variables
Results N, (%) F ¼ 83 (53%) M ¼ 73 (47%) Yes ¼ 21 (14%) No ¼ 133 (86%) MRD ¼ 66 (42%) MUD ¼ 90 (58%) BM ¼ 30 (19%) PBSC ¼ 125 (80%) BM þ PBSC ¼ 1 (1%) CR1 ¼ 69 (44%) CR2 ¼ 64 (41%) Advanced/active ¼ 23 (15%) RIC ¼ 85 (54%) MAC ¼ 71 (46%) Yes ¼ 84 (54%) No ¼ 72 (46%) CSA þ MTX ¼ 77 (49%) CSA þ MMF ¼ 41 (27%) CSA alone ¼ 28 (18%) D þ /R þ ¼ 48 (31%) D þ /R- ¼ 14 (9%) D-/R þ ¼ 52 (34%) D-/R- ¼ 39 (26%)
Recipient gender Female donora`male recipient Donor source Cell source Disease status at allogeneic HCT Preparative regimen intensity ATG use GVHD prophylaxis Donor/recipient CMV serology
Abbreviations: F: female, M: male, MRD: matched-related donor, MUD: matched-unrelated donor, RIC: reduced-intensity conditioning, MAC: myeloablative conditioning, CSA:
[P283]
Table 1.
cyclosporine A, MTX: methotrexate, MMF: mycophenolate mofetil, D: allograft donor, R: allograft recipient Conclusion: AML disease status at allografting remains the most important predictor of post-allogeneic HCT outcomes despite expression of mutated NPM1. Survival outcomes are better when patients are transplanted in CR14CR24advanced/active. The impact of other molecular abnormalities in conjunction with NPM1 is yet to be established. Also, a future matched-controlled analysis comparing AML patients with mutated vs. wild-type NPM1 expression in the setting of allogeneic HCT is warranted. Disclosure of Interest: None declared. P283 Sorafenib Maintenance Appears Safe and Improves Clinical Outcomes in FLT3-ITD Acute Myeloid Leukemia after Allogeneic HCT A. Antar1, M. Kharfan-Dabaja2, R. Mahfouz3, A. Bazarbachi1,* 1 Internal Medicine/Hematology-Oncology, American University of Beirut , Beirut , Lebanon, 2Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, United States, 3Pathology and Laboratory Medicine, American University of Beirut , Beirut , Lebanon Introduction: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) gene is one of the most frequently observed genetic alterations in AML with an incidence of about 20–30%. FLT3-ITD is significantly associated with a poor outcome and it is recommended that patients harboring this mutation should undergo allogeneic hematopoietic cell transplantation (allo-HCT). Sorafenib is a tyrosine kinase inhibitor with activity against RAF, VEGF, and FLT3-ITD. It has been used off-label in FLT3-ITD AML. Here, we report the successful use of sorafenib following allo-HCT in patients with FLT3-ITD AML. Materials (or patients) and methods: Six male patients (median age ¼ 50(32-58) years) with normal karyotype FLT3ITD AML at diagnosis received sorafenib as post-transplant maintenance therapy (n ¼ 5) or as salvage therapy following a post allo-HCT relapse (n ¼ 1). Patient #6 was started on sorafenib in combination with chemotherapy for early relapse prior to transplant and was transplanted in CR2. The remaining 5 patients were transplanted in CR1 and received sorafenib post-transplant as maintenance therapy. Patients’ characteristics and treatment details are summarized in Table 1. Results: Sorafenib was well tolerated. Interestingly, skin GVHD grade II was observed in 5/6 patients and generally occurred within few days after initiation of sorafenib, but responded promptly to corticosteroids in all patients. No other manifestations of acute GVHD were observed out of the ordinary. All 6 patients are still alive and in complete remission with a median follow-up of 16 months (range, 10-29) since first induction, and a median follow-up of 12 months (range, 4-20) since initiation
Characteristics of patients and their treatment
Patient nb.
1
2
3
4
5
6
Molecular ( þ ) other than FLT3 ITD Induction
–
NPM1
–
–
NPM1, CEBPa
–
Daunorubicin þ Ara-C (3 þ 7) FB4 þ ATG 1Ctx,allo-sct,2Ctx
Idarubicin þ Ara-C FB3 þ ATG 2Ctx, allosct Post (71)
Daunorubicin þ Ara-C (3 þ 7) FB3 þ ATG 3Ctx,allo-sct
Daunorubicin þ Ara-C (3 þ 7) FB4 þ ATG 2Ctx, allo-sct
Daunorubicin þ Ara-C (3 þ 7) FB4 þ ATG 4Ctx,allo-sct
Daunorubicin þ AraC (3 þ 7) FB4 þ ATG 4Ctx
Post(35)
Post (40)
Post (30)
Pre(70)
Conditioning Therapy sequence before sorafenib Day on Sorafenib prepost HSCT Sorafenib dose Side effects (grade) Dose modification
Post (150) 400 mg BID Skin GVHD ( II) Liver toxicity 200 mg BID
400 mg BID None
400 mg BID
400 mg BID
400 mg BID
400 mg BID
Skin GVHD (II)
Skin GVHD (II)
Skin GVHD ( II)
No
No
No
No
Skin GVHD (II) Myocardial infarction 200 mg BID
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of sorafenib. Remarkably, all patients are in molecular remission. Stem cell source for all patients : peripheral blood ; All donors were matched related donor; all patients received cyclosporine as GVHD prophylaxis; FB4: 5 days fludarabine plus 4 days busulfan(130 mg/m2/day); FB3: 5 days fludarabine plus 3 days busulfan (130 mg/m2/day); ATG: anti-thymoglobuline; Ctx: chemotherapy. Conclusion: These results suggest that sorafenib appears to be an effective maintenance therapy post allo-HCT in FLT3-ITD AML with achievement of durable complete responses. This result may suggest an immuno-modulatory effect of sorafenib in the post transplant setting and warrant a broader clinical evaluation of the use of maintenance sorafenib in FLT3-ITD AML. Disclosure of Interest: None declared. P284 Value of pre-transplantation minimal residual disease in acute myelioid leukemia and myeloablative allogeneic hematopoietic cell transplantation M. Kwon1, A. C. Franco Villegas1,*, P. Balsalobre1, I. Bun˜o1, C. Martinez-Laperche 1, J. Anguita 1, A. Perez Corral 1, C. Pascual 1 , D. Serrano1, J. Gayoso1, J. L. Diez1 1 Hematology, University General Hospital Gregorio Maranon, Madrid, Spain Introduction: Relapse remains the main cause of treatment failure in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (SCT). The detection of minimal residual disease (MRD) in AML has improved in the past years with multiparametric flow cytometry (MFC) and molecular analysis (RT-PCR). However the prognostic impact of pre-transplantation MRD and the outcome after SCT is not well established. The aim of this study was to evaluate pre-transplantation MRD in patients in first complete remission (CR1) undergoing myeloablative allogeneic SCT. Materials (or patients) and methods: Consecutive adult patients with AML in CR1 after intensive chemotherapy with available MRD determination before transplant who underwent myeloablative SCT were retrospectively analyzed. MRD was studied by 4-color MFC on bone marrow aspirates, and quantitative RT-PCR (NPM1,WT1,MLL) on bone marrow and/or peripheral blood samples obtained within thirty days before transplant. Results: 46 patients consecutively transplanted in our institution between 1999 and January 2014 were included (Table 1).
Twenty-eight showed negative MRD pre-transplant whereas 18 showed positive values. Pre-transplant MRD negative patients included a higher proportion of high-risk cytogenetics LMA and HLA-identical siblings as stem cell source (Table 1). Within the MRD-positive group, 11/18 (61%) patients showed MRD-positive values by MFC: in 7 cases MRD was also detected by PCR, only 1 showed negative PCR and in the remaining 3 cases, PCR was not available. On the other hand, in 7/18 (38%) patients MRD was not detected by MFC, however, PCR detected MRD in all of the cases. With a median follow-up of the whole series of 35 months, 3-years estimates of overall survival were 66% (95% CI, 44.5-87) vs 41% (95% CI, 15-66.5) for MRD-negative and MRD-positive patients (P ¼ 0.12), respectively. Disease free survival was 68% (95% CI, 48-88) vs 36% (95% CI, 10-61) (P ¼ 0.06) whereas cumulative incidence of relapse was 23% (95% CI, 10-51) vs 31% (95% CI 14-67), respectively (P ¼ 0.54). In the MRD-negative group, cause of death was toxicity in 7% of the cases and relapse in 17%, while in the MRD-positive group, 22% of patients died due to toxicity and 27% due to relapse. Conclusion: Our data shows that the presence of MRD before allogeneic SCT in patients with AML in CR1 is associated with a significant worse DFS rate compared to patients with negative MRD, as well as a tendency towards a worse overall survival. The detection of MRD by MFC correlates with the detection by PCR in most of the cases. However, in a significant group of patients, MRD was detected only by PCR. This could be related to differences in sensitivity between both methods. Further studies including larger series are needed to confirm these observations. Disclosure of Interest: None declared. P285 Prognostic Impact of Pretransplantation Minimal Residual Disease detection for flow Cytometric, on Outcome of Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia. A Single Center Experience in Colombia over 14 Years A. A. Borda1,*, I. Cordoba1, V. Abello1, C. Rosales1, M. Rosales1, J. Figueroa1, E. Pedraza1, H. Esguerra1, M. Osuna2, A. Villegas1 1 Unidad de trasplante de me`dula o`sea, Clinica de Marly, 2 Hematologı`a, Hospital de San Jose, Bogota, Colombia Introduction: The accumulated evidence from studies of multiparameter flow cytometric MRD (MFC-MRD) assessment in AML leaves little doubt that this method of MRD detection can be used to risk stratify both younger and older patients at treatment time points. Persistence of disease or high levels of pretransplantation minimal residual disease (MRD) have been reported to predict disease relapse after Allogeneic bone marrow transplantation (BMT). The prognostic impact of MFCMRD is strong enough to have emerged despite study differences in the MFC assays and the limitations of now outdated restricted antibody panels. Aims: To determine the value of Minimal Residual Disease (MRD) assessed by Multiparameter Flow Cytometry (MFC) pretransplantation Allogeneic BMT, in predicting outcome in patients with acute myeloid leukemia (AML). Materials (or patients) and methods: We performed a retrospective analysis the predictive value of MRD assessment by MFC pre trasnplantation alogeneic in 119 patients (diagnosed AML treated between 2000 and 2014 submitted at our institution who had available MRD assessment). MRD by MFC on bone marrow specimens obtained approximately 30 days before transplantation. The detection threshold for defining pre transplantation positive MRD was 40.3% . Results: Of the 119 patients, 80 (67%) were in complete remission (CR1) , 31 (26%) CR2 and 4 CR2 8 (6%). Their median age was 38 years (Range, 10-64). Hyperleucocytosis in 39 (32%) and Cytogenetics was favorable risk in 32 (26%), intermediate risk in 39 (32.%), adverse risk in 35 (29%) and unknown in 13 (14%). There were a total of 44 deaths and 17 relapses; these contributed to the probability estimates for
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overall survival (OS) and disease free survival (DFS), stratified by MRD status and shown in figure 1. The median follow-up after BMT among survivors was 8.3 years (range, 6.9 to 9,6 years). The 7.5-years estimates of OS for MRD-positive and MRD-negative patients were 43.1% (range, 23,2% to 58,6%) and 68% (range 56% to 78.3%), respectively, and the 7,5 year estimates for DFS for MRD-positive and MRD-negative patients were 40.5% (range 21.4% to 52.6% ) and 56% (range 42.5% to 65.8%) respectively. After adjustment for various covariates, age, cytogenetics risk, hyperleucocytosis, secundary AML, the hazard ratios of MRD positive versus MRD negative were 2.06 (range 1.52 to 6.24; P ¼ 0,003) for overall mortality, 3.45 ( range 2.14 to 7.32; P ¼ 0.014) for DFS. Conclusion: That detection of MRD pre transplantation define a population of patients with AML who are at higher risk for adverse outcome, even after adjusting for other factors that influence post-BMT outcome. Disclosure of Interest: None declared. P286 Mild chronic GvHD may alleviate the poor prognosis associated with FLT3-internal tandem duplication in adult patients with AML following alloHSCT with myeloablative conditioning in first complete remission B. Nasilowska-Adamska1,*, A. Czyz2, K. Halaburda1, A. Tomaszewska3, M. Markiewicz4, P. Rzepecki5, B. Piatkowska-Jakubas6, M. Paluszewska7, R. Szydlo8, I. Solarska3, K. Borg1, S. KyrczKrzemien4, M. Komarnicki2, A. Skotnicki6, W. W. Jedrzejczak7, K. Warzocha1 1 Institute of Hematology and Transfusion Madicine, Warsaw, 2 K.Marcinkowski University of Medical Science, Poznan, 3Institute of Hematology and Transfusion Medicine, Warsaw, 4Silesian Medical Institute, Katowice, 5Military Medical Institute, Warsaw, 6 Collegium Medicum of Jagiellonian University , Krakow, 7Warsaw Medical University, Warsaw, Poland, 8Hammersmith Hospital, London, United Kingdom Introduction: Internal tandem duplication (ITD) of FLT3 is the most common mutation in AML with the highest frequency in patients with normal karyotype. The aim of this study was to estimate the significance of FLT3-ITD at diagnosis on the outcome of alloHSCT. Materials (or patients) and methods: A total of 140 patients at median age of 38 years (range 18- 68) with the analysis of FLT3-ITD performed at diagnosis who underwent first alloHSCT after myeloablative conditioning were included in this retrospective study. All patients were transplanted in first complete remission (CR) of AML between the years of 2006-2013 in six Polish centers. Results: FLT3-ITD was detected in 42/140 (30%) patients. Compared with FLT3-ITD-negative patients, FLT3-ITD positive patients had higher median white blood cells count (WBC) at
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diagnosis (71x109/l vs 28x109/l, Po0,001). Other characteristics were similar in the FLT3-ITD positive and negative groups of patients. At three years, relapse incidence (RI) was significantly higher in AML patients with intermediate or normal karyotype and WBC count 425 x109/L at diagnosis [12,7% (95%CI 5-29%) vs. 51.6% (95%CI 35-68%), Po0,001] or FLT3-ITD positive [20.4% (95%CI 11-34%) vs.52.9% (95%CI 33-72%), P ¼ 0,002] in univariate analysis. In multivariate analysis, we found that these two variables also significantly influenced RI following alloHSCT in AML patients with normal or intermediate karyotype [WBC425 x109/L (HR 4,2; 95%CI, 1,6-10,8, P ¼ 0,003), FLT3-ITD (HR 2,2; 95%CI, 0,99-5,0, P ¼ 0,053)]. Three-year leukemia free survival (LFS) following alloHSCT did not significantly differ between FLT3-ITD positive and negative groups, although, we found significant difference in LFS between patients FLT3-ITD positive and negative and normal or intermediate karyotype, respectively 44,7% (95% CI 26,3-64,7%) and 67,6% (95% CI 54,5-78,4%), P ¼ 0,06. Threeyear overall survival (OS) did not significantly differ between FLT3-ITD positive and negative groups, even in the group of patients with normal and intermediate karyotype. We also found that in patients who experienced chronic GvHD (cGvHD), the RI was significantly lower than in patients not affected with cGvHD [14,2% (95% CI 6-30) vs. 37,8% (95%CI 2750), P ¼ 0,041]. It is interesting, that patients with mild cGvHD had significantly lower RI when compared to patients with moderate or severe grade of cGvHD or those who did not experienced cGvHD, respectively 4,8 % (95%CI 1-25%) vs. 36,0% (95%CI 16-62%) vs. 27,8% (95%CI 18-41%), P ¼ 0,032. We did not observe any differences in LFS in accordance to overall incidence of cGvHD, but we found that mild cGvHD may positively influence LFS when comparing to the group of patients with no cGvHD or moderate/severe grade of cGvHD [respectively, 88.4% (95%CI 65-97%) vs. 67.5% (95%CI 55-78%) vs. 61.7% (95%CI 35-83%), P ¼ 0,06]. Conclusion: We observed that FLT3-ITD was harboring a poor prognosis in AML patients who underwent alloHSCT with myeloablative conditioning in first CR, mainly in those with intermediate or normal cytogenetic profile at diagnosis and significantly increased risk of relapse. Therefore, patients with high molecular risk AML may not be curable even following alloHSCT, unless they develop symptoms of mild cGvHD and concomitant GvL which may decrease RI and improves LFS. Disclosure of Interest: None declared. P287 Early Cumulative Incidence of Relapse in 80 Acute Myeloid Leukemia patients after chemotherapy and Transplant post-consolidation treatment: prognostic role of postinduction WT1 D. Capelli1,*, I. Attolico2, F. Saraceni1, G. Mancini1, S. Mancini1, A. R. Scortechini1, M. Montanari1, I. Scortechini1, N. Viola3, S. P. Pascale2, M. Chiarucci1, P. Leoni1, A. Olivieri1 1 Ospedali Riuniti di Ancona, Clinica di Ematologia, Torrette di Ancona, 2Divisione di Ematologia, Ospedale San Carlo, Potenza, 3 Ospedali Riuniti di Ancona, Servizio di Immunologia, Torrette di Ancona, Italy Introduction: Post consolidation minimal residual disease (MRD) evaluation represents a strong prognostic factor for AML outcome, while the significance of post induction MRD is still uncertain. Materials (or patients) and methods: We evaluated post induction and post consolidation bone marrow MRD in 80 AML patients aged in median 57 years (range: 17-80) with 16 months median follow-up (range 2-55). We analysed abnormal leukemia immunophenotype by multiparameter flow cytometry (MPFC) and WT1 by RT-PCR as described by Buccisano et al and Cilloni et al. The ELN cytogenetic molecular risk was determined in 62 patients (24 favorable, 24 INT-1, 14 unfavorable). WT1 was þ ve in 63/75 patients (85%) at diagnosis (median 1,056; range:259-232,056), in 10/59
P288 Comparison of The Results of Acute Leukemia Patients Who Had Undergone Haploidentical and HLA-I˙dentical Allogeneic Stem Cell Transplantation: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, C. Bal3, I. Dora1, B. Kosan1, E. Er1, B. Sarıtas1, N. Baskan1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, 3 Biostatistics Department, Osmangazi University, Kocaeli, Turkey
(16,9%) post induction (median 21; range:0,6-134.633) and in 8/50 (16%) post consolidation (median 18,6; range:0,8-45.338). MPFC MRD was þ ve in 27/56 (48.2%) patients after induction and in 16/44 (36.4%) after consolidation. Results: We analysed overall, 3, 12 month Cumulative Incidence of Relapse (CIR) by MRD status adjusted by ELN risk, gender, secondary disease, hyperleukocytosis, age and kind of post consolidation treatment. 68 patients achieved CR, 27 patients relapsed in a median of 6.6 months (1-34 months) with a 45% CIR: 12 patients at 3 months (13%), 23 at 12 months (33%). Post consolidation treatment consisted of chemotherapy in 34, Autologous and Allogeneic Tansplant in 32 patients, performed after 1 induction (37 high dose Cytarabine based, 43 standard dose Cytarabine based) and 1 high dose Cytarabine consolidation course. We observed 40%, 24.3% and 25.5% 12 month CIR after chemotherapy, Autologous and Allogeneic Transplant respectively. We also observed a 12 month CIR of 14.6% in favorable, compared to 23.3% in NPM-FLT3-, 57% in FLT3 þ and 61.6% in unfavorable ELN risk patients respectively. Patients with WT1 þ ve post induction and consolidation had a 12 month CIR of 90% and 62.5% respectively. Patients with MPFC þ ve post induction and consolidation had a 12 month CIR of 51.5% and 22.7% respectively. Multivariate analysis identified post induction WT1 þ ve status and not MPFC MRD status as the main predictor of 12 month CIR. Patients with WT1 þ ve after induction had a 14.3 RR to relapse at 12 months (Po0.0001) in comparison with WT1 -ve patients (Figure 1). Hyperleukocytosis (WBC count 450,000/mcl at diagnosis) and age460 yrs also significantly predicted 12 month CIR with a RR of 7.15 (P ¼ 0.004) and 12.3 (P ¼ 0.001) respectively. Conclusion: In conclusion post induction WT1 positivity confirmed its prognostic significance in our series, indipendently from ELN risk and post consolidation treatment, targeting a subset of early relapsing patients with an extremely poor outcome. References: Buccisano F, Maurillo L, Del Principe MI et al. Prognostic and therapeutic implications of minimal residual disease detection in acute myeloid leukemia. Blood. 2012; 119: 332–341. Cilloni D, Renneville A, Hermitte F et al. Real-time quantitative polymerase chain reaction detection of minimal residual disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study. J Clin Oncol. 2009; 27: 5195–5201. Disclosure of Interest: None declared.
Introduction: HLA-identical hematopoietic stem cell transplantation (HSCT) is now considered as an established therapeutic modality in several hematological malignancies. However, only 30-50% of the patients have a HLA-identical donor. In the present study, we report and compare the results of acute leukemia patients who were in complete remission at the time of transplantation and who had undergone HLAidentical or haploidentical hematopoietic stem cell transplantation. Materials (or patients) and methods: We analyzed retrospectively, the results of acute leukemia patients who were in complete remission at the time of transplantation and who had undergone allogeneic stem transplantation between July 2010 and December 2013. No T cell depletion were performed. Characteristics of the patients are shown in Table 1. Results: Characteristics of the patients in the 2 groups were similar (Table 1). Documented viral infection rate was higher in the haploidentical HSCT group in comparison to HLA-identical HSCT group (80% vs 50% respectively, P ¼ 0.003). Median hospitalization duration was 35 days in haploidentical HSCT group and 28 days in the HLA-identical HSCT group (Po0,001). Relapse and mortality rates (First 100 day, 1 year, 2 years) were similar in the 2 groups. Disease free survival and overall survival of haploidentical HSCT group and HLA-identical HSCT group did not differ significantly (P ¼ 0.46, P ¼ 0.42, respectively). Clinical results are shown in Table 2. Conclusion: According to our results, disease free survival and overall survival are similar in haploidentical and HLA-identical HSCT. However, in haploidentical HSCT time to neutrophil and thrombocyte engratment and hospitalization durations are longer, viral infection rates and GVHD rates are increased when compared to HLA-identical HSCT. More immunosuppressive drug usage is obligatory in haploidentical HSCT. Increased rate of viral infections and GVHD and longer hospitalization durations leads to increased costs in haploidentical HSCT and emphasizes the importance of experience in disease management. Disclosure of Interest: None declared. P289 Prospective study on 220 neutropenic episodes in 109 AML patients. Role of bed-side ultrasound (BUS) in neutropenic enteroclitis (NEC): early diagnosis, incidence and survival with different chemotherapy regimen E. Benedetti1,*, R. Giubbolini2, R. Morganti1, I. Bertaggia1, B. Bruno3, P. Lippolis4, F. Caracciolo5, E. Orciuolo1, G. Buda1, F. Cerri6, D. Caramella6, M. Petrini2 1 Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, 2Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Hematology Unit Ospedale S. Chiara Pisa, Pisa, 3U.O Ematologia Trapianti di midollo, University of Torino, Torino, 4UO Chirurgia D’Urgenza , Azienda Ospedaliero-Universitaria Pisana, 5U.O Ematologia Trapianti di midollo, Hematology Unit Ospedale S. Chiara Pisa, 6 Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, U.O. Radiologia Universitaria Pisa, Pisa, Italy Introduction: Neutropenic enterocolitis (NEC) is a life threatening complication of leukemic patients (pts) treated with chemotherapy (CHT) with mortality rate up to 50%. It is
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[P288]
characterized by abdominal pain (AP), fever (F) and diarrhoea (D). Ultrasound (US) was used to evaluate bowel-wall thickening (BWT), and 44 mm is considered diagnostic of NEC. Early diagnosis is crucial to start conservative medical management (CMM) which appears the optimal strategy for most cases. Objective: evaluate prospectively if US can detect early signs of NEC and guide a prompt treatment (CMM or surgical) in order to reduce mortality and to evaluate the impact of different CHT regimens on mucosal damage and NEC occurrence. Materials (or patients) and methods: In the last 7 years all AML pts admitted in our center undergoing CHT were enrolled (n ¼ 109). Abdominal US was performed, baseline before treatment, and as only one symptom (or a combination) appeared within 12h from onset: F and/or D and/or AP in CHTrelated neutropenic pts. Results: N ¼ 220 chemotherapy-related neutropenic episodes (NE) occurred in 109 pts. N ¼ 17 NEC were diagnosed (7.7 %). N ¼ 2 pts had 2 episodes of NEC and both survived. CHT regimens received (number of cycles/NEC episodes) was: 3 þ 7 (Idarubicin þ ARAC) N ¼ 62/10, Idarubicin (AML-M3) N ¼ 9/1, 3 þ 3 þ 5 (Idarubicin,VP-16,ARAC) N ¼ 48/3; 2 þ 5 (Idarubicin þ ARAC) N ¼ 24/1; Clofarabine (40mg) N ¼ 5/0; Clofarabine (20mg) 14/0; Clofarabine þ ARA-C (Cofa 20mg) N ¼ 18/1; FLANG N ¼ 13/1; HD-ARA-C (3gr/mq for 3 consecutive days) N ¼ 26/0. Overall 3 pts died out of 17 NEC episodes (17.6%). All pts were promptly treated as soon as BUS was diagnostic of NEC. Median time to response from beginning of CMM was 24h and the first sign of was a decrease in AP followed by F, D and BWT. Amelioration of symptoms occurred with pts still neutropenic. Symptoms at Dx were:F þ AP þ D N ¼ 9, F þ D N ¼ 1, F þ AP N ¼ 1, Dia þ AP N ¼ 4,D N ¼ 0, F N ¼ 0, AP N ¼ 2.
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Fever at Dx was absent in N ¼ 6 episodes (35%). Statistically (St) CHT regimens mostly associated with NEC were: 3 þ 7 odds ratio (OD) 6.00 (Po0.0001), 3 þ 3 þ 5 OD ¼ 1.75 (Po0.0001). There was not a St significant association of the following CHT regimens with NEC occurrence: Clofarabine (20 or 40mg) N ¼ 0 NEC, 2 þ 5 (P ¼ 0.125), Idarubicin (AML-M3) (P ¼ 0.111), Clofa 20mg þ ARAC (P ¼ 0.167), HD-ARAC N ¼ 0 NEC, FLANG (P ¼ 0.143). The association of ARAC to Clofarabine vs Clofarabine (20 or 40mg) alone was not St significant in NEC occurrence (P ¼ 0.9). The likelihood of NEC Dx in a discriminant model (Bayes theoreme) for pts with BWT and AP ¼ 100%, AP þ D ¼ 100%, AP þ D þ F ¼ 100%, AP þ F ¼ 100%. Conclusion: BUS allowed to detect early signs of NEC and to start prompt treatment in this life threatening complication, with a 76% survival rate. With BUS pts do not live the isolation room. Early diagnosis and intervention allowed to reduce mortality. Images of BUS and CT were superimposable. Fever is not a condition sine qua non for NEC diagnosis. Different chemotherapy regimens do have a different impact on mucosal damage. A prompt BUS in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical) eventually reducing mortality. Disclosure of Interest: None declared.
P290 BID, BAK AND BCL-XL expression in childhood acute lymphoblastic leukemia bone marrow M. Panagouli1, G. Martimianaki1, M. Pesmatzoglou1, E. Stiakaki1,* 1 Dept of Pediatric Hematology-Oncology, University Hospital of Heraklion, University of Crete, Heraklion, Greece Introduction: The apoptosis which may be involved in the pathogenesis of acute lymphoblastic leukemia (ALL) is known to be regulated by the receptor-mediated (extrinsic) and the mitochondria-mediated (intrinsic) pathway. The latter is controlled by the family of BCL-2 proteins, which are critical regulators of the apoptotic response with pro-apoptotic (BAX, BAK) and anti-apoptotic (BCL-2, BCL-XL, MCL-1) members. BAX and BAK play a pivotal role to the pro-apoptotic activity of BCL-2 family regulated by anti-apoptotic members of BCL-2 and BCL-XL while BID is considered one of the main activators of apoptosis. Aim: The quantitative determination of the expression levels of anti-apoptotic BCL-XL, and proapoptotic molecules BAK and BID in bone marrow (BM) of children with acute lymphoblastic leukemia at diagnosis and after remission achievement and of children with Solid Tumours (ST) without bone marrow involvement. Materials (or patients) and methods: Twenty eight children (15 boys, median age 7,68±1,06 years) with ALL (23 B lineage ALL, 5T ALL), 25 children at diagnosis (ALLd), 23 after remission was achieved (ALLr), and 38 with Solid Tumors at diagnosis without bone marrow involvement, were studied. Total RNA was isolated from bone marrow mononuclear cells. The mRNA levels corresponding to BCL-XL, BAK, and BID were quantified by real-time polymerase chain reaction (PCR) analysis using GAPDH as normaliser. All reactions were performed in duplicates. For quantification of the genes a standard curve was made by serial dilutions of a reference line cDNA (Tanoue). Results: The expression levels of anti-apoptotic BCL-XL were determined significant higher in ALL blasts at diagnosis compared with remission (ALLdvsALLr:3,16vs0,97, P ¼ 0,00001), as well as with ST(ALLdvsST:3,16vs0,64, P ¼ 0,000000013). The expression levels of BID were determined low at diagnosis of ALL and high enough in remission with statistically significant difference (ALLdvsALLr:4,57vs18,07, P ¼ 0,000001), result which is in accordance with its apoptotic role. High levels were also observed in ST without BM involvement (ALLdvsST:4,57vs10,329, P ¼ 0,003). Concerning the expression of apoptotic BAK gene, the levels were found statistically significant higher at ALLd compared with ALL in remission (ALLdvsALLr:4,73vs1,84, P ¼ 0,001). Similar high levels were determined in Solid Tumors with statistically significant differences compared with ALLr (STvsALLr:5.44 vs 1,844, P ¼ 0,011). Conclusion: The over-expression of BCL-XL at ALL diagnosis seems to be suppressed by the induction treatment and remission achievement. The expression of BID is amplified in remission as a pro-apoptotic molecule stimulating apoptosis. The role of BAK levels at diagnosis and remission in respect to its pro-apoptotic function warrants further investigation. References: 1. Sawata S, Suyama E and Taira K. A system based on specific protein–RNA interactions for analysisof target protein–protein interactions in vitro: successful selection of membrane-bound Bak–Bcl-xL proteins in vitro.Protein Eng Des & Sel 2004; 17: 501-508. 2. Addeo R, Caraglia M, Baldi A, D’Angelo V. Prognostic Role of bcl-xL and p53 in Childhood Acute Lymphoblastic Leukemia (ALL). Cancer Biology & Therapy 2005; 4: 32-38. 3. Du H, Wolf J, Schafer B, Moldoveanu T, et al. BH3 Domains other than Bim and Bid can Directly ActivateBax/Bak. Journal of Biol Chem 2011; 286: 491–501. Disclosure of Interest: None declared.
P291 Allogeneic haematopoietic stem cell transplantation in patients older than 65 years with high risk acute myeloid leukemia: results in 46 consecutive patients E. Sala1,*, M. G. Carrabba2, C. Messina1, F. Pavesi1, E. Xue1, B. Gentner1, L. Vago1, M. Morelli1, A. Assanelli1, S. Marktel1, M. T. Lupo-Stanghellini1, C. Corti1, J. Peccatori1, F. Ciceri1, M. Bernardi1 1 Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, 2Unit of Hematology and Bone Marrow Transplantation, San Raffaele Scientific Institute, Milan, Italy Introduction: Allogeneic haematopoietic stem cell transplantation (allo-HSCT) from related or unrelated donors is the only curative option for patients (pts) with high risk acute myeloid leukemia (AML). Most of these pts are older than 60 years. Elderly pts have been excluded from allo-HSCT option until recently. The reduction of therapy-related complications and the introduction of reduced-intensity conditioning regimens has increased the upper age limit. Encouraging results of alloHSCT in pts up to the age of 70 years have been reported. Materials (or patients) and methods: We conducted a restrospective analysis of 46 consecutive pts, 65 yrs of age or older, with high risk AML, who received allo-HSCT at our institute between 2004 and 2014. Twenty pts out of 46 had a diagnosis of de novo AML (adverse or intermediate-2 risk profile according to ELN stratification was evaluated in 14/20, while 6 had a chemorefractory disease), 23/46 had AML secondary to previous Myelodisplastic or Myeloproliferative disorder and finally 3 pts had a diagnosis of therapy-related AML. Data were reviewed from the local database and updated at last follow up for pts still alive with a minimum follow up of 6 months. Results: Between August 2004 and July 2014, 46 pts, median age 66 years (range 65-78) underwent allo-HSCT. At transplant 28 pts were in complete remission (CR), 18 were in persistence of disease (PD). The stem cells source was in 8 (17%) cases a matched related donor (MRD), 14 (30%) a matched unrelated donor (MUD), 23 (50%) a haploidentical donor (HAPLO), in 1 case (3%) cord blood (CB). All pts transplanted from an adult donor received mobilized peripheral blood stem cells. Conditioning regimen was a reduced toxicity combination Treosulfan-based in most of the pts (36 out of 46, 78%). Graft versus Host Disease (GvHD) prophylaxys was cyclosporine þ methotrexate in 19 cases, rapamycine þ mofetil mycophenolate in 24 cases, ex vivo T-cell depletion in 3 cases. Median follow-up after transplant was 328 days (range 13-2050). Early deaths after transplant (before 30 days) were 4 (8.6%), 1 for disease progression and 3 for infection during aplasia. Engraftment rate was 97% (41/42 pts). Severe acute GvHD incidence (grade III-IV) during follow-up was 9% (4 out of 42 evaluable pts with at least 100 days of follow up), while severe chronic GvHD was detected in 3 pts out of 42 (7%). At last follow-up 17/46 pts (37%) are alive and 16 in CR, 29 pts (63%) were dead: 12 died because of disease relapse/progression, 16 for causes related to transplant toxicities (TRM), 1 patient was lost at follow up. The overall survival (OS) from diagnosis was 80% at 1 year and 38% at 3 years, while the OS from allo-HSCT was 55% at 1 year and 36% at 3 years, EFS at 1 year from alloHSCT was 45%. OS 1 year after allo-HSCT was 69% for pts who were in CR at transplant and 33% for pts who were in PD (p 0.0036). TRM at 1 year was 32%. Conclusion: Allo-HSCT from both matched related and alternative donors is feasible and potentially curative in pts older than 65. Disclosure of Interest: None declared.
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P292 Determination of copy number variation (CNV) helps in assessing the risk factor of response to HSCT and standard chemotherapy in AML patients E. Jaskula1,2,*, M. Mordak-Domagala1, M. Sedzimirska1, A. Sobczynska-Konefal1, J. Lange1, N. Surgut1, D. Owczarek1, A. Siemienska1, A. Lange1,2 1 Lower Silesian Center for Cellular Transplantation with National Bone Marrow Donor Registry, 2L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland, Wroclaw, Poland Introduction: For the purpose of this study, copy number variations were determined with the use of Agilent Technologies microarray plates (ShurePrint G3 Cancer CGH þ SNP 4x180 K) which were read after the completion of hybridization in the Roche instrument (NimbleGen MS200 Microarray Scanner) with use of Nexus Copy Number Software (BioDiscovery). Gains and losses were summarized together, giving the number of CNV in tested samples. Materials (or patients) and methods: Baseline characteristics of the cohort – transplanted (non-transplanted): F/M: 13/14 (29/33), median age: 42 (60), karyotype – favorable/other: 2/25 (9/43), Flt3positive/negative: 9/18 (18/34), HSCT – myeloablative/RIC: 22/5 non-transplanted – fit: standard intention to treat/unfit – low dose AraC - 42/10. Results: Overall survival was significantly better in the transplant group as compared to the standard chemotherapy group: 0.45 vs. 0.09, Po0.04 at 2-year cut-off point. Survival of patients assessed 18 months post-HSCT was better in the group with CNVs not exceeding 20 as compared to those with higher values (9/6 vs. 4/8, n.s.) irrespective of the presence of karyotype abnormalities. Survival of non-transplanted patients was significantly affected by the presence of CNVs above 20 (Po0.039, log rank test) (Figure 1). The adverse effect of CNVs above 20 was seen irrespective of the presence of FLT3 mutation in both transplanted and nontransplanted groups. Conclusion: Higher numbers of CNV in AML patients may assist both the decision-making process as to the transplantation, and – if transplantation is performed – advance planning
of the post-transplant treatment modalities knowing the CNVassociated risk factor of relapse. Supported by INNOMED/I/1/NCBR/2014 CellsTherapy NCBiR grant. Disclosure of Interest: None declared. P293 Is allogeneic stem-cell transplantation an option for patients with acute leukemia and myelodiplastic syndrome transplanted with active disease? A center experience E. Pe´rez-Lo´pez1,*, O. Lo´pez-Godino1, M. Cabrero1, J. Labrador2, F. Sa´nchez-Guijo1, L. Va´zquez1, B. Vidriales1, L. Lo´pez-Corral1, D. Caballero1 1 Hospital Universitario de Salamanca, Salamanca, Spain, 2 Hematology department, Hospital Universitario de Salamanca, Salamanca, Spain Introduction: The prognosis of patients with refractory/ relapsed acute leukemia (rAL) or myelodisplastic syndrome (MDS) is very poor. Although patients with refractory disease are not good candidates for allogeneic stem cell transplantation, this could be the only curative treatment for these patients. Unfortunately, reports of long-term follow up in this population and doubt exist about if this strategy should be offer to these patients The purpose of the present study is to evaluate the outcomes and efficacy of salvaged HSCT for rAL and MSD in our centre. Materials (or patients) and methods: We examined retrospectively 74 patients with rAL (myeloid & lymphoblastic n ¼ 36/10) and MDS (n ¼ 28) who underwent allogeneic transplantation at our centre since 1999 to 2013 with active disease at transplant. Feasibility and efficacy of HCT was analyzed in terms of TRM, event free survival (EFS) and overall survival (OS). Results: The main characteristics of the patients and transplant were: median age 55 years (14-69), status disease at transplant: refractory 37 (50%), relapse 17 (23%), untreated 8 (10%) and aplastic 12 (30%) with a median blasts of 10% (572) in those with burdern leukaemia. Median time from diagnosis to transplant was 7 months (1-160).
[P292]
Figure 1.
Overall survival of non-transplanted AML patients on chemotherapy.
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Conditioning regimen was myeloablative in 22 (30%) and reduced intensity (RIC) in 52 (70%) 8 patients (11%) have received a prior transplant, 4 of them an allogeneic transplant. The source was peripheral blood in all but 5 (3 bone marrow and 2 cord blood). The donor was: related in 45 (60%), unrelated in 23 (32%) and 6 haploidentical (8%). Graft-versushost disease (GVHD) prophylaxis was calcineurin inhibitorsregimens ( þ MMF or þ methotrexate) in 38 (52%) and tacrolimus and rapamicine in 25 (34%), and other inmunosupression in 10 (14%). All the patients but one engrafted. The incidence of acute-GVHD (aGVHD) was 61% (14% grade III-IV) with median onset of 26 days (8-130); in evaluable patients, chronic-GVHD (cGVHD) was 45% (24% severe) with a median onset of 163 days (84-451). Regarding response to transplant at day þ 100: 43 patients (58%) achieve complete remission (CR), 13 (17%), progression disease and 18 (24%) were death previously, mostly from relapse (n ¼ 12). From those patients without CR at þ 100, 11/ 13 death because relapsed disease. With a median follow up of 10 months, the estimated OS and EFS at 1 year was 50% and 38%, respectively. In the univariate analysis, achieve CR at þ 100 and development of cGVHD (yes/no and grade) were associated with better OS (Po0.05); these variables remained in the multivariate analysis. The same factors were associated with better EFS in the univariate and multivariante analysis. Status disease at transplant and leukaemia burden has not significance in both OS and EFS. TRM was 17% (10% at day þ 100). The main cause of death was progression disease (n ¼ 29 40%). From those patients alive who achieve CR at þ 100, 31% of them (23/74) remained in CR. Conclusion: Our results indicate that patients with rAL or MDS can benefit from HSCT even with high leukemia burden. We have shown that HSCT can induce long-term disease remission and possibly cure in those who developed cGVHD especially who achieve CR at þ 100. Disclosure of Interest: None declared. P294 Maintenance treatment with the association of deferasirox and Vitamin D improves survival in AML after allogeneic hematopoietic stem cell transplantation E. Paubelle1,*, A. Brignier2, F. Lemonier1, A. Marc¸ais1, D. Sibon1, R. Delarue1, O. Hermine1, F. Suarez1 1 Hematology, Hoˆpital Necker, 2Cellular Therapy, Hoˆpital Saint-Louis, Paris, France Introduction: Currently, the most efficient curative treatment for patients with Acute Myeloid Leukemia (AML) is allogeneic hematopoietic stem cell transplantation (HSCT). However, many patients relapse because of the presence of quiescent leukemia initiating cells (LIC). It has been shown that deferasirox(DFX) is able to induce AML cells apoptosis in vitro through inhibition of the NF-kB pathway. In addition, our group has shown that iron chelators are able to promote in vitro monocyte differentiation in both normal hematopoietic progenitors and primary AML cells and that iron chelators act synergistically with vitamin D (VD) to promote cell differentiation. Morerover, both iron chelators and VD analogues have a favorable safety profile and are often used in this patient population. We report here the outcome of patients who receivedDFX-VD combination therapy as maintenance treatment after allogeneic HSCT for AML. Materials (or patients) and methods: From June 2007 to June 2014, 33 patients diagnosed with AML and receiving allogeneic HSCT in Necker Hospital were included in the study.DFX-VD maintenance was left to the discretion of the referring physician. Overall, 10 patients received DFX-VD. The dose of DFX was adapted on ferritin and creatinine levels. DFX was initiated at 20–30 mg/kg per day and VD at 100,000 units orally weekly. Overall survival (OS) was defined as the time from the first day of induction to death from any cause and
was censored at the date of last visit. Survival was estimated by the Kaplan-Meier method and was comparisons were performedwith the log-rank test. Multivariate analysis was performed using Cox proportional hazard model. Statistical significance was defined as p-value o0.05. Results: Median age was 54 and sex ratio 1.4. According to molecular and cytogenetic prognosis group, 2 patients (6%) had favorable risk, 26 (79%) intermediate and 11 (33%) unfavorable risk. Risk data were not available for 4 (12%) patients. At diagnosis, medium serum ferritin was of 603ng/mL and medium transferrin saturation of 52%. Baseline characteristics did not differ between the 10 patients who received DFX-VD maintenance and the 23 patients who did not. Medium time between HSCT and the beginningof DFX-VD was of 121 days with a median duration of 19 months without significant adverse event.Incidence of graft versus host disease was similar. Relapse rate was 43.5 and 10%, P ¼ 0.06 for control and DFX-VD patients respectively, and treatment related mortality of 13 and 20%, P ¼ 0.62. DFX-VD maintenance treatment improved significantly survival as median OS had not been reached versus 20.1 months in no maintenance group, P ¼ 0.04. In multivariate analysis, only maintenance treatment with DFX-VD had a favorable impact with aHazardRatio of 0.2 (0.1 – 0.9), P ¼ 0.03. Conclusion: Based on this small observational study, DFX-VD maintenance treatment may improve overall survival after allogeneic HSCT in AML patients by preventing relapse.As shown for As2O3 and retinoids, VD might affect LICs. Therefore, further studies should evaluate the impact of DFX-VD on LIC. These encouraging results from this retrospective study need to be validatedin a large randomized prospective multicenter study. Disclosure of Interest: None declared. P295 Chemotherapy followed by donor stem cell infusion provides a better survival than chemotherapy alone for patients with acute leukemia relapsed after allogeneic hematopoietic stem cell transplantation Y. Zhao1, Y. Luo1, Y. Tan1, J. Shi1, W. Zheng1, H. Huang1,* 1 Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Introduction: Relapse remains a major obstacle to the success of allogeneic hematopoietic stem cell transplantation (HSCT)in acute leukemia, yet little is known about the relevant prognostic factors after relapse, and the best treatment strategy has not been demonstrated. Thus, this retrospective study was designed to compare the treatment efficacy of chemotherapy alone and chemotherapy followed by donor stem cell infusion (DSI) in patients with relapsed leukemia after HSCT, and also to investigate the prognostic factors in these patients. Materials (or patients) and methods: Forty-nine patients who relapsed after HSCT at the First Affiliated Hospital of Zhejiang University between January 2006 and August 2013 , were nonrandomly assigned to treatment with chemotherapy alone (n ¼ 29) or chemotherapy followed by DSI group (n ¼ 20) according to the patient’s own choice. Results: The 1-year postrelapse overall survival (OS) rate and diseasefree survival (DFS) for entire cohort was 16.5% and 14.3%, respectively. In patients receiving chemotherapy followed by DSI, complete remission rate was significantly higher (40.0% vs. 13.8%, P ¼ 0.036), and 1-year overall survival (OS) and disease-free survival (DFS) was significantly improved (1-year OS: 32.3% vs. 10.3%, P ¼ 0.005; 1-year DFS: 25.8% vs. 6.9%, P ¼ 0.005), compared with patients receiving chemotherapy alone. For the patients with acute myeloid leukemia (AML), chemotherapy followed DSI was superior to chemotherapy alone, with 1-year OS and PFS rates of 56.3% vs. 11.1% (P ¼ 0.026) and 56.3% vs. 11.1% (P ¼ 0.038), respectively, however, for the patients with acute lymphoid leukemia
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(ALL), the benefit of DSI was not obvious (1-year OS: 10% vs 10%, P ¼ 0.175;1-year DFS: 10% vs 5%, P ¼ 0.182). There was no significant differences in treatment related mortality (TRM) between the two intervention groups (34% vs 33.7%, P ¼ 0.712). Risk factors for mortality after relapse included shorter time to relapse, disease type (ALL), higher marrow blast cells at relapse, intervention group (chemotherapy alone), and no chronic graft-versus-host disease (cGVHD) occurring after intervention, but only cGVHD occurring after intervention was an independent predictor after multivariate analysis (RR ¼ 0.425, P ¼ 0.043). The 1-year OS and DFS was significantly improved in the patients harboring cGVHD after intervention compared with no cGVHD patients (1-year OS: 41.9% vs 6.3%, P ¼ 0.00; 1-year DFS: 41.9% vs 0%, P ¼ 0.00). Conclusion: The study suggest that chemotherapy followed by DSI had a stronger anti-leukemic effect and provided a better survival in relapsed leukemia after HSCT, especially for those AML. Furthermore, cGVHD occurring after intervention was associated with a longer survival for relapsed patients. Disclosure of Interest: None declared. P296 Clinical outcomes of CLAG/CLAGM or fludarabine based chemotherapy following allogeneic SCT in relapsed/ refractory acute myeloid leukemia patients H. Park1,*, Y. Koh1, J.-H. Youk1, I. Kim1, S. Park1, J.-O. Lee2, S.-M. Bang2, S.-S. Yoon1 1 Seoul national university hospital, Seoul, 2Seoul National University Bundang Hospital, Seongnam, Korea, Republic Of Introduction: Substantial number of acute myeloid leukemia (AML) patients does not respond to induction chemotherapy (refractory) or relapse after achieving complete remission (CR). In relapsed/refractory AML, therapeutic options are still undefined and unsatisfactory. We intended to reveal the efficacy of new agents followed by allogeneic stem cell transplantation (ASCT) in relapsed/refractory AML patients. New agents included cladribine (CLAG/CLAGM) and fudarabine (FLAG/FLAGI/mFLAI). Materials (or patients) and methods: Relapsed/refractory AML patients who were treated with cladribine or fludarabine based chemotherapy at two institutions (Seoul National University Hospital and Seoul National University Bundang Hospital) were retrospectively reviewed. We analyzed receive of ASCT, relapse free survival (RFS), and overall survival (OS). Results: A total of 118 patients (Male : Female ¼ 71:47, median age ¼ 53 years) were analyzed; 66 patients were treated with CLAG/CLAGM and 52 patients were treated with FLAG/FLAGI/ m-FLAI. Twenty-nine patients underwent ASCT after reinduction chemotherapy; The 19 patients after CLAG/CLAGM and 10 patients after FLAG/FLAGI/mFLAI. The median OS of patients who received CLAG/CLAGM was 13.5 months; 39.1 months for ASCT group and 12.9 months for non-transplant group (P ¼ 0.238). For patients who achieved complete remission (CR) after CLAG/CLAGM, significantly better OS was observed in patients who underwent ASCT post-CLAG/CLAM chemotherapy compared to those who did not (median OS not reached vs. 12.3 months, P ¼ 0.036). In fludarabine-based chemotherapy group, the median OS was 15.1 months; 30.0 months for ASCT group and 14.7 months for non-transplant group (P ¼ 0.089). For patients who achieved CR after fludarabine-based chemotherapy, better OS was observed in patients who underwent ASCT after chemotherapy (median OS 30.0 months vs 14.7 months. P ¼ 0.046). As for RFS (CR patients ¼ 57, nonCR patients ¼ 36, not evaluated patients ¼ 25), the probability of 2yr RFS was 61.9%, the median RFS was not reached in CLAG/CLAGM group. 2Yr RFS rate was 77.8% for ASCT group, and was 55.6% for non-transplant group (P ¼ 0.144). Similarly, in FLAG/FLAGI/ mFLAI group, no significant difference in 2yr RFS rate according to subsequent ASCT was observed (62.5% vs 14.7%, P ¼ 0.184). The probability of 2yr RFS was 14.7 % in non-transplant group, was 62.5% in ASCT group.
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In cladribine-based chemotherapy groups, CR rate was 61.5%, which was not significantly different with fludarabine-based group (61.0%) (P ¼ 0.562). Also, the median OS of ASCT group after CR was not significantly different between two groups (median OS 31.0 months (cladribine group) vs 30.0 months (fludarabine group). P ¼ 0.911). Conclusion: For patients who receive reinduction chemotherapy using cladribine or fludarabine, consolidation ASCT is definitely necessary to improve survival outcome. Disclosure of Interest: None declared. P297 Allogeneic transplantation for acute lymphoblastic leukemia (ALL) in adults 2008-2013: results from Denmark I. Schjoedt1,*, D. Hovgaard1, L. Vindeløv1, L. Friis1, N. Smedegaard Andersen1, S. Lykke Petersen1, H. Sengeløv1 1 Hematology, RIGSHOSPITALET , Copenhagen, Denmark Introduction: The study is a retrospective single-center analysis of survival from allogeneic transplantation for B-ALL or T-ALL from Rigshospitalet University Hospital, Copenhagen over a six-year period. Materials (or patients) and methods: Data from all ALLpatients transplanted at Rigshospitalet University Hospital, the only Danish ALL- transplant center during the period 20082013, were retrospectively evaluated. Patients with an adult donor and myeloablative conditioning received Etoposide 60 mg/kg x 1 and total body irradiation (TBI) 12 Grey. If TBI was not to be given, patients received Etoposide 60 mg/kg x 1 and Busulphane 3.2 mg/kg x 5 days instead. If the donor source was UCB, conditioning consisted of Fludarabine 25 mg/m2 x 3 days, and Cyclophosphamide 60 mg/kg x 2 days plus TBI 12 Gy. Non-myeloablative conditioning consisted of Fludarabine 30 mg/m2 x 3 days plus TBI 2 Gy. No CNS-irradiation was given as part of the conditioning regimen. Rejection- and gvh profylaxis was MTX and Cyclosporine for the myeloablative conditioning or Tacrolimus and Mycophenolate Mofetil for the non-myeloablative conditioning. Results: In total 63 patients, 47 (75%) with B-ALL and 16 (25%) with T-ALL were transplanted during this period. Of these 41 (66%): 31 B and 10 T were transplanted in 1.CR, whereas 22 (33%): 16 B and 6 T were in a 2. or later remission. The male:female ratio was 2:1. The median age was 20 and range 18-63. Most patients (59 (94%)) had a myeloablative conditioning, as the non-myeloablative conditioning (4 (6%)) for ALL was not conducted at Copenhagen until 2013. The median observation time was 878 days (81 – 2200 days). Overall survival was 72% for all 63, with a plateau reached after approximately 800 days. 16 patients died, 5 from relapse (8%), 7 from gvhd (11%), 3 from infection (5%) and 1 (2%) from a non-hematologic cancer. The stem cell source had no significant impact on overall survival. 39 were transplanted with bone marrow, 20 with peripheral blood stem cells, and 4 with double umbilical cords.
There was no significant difference in survival according to donor source, 51 (81%) had a matched unrelated (MUD) donor and 12 (19%) a sibling donor. Conclusion: This retrospective single-center study demonstrates excellent survival for patients with either B-ALL or T-ALL, treated in Denmark over the six-year period from 20082013. Compared to other published data, overall survival is high and a true plateau is reached after approximately two years. The intensive pre-transplant nordic (Nopho) treatmentregimen for younger patients, the short delay from treatment to transplantation and the timely well-structured CMV-survey are important contributing factors, however several other factors may influence these positive results. Disclosure of Interest: None declared. P298 Second Haploidentical Hematopoietic Stem Cell Transplantation with Treosulfan-based Conditioning Regimen for Acute Leukemia in Relapse J. Peccatori1,*, L. Crucitti1, R. Greco1, M. T. Lupo Stanghellini1, L. Vago2, N. Cieri3, F. Giglio1, M. Morelli1, A. Assanelli1, M. Marcatti1, M. Carrabba1, S. Marktel1, T. Perini1, F. Lorentino1, S. Girlanda1, C. Corti1, M. Bernardi1, F. Ciceri1 1 Unit of Hematology and Bone Marrow Transplantation, 2Unit of Molecular and Functional Immunogenetics, 3Experimental Hematology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy Introduction: In the last years, HSCT using haploidentical T-replete source has become more and more popular, thanks to acceptable rates of GvHD and NRM. Acute leukemias (AL) relapsing after allo-HSCT have a dismal prognosis. Strategies to prevent post-transplant disease recurrence are limited. We evaluated the outcome of haploidentical transplant performed as second transplant (HSCT2) for patients with AL, relapsing after a first allo-HSCT (HSCT1). Materials (or patients) and methods: We analyzed 42 patients (median age 48 years) who underwent to HSCT2 from a haploidentical donor, different from the previous one, between 2007 and 2013 in our Institute. Patients suffered of myeloid AL (n ¼ 31), lymphoblastic AL (n ¼ 9) or biphenotypic AL (n ¼ 2) relapsing after HSCT1 (matched related: 21; unrelated: 17; mismatched related: 4; cord blood: 4). Thirtyseven patients (88%) received re-induction: 13 of them achieved complete remission (CR), 16 did not respond and 8 patients were transplanted in aplasia. In 5 cases HSCT2 was performed without re-induction therapy, in active disease (AD). Median HCT-Co-morbidity Index was 2 (range 0-6). All patients received treosulfan (14 g/m2 for 3 days) and fludarabine (30 mg/m2 for 5 days) as part of preparative regimen for HSCT2; conditioning was intensified with total body irradiation (4 Gray) in 18 (43%) or with melphalan (70 mg/m2 for 2 days) in 6 patients (14%). GvHD prophylaxis consisted of mycophenolate mofetile and sirolimus combined with pre-transplant ATG-Fresenius (n ¼ 36) or with posttransplant cyclophosphamide (n ¼ 6). Unmanipulated PBSCs were used as graft source in all cases. Results: Three patients (7%) died during aplasia, all the others achieved neutrophil engraftment (n ¼ 39, 93%), with no primary graft rejection. Complete remission at day þ 30 was reached in 37 out of 39 evaluated cases (95%). Estimated OS and LFS rates at 1 year from HSCT2 were 37% ± 8% and 35% ± 7%, respectively. One-year RI was 32% ± 7% and NRM to 31% ± 7% (7 patients died for infectious, 5 for GvHD and 1 for CNS toxicity). Sixteen patients (38%) suffered of acute GvHD grade II-IV and 20 (47%) of chronic GvHD. Disease status at HSCT2 (AD vs CR) had no impact on OS (HR:1.26, 95% CI 0.582.76, P ¼ 0.564) and relapse (HR:1.35, CI 95% 0.46-3.76, P ¼ 0.61). Conversely, time of remission from HSCT146 months provided the better outcome in terms of estimated OS at 1 year (50% ± 10%) compared to patients with shorter duration of remission (18% ± 9%) (HR:0.49, 95% CI 0.24-1.02, P ¼ 0.05) and reduced risk of relapse (HR:0.28, 95% CI 0.11-
0.75, P ¼ 0.011). After a median follow-up time of 22 months (range 6-68), 11 patients (26%) were alive with ongoing leukemia remission. Conclusion: These results demonstrate the feasibility of second allo-HSCT from a haploidentical donor as treatment for AL relapse, showing rates of OS, LFS and NRM comparable to the reported in literature for HSCT2 from matched related or unrelated graft. Interestingly, disease status at HSCT2 did not influence the outcome of transplant. While, in accordance with previous reports, lasting time of remission after HSCT1 is the major factor involved in predicting outcome of a second transplant due to an increased relapse incidence, reflecting diseases biologically different. If confirmed in a larger cohort, these data could be useful in decision making after HSCT1 failure for disease recurrence. Disclosure of Interest: None declared. P299 The double-negative NPM1/FLT3-ITD mutation shows long term outcomes after allogeneic transplant similar to those of the ‘favourable’ risk group according to the ELN risk classification, distinct from other intermediate-I risk in normal-karyotype AML J.-S. Ahn1,*, H.-J. Kim1, Y.-K. Kim1, S.-H. Jung1, D.-H. Yang1, J.-J. Lee1, N. Y. Kim2, S. H. Choi2, M. D. Minden3, C. W. Jung4, J.-H. Jang4, H. J. Kim5, J. H. Moon6, S. K. Sohn6, J.-H. Won7, S.-H. Kim8, D. D. H. Kim3 1 Department of Hematology-Oncology, 2Genomic Research Center for Hematopoietic Diseases, Chonnam National University Hwasun Hospital, Jeollanam-do, Korea, Republic Of, 3Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada, 4Division of Hematology-Oncology, Samsung Medical Center, 5Department of Hematology, The Catholic University of Korea, Seoul, 6 Department of Hematology-Oncology, Kyungpook National University Hospital, Daegu, 7Department of Hematology-Oncology, Soon Chun Hyang University Hospital, Seoul, 8Department of Hematology-Oncology, Dong-A University College of Medicine, Busan, Korea, Republic Of Introduction: The prognostic significance of the European LeukemiaNet (ELN) molecular risk classification was examined in patients with normal-karyotype acute myeloid leukaemia (NK-AML) after allogeneic haematopoietic cell transplantation (HCT). Materials (or patients) and methods: In total, 121 patients received allogeneic HCT for NK-AML and were evaluated for ‘‘fms-related tyrosine kinase 3-internal tandem duplication’’ (FLT3-ITD), nucleophosmin 1 (NPM1), and ‘‘CCAAT/enhancer binding protein a’’ (CEBPA) mutations in diagnostic samples, and analysed in terms of the long-term outcomes following HCT. Results: The prevalences of FLT3-ITDpos, NPM1mut, and CEBPAmut were 32.2%, 43.8%, and 29.2%, respectively. The 5year OS in the favourable (n ¼ 60) versus the intermediate-1 group (n ¼ 61), according to the original ELN risk, was 69.0% versus 48.1% (P ¼ 0.038). Among the intermediate-1 group, the 5-year OS was 57.6% in NPM1wild/FLT3-ITDneg (n ¼ 25), 45.8% in NPM1mut/FLT3-ITDpos (n ¼ 24), and 28.1% in NPM1wild/FLT3ITDpos (n ¼ 12) patients. Thus, we reclassified the doublenegative NPM1wild/FLT3-ITDneg group to the favourable from the intermediate-1 risk group. With this revised ELN risk classification, the 5-year OS was 65.2% in the favourable (n ¼ 85) and 40.4% in the intermediate-1 groups (n ¼ 36; P ¼ 0.004). Thus, reclassification of the NPM1wild/FLT3-ITDneg double-negative group into the favourable risk group improved the prognostic stratification power of the ELN risk classification significantly (Po0.001). Conclusion: A revised ELN risk classification, reclassifying double-negative patients into the favourable risk group, can improve risk stratification in NK-AML patients following allogeneic HCT. Disclosure of Interest: None declared.
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P300 Myeloablative Haploidentical Stem Cell Transplantation (MAC-HAPLO) With Post-Transplant Cyclophosphamide (PTCy) As Gvhd Prophylaxis In High Risk Leukemias/ Myelosdisplastic Syndromes (MDS) J. Gayoso1,*, P. Balsalobre1, M. Kwon1, A. Sampol2, P. Montesinos3, D. Serrano1, P. Herrera4, M. J. Pascual5, A. Bermu´dez6, C. Castilla-Llorente7, L. Bento2, D. Champ1, A. Pe´rez-Corral1, E. Buces1, J. L. Dı´ez-Martı´n1 on behalf of Grupo Espan˜ol de Trasplante Hemopoye´tico (GETH) 1 Hematology, HGU Gregorio Maran˜o´n, Madrid, 2Hematology, Hosp. Son Espasses, Palma Mallorca, 3Hematology, Hosp. La Fe´, Valencia, 4Hematology, Hosp. Ramo´n y Cajal, Madrid, 5Hematology, CHU Ma´laga, Ma´laga, 6Hematology, Hosp. Marque´s de Valdecilla, Santander, 7Hematology, Hosp. Morales Messeguer, Murcia, Spain Introduction: Allogeneic transplantation is the only curative option for patients with high risk leukemias or MDS. Only one third of them have an HLA identical sibling donor and around 60-70% will find an unrelated donor, that´s why haploidentical stem cell transplantation (HAPLO-HSCT) offers a therapeutic option to most of these patients. Myeloablative conditioning (MAC) used to obtains better disease control than reduced intensity conditioning regimens (RIC), but with higher toxicity, rendering long term similar results Materials (or patients) and methods: We retrospectively evaluated the results in patients diagnosed with high risk leukemias or MDS of our MAC-HAPLO regimens (Fludarabine 30 mg/m2 x5 days (-6 to -2), Cyclophosphamide14,5 mg/kg x2 days (-6 to -5), IV Busulfan 3,2 mg/kg x 3 days (BUX3) on days 4 to -2, or Fludarabine 40 mg/m2 x4 days (-6 to -2) and IV Busulfan 3,2 mg/kg x 4 days (BUX4)) with GVHD prophylaxis based on PTCy (50 mg/kg on days þ 3 and þ 4) and a calcineurin inhibitor plus mycophenolate from day þ 5, performed in GETH centers. Results: From Feb-2011, 24 MAC-HAPLO have been done in 7 centers. Median age was 37 years (15-65), 58% were males and all were in advanced disease phase or presented high risk features (AML 16/ALL 4/MDS 2/ CML-BC 1/ CMML 1). Previous HSCT had been employed in 21% (autologous in 1, allogeneic in 4), and in 79% the HAPLO-HSCT was their first transplant. Disease status at HAPLO-HSCT was morphologic CR in 83%, but with persistent disease (morphologic or MRD þ by flow or molecular markers) in 67%. BM was the graft source in 3 patients (12.5%) and PBSC in 21 (87.5%), non T-cell depleted in all cases. The haploidentical donor was the patient´s mother (21%), father (12.5%), siblings (41.5%) or offspring (25%). MAC regimen was BUX3 in 8 (33%) and BUX4 in 16 patients (67%). Median infused CD34 þ cells were 4,93x10e6/kg (3,20-8,43). Median neutrophils engraftment was reached at day þ 16 (1329) and platelets 420K at day þ 27 (11-131). Complete chimerism was obtained at a median of 22 days (13-44) in 21 evaluated patients. Cumulative incidence (CI) of non-relapse mortality (NRM) was 21.5% at 1 year. CI of grade II-IV acute GVHD was 45.5% at day þ 100, and grade III-IV was 9%. CI of chronic GVHD at 1 year was 35%, being extensive in 8% . No differences in acute or chronic GvHD CI were seen when comparing BUX3 against BUX4. After a median follow-up of 15 months (3-31), estimated 18-months event-free survival (EFS) and overall survival (OS) were 63% and 75% respectively. CI of relapse or progression was 19.5%. No significant differences in NRM, EFS, OS and relapse incidence were detected between BUX3 and BUX4. The effect of CR prior to MAC-HAPLO has not been apropiately assesed due to the limited number of events in our series Conclusion: IV Busulfan based MAC-HAPLO with PTCy in the treatment of high risk leukemias and MDS offers good disease control with manageable toxicity, with either BUX3 or BUX4. Disclosure of Interest: None declared.
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P301 Safety and efficiency of bortezomib to improve GVHD control in TCR-alpha/beta depleted transplantation in children with acute myeloid leukemia L. Shelikhova1,*, M. Maschan1, D. Balashov1, J. Skvortsova1, E. Boyakova1, V. Kalinina2, D. Shasheleva1, M. Persiantseva1, G. Novichkova1, A. Maschan1 1 Hematopoietic stem cell transplantation, 2Molecular Biology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: Relapse of malignancy and graft-versus-host disease (GvHD) remains the most important cause of failure after allogeneic transplantation for acute leukemia in children. Negative depletion of a/b( þ ) T cells and CD19 þ B lymphocytes is a new technology of graft manipulation, which improved GVHD control and immune reconstitution in our pilot trial. Bortezomib, a proteasome inhibitor capable of direct antitumor effects, has shown activity in GVHD prevention and therapy. In vitro data suggest that bortezomib sensitizes tumor cells to the cytotoxicity of NK cells. In this pilot trial we evaluated the safety and effects of bortezomib added to our backbone of intensive chemotherapy-based conditioning and TCR-alpha/beta depletion in haploidentical and matched unrelated transplantation (MUD). Materials (or patients) and methods: A total of 19 pediatric patients with acute myeloid leukemia (AML), 7 female, 12 male, median age 10,6 years, underwent allo HSCT between January 2014 and august 2014. Eight patients were transplanted from haploidentical donors and 11 from MUD. Patients were divided in 2 groups according to remission status: complete remission (CR) - 13 pts, active disease (AD) - 6 pts. All patients with AD were transplanted from haploidentical donor. Preparative regimen included Fludarabine 150 mg/m2, Treosulfan 42 g/m2, Melphalan 140 mg/m2, ATG (rabbit, Thymoglobuline) 5 mg/kg and rituximab 100mg/m2. Bortezomib 1,3mg/m2 was used at days -5,-2, þ 2, þ 5. PBSC were depleted of TCR-alpha/beta and CD19 þ B-cell according to manufacturer’s recommendations. The median dose of infused CD34 þ cells was 7,2x106/kg (range 4,2-17), TCR a/b -13,8 x103/kg (range 0,6-68). No additional post-transplant immune suppression was used in 18 pts, 1 pt received additional tacrolimus till day þ 30. Results: Primary engraftment was achieved in 16 of 19 pts, the median time to neutrophil and platelet recovery was 13 days. Two pts transplanted in AD had active disease on day þ 30, 1 pt in active disease had primary graft failure with no evidence of leukemia and was retransplanted from MUD. None of the patients died of transplant-related cause, pTRM - 0%. No patient had significant acute organ toxicity (renal, cardiac, hepatic, bladder, CNS). Febrile neutropenia was registered in 5 (26%), mucositis grade II - in 5 pts (26%), severe infection, requiring inotropic support, developed in one case (5%). Bortezomib-induced peripheral neuropathy (BINP) grade 1 was observed in 3 pts (15%), dose reduction was required in 1 pt (5%). Cumulative incidence of aGvHD grade II was 5,9% (95% CI: 039), no case of grade III-IV aGvHD was observed. Posttransplant steroids were administered to 1 (6%) pt. With a median follow-up of 5,8 months (3-9) 15 of 19 pts are alive (3 transplanted in AD, 12 in CR, P ¼ 0,029), 12 disease-free. Disease status at HSCT significantly influenced the relapse rate: cumulative incidence of relapse was 27 % in CR patients and 83% in AD (Grey test P ¼ 0,001). Conclusion: We demonstrate that bortezomib can be safely administered to children with AML in the context of treosulfan/melphalan chemotherapy with minimal neurologic and low visceral toxicity. We suggest that peritransplant administration of bortezomib might further improve GVHD control provided by depletion of TCR-alpha/beta lymphocytes from the graft and allow for immune-suppression-free posttransplant environment. Disclosure of Interest: None declared.
P302 TCRab þ /CD19 þ -depletion in hematopoietic stem cells transplantation from matched unrelated and haploidentical donors following treosulfan-based conditioning in pediatric acute lymphoblastic leukemia patients L. Shelikhova1,*, M. Maschan1, D. Balashov 1, J. Skvortsova1, V. Kalinina2, D. Shasheleva1, G. Novichkova1, A. Maschan1, M. Ilushina1 1 Hematopoietic stem cell transplantation, 2Molecular Biology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: Graft-versus-host disease (GvHD) and GVHDassociated morbidity and mortality are major obstacles to the success of transplantation from unrelated and haploidentical donors. Negative depletion of a/b( þ ) T cells and CD19 þ B lymphocytes is a new technology of graft manipulation with a potential to improve GvHD control and immune reconstitution. Materials (or patients) and methods: A total of 34 pediatric patients with acute lymphoblastic leukemia (T-ALL- 16, B-ALL18, 13 female, 19 male, median age 8,6 years) underwent allogeneic HSCT between May 2012 and december 2013. Thirteen - from haploidentical donor and 21 from matched unrelated donor. Preparative regimen included Fludarabine 150 mg/m2, Treosulfan 42 g/m2, Melphalan 140 mg/m2 and ATG (horse, ATGAM) at 25 mg/kg/day, d -8,-9 (11 pts) or d -5,-4 (18 pts), or (rabbit, Thymoglobuline) at 2,5mg/kg/day, d -5,-4 (5pts). PBSC were depleted of TCR-alpha/beta and CD19 þ B-cell on CliniMACS Plus, Miltenyi Biotec, according to the manufacturer’s recommendations. The median dose of infused CD34 þ cells was 9,1x 106/kg (range3,9-18), TCRa/b 17 x103/ kg (range 0,7-190). Seven pts recieved no GvHD prophylaxis after HCT, tacrolimus (tacro) and short methotrexate (Mtx) were used for 24 pts, tacro - 2 pts, Mtx - 1 pts. Patients were divided in 4 groups according to remission status: CR1 (11pts), CR2 (16 pts), CR42 (6pts.), active disease (AD) (1pt.) and according to CNS involvemen: CNS þ (10 pts), CNS - (24pts). Results: Primary engraftment was achieved in 33 of 34 pts, the median time to neutrophil and platelet recovery was 13 and 14 days, respectively. Early mortality was low with a 100-day pTRM - 2,9% (95% CI: 0,2-20), 2-year pTRM – 14,9% (95%CI: 836). The 2 early deaths included bacterial sepsis and viral infections, three late: bacterial sepsis in 2 pts. and rhinocerebral mucormycosis in 1pt, all with extensive chronic GVHD. Cumulative incidence of acute GvHD grade Z II was 29% (95% CI: 17-49), grade III–IV 9 % (95%CI: 3-26). No correlation between graft composition and aGVHD was noted. Cumulative incidence of relapse at 2 year is 48,6% (95%CI: 34-69). Two year pEFS is 36,5% (95%CI: 19-53), 2-year pOS - 39% (95%CI: 17-61). There was no significant difference in survival and relapse rate
according leukemia subtype and donor type. Patient’s CNS status had a trend to influence significantly the relapse rate: pRFS for CNS (-) 59% (95%CI: 37-80), for CNS ( þ ) 34% (95%CI: 13-65), P ¼ 0,054. Conclusion: We confirm that depletion of TCR-alpha/beta and CD19 lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in ALL patients. All major outcomes were equivalent between transplantation from unrelated and haploidentical donor. The anti-leukemic activity of this regimen was obviously not optimal. We suggest that TCRalpha/beta depletion in ALL should be further explored in combination with TBI-based conditioning regimen and/or novel targeted immunotherapy approaches. Disclosure of Interest: None declared. P303 Exon 23 DNA methyltransferase 3A gene mutations in patients with acute myeloid leukemia receiving hematopoietic stem cell transplantation after non myeloablative conditioning L. B. Bonifacio1,*, L. Schmalbrock1, M. Bill1, M. Jentzsch1, K. Schubert1, K. Wildenberger1, L. Kloss1, H. Weidner1, T. Gaber1, W. Po¨nisch1, V. Vucinic1, G.-N. Franke1, T. Lange1,2, M. Cross1, G. Behre1, D. Niederwieser1, S. Schwind1 1 Department of Hematology, Medical Oncology and Hemostaseology, University of Leipzig, Leipzig, 2Hematology, Asklepios Hospital , Weissenfels, Germany Introduction: Some studies revealed a negative impact of mutations (mut) in the DNA methyltransferase 3A (DNMT3A) gene on outcome in acute myeloid leukemia (AML) subgroups. However, the prognostic impact of DNMT3A mut in patients (pts) receiving hematopoietic stem cell transplantation after non myeloablative conditioning (NMA-HCT) is unknown. Here, we determined the prognostic significance of exon 23 DNMT3A mut in NMA-HCT treated AML pts. Materials (or patients) and methods: We analyzed the coding region of exon 23 of the DNMT3A gene by Sanger sequencing of genomic DNA from pre-treatment samples of 104 AML pts (median age 64.5 years (y), range 47-75y). 67 (64%) pts had de novo AML. A normal karyotype (NK) was found in 50 (48%) pts. All pts underwent NMA-HCT (Fludarabine 30mg/m2 from day -4 to -2 followed by 2 Gy total body irradiation at day 0). 17 (16%) pts had a matched related, 57 (55%) pts a matched unrelated & 30 (29%) a mismatched unrelated donor. All pts were also characterized for NPM1, CEBPA, IDH1 & IDH2 mut & the presence of FLT3-ITD. Pts were classified according to the European LeukemiaNet (ELN) cytogenetic risk groups: Favorable n ¼ 20 (19%), Intermediate-I n ¼ 32 (31%), Intermediate-II n ¼ 23 (22%) & Adverse n ¼ 28 (27%). Median follow-up was 2.8y. The prognostic
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impact on Overall Survival (OS) & Event-Free Survival (EFS) & associations with clinical parameters & molecular aberrations were evaluated. Results: In our pts set, 17 (16.3%) pts had an exon 23 DNMT3A mut at codon R882 (n ¼ 16) or P904 (n ¼ 1). The presence of exon 23 DNMT3A mut was significantly associated with older age (P ¼ .04) & de novo AML (P ¼ .11) by trend. Remarkably, 14 out of the 17 exon 23 DNMT3A mut pts had at least one mutation in the NPM1, CEBPA, IDH1 or IDH2 gene or a FLT3-ITD. Exon 23 DNMT3A mut were significantly associated with the presence of NPM1 mut (P ¼ .01), especially in pts with NK (Po.001). Exon 23 DNMT3A mut significantly associated with the occurrence of IDH1 mut (P ¼ .01) in the whole pts set. We did not find any differences in exon 23 DNMT3A mutation frequency in the four ELN genetic groups. Furthermore, there was no impact of exon 23 DNMT3A mut on OS (P ¼ .71; Figure A) or EFS (P ¼ .48; Figure B) in our pts set. Conclusion: In conclusion, there was no significant impact of exon 23 DNMT3A mut status on outcome of AML pts treated with NMA-HCT. This study suggests that NMA-HCT might abrogate the negative impact of DNMT3A mut in some AML subgroups. Disclosure of Interest: None declared.
Aplastic anaemia P304 Donor lymphocyte infusions (DLI) for mixed chimerism in patients with severe aplastic anemia A. Bacigalupo1,*, A. Ghiso1, A. M. Raiola1, F. Gualandi1, A. Dominietto1, R. Varaldo1, S. Bregante1, C. Di Grazia1, T. Lamparelli1, S. Luchetti1, S. Geroldi1, E. Tedone1, F. Galaverna1, M. T. van Lint1 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: Patients with acquired severe aplastic anemia (SAA) are often found to have mixed chimerism (MC ) both in unfractionated bone marrow cells (BM) and in CD3 þ selected peripheral blood cells (CD3 þ PB). MC can be transient, permanent or progressive (Lawler et al BJH 2009). Progressive MC predicts graft rejection. The role of donor lymphocyte infusions (DLI) in treating MC in SAA patients is unclear and poorly reported. Materials (or patients) and methods: In this study we analyze the outcome of 14 SAA patients receiving DLI for the treatment of mixed chimerism. Patients.The median age was 28 years (23-53). The donor was an HLA identical sibling (n ¼ 11) or an alternative donor (n ¼ 3). The median interval between transplant and the first DLI was 161 days (51-2263). Only 4 patients were given DLI beyond 1 year from BMT: all four had a sudden fall in platelet counts, together with MC. Five patients have severe thrombocytopenia; one patient had severe neutropenia, four patients were anemic. DLI were started at 10^6/kg CD3 þ cells for SIB, and 10^5/kg for alternative donors. DLI were always given together with cyclosporin A (CyA). All paients had mixed chimerism in both BM and PBCD3 þ cells: the median donor chimerism at the time of DLI was 89% for BM ( 21-99%) and 36% for PBCD3 þ (14- 92%). Results: Patients received a median of 4 DLI (range 1-10). MC chimerism was converted to complete donor chimerism (100%) in unfractionated BM cells in 9/14 patients (64%) ; in PBCD3 þ cells it became complete donor in 7/14 (50%). The median BM donor chimerism went from 89% (average 79%), to a median of 100% (average 95%). The median PBCD3 þ donor chimerism went from 36% (average 44) to 63% (average
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79%). Four patients who were cytopenic, recovered their counts. Acute GvHD grade I occurred in 3 patients (21%) and grade III in 1 patient (7%). Two patients died (14%) : one of GvHD and one of graft failure, despite full donor chimerism and repeated infusions of donor marrow cells. Twelve patients are alive and well at a median interval of 2155 days (215-6306) from transplant. The 10 year actuarial survival of this group of patients is 77%. Three patients are off CyA (21%), 9 patients are still on CyA (64%). At last follow up 11/14 patients have normal peripheral blood counts. Conclusion: DLI , administered together with CyA , because of mixed chimerism with or without cytopenia , can induce complete donor chimerism in over 50% of the patients. A proportion of these can be taken off immunosuppression. Several patients remain permanent MC especially in PBCD3 þ cells. The risk of lethal GvHD, in the current series 7%, should be taken in to account when considering DLI for the treatment of mixed chimerism. Disclosure of Interest: None declared. P305 Bone marrow transplantation (BMT) for acquired severe aplastic anemia A. Bacigalupo1,*, A. Ghiso1, A. M. Raiola1, F. Gualandi1, A. Dominietto1, R. Varaldo1, S. Bregante1, C. Di Grazia1, T. Lamparelli1, S. Luchetti1, S. Geroldi1, E. Tedone1, M. T. van Lint1 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: The outcome of transplants for SAA has improved over the past decades. This study was done to assess changes in BMT protocols, possibly relevant for patients with acquired severe aplastic anemia (SAA) in the periods 1991-2000, 2000-2009, 2010-2014 Materials (or patients) and methods: 103 SAA patients were grafted betweeen 1991 and 2014. Median age was 29 years (962). The donor was an identical sibling (63%), a mismatched relative (10%), a matched unrelated donor (UD) (25%), a cord blood unit (2%). The number of patients in the 3 periods was 40, 41, and 22. The proportion of alternative donor was 10%, 49%, 64%. The conditioning regimen was cyclophosphamide 200 (CY200), FLU CY low dose (FC-LD), FLU CY high dose and TBI 200 rads. GvHD prophylaxis was Cyclosporina and methotrextae for all patients. UD grafts also received antithymocyrte globulin 3m,75 mg/kg x2 (Thymoglobulin , Sanofi). The source of stem cells was bone marrow (n ¼ 99), peripheral blood (n ¼ 2) and cord blood (n ¼ 2). Results: More patients in the first period received CY200, more patients in the last period received a combination of fludarabine CY (FC) or FC with TBI200, due to a change in donor type, with over 60% of donors other than SIBS (Table 1). Table 1. 19901999
20002009
20102014
N¼ Median age yy range Alternative donors CY 200 GvHD II-IV
40 23 16-60 10%
41 29 9-53 49%
22 28 17-62 64%
66% 20%
22% 12%
11% 22%
Causes of Death Rejection Infections GvHD MOF Alive
2.5% 10% 10% 10% 60%
5% 15% 2% 2.5% 68%
9% 0% 0% 0% 91%
P
0.6 o0.0001 o0.0001
o0.0001 o0.03
Patients surviving in the 3 periods were as follows 60%, 68%, 91%. Causes of death stratified for transplant period, showed no reduction in death due to rejection, but a reduction of infections (from 10% to 0%), of GvHD (from 10% to 0%) and multiorgan failure (from 10% to 0%). The actuarial 4 year survival for the 3 groups is 60%, 67%, 90%. The infectious cause of death in the first period were Pseudomonas (n ¼ 2), Serratia (n ¼ 1) Aspergillus (n ¼ 1); in the second period the etiology was Aspergillus (n ¼ 1), Pseudomonas þ EBV (n ¼ 1), fusariosis (n ¼ 1), EBV alone (n ¼ 1), bacterial sepsis (n ¼ 2). Conclusion: Despite a significantly greater proportion of alternative donors in recent years, the outcome of transplants for aquired SAA has significantly improved: the reason for improvement is not entirely clear: there has been a reduction of infections, possibly due to better diagnosis - early treatment, including mabthera prophylaxis of EBV for unrelated transplants, but also to the widespread use of new antifungal agents. Disclosure of Interest: None declared. P306 Allogeneic hematopoietic stem cell transplantation in severe aplastic anemia – low incidence of graft versus host disease and excellent results with graft failure rescue F. Moita1,*, B. Velosa Ferreira1, M. J. Gutierrez1, G. Teixeira1, I. Sousa Ferreira1, N. Miranda1, M. Abecasis1 1 Bone Marrow Transplant Unit, Instituto Portugueˆs de Oncologia de Lisboa, Francisco Gentil, Lisbon, Portugal Introduction: Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains the only curative therapy for patients with aplastic anemia (AA). It is, however, only recommended as first line treatment in young patients with a matched sibling donor (MSD), while alloHSCT from alternative donors is only indicated after failure of immunosuppressive therapy (IST). Materials (or patients) and methods: A retrospective analysis of all patients with the diagnosis of severe acquired AA that underwent alloHSCT in our institution, between 1987 e 2014, was conducted. Results: Fifty alloHSCT were performed, in a total of 40 patients. The median age at transplantation was 15,8 years (range 3,3 - 55,8 years). Thirty-four cases received alloHSCT from a MSD and 16 cases from a matched unrelated donor (MUD) (13 with a 9/10 HLA match; 3 with a 10/10 HLA match). Bone marrow was the chosen stem cell source in 42 cases. T-cell depletion with ATG or alemtuzumab was used in association with the conditioning regimen in 70% of cases. The median time between diagnosis and alloHSCT was 7 months (3 for MSD and 11,5 months for MUD). Fifteen patients received IST prior to alloHSCT. Extensive (grade 3/4) acute graft versus host disease (GVHD) was documented in only one patient. Chronic GVHD occurred in 34,2% patients, with an incidence of 32,1% in MSD and 40% in MUD. Severe chronic GVHD was only documented in 2 patients (7%), both transplanted with a MSD. Graft failure was documented in 11 alloHSCT (8 patients), at a median time of 5 months after alloHSCT, with no significant difference between donor source (20,6% in MSD and 26,7% in MUD). The incidence of graft failure was superior in patients that did not receive T-cell depletion in the conditioning regimen (33,3% versus 17,1% for T-cell depleted alloHSCT). Seven of these 8 patients proceeded to a subsequent alloHSCT. All of the re-transplanted patients are alive, with a median follow-up of 11 years after the last alloHSCT. Four patients died, all due to infectious complications, at a median time of 4,5 months after alloHSCT. The median followup for living patients is 12 years (minimum 0,5; maximum 26,6 years). The median overall survival was not reached and the overall survival at 12 years is 88,4% (CI 95%: 78,2%4100%). Conclusion: In this small series, alloHSCT was associated with excellent long term outcomes, with no significant differences between MSD and MUD in terms of extensive GVHD and graft
failure. The low mortality and morbidity make it a very appealing procedure for this disease, particularly when compared with the outcome of IST. In conclusion, our data support the use of alloHSCT as first line treatment in young patients with severe AA, even in the absence of a MSD. Disclosure of Interest: None declared. P307 Outcome of allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome or leukemia evolving from aplastic anemia I. Golubovskaya1,*, E. Morozova1, Y. Rudnickaya1, T. Rudakova1, V. Ovechkina1, A. Rats1, A. Kulagin1, B. Afanasyev1 1 First Pavlov State Medical University of St. Petersburg, Raisa Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, St Petersburg, Russian Federation Introduction: Long-term survivors of acquired aplastic anemia (AA) have an increased risk of transforming into myelodysplastic syndrome or acute myeloid leukemia (MDS/AML) after immunosuppressive therapy. Allogeneic stem cell transplantation (HSCT) is optimal option to improve prognosis in patients with secondary MDS/AML evolving from aplastic anemia. The number of patients transplanted to date is limited and impact of post-transplant therapy has not been extensively studied. Overall survival (OS) in this extremely unfavorable cohort of patients is significantly lower than in patients with de novo MDS/AML. Reduced intensity conditioning regimen (RIC) and myeloablative conditioning regimen (MAC) result in similar OS in MDS/AML (Kro¨ger et al., ASH 2014). The aim of our study was to analyze the effectiveness of HSCT in secondary MDS/ AML after AA. Materials (or patients) and methods: We analyzed 14 patients with MDS/AML transformation after treatment of acquired aplastic anemia who underwent allo-HSCT between September 2007 and January 2014 from matched related (n ¼ 5), matched unrelated (n ¼ 7) or haploidentical donor (n ¼ 2). Primary diagnoses were severe AA (n ¼ 6) and non-severe AA (n ¼ 8). The median time between primary diagnoses and transformation to MDS/AML was 33 months (range 7-204). Monosomy 7 was detected in 8 (57%) patients. The median age at HSCT was 19 years (range 10-37) and the median time from diagnosis MDS/AML to HSCT was 6 months (range 1-18). Eight patients failed to respond to previous chemotherapy or hypomethylating therapy (refractory disease), 9 patients were heavily transfused and the median number of RBC and PLT transfusions was 40 and 47 units, respectively. The conditioning regimen was fludarabine-containing RIC in 12 (86%) and MAC in 2 (14%) cases. The median dose of Infused CD34 þ cell was 3,3#106/kg (range 1-9). Results: Sustained engraftment was achieved in 8 of 14 patients (57%). One patient died of acute GVHD Grade IV. Two patients with refractory MDS (RAEB-2, CMML) after MAC relapsed at day þ 60 and þ 350 and received donor lymphocyte infusion (DLI) in combination with 5-azacytidine as further therapy. Despite of second HSCT both patients died of disease progression. Six patients who failed to engraft received second HSCT from the same (n ¼ 2) or alternative donor (n ¼ 4). All of these patients had refractory disease and were heavily transfused (median number of RBC and PLT transfusions was 45 and 44 units vs 15 and 20 units in engraftment group, respectively). They died of infectious complications due to primary graft failure (n ¼ 5) or acute GVHD Grade IV (n ¼ 1). Five patients (36%) are alive with the median follow-up of 30 months (range 11-88). Four of them received 2 cycles of hypomethylating agents as a prophylaxis of relapse after HSCT. All survivors are in complete remission with signs of limited chronic GVHD. Conclusion: Primary graft failure and disease relapse remain a significant problems that have an impact on the outcome of HSCT in MDS/AML evolving from AA. Higher incidence of graft failure after HSCT may be associated with lack of immunoablation in fludarabine-containing RIC in heavily transfused patients.
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Further optimization of conditioning regimens can be a facility for improvement of HSCT results. Our limited experience suggests that post-transplant hypomethylating therapy may be a promising option for the better disease control. Disclosure of Interest: None declared. P308 Ex-vivo T-cell depleted peripheral blood stem cell transplantation from HLA-matched sibling donors for the treatment of severe aplastic anemia I. Cano-Ferri1,*, F. Moscardo1, J. Sanz1, M. A. Dasi1, P. Solves1, I. Lorenzo1, L. Senent1, P. Montesinos1, F. Lopez-Chulia1, A. Lancharro1, J. Martinez1, N. Carpio1, R. Rodriguez-Veiga1, M. A. Sanz1, G. Sanz1 1 HOSPITAL UNIVERSITARIO LA FE, Valencia, Spain Introduction: Bone marrow is the preferred stem cell source for allogeneic transplantation (allo-SCT) in acquired severe aplastic anemia (SAA) due to a lower risk of graft-versus-host disease (GVHD). However, it has not been compared with exvivo T-cell depleted peripheral blood (TCD-PB). Materials (or patients) and methods: Between July 1996 and March 2014, 22 consecutive patients with SAA underwent alloSCT with TCD-PB grafts. Graft manipulation consisted of a CD34 positive selection followed by a T-cell add-back to achieve 3x105/kg. All patients received Cyclophosphamide 200 mg/kg y anti-thymocite globulin (ATG) for conditioning regimen and cyclosporine A (CSA) combined with long-course prednisone for GVHD prophylaxis. Results: Median age was 35 years (range, 3-63) and 10 (45%) were male. Prior immune suppressive therapy with ATG and CSA was administered in 8 patients (36%). Median CD34 þ cells and CD3 þ cells were 5.1x106/kg and 3.6x105/kg, respectively. Female donor to male recipient combination was present in 4 cases (18%). Sixteen donor/recipient pairs (73%) tested positive for cytomegalovirus (CMV) serostatus. Two patients initiated allo-SCT with uncontrolled infection and died on days þ 1 and þ 2. All remaining patients engrafted at a median time of 12 days (range, 10-14). Cumulative incidence (CI) of neutrophil recovery was 91%. An additional patient died on day þ 21 due to cerebral hemorrhage. All the remaining patients had platelet recovery at a median time of 18 days (range, 8-53). CI of platelet recovery was 86%. Two patients developed grade 1 acute GVHD while no patients showed acute GVHD grade Z2. Two patients had limited and 1 patient had extensive chronic GVHD. Only 1 patient remains on immunosuppressive therapy. CMV reactivation was observed in 4 patients but none of them developed organ disease. After a median follow-up of 90 months (range, 8-184), no relapses were observed and all patients maintained stable blood counts. 8-year CI of non-relapse mortality was 14%. 8-year disease-free and overall survivals were 86%. Conclusion: Allo-SCT from HLA-matched sibling donors using ex-vivo T-cell depletion achieved excellent short and longterm outcomes with low GVHD and good quality of life in patients with SAA. This procedure should be compared with bone marrow allo-SCT. Disclosure of Interest: None declared. P309 Allogeneic Hematopoietic Stem Cell Transplantation in Adult Patients with Paroxysmal Nocturnal Hemoglobinuria: Single Centre Experience M. Merter1,*, U. Sahin1, E. Atilla1, S. Civriz Bozdag1, S. Kocak Toprak1, M. Kurt Yu¨ksel1, K. Dalva1, P. Topcuoglu1, O. Arslan1, M. Ozcan1, T. Demirer1, O. Ilhan1, H. Akan1, M. Beksac1, N. Konuk1, G. Gurman1 1 Hematology Department, Ankara University Medicine Faculty, Ankara, Turkey Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hemopoietic stem cell disorder which is
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characterized by complement-mediated hemolytic anemia, thrombophilia and bone marrow failure. Despite the introduction of eculizumab, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still the only curative treatment. Materials (or patients) and methods: In this retrospective study we assessed 9 PNH patients who received HSCT in our transplantation unit between 2005 and 2014. Median age of patients was 29 (range 21-40). Three of patients were male and 6 of patients were female. Results: Four patients had a history of treatment with eculizumab before transplantation. There were hemolytic component in 8 patients, venous thrombosis in 3 patients and bone marrow failure in 1 patient. Eight patients were transfusion dependent. All donors were HLA-identical (8 siblings and 1 unrelated). Median age of donors was 24(1746). Median time from diagnosis to transplantation was 11(2180) months. Seven patients received a reduced intensity conditioning (RIC) consisting of fludarabin, cyclophosphamide and ATG (Flu-Cy-ATG), one patient received a myeloablative conditioning consisting of cyclophosphamide and ATG (CyATG) and one patient received a myeloablative conditioning consisting of busulfan, cyclophosphamide and ATG(Bu-CyATG). We used peripheral blood stem cells in 3 patients and bone marrow in 6 patients as stem cell source. Two patients who had a history of thrombosis, died due to sepsis during the aplasia period of the transplantation. Transplant-related mortality for our patients was 22%. There was primary engraftment failure in one patient (11%). The transfusion need disappeared in this patient after the transplantation. As of December 1 2014, 7 patients are alive. Six patients have been complete hematologic recovery with no evidence of PNH following a median follow-up of 69 months (18-109 months). No thromboembolic event has been observed in early and long-term follow-up in these six patients. Besides, we did not see any TE event in the one patient with primary engraftment failure. Conclusion: Our findings confirm that hematopoietic stem cell transplantation is a definite curative treatment for PNH and Flu-Cy-ATG is a feasible regimen for transplantation. In patients who have a history of thromboembolism, other treatment strategies may be more appropriate rather than transplantation. Disclosure of Interest: None declared. P310 Outcome of allogeneic hematopoietic stem cell transplantation in pediatric patients with acquired severe aplastic anemia: a Turkish multicenter study U¨. Koc¸ak1,*, A. Ku¨pesiz2, S. Aksoylar3, V. Hazar4, M. Karaku¨kc¸u¨5, ¨ zbek8, T. ˙Ileri9, M. Elli10, S. Kansoy3, G. Karasu6, V. Uygun7, N. Y. O ¨ ztu¨rk11, E. U¨nal5, Z. Kaya1, S. Go¨zmen3, M. Ertem9, S. Çakı G. O ¨ zyu¨rek13, A. Akc¸ay 11, T. Fıs¸gın13, E. U¨nal9, Kılıc¸12, B. Tunc¸8, E. O 14 O. Gu¨rsel , D. Atay15, M. A. Yes¸ilipek6 on behalf of Turkish Pediatric Bone Marrow Transplantation Study Group (TPBMT-SG) 1 Pediatric Hematology and BMT Unit, Gazi University School of Medicine, Ankara, 2Pediatric Hematology/Oncology and BMT Unit, Akdeniz University School of Medicine, Antalya, 3Pediatric Oncology and BMT Unit, Ege University School of Medicine, ˙Izmir, 4 Pediatric Hematology/Oncology and BMT Unit, Medipol University School of Medicine, ˙Istanbul, 5Pediatric Hematology/ Oncology and BMT Unit, Erciyes University School of Medicine, Kayseri, 6Pediatric BMT Unit, Bahcesehir University School of Medicine Medical Park Goztepe Hospital, ˙Istanbul, 7Pediatric Hematology/Oncology and BMT Unit, Bahcesehir University School of Medicine Medical Park Antalya Hospital, Antalya, 8 Pediatric Hematology/Oncology and BMT Unit, Ankara Child Health and Diseases Hematology and Oncology Training and Research Hospital , 9Pediatric Hematology and BMT Unit, Ankara University School of Medicine, Ankara, 10Pediatric Oncology and BMT Unit, Ondokuz Mayıs University School of Medicine, Samsun, 11 Pediatric BMT Unit, Acıbadem University Atakent Hospital, 12 Pediatric BMT Unit, Go¨ztepe Medical Park Hospital, ˙Istanbul, 13 Pediatric BMT Unit, Bahcesehir University School of Medicine
Medical Park Samsun Hospital, Samsun, 14Pediatric Hematology and BMT Unit, Gulhane Military Medical Academy, Ankara, 15 Pediatric BMT Unit, Medical Park Bahcelievler Hospital, ˙Istanbul, Turkey Introduction: Acquired severe aplastic anemia (SAA) during childhood is a rare bone marrow failure which is classically treated with allogeneic hematopoietic stem cell transplantation (HSCT) from a sibling donor if available. Although the second option currently is immunosuppressive therapy (IST), with a merely alternative donor (AD) search when IST fails, both the high rate of failure and long term complications are undesirable particularly in the pediatric population that have a long life expectancy. The outcome of HSCTs performed in a country between Asia and Europe has not been previously investigated. Materials (or patients) and methods: The outcome of 121 children with acquired SAA that were transplanted in 12 pediatric centers in Turkey between 1999-2014 was retrospectively analyzed. Results: There were 121 patients [48 (39.7%) male, 73 (60.3%) female] with a median age of 11 years (1-20 years). The donor was HLA matched related (MRD) in 86 (71%) (71 matched sibling, 14 related family donor), alternative (AD) in 35 (29%) (23 matched unrelated, 5 mismatched unrelated, 7 mismatched family donors) patients. Graft source was bone marrow in 62 (51.2%), peripheral stem cells in 51 (42.1%), cord blood in 4 (3.3%) and both bone marrow and peripheral stem cells in 4 (3.3%) patients. The median time to neutrophil recovery was 16 days (8-34 days) in MRD, 15 days (8-45 days) in AD transplants. Graft failure was observed in 14 patients (11.6%) (6 MRD, 8 AD). No secondary malignancy was observed in any of the patients. The median time to transplant from the diagnosis was 5.1 months (0-98.6 months) in MRD, 21.2 months (8-119 months) in AD transplants. Graft versus host disease (GVHD) prophylaxis was with cyclosporine alone or combined in all patients. AD transplants were complicated with more grade II/IV acute GVHD (aGVHD) (25% vs 4.7%) than MRD transplants. There was no grade III/IV aGVHD in MRD transplants and only 1.2% limited and 1.2% extensive chronic GVHD (cGVHD) whereas there were 11.1% III/IV aGVHD, 2.8 % limited and 5.6% extensive cGVHD in AD transplants. Five year event free survival was 84.7% in MRD, 61.1% in AD transplants (Figure 1). Five year overall survival in patients transplanted from a MRD was 92.9 % with a median follow up 4.1 years (0.3-15 years) whereas it was 69.4 % in AD with a median follow up 3.7 years (1.3-14 years) (Figure 2). Graft failure and aGVHD was associated with poor survival in all transplants (Po0.001). Conclusion: This is the first report of pediatric patients with acquired SAA from Turkey presenting that outcome of upfront MRD transplants are comparable to that of the developed
countries. The relatively lower outcome of patients that were transplanted from ADs on the other hand might be due to the occurrence of more aGVHD or the longer time elapsed from diagnosis to transplant. We believe that improving supportive care and earlier availability of unrelated donors would lead to better results in AD transplants as well. Disclosure of Interest: None declared. P311 Hematopoietic Stem Cell Transplantation for Aplastic Anaemia : Outcomes from Argentine Group of Haematopoietic Stem cell transplant (GATMO) V. Milovic1,*, A. Basquiera2, J. Garcı´a2, S. Formisano2, R. Ferna´ndez2, G. Jaimovich2, G. Kuminsky2, J. Milone2, M. Rivas2, S. Yantorno2, J. Jarchum2, M. Rizzi2, J. Martı´nez Rolo´n2, C. Foncuberta2, R. Burgos2, G. Balladares2, S. Saba2, G. Drelichman2, J. Real2, L. Feldman2, J. E. Roberti1 1 Hospital Aleman, 2GATMO, Buenos Aires, Argentina Introduction: Acquired aplastic anaemia (AAA) is a serious and rare disease characterised by peripheral blood pancytopenia, bone marrow aplasia or hypoplasia. For patients with severe and very severe AAA younger than 40 years of age, hematopoietic stem cell transplantation (HSCT) is the treatment of choice. The use of ATG/Cyclosphosphamide (Cy) conditioning regimen has been associated with a 5-year survival rate of 60-90%. Since 2002, rATG is the only available ATG in the Argentine market. We describe the outcomes of HSC transplantation (tx) in pts with AAA, who received rATG/ Cyclophosphamide (Cy) as conditioning regimen Materials (or patients) and methods: Retrospective cohort study. We analysed data from 84 transplants performed in 82 pts between 1998-2014 in 11 Argentine centres. Patients: Mean age at transplant was 19.264±10.09 years; 46(55%) pts were o20 years old, and 36(43%) were 420 years old. Median interval from diagnosis to transplant was 141(21-4429) days. Nineteen patients (25%) had received a previous treatment, 3(3.4%) were second transplants; 74(88%) patients received HSC from related donors; the source of HSC was: bone marrow in 56(68%), and peripheral blood in 21(25.6%) patients. Mean infused cell dose was 4.3±3x106/kg. Results: Median time to neutrophil count41000/mm3 was 20(9-42) days, and to platelet count 420000/mm3 was 23(11108) days. Four (4.8%) patients had early rejection and 4(4.8%) had late graft rejection, with a median time to rejection of 371(137-745) days in the late rejection group. Seven (8.3%) patients developed cGvH, with no significant difference according to SC source. With a median follow-up of 823(14429) days, 23(28%) patients died; the main cause of death was infection in 15(71%) cases. The overall survival at 100, 365 and 4429 days was 89%(CI80-94), 79%(CI68-86) and 67%(CI54-
[P310]
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77), respectively, without statistically significant difference between age groups. Conclusion: Most patients with AAA were able to receive SCT as the first line therapy, with a low early graft rejection rate. Overall survival and cGvH rate are similar to those reported internationally. Disclosure of Interest: None declared. P312 Immunosuppressive treatment combined with cord blood infusion for patients with severe aplastic anemia Y. Cai1,*, C. Wang2, J. Yang2, J. Jiang2 1 hematolgy, 2hematology, Shanghai General Hospital, Shanghai, China Introduction: Treatment of acquired severe aplastic anemia (SAA) consists of immunosuppressive therapy and allogeneic stem cell transplantation. Patients usually receive immunosuppressive therapy (IST) as first-line treatment. The median time to transfusion independence was 96 days. Patients experienced prolonged neutropenia, a high rate of infection. The response of IST was 70-80%. Cord blood transplantation (CBT) has become a promising therapeutic option for patients with nonmalignant diseases.Unrelated donor CBT has not yet been recommended for SAA patients. Doctor SUN et al reported a single-center experience in treating 18 patients with SAA who received unrelated CBT. Their conditioning regimen composed of cyclophosphamide, rabbit antithymocyte globulin and fludarabine. All patients were given a combination of cyclosporine (CsA) and mycophenolate mofetil for GVHD prophylaxis. Cord Blood failed to engraft in all cases, but may facilitate autologous recovery and improve survival with low risk of transplantrelated mortality. Materials (or patients) and methods: We report a singlecenter experience in treating 12 patients with SAA who received immunosuppressive treatment combined with unrelated cord blood infusion and Cord Blood may facilitate neutrophil recovery. Between May 2010 and January 2014, 12 patients (3 male and 9 female) were enrolled in a study. The median age at diagnosis was 18 (5-55) years. Seven patients were newly diagnosed and 5 patients received cyclosporine±testosterone /undecanoate before. All patients were treated with fludarabine at 30mg/m2/day for 5 days without CTX , 6 cases received Thymoglobuline at 2-3 mg/kg/day for 5 days.And another 6 cases received ATGFresenius at 5-6 mg/kg/day for 5 days.All patients were treated with cyclosporine. Cord unit selection was based on the best HLA compatibility, with at least four of six loci matching. The median nucleated cell was 2.8 (1.9-14.2) 107 cells/kg. And CD34 positive cell was 0.9 (0.4-3.6) 105 cells/kg. Unrelated cord blood was infused on d0. Results: The median time to ANCZ0.5 109 /L was 20 (11 to 66)days .It may be favorable to control infection. And the median time to plateletsZ20 109/L was 96 (12 days-7 months)days. Only 1 patient acquired unrelated cord stem cell engraftment. The remaining 11 patients experienced graft rejection and autologous recovery. One patient achieved 95-98.5% donor cells continuously. One case showed 95-98.5% donor cells in 1 month and donor cells declined to 0% in 3M. Four cases showed11.6-68.9% donor cells on day 14 and declined to 0% on day 28.The remaining 6 cases showed no donor cells at all test points. Eleven patients have survived for 11 – 48 months (median follow-up was 14 months). CR and PR was 7 and 5 cases, respectively. Three cases relapsed in 4 months, 6 months and 24 months, respectively. Among them, 2 cases relapsed after termination of immunosuppressant. The median follow-up was 14 months. One patient relapsed at 2 years for termination of immunosuppressant and died of
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infection in 3 years. 1-year and 2-year survival rates were 100%. 3-year survival rate was 91.7%. Conclusion: Compared to traditional immunosuppressive treatment, IST plus unrelated CBT achieved faster hematopoiesis recovery and reduced death caused by infection.Compared to stem cell transplantation, IST plus unrelated CBT might facilitate autologous recovery, minimize transplantrelated mortality and have no GVHD. Disclosure of Interest: None declared. P313 Determination of effective blood concentrations of cyclosporine in the treatment of severe aplastic anemia in children M. Philippe1, E. He´nin2, Y. Bertrand3,*, D. Plantaz4, S. Goutelle5, N. Bleyzac1 1 Pharmacy Department, Pediatric Hematology and Oncology Unit, IHOP, 2Departement of Clinical Pharmacology & Clinical Trials, Hospices Civils de Lyon, 3Transplantation Unit, Pediatric Hematology and Oncology Unit, IHOP, Lyon, France, Lyon, 4 Pediatric Hematology and Oncology Unit, CHU Grenoble, Grenoble, 5Pharmacy Department, Groupement hospitalier de ge´riatrie, Lyon, France Introduction: Optimal immunosuppressive therapy in acquired severe aplastic anemia (SAA) remains to be refined, especially cyclosporine (CsA) use. Current recommendations state that CsA trough blood concentrations (TBC) should be maintained between 200 and 400 ng/mL despite the lack of supporting data. This study aimed at quantifying relationships between exposure to CsA and hematological response, and at determining the optimal CsA TBC target. Materials (or patients) and methods: Twenty-three pediatric patients with SAA treated with CsA were retrospectively analyzed. Nonlinear mixed effects modelling approaches were used to develop a Pharmacokinetic-Pharmacodynamic model. The pharmacokinetic model described the relationships between CsA doses and TBC. The pharmacodynamic model allowed to estimate boundaries for optimal CsA effects, neutrophils being used as biomarker of response. A time-toevent model linked effective concentration to time-totherapeutic success. Simulations were subsequently performed using the model to evaluate the chance of therapeutic success according to various CsA TBC target, and thus estimate the optimal CsA exposure. Time-to-therapeutic success was externally predicted in two additional patients not included in the model building. Results: Twenty-three pediatric patients with SAA were included. The median (min-max) age and weight were: 8.5 (8-15) years, and 34 (9.8-79.3) kg, respectively. Fifteen out of 23 patients responded to the IST (65.2%) with a median time of response of 69 days (min 19- max 182). CsA TBC were adequately described by a two-compartment model with firstorder absorption, a lag-time and a linear elimination. The optimal target range of CsA TBC was estimated between 87 and 120 ng/mL (relative standard erroro5%). Model-based simulations showed that CsA TBC should be maintained around 100 ng/mL to expect a maximal response rate; higher TBC would be associated with lower response rate. Using the model, time-to-therapeutic success could adequately be predicted for two new patients. Conclusion: This original modelling approach was successful in describing the relationship between CsA TBC and the neutrophil response in patients with SAA. While further research in a larger population is necessary to confirm our findings, this work suggest that a CsA TBC target of 100 ng/mL, much lower than that currently recommended, would be associated with a better response rate in children with SAA. Disclosure of Interest: None declared.
Lymphoma
Table 1. Treatment-relative mortality (TRM) and 2-years Overall Survival (OS) of children with pediatric lymphoma who underwent allogeneic stem cell transplantation.
P314 Hematopoietic stem cell transplantation for refractory or recurrent Non-Hodgkin Lymphoma in children and adolescent. A retrospective study from the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) A. Spiegel1,*, N. Rauss2, Y. Bertrand2, J.-H. Dalle3, G. Michel4, F. Rialland5, P. Schneider6, C. Jubert7, J. Kanold8, L. Brugie`res9, P. Lutz1, C. Paillard1 1 Service d’onco-he´matologie pe´diatrique, Centre Hospitalier Universitaire de Strasbourg, STRASBOURG, 2IHOP, Centre Hospitalier Universitaire de Lyon, LYON, 3Service d’he´matologie pe´diatrique et de greffe de moelle osseuse Robert Debre´, APHP, PARIS, 4Service d’he´matologie pe´diatrique et de greffe de moelle osseuse, Centre Hospitalier de Marseille, MARSEILLE, 5 Centre Hospitalier Universitaire de Nantes, NANTES, 6Centre Hospitalier Universitaire de Rouen, ROUEN, 7Centre Hospitalier Universitaire de Bordeaux, BORDEAUX, 8Centre Hospitalier Universitaire de Clermont-Ferrand, CLERMONT-FERRAND, 9Institut Gustave Roussy, AP-HP, PARIS, France Introduction: Despite excellent outcome with current riskadapted combined modality first-line treatment for children with non Hodgkin lymphoma (NHL), treatment of patients failing first-line therapy or relapsing remains a major challenge. Allogeneic HSCT have become part of salvage therapy strategies for these patients. The potential benefit of a graft-versus-lymphoma effect has been counterbalanced by high transplant related mortality in this population. The purpose of the current study is to describe outcomes in a large cohort of pediatric patients with lymphoma who received an allo-HSCT in recent years. Materials (or patients) and methods: Clinical data were prospectively collected using ProMISe (Project Manager Internet Server), an internet-based data registry system shared by all centers of the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC). Between 2000 and 2014, this French retrospective multicenter study involved 98 consecutive patients under age of 18 years who received a first allo-HSCT for NHL. Their clinical features as well as their therapies and evolution are described and compared to results of literature. Results: Sex ratio is about 1:2. Median age of diagnosis is 8,2 years. There are three major subgroups of lymphomas: Anaplastic large-cell lymphomas (ALCL) with T or null phenotype (35 cases), Lymphoblastic lymphomas (LBL) (18 cases), Mature B-cell lymphomas including Burkitt lymphoma (BL) (11 cases) and diffuse large B cell lymphoma (DLBCL)(15 cases). Data are incomplete for 19 children. Median age of HSCT is 9.8 years old and median time of allogeneic stem cell transplantation to diagnosis is 417 days. Complete remission (CR) before HSCT is reached in 55/79 cases mainly CR2 (37/55 cases). Myeloablative conditioning regimen is described in 71/ 79 cases. Total Body Irradiation (TBI) with myeolablative dose is used in 71% of patients. The use of bone marrow is most frequent (41/79 cases). Sibling donor is found in 1/3 cases. Engraftment is obtained in 87% of patients and median time of neutrophils recovery is 21 days. Acute graft versus host disease is observed in 45/79 cases mainly stage I (17/45 cases) and stage II (15/45 cases). Chronic graft versus host disease is noticed in 12/79 cases. 31/79 patients are death, mainly due to disease progression or relapse (16/31cases). For all patients, toxicity related mortality is 17.7% (14/79 cases). 2-year overall survival is 60.8%. Conclusion: The present data and previous studies clearly demonstrate that allogeneic HSCT may be curative for selected patients with NHL. Several questions remain to be determined, among them the optimal time point for transplantation and choice of the best conditioning regimen.
TRM All patients ALCL LBL BL&DLBCL
17.7% 20% 27.8% 7.7%
(14/79) (7/35) (5/18) (2/26)
2-years OS 60.8% 71.4% 33.3% 69.2%
(48/79) (25/35) (6/18) (18/26)
Some patients may be selected for RIC regimens that are associated with lower TRM. Disclosure of Interest: None declared. P315 Autologous stem cell transplantation for children and adolescents with relapsed and refractory Hodgkin’s lymphoma A. Hedayati Asl1,* on behalf of Mehrvar A, Zangooei R, Faranoush M, Dinarooni P, Olad E, Jenabzadeh M, Athari G, Fallah V, Haghighi M 1 Pediatric Hematology-Oncology, MAHAK, Tehran, Iran, Islamic Republic Of Introduction: Despite the generally excellent prognosis of children and adolescents with Hodgkin’s lymphoma, approximately 20% of patients relapse. High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a recognized treatment option for patients with relapsed Hodgkin’s lymphoma. This study evaluates the results and outcome of non-cryopreserved autologous stem cell transplant of 23patients with Hodgkin lymphoma. Materials (or patients) and methods: Twenty-three patients age 5 to 25 years (median 14.5 years, M/F ¼ 17/6), with relapsed, refractory or poor prognosis HD, underwent ASCT in our hospital (from 2012-2014). Status at transplant was: second complete remission (CR2): n ¼ 13; further CR (CR 42): n ¼ 8, partial remission (PR): n ¼ 2. Twenty-three patients received chemotherapy- based conditioning regimens: cyclophosphamide, carmustine and etoposide (CBV): 6, CCNU, etoposide, cytarabine and melphalan (CEAM): 17, Peripheral blood (PB) was the source of progenitor cells in 23 patients. All patients engrafted. Results: The median mononuclear cell dose was 5.4 108/ kg. The median time to reach absolute neutrophil count 4 0.5 109/L was 11 days, and the median time to platelet count 4 20 109 was 13 days. Grade 2 and grade 3 mucositis was seen in 60% our patients. Transplant-related mortality at 100 days not occurred. Only two patient relapsed 15 and 18 months after transplant (mean 16.5 m.). With a median followup of 19.5 months (5-29 months) after transplant the eventfree survival were 85%. All patients remain alive. No significant different between CBV group vs. CEAM group in engraftment day. Conclusion: High-dose therapy with stem cell rescue can lead to durable remissions in children and adolescents with advanced HD. Future investigations should focus on strategies designed to decrease relapse after auto-transplantation, particularly in patients at high risk for relapse. Our analysis suggests that these regimens (CEAM, CBV) are feasible in pediatric patients with acceptable engraftment and toxicity. Disclosure of Interest: None declared.
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P316 Application of LYSA risk factors in patients with relapse/ refractory Hodgkin Lymphoma undergoing Autologous Stem Cells Transplantation: a retrospective single center study from Argentina A. Zenatti1, A. L. Basquiera1,2,*, P. Garcı´a1, G. Sturich1, L. Guanchiale1, J. C. Damonte1, G. Caeiro1, P. Abichain1, C. Hollman1, A. Berretta1, J. J. Garcı´a1,2 1 Hematology and Oncology, Hospital Privado Centro Me´dico de Co´rdoba, 2Instituto Universitario de Ciencias Biome´dicas de Co´rdoba (IUCBC), Co´rdoba, Argentina Introduction: Patients with relapse/refractory (R/R) Hodgkin Lymphoma can achieve long-term remission with high dose therapy followed by autologous hematopoietic stem cell transplantation (ASCT). However, a number of patients will relapse after ASCT and for this group of patients a further treatment after ASCT may be necessary. Thus, identification of these patients before ASCT is the paramount importance. An expert panel from the Lymphoma Study Association (LYSA) defined a risk stratification for patients with R/R Hodking Lymhoma. This classification took into account three prognostic factors considering the setting of the relapse previous ASCT: primary refractory disease, early relapse (less than 12 months after end of treatment) and stage III/IV at relapse. Patients were separated into three risk groups: standard, intermediate and high risk of relapse after ASCT. Materials (or patients) and methods: Our objective was to apply the LYSA classification in all patients with R/R Hodgkin lymphoma underwent ASCT in the Hospital Privado de Co´rdoba, Argentina. We reviewed clinical data of 97 patients who received an ASCT between 1991 and 2013. 91 out of 97 patients were included in the analysis (64 men; median age at ASCT 25 years, range 5-62). Results: Distribution of risk factors was as follow: 31 patients were refractory, 30 had early relapse and 49 patients relapsed as advance stage. According to LYSA recommendations 15/91 patients were standard risk, 45/91 were intermediate risk and 31/91 were high risk. Conditioning regimen was CVB (n ¼ 39; 43%) and BEAM (n ¼ 52; 57%). Median follow-up of the surviving patients was 22.4 months after transplantation. Cumulative incidence of non-relapse mortality was 4% at 6 months. Relapse occurred in 38 patients after ASCT in a median time of 5.1 months. We found a statistically difference between the groups in terms of five-year overall survival (OS) (93% vs. 67% vs. 55%; P ¼ 0.035) and cumulative incidence of relapse (13% vs. 47% vs. 53%; P ¼ 0.022) for standard, intermediate and high risk respectively. Conclusion: Our series contribute to demonstrate that risk factors at relapse can identify patients with adverse outcome after ASCT. For these patients additional treatment strategies may be considered. Disclosure of Interest: None declared. P317 The role of pre-transplant positron emission tomography (PET), in patients with Hodgkin disease undergoing a haplo transplants with post-transplant cyclophosphamide C. Marani1, A. M. Raiola1, A. Dominietto1, R. Varaldo1, F. Gualandi1, C. Di Grazia1, T. Lamparelli1, S. Bregante1, M. T. van Lint1, F. Galaverna1, A. Ghiso1, A. Bacigalupo1,* 1 U.O. EMATOLOGIA TRAPIANTO di MIDOLLO, IRCCS - OSPEDALE SAN MARTINO-IST, Genoa, Italy Introduction: Patients with Hodgkings disease (HD) relapsing after an autologous stem cell transplants, or after second/third line therapy, represent a group of patients with a very dismal outcomes. The Baltimore/Seattle groups have reported encouraging results in these high risk HD patients, using a non myeloablative (NMA) conditioning regimen followed by unmanipulated haploidentical bone marrow transplantation (haplo-BMT) and high dose post-transplant cyclophosphamide (PT-CY) (Lutznik 2008). Recently, we have confirmed these data
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(Raiola 2014). However, relapse after allogenic BMT continued to be major problem. Materials (or patients) and methods: We hypothesized that, in HD patients treated with the Baltimore protocol, positron emission tomography (PET) status before a haplo-BMT would be predictive of relapse. Patients with a negative PET scan or with a reduction in the number of hypermetabolic areas after salvage chemotherapy were defined as ‘‘chemosensitive’’; patients with an unmodified or enhanced PET signal after salvage chemotherapy were considered ‘‘chemorefractory’’. Patients. 32 patients with relapsed HD received a haplo-BMT between September 11th 2009 and March 4th 2014. The donor was a haploidentical relative in all cases. All patients had relapsed after an autologous graft and underwent a PET scan before transplant. The median age was 31years(18-59). For patients classified as ‘‘chemosensitive’’ (n ¼ 21) the PET scan was negative in 12 and was borderline positive in 9. For patients classified as ‘‘chemorefractory’’ (n ¼ 11) the PET scan was strongly positive. Results: All patients engrafted with 1 exception, a patient who showed autologous recovery. The median day for 500 neutrophils was 18 days (14-26) for chemosensitive and 18 days (13-25) for chemorefractory patients. Acute GvHD grade IIIV was diagnosed in 24% and 18% of chemosensitive/ chemorefractory patients (P ¼ 0.1). Moderate severe chronic GvHD was diagnosed respectively in 19% and 18% (P ¼ 0.9). Transplant related mortality (TRM) was 0% for chemosensitive and 9% for chemorefractory patients. With a median follow-up of 2 years for surviving patients , 16 have relapsed (50%): the relapse rate was 29% in chemosensitive vs 91% in chemorefractory patients (P ¼ 0.0008). The 2-year progression free survival is 63% for chemosensitive patients and 0% for chemorefractory patients (P ¼ 0.0001). The overall survival is respectively 86% and 71% (P ¼ 0.1) Conclusion: This study proves that a pre-transplant ‘‘chemosentive’’ PET-based status is essential to achieve long term remissions in patients with HD undergoing a haplo BMT with PT-CY. Disclosure of Interest: None declared. P318 Efficacy of upfront autologous stem cell transplantation and safety of high dose ranimustine regimen (MEAM) in the treatment of diffuse large B cell lymphoma A. Nakaya1,*, S. Fujita1, A. Satake1, Y. Azuma1, Y. Fujita1, M. Hotta1, H. Yoshimura1, K. Ishii1, T. Ito1, S. Nomura1 1 First Department of Internal Medicine, Division of Hematology and Oncology, Kansai Medical University, Osaka, Japan Introduction: In patients with diffuse large cell lymphoma (DLBCL) classified as high-intermediate risk or high risk on the international prognosis index (IPI), the efficacy of high-dose chemotherapy (HDT) together with autologous stem cell transplantation (ASCT) during the first remission remains controversial in the rituximab era. In our institute, patients with DLBCL in both the high-intermediate and high IPI groups initially underwent R-CHOP followed by HDT/ASCT in an upfront setting. Various high-dose conditioning regimens have been developed for treatment prior to ASCT. However to date an optimal conditioning regimen with the lowest toxicity and highest therapeutic efficacy has not been established. Conditioning HDC for ASCT is widely performed using the BEAM (BCNU[carmustine]/VP-16/ARA-C[cytarabine]/L-PAM[melphalan]) regimen. However, one of the components of the BCNU regimen has not been approved by the Japanese National Health Insurance scheme and has been replaced with the alkylating agent ranimustine, MCNU; this revised regimen has been termed the MEAM regimen (MCNU/VP-16/ARA-C/L-PAM). The MEAM regimen is one of the most frequently used conditioning regimens for lymphoid malignancies in Japan. However, there is very limited information regarding its safety profile.
Materials (or patients) and methods: To investigate if upfront HDT/ASCT could improve the prognosis of high risk DLBCL patients, we retrospectively analyzed 46 patients who had all been treated with HDT/ASCT in both upfront and salvage settings. Furthermore, we examine the safety and feasibility of the MEAM regimen. Results: Forty six DLBCL patients underwent ASCT following treatment with the MEAM regimen between 2006 and 2013 at Kansai Medical University Hospital. Twenty four patients received upfront HDT/ASCT and twenty two patients received salvage therapy. Median patient age was 58.4 years in the upfront setting and 54.1 years in the salvage group. In the upfront setting, with the exception of one patient, 23 patients had a high-intermediate or high risk IPI. All patients in the upfront setting were diagnosed as having DLBCL, whereas only three patients in the salvage group were diagnosed as having DLBCL that had transformed from follicular lymphoma. Three-year overall survival (OS) and progressionfree survival (PFS) in the upfront setting were 95.0 %(P ¼ 0.00249) and 83.3 %(P ¼ 0.11), respectively. In the salvage group, 3-year-OS and PFS were 53.6% and 65.6%, respectively. The major toxicities were anorexia, nausea, diarrhea, and febrile neutropenia (FN). In the salvage group, two patients died from regimen-related toxicities. One patient became fatal because of FN and the other died of interstitial pneumonia. Therapy related mortality was 4.3%. Vascular sarcoma and squamous cell carcinoma in skin were observed as secondary malignancy. Conclusion: Upfront HDT/ASCT in patients with high-risk DLBCL is feasible and can improve therapeutic outcome; and the MEAM regimen could be a tolerable, effective and curative conditioning regimen regarding ASCT. Disclosure of Interest: None declared. P319 Outcome of Hodgkin’s Lymphoma Patients Relapsing after Allogeneic Hematopoietic Stem Cell Transplantation B. Sarina1,*, A. Vai1, S. Garcia2, A. Granata2, R. Crocchiolo1, S. Bramanti1, D. Coso2, R. Bouabdallah2, F. Broussais2, L. Giordano1, L. Morabito1, A. Santoro1, D. Blaise2, L. Castagna1 1 Humanitas Cancer Center, Rozzano (Mi), Italy, 2Institut Paoli Calmettes, Marseille, France Introduction: Relapse in Hodgkin’s lymphoma (HL) patients (pts) who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still high and no standardized treatment is currently available. Materials (or patients) and methods: We retrospectively analyzed 99 consecutive HL pts who underwent allo-HSCT at two European transplant centers between 1999 and 2013. Treatment choice at relapse after allo-HSCT was made by treating physician, mainly based on previous therapies, relapse characteristics and clinical condition. The aim of this study was to describe the outcome of HL pts relapsing after allo-HSCT. Results: The median pts’ age was 30 (range: 16-62). 77 (78%) pts were in complete (CR) or partial response status before allo-HSCT. More than 90% of the pts received a previous autologous HSCT. 47 (47%) pts received allo-HSCT from an HLA-identical sibling donor, 14 (14%) pts from an unrelated donor, 36 (36%) from a haploidentical donor and 2 (2%) from cord blood units. 35 out of 99 (35%) pts relapsed or progressed after allo-HSCT. The characteristics and therapies of the relapsed pts are shown in Table 1. The median time to relapse or progression after allo-HSCT was 4 months (0-18). 4 (11%) pts were candidate to supportive therapy, because of poor performance status. 6 (17%) pts underwent haploidentical transplantation following relapse and their data were censored at the time of transplant. After a median follow-up (FU) of 29 (6-101) months from relapse, overall survival (OS) was 34% and progression-free survival (PFS) was 26%. At the last follow-up among the 15 pts who received DLI (DLI group), 7 pts (47%) were alive and 6 pts in CR (40%), meanwhile among the 15 pts who did not receive DLI (no-DLI group), 5
Table 1. Characteristics and therapies of patients relapsing/ progressing after allo-HSCT n ¼ 35 Patients treated Median time to relapse (months) Acute graft-versus-host disease Before relapse After DLI
30 (86%) 4 (0-18) 14 (40%) 11 2
Number of therapy lines after relapse 1 2 42
3 (9%) 13 (37%) 14 (40%)
Treatment at relapse DLI DLI þ RT DLI þ CT DLI þ Brentuximab Brentuximab ± CT CT RT
15 (43%) 2 8 5 2 (6%) 10 (29%) 3 (9%)
(33%) pts were alive and 2 (13%) pts in CR (P ¼ 0.11 and P ¼ 0.03, respectively). Conclusion: The prognosis in relapsed HL pts is still poor. However, DLI group seem to compare favourably with no-DLI group. On the basis of these results, new therapeutical approaches, including prophylactic or pre-emptive strategies, are warranted. Disclosure of Interest: None declared. P320 Comparison of HLA Identical and Haploidentical Hematopoietic Stem Cell Transplantation in Non Hodgkin Lymphoma patients: A Single Center Experience D. Cekdemir1,*, E. Birtas Atesoglu2, I. Dora1, B. Kosan1, E. Er1, N. Baskan1, S. Kural1, E. Gucyener1, M. Sengezer1, N. Tiryaki1, Z. Gulbas1 1 Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, 2Department of Hematology, Kocaeli University, Kocaeli, Turkey Introduction: Allogeneic hematopoietic stem cell transplantation (AHSCT) is an established therapeutic modality in NonHodgkin lymphoma (NHL) patients relapsing after autologous stem cell transplantation. In the present study, we compare the results of Non-Hodgkin lymphoma patients who had undergone haploidentical or HLA identical allogeneic hematopoietic stem cell transplantation in our center. Materials (or patients) and methods: We analyzed the results of 14 NHL patients who had undergone haploidentical hematopoietic stem cell transplantation and 17 NHL patients who had undergone HLA identical hematopoietic stem cell transplantation between July 2010 to December 2013. 50% response to previous chemotherapy were considered as the eligibility criteria for transplantation. While,71,4% of the patients had active disease prior to transplantation in haploidentical AHSCT group, 70,6% of the patients in the HLA identical AHSCT group had active disease. No T cell depletion were performed. Cyclophosphamide, Cyclosporine and Mycophenolate mofetil were used for GVHD prophylaxis. Posacanazole and acyclovir were started at the day of transplantation and piperacillin-tazobactam were started when neutropenia ensues prophylactically. Characteristics of the patients are shown in Table 1.
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Results: Characteristics of the patients are shown in Table 1 and clinical results are shown in Table 2. There were no statistically significant difference between overall survival and disease survival of haploidentical AHSCT patients and HLA identical AHSCT patients (P ¼ 0,66, P ¼ 0,95, respectively). Kaplan-meier overall survival and disease free survival curves of the haploidentical AHSCT and HLA identical AHSCT groups are shown in Figure 1. Conclusion: For patients without HLA-identical or unrelated donor and relapsing after autologous hematopoietic stem cell transplantation, haploidentical hematopoietic stem cell transplantation could be an effective mode of treatment. Disclosure of Interest: None declared.
P321 Comparison of total body ultrasonography with total body computerized tomography (CT) and 18-Fluorodeoxyglucose positron emission tomography (PET/CT) to detect recurrence of disease in patients with Hodgkin and non-Hodgkin lymphoma E. Benedetti1,*, F. Martini1, R. Morganti1, F. Caracciolo1, N. Cecconi1, G. Cervetti1, D. Caramella2, L. Rigacci3, M. Petrini4 1 Depart. of Oncology, Hematology Unit Ospedale S. Chiara Pisa, 2 Depart. of Oncology, U.O. Radiologia Universitaria Pisa, Pisa, 3 Universita’ di Firenze, U.O Ematologia Firenze , Firenze, 4Depart. of Oncology, U.O. Ematologia Pisa, Pisa, Italy Introduction: Patients with lymphoma may relapse with asymptomatic relapse in approximately 30% of cases. Total body CT (TB-CT) scan is the NCCN recommended imaging technique with a biannual basis frequency for the first 5 years. PET is strongly recommended for 18 fluorodeoxyglucose (18FDG) avid lymphoma such as Hodgkin lymphoma (HD). Nevertheless radiation exposure during follow up is becoming a concern. The development of new generations ultrasound sonographer may help in the follow up of lymphoma patients, with less radiation exposure and less expenses.
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Materials (or patients) and methods: In our Center we retrospectively evaluated our Lymphoma data-base and in the last 4 years, we selected 150 lymphoma patients (pts) who experienced relapse and received chemotherapy. Out of the 150 pts, 50 relapsed pts were identified who were investigated with both Ultrasound (US) and TB-CT (N ¼ 43) or PET(N ¼ 7). The examinations were performed within 2 weeks from each other. US was performed double blinded, always before CT or PET. US was accompanied by a chest X ray (CXR) in most cases during the routine follow up. Pts were assessed on a 3 monthly bases. Total body US assessed: superficial nodes (S): laterocervical, supra and sub clavicular, axillary, and abdominal (A) and, if possible, anterior mediastinal (M) lymph nodes (Ly) stations. As a control group N ¼ 50 randomly chosen complete remission (CR) pts out of the 150 pts were analyzed. Results: Histologies of the 50 pts were as follows: HD N ¼ 10, Follicular (Fol) NHL N ¼ 9, Marginal (MZL) N ¼ 3, MALT N ¼ 3, Mantle cell (MCL) N ¼ 11; not specified N ¼ 2, DLBCL N ¼ 9, Burkitt N ¼ 1; T-NH N ¼ 2. In 32 cases there was a concordance between US and CT or PET/CT regarding S and A Ly. One case CT scan revealed M relapse in respect to US. In 9 cases of contemporaneous M þ A þ S involvement CT was superior to detect only M involvement. In 3 cases CT or PET/CT revealed lung and or bone involvement with concordance with US in S and A ly. Out of 3 cases of negative US 2 positive cases of CT scan regarded M involvement and 1 A Ly. Nevertheless, overall, there was not a statistically (ST) significant difference in detection of lymphoma relapse between US and CT or PET/ CT (P ¼ 0.186).Pts have been stratified according to BMI (BMI4 ¼ 25 and o25): BMI does not influence concordance in relapse detection between US and CT or PET/CT (P ¼ 0.8). There was not a ST significant difference in relapse detection according to Histologies: HD vs Follicular NHL (P ¼ 0.650), HD vs MCL (P ¼ 0.183), Fol vs MCL (P ¼ 0.617),Fol vs DLBCL (P ¼ 0.9). In the CR control group: N ¼ 44/50 there was concordance US vs CT or PET. In N ¼ 3 pts PET was a false positive. In N ¼ 1pts/50 US was positive vs neg CT and the pts relapsed 3 months later. N ¼ 2 pts US revealed increased and dishomogeneous spleen vs normal CT and the pts experienced relapse 2.8 months later. In CR pts US specificity is 100%. Conclusion: In our retrospective study TB-US was helpful to detect lymphoma relapses without a St significant difference in respect to CT and PET/CT. US allows a close follow up, it is radiation-free, easily performed also in renal insufficiency pts in whom CT contrast media may be contraindicated, and is less expensive. Further prospective studies are warranted in order to assess its usefulness in lymphoma patients. Disclosure of Interest: None declared. P322 A Potential opportunity for improving outcomes of allogeneic hematopoietic stem cell transplantation in relapsed / refractory classic Hodgkin lymphoma E. Borzenkova1,*, A. Alyanskiy 1, M. Popova1, I. Osipov1, E. Kondakova1, M. Ivanova1, E. Babenko1, O. Slesarchuk1, A. Kozlov1, S. Bondarenko1, N. Mikhaylova 1, B. Afanasyev1 1 Raisa Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation First Pavlov State Medical University of St. Petersburg, St.-Petersburg, Russian Federation Introduction: The relapsed/refractory classical Hodgkin Lymphoma (r/r-cHL) pts comprise about 30% of overall cHL patient’s population. High-dose chemotherapy followed by autologous stem cell transplantation induces long-term response in 50% of pts. Brentuximab-vedotin (BV) is an antiCD30 drug-antibody conjugate, which allowed achieving good response in majority of r/r-cHL pts. In pts, who had relapse/PD after auto-HSCT, allo-HSCT could be a therapeutic option. The outcome of allo-HSCT in pts with active disease is poor. Most of the pts have relapse or PD of cHL early allo-HSCT before "graft versus cHL" effect appears.
The aim of this study was to estimate efficacy and safety of BV administration before and after allo-HSCT in pts with r/r-cHL. Materials (or patients) and methods: From Jan 2002 to Nov 2014, 37 pts with r/r-cHL have undergone RIC allo-HSCT: Fludarabine/Melphalan or Busulfane (n ¼ 22), Fludarabine/ Bendamustine (n ¼ 13) or Fludarabine/2GyTBI (n ¼ 2). GVHD prophylaxis included calcineurin inhibitors (ICN) and MTX or MMF (from MRD), and ATG (from MUD) or cyclophosphamide, ICN and MMF (from MUD or MMUD). Primary objective was to estimate ORR to BV before alloHSCT and for relapse/PD therapy in the early period after alloHSCT. Secondary objective was to estimate TRM, OS and PFS at 100 days after allo-HSCT and at 180 days after relapse/PD onset. BV was administrated at a dose 1.8 mg/m2 (IV) every 3 weeks. Results: BV as a )bridge* to allo-HSCT, was used in 10 pts with progression of cHL with median number of cycles 6 (1-9). The control group consisted of 27 pts who received CT before alloHSCT with median number of cycles 2 (1-4). Relapse or PD after allo-SCT was observed in 14 pts. Three pts didn’t receive any therapy. BV after allo-HSCT was administrated in 5 pts: BV þ DLI (n ¼ 3), BV þ CT (n ¼ 1), BV alone (n ¼ 1). Control group included 5 pts with CT þ DLI administration after alloHSCT and 1 pts with DLI alone. The ORR of BV administration before allo-HSCT was 60% (including 30% who achieved PET – negative state) vs 38% in CT group. ORR of BV administration after allo-HSCT for relapse therapy ( þ /- DLI/CT) was 60% (3 of 5 pts achieved PET-negative status) vs 16% in CT group. PFS at 100 days after allo-HSCT was 60% in BV group and 48.1% in CT group (P ¼ 0,2). OS at 100 days after allo-HSCT was 100% in BV group vs 66,7% in CT group (P ¼ 0,048). TRM at 100 days after allo-HSCT was 0% in both groups. PFS2 at 180 days after relapse or PD was 60% in BV group and 30% in control group. OS at 180 days after relapse or PD was 80% and 16,7%, respectively. We did not observe toxicity grade 3-4 after BV administration. The pts treated with BV before and after allo-HSCT had the highest rate of overall survival at 2 years after allo-HSCT – 83,3%, followed by pts who received BV only before allo-HSCT – 75%. Two-years OS in CT group was 44,0%. Conclusion: BV has high clinical efficacy in patients with r/rcHL, both before and after allo-HSCT. Use of BV as a )bridge* to allo-HSCT may help achieve optimal disease status, which improves the results of allo-HSCT. BV could be safely administrated for relapse or PD treatment after allo-HSCT and was also associated with better PFS and OS. Prospective studies on BV with allo-HSCT are needed. Disclosure of Interest: None declared. P323 Excellent outcome of reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation in patients with Se´zary Syndrome F. Onida1,*, G. Saporiti2, D. Vincenti2, C. Annaloro2, E. Tagliaferri2, B. Motta2, S. Alberti Violetti3, E. Berti3, A. Cortelezzi1 1 Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico - University of Milan, 2Hematology, 3Dermatology unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Introduction: Patients with Se´zary Syndrome (SS) have a dismal life-expectation, with reported median survival ranging from 24 to 48 months and only 25 % of patients surviving beyond 5 years from diagnosis. Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only curative treatment option for patients with SS, with reduced-intensity (RIC) or non-myeloablative (NMA) regimens as the generally preferred conditioning strategies due to significantly lower morbidity and mortality in comparison to myeloablative conditioning. Materials (or patients) and methods: In our BMT Center, since April 2004 to June 2014 eight patients with refractory SS patients (5 males and 3 females) underwent allo-HSCT after a
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pentostatin/TBI200 NMA or a fludarabine/melphalan RIC regimen. Graft-versus-Host Disease (GvHD) prophylaxis included CsA and MMF ( þ ATG in MUD transplants). Kaplan-Meier survival cumulative probability estimates were calculated from the time of transplant. Results: Median age at the time of transplant was 61 years (range 43-66). Median time from diagnosis to transplantation was 34 months (range 13-252) and median number of previous treatment lines was 6 (range 2-8). Donors were HLA-identical sibling in 2 patients and HLA-matched unrelated in 6 (fully matched in 3 and with at least 1 allelic mismatch in 3). Source of stem cells was peripheral blood in all patients. Full donor chimerism was obtained within 3 months in all patients but one, who eventually experienced a graft failure and autologous hematopoietic reconstitution. Acute GvHD occurred in 3 patients (2 grade I-II, 1 grade III), whereas chronic GvHD was observed in 2 patients, of limited stage in both. Overall, clinical CR was obtained and maintained in 7 out of 8 patients. With a median follow-up of 25 months (range 5-128), all the 8 patients are currently alive with an estimated DFS at 5 years of 87% (Fig.1). Worth mentioning, a complete blood and marrow disease clearance was achieved also in the patient who did not show allogeneic engraftment, possibly as a result of the immunological mechanisms also responsible for the graft failure. Conclusion: In conclusion, although on a limited number of patients, our experience confirm the value of RIC/NMA alloHSCT as a therapeutic strategy for high-risk patients with refractory Se´zary Syndrome, showing an excellent disease-free survival rate and supporting the existence of a clinically relevant graft-versus-lymphoma effect in inducing and maintaining remission. Disclosure of Interest: None declared. P324 Autologous Stem Cell Transplantation in Refractory Hodgkin Lymphoma G. D. Petrova1,*, K. Melkova1, T. Chernyavskaya1, N. Gorbunova1, V. Kostrykina1 1 stem cell transplantation, Federal State Scientific Institution "Russian Cancer Research Center named N.N.Blokhin", Moscow, Russian Federation Introduction: Today the role of single or double Autologous Stem Cell Transplantation (ASCT) in patients (pts) with refractory Hodgkin Lymphoma (HL) is not clearly understood. We evaluated the feasibility and efficacy of single and double ASCT in pts with refractory to induction chemotherapy HL. Materials (or patients) and methods: Between 2007 and 2014 62 pts (34 female) aged 16-52 (median 28) with biopsy
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proved HL who never achieved complete remission (CR) were enrolled in study. All pts received salvage therapy with stem cell mobilization, after which 39/62 (63%) patients achieved disease control (DC) (5CR, 15PR, 19SD) and 23/62 (37%) pts had PD. Stem cell collection was effective in all 62 pts and 26/ 62(42%) pts had stem cell harvest adequate for 2 ASCT. Of the 62 pts 9 (15%) did not receive ASCT. Results: First ASCT was administered to 53 pts (27 female), 10 pts received 2 ASCT. Median follow-up was 3 years (range 1-7). At the time of first transplantation 79% pts had more than minimal disease (Z2 cm), 53% pts had extranodal involvement and 21% pts had B-symptoms. The time interval between diagnosis setting and 1ASCT was 5-124 mo (median 19). Number of previous treatment cycles was 6-27 (median 11), lines - 1-7 (median 3). Induction radiotherapy was performed in 55% pts. Disease status at 1ASCT: 3CR, 15 PR, 17 SD, 18 PD. After 1ASCT DC was achieved in 68% pts (13CR, 8PR, 15SD), 32% pts (17) with PD were not considered to be off the study. Equal number of patients (9 in each group) with PD at 1ASCT had DC (1CR, 1PR, 7SD), and PD. In a period of 3-9 mo (median 6) 10 pts received 2ASCT. At time of 2ASCT equal number of patients - 5/10 (50%) had chemoresistant (2SD, 3PD) and chemosensitive disease (5PR). Results of 2ASCT in pts with PD were very poor: two patients died due to treatmentrelated toxicity and in one case there was further PD. Median overall survival (OS) in a group of 17/53 (32%) pts with PD after 1ASCT was only 1,5 years and didn’t differ in groups with 2ASCT and alternative treatment (P ¼ 0,8). After 2ASCT among 8 alive pts DC was achieved in 88% (5CR, 1PR, 1SD), 1PD; estimated 5-year OS and progression free survival (PFS) were 50% and 38% respectively. Estimated 5-year OS and PFS for all 53 pts were 42% and 26% respectively. Estimated 5-year OS for 23/53(43%) pts with PR/SD after 1ASCT OS was 100% in a group of 6 pts with 2ASCT and 12% in a group of 17 pts without 2ASCT (P ¼ 0,006). Estimated 5-year OS for 13/53 (24%) pts with CR after 1ASCT was 100%.Univariate analysis identified that pts had a superior outcome if they had interval between diagnosis setting and 1ASCT less than 5 years, had chemosensitive disease and less than 14 cycles or 5 lines of previous therapy at time of 1ASCT and achieved CR/PR after 1ASCT. Conclusion: ASCT is an effective treatment option for pts who never achieved CR. Double ASCT proved to be highly effective - according to an intention to treat analysis the number of patients with CR increased from 25% after the 1ASCT therapy to 34% after the 2ASCT. Second ASCT is a treatment of choice for pts with PR/SD after 1ASCT. Second ASCT doesn’t improve outcome of pts with PD after 1ASCT. Alternative variants of treatment are needed for this patient group. Disclosure of Interest: None declared.
P325 Analysis of clinical outcomes and prognostic factors in 191 patients with diffuse large B cell lymphoma who underwent autologous stem cell transplantation at any time H.-J. Shin1,*, D. H. Yoon2, H. S. Lee3, S. Y. Oh4, D. H. Yang5, H. J. Kang6, S. Y. Chong7, Y. Park8, Y. R. Do9, S.-N. Lim10, J.-C. Jo11, W. S. Lee12, M.-K. Song1, J. S. Chung1 1 Pusan National University Hospital, Busan, 2Asan Medical Center, Seoul, 3Kosin University College of Medicine, 4Dong-A University College of Medicine, Busan, 5Chonnam National University Hwasun Hospital, Hwasun, 6Korea Cancer Center Hospital, Seoul, 7Bundang Cha Hospital, Bundang, 8Korea University College of Medicine, Seoul, 9Dongsan Medical Center, Daegu, 10Haeundae Baek Hospital, 11Ulsan University Hospital, 12 Busan Paik Hospital, Busan, Korea, Republic Of Introduction: The exact timing of autologous stem cell transplantation (ASCT) for patients with diffuse large B cell lymphoma (DLBCL), especially with high-intermediate and high IPI, has been still conflicted. Recent randomized trials revealed upfront ASCT improved progression-free survival (PFS), however, the advantage in PFS does not translate in overall survival (OS) benefit. Materials (or patients) and methods: We retrospectively collected and reviewed the clinical data of 191 patients with DLBCL who underwent ASCT at any time. Results: Median age was 50 years (range, 15-65 years) and male to female ratio was 1.16:1. The median follow up duration was 39 months (range, 5-210 months). The estimated 4-year OS rate was 64.0% ± 3.8%. In analysis with disease status at time of ASCT, patients with first complete remission (CR) had excellent OS (4-year OS rate, 79.9% ± 5.8%) (Po0.001). Patients with first partial remission (PR) and second CR (4-year OS rate, 60.3% ± 9.2% vs. 73.8% ± 6.9%) showed similar OS rates. The estimated 4-year OS rate was only 52.9% ± 8.2% in patients with second PR. In the subgroup analysis, for low/low-intermediate risk age-adjusted (AA) IPI patients, first CR/PR revealed favorable OS (estimated 4-year OS rates of 2 groups were more than 90%) while 4-year OS rate of second CR was superior compared to second PR (4-year OS rate, 83% ± 9.0% vs. 44.1% ± 10.9%) (Po0.001). It reflected in patients with low/low-intermediated AA-IPI, it is important to make CR prior to ASCT after relapse. For high-intermediate/high risk AAIPI patients, 4-year OS rate for patients with first CR was 77.0% ± 7.4% compared to those with first PR who had 48.5% ± 11.0% of 4-year OS rates (P ¼ 0.002). Therefore, for patients with high-intermediate/high AA-IPI, achievement of CR before
upfront ASCT is very important to prolong survival. For upfront ASCT, survival graphs showed well-validated as each AA-IPI risk group (Po0.001) while OS was not different between each AA-IPI risk group for salvage ASCT after relapse. This means AA-IPI at diagnosis can predict survival outcome only in patients who underwent upfront ASCT not salvage ASCT. OS was similar between BuCy-based and classic BEAM/BEAC conditioning regimens. Notably, BuCy-based conditioning regimens showed improved OS compared to BEAM/BEAC conditioning regimens in patients who were exposed to rituximab prior to ASCT (4-year OS rate of upfront ASCT, 78.1% ± 6.2% vs. 66.2% ± 14.0%; 4-year OS rate of salvage ASCT, 72.7% ± 9.0% vs. 50.0% ± 17.7%). It shows BuCy containing conditioning regimen may be appropriate compared to classical regimens in R-CHOP era compared to CHOP chemotherapy. Age older than 48.5 years, disease status at time of ASCT, and best response for ASCT revealed independent prognostic factors for DLBCL patients who underwent ASCT at any time. Bulky mass and bone marrow involvement did not impact on survival. Conclusion: In current study, it is suggested that achievement of first CR followed by upfront ASCT is the most important strategy to prolong OS in high risk (high-intermediate/high IPI) patients with DLBCL who is going to undergo ASCT. Disclosure of Interest: None declared. P326 Autologous-stem cell transplantation in Hodgkin’s Lymphoma: the Tuscany experience L. Iovino1, I. Donnini2,*, L. Rigacci2, E. Orciuolo1, B. Puccini2, S. Kovalchuk2, L. Rigacci2, F. Mazziotta1, B. Puccini2, A. Fabbri3, E. Cencini3, M. Bocchia4, M. Petrini1, A. Bosi2 1 Hematology, University of Pisa, Pisa, 2Hematology, University of Florence, Florence, 3Hematology, University of Siena, 4Hematology, University of Florence, Siena, Italy Introduction: Hodgkin Lymphoma (HL) is a very chemosensitive disease. Near a 35% of patients results refractory after a first-line regimen (primary refractory), or relapse after having achieved complete remission. In this cases, high-dose therapy and autologous stem cell transplantation (ASCT) are recommended as salvage treatment. The objective of the this study was to investigate the results of ASCT for HL during recent years and to identify variables predictive for outcome. Materials (or patients) and methods: For this retrospective study, patients aged between 18 and 70 who were treated with a first ASCT between 2009 and 2013 in three Tuscan
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Centers (Firenze, Pisa, Siena) were eligible. Baseline patient’s, disease’s, and transplant’s data were collected from MED-A forms. Centers with potentially eligible patients were contacted to provide additional treatment and follow-up information including a written histopathology report. Statistical univariate analysis used log rank’s test to assess the impact of baseline characteristics on survival endpoints. Results: 32 patients with HL were eligible for this analysis. Fifty-three percent were male, median age was 35 years (range 19-50). Twenty-eight percent of the patients presented with advanced Ann-Arbor stage at diagnosis, and 37% had B symptoms. Twenty-eight percent of the patients received more than two treatment lines prior to transplant. Remission status at the moment of ASCT was complete remission (CR) in 44% of the patients, partial remission (PR) in 41%, and refractory disease in 15%. Second line therapy was a high-dose of ARA-C with a platinum-based drug (oxaliplatinum and cisplatinum) in 47% of patients, IGEV in 47% of patients, 6% received another second line scheme. Response after ASCT was CR in 81% of patients, 9% achieved a PR, 6% of patients did not achieved a response (stable disease or progression). One patients died in first 30 days for transplant-related mortality (TRM). At the last follow-up, 84% of patients were alive. Two of them were alive but relapsed after the achievement of CR. With a median follow-up of 42 months, the 5-years overall survival (OS) and progression-free survival (PFS) for the whole series were 82% and 80%, respectively. The trend of survival curves for OS and PFS are notably identical, as confirmation of the impact of relapse on the OS (Figure 1). Five-year OS and PFS was better in patients who achieved a CR before ASCT, but not statistically significant, whereas the response after ASCT affected distinctly both OS than PFS (Po0,0001). The type of second line (DHA-platinum, IGEV or others) did not affect OS and PS, as the number of previous lines, the Ann-Arbor stage and the presence of B symptoms. Conclusion: In conclusion, this study confirms the feasibility of ASCT in HL and his benefit as salvage therapy, with a role in prevention of relapse. High-dose therapy is effective when the bulk of disease is notably reduced, independently of number and type of previous lines of therapies. New drugs (i.e. Brentuximab-vedotin) can improve the rate of CR sparing the toxicity of a heavy pre-treatments with conventional drugs. Disclosure of Interest: None declared. P327 The influence of rituximab, high dose therapy followed by autologous stem cell transplantation and age in patients with primary CNS-lymphoma M. Madle1, I. Kra¨mer1,*, N. Lehners1, M.-A. Schwarzbich1, P. Wuchter1, G. Egerer1, A. D. Ho1, M. Witzens-Harig1 1 Department of Hematology , University of Heidelberg, Heidelberg, Germany Introduction: For patients with diffuse large B-cell lymphoma without the involvement of the CNS, the addition of rituximab to standard chemotherapy has significantly improved survival. Thus far, there have been no prospective randomized trials evaluating the impact of rituximab as part of the primary treatment of primary CNS-lymphoma (PCNSL). Materials (or patients) and methods: In this single-center, retrospective analysis, a total of 79 PCNSL-patients treated in our institution between 2000 and 2011 were included. Beside first-line chemotherapy with or without rituximab, we evaluated the impact of age (r/4 60 years), autologous stem cell transplantation (ASCT þ /-) and other factors upon overall survival (OS) and progression-free survival (PFS). Results: In patients treated with rituximab (n ¼ 27), 3-year OS was 77.8% (95%4CI: 62-93%). In contrast, in patients treated without rituximab (n ¼ 52), 3-year OS was only 39.9% (CI: 2753%, Figure 2). The difference in OS was significant in the univariate (P ¼ 0.002) as well as the multivariate analysis (P ¼ 0.049, Hazard ratio (HR) ¼ 0.248). In the rituximab group, 80.8% were free of progression at the date of analysis (median
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not reached), whereas median PFS in the group without rituximab was only 17 months (CI: 8-26), (P ¼ 0.001). Patients r 60 years of age (n ¼ 28) had a 3-year OS of 78.2% (CI: 6394%) and a median PFS of 75 months, in patients 4 60 years (n ¼ 51) 3-year OS was 38.7% (CI: 25-52%) and median PFS was 39 months (CI: 6-72). Patients who received high dose therapy and autologous stem cell transplantation (ASCT) had a 3-year OS of 85.2% (CI: 72-99%) and 65.1% were alive up to the time of analysis (range 9-131 months). Without ASCT median OS was only 16 months (CI: 11-21) and 3-year OS was 35.2% (CI: 22-48%). Age and ASCT were significantly associated with better OS in univariate (P ¼ 0.002 and P ¼ 0.001) as well in multivariate analysis (P ¼ 0.004, HR ¼ 0.023 and P ¼ 0.001, HR ¼ 0.014). Conclusion: Rituximab treatment, ASCT and age are independent prognostic factors for overall survival in the first-line treatment of PCNSL. Disclosure of Interest: M. Madle: None declared, I. Kra¨mer Funding from: Gilead Sciences GmbH Germany, N. Lehners: None declared, M.-A. Schwarzbich: None declared, P. Wuchter Employee of: Sanofi and ETICHO, G. Egerer: None declared, A. D. Ho: None declared, M. Witzens-Harig: None declared. P328 Factors predicting early immune recovery after autologous transplantation in patients with non-Hodgkin lymphoma J. Valtola1,*, V. Varmavuo1, A. Ropponen2, H. Kuitunen3, O. Kuittinen3, L. Keskinen4, E.-R. Savolainen5, R. Silvennoinen1, T. Kuittinen1, T. Nousiainen1, J. Pelkonen2, P. Ma¨ntymaa6, E. Jantunen1 1 Department of Medicine, Kuopio University Hospital, 2Department of Clinical Microbiology, University of Eastern Finland, Kuopio, 3Department of Medicine, Oulu University Hospital, Oulu, 4 Department of Oncology, Tampere University Hospital, Tampere, 5Laboratory of Northern Finland, Oulu, 6Laboratory of Eastern Finland, Kuopio, Finland Introduction: Rapid lymphocyte recovery on day 15 (ALC15) after auto-SCT has been associated with improved outcome in patients with non-Hodgkin lymphoma (NHL). The chosen mobilization method affects the graft composition and earlier evidence indicates that various graft constituents may alter the course of immune recovery and outcome post-transplant. Limited prospective data is available assessing the possible factors predicting early immune recovery after auto-SCT. Materials (or patients) and methods: Seventy-one patients with NHL were included into this prospective study. There were 34 males and 37 females with a median age of 62 years. The most common histology was diffuse large B-cell lymphoma (DLBCL) (44%) and 79% of the patients were in first complete or partial remission at the time of the auto-SCT. All patients were mobilized with chemotherapy plus G-CSF. In addition, plerixafor was given for 24 patients. Cryopreserved graft samples were analyzed with flow cytometry for CD34 þ cells, T and B lymphocytes and NK cells. Complete blood counts were evaluated at þ 15 days and 1 month posttransplant and a flow cytometry of lymphocyte subsets (T, NK, B) was performed one month after the graft infusion. Associations of various factors (age, gender, histology, disease status, use of plerixafor or rituximab, graft CD34 þ counts and lymphocyte subsets) to various cut-off levels of ALC15, ALC30 and lymphocyte subsets at 1 month post-transplant were evaluated. Results: Histology of DLBCL (P ¼ 0.006) and the use of plerixafor (P ¼ 0.016) were associated with ALC15 Z0.5 x 109 /L. Also the total number of infused T lymphocytes, the amount of CD3 þ CD4 þ and NK cells in the graft correlated with ALC15 Z0.5 x 109 /L. ALC30 Z1.0 x 109/L was associated with prior rituximab use and the number of CD3 þ (P ¼ 0.024) and CD3 þ CD4 þ cells (P ¼ 0.018) in the graft. Blood CD3 þ CD4 þ level of Z0.3 x 109/L (lower normal range) at one month was associated with the number of CD3 þ CD4 þ cells in the graft (P ¼ 0.022). The NK cell level of Z0.2 x 109 /L in the
blood at one month was associated with male gender (P ¼ 0.005), the use of plerixafor (P ¼ o0.001) and the amounts of CD3 þ (P ¼ o0.001), CD3 þ CD4 þ (P ¼ 0.001), CD3 þ CD8 þ (P ¼ 0.002) and NK cells (P ¼ o0.001) in the infused grafts. Conclusion: The number of CD34 þ cells infused does not affect the early immune recovery. The amount of various lymphocyte subsets in the grafts seems to be of greater importance. Patients receiving plerixafor after chemomobilization appear to have more rapid immune recovery especially if defined with ALC15 Z 0.5x 109/L or NK cells Z 0.2 x 109/L at 1 month post-transplant. The potential effects of early immune recovery to post-transplant infectious complications and longterm outcome will be evaluated in the ongoing GOA study (Graft and Outcome in Autologous stem cell transplantation). Disclosure of Interest: J. Valtola: None declared, V. Varmavuo: None declared, A. Ropponen: None declared, H. Kuitunen: None declared, O. Kuittinen Employee of: Received lecture fees from Roche and Mundipharma, L. Keskinen Employee of: Received lecture fees from GlaxoSmithKline, E.-R. Savolainen: None declared, R. Silvennoinen Employee of: Received lecture fees from Sanofi, T. Kuittinen: None declared, T. Nousiainen: None declared, J. Pelkonen: None declared, P. Ma¨ntymaa: None declared, E. Jantunen Employee of: Received lecture fees from Genzyme and Sanofi, Conflict with: Participated in EU Leadership meeting organized by Genzyme as well as Medical Advisory Board meeting organized by Genzyme. P329 The Efficacy of CD34 cell mobilization regimens in patients with lymphoma and myeloma at a single institution J. Romejko-Jarosinska1,*, P. Lidia1, L. Targonski1, M. Szymanski1, E. Mroz Zycinska1, Z. Pojda1, J. Walewski1 1 Maria Sklodowska Curie Memorial Cancer Centre, Warsaw, Poland Introduction: The outcome of autologous hematopoietic cell transplantation (auto-HCT) is essentially related to CD34 cell dose with the generally accepted minimum of 2x10^6 per kg of patient body weight. Patients who are unable to reach this threshold are usually deprived of this potentially curative procedure. We retrospectively evaluated efficacy of a number of mobilization procedures at our institution and analyzed factors predicting the poor harvest Materials (or patients) and methods: We reviewed reports of the 220 mobilization procedures in a total of 202 adult patients with Hodgkin lymphoma (48), DLBCL (51), mantle cell lymphoma (37), myeloma (43), and other lymphoma subtypes (21), who were planned for auto-HCT between January 2012 and November 2014. Median (range) age was 51 (19-69), and 47 patients were 60 y/o or more. The following mobilization regimens were used: G-CSF þ high dose cyclophosphamide (n ¼ 97), G-CSF þ high or intermediate dose cytarabine (n ¼ 96), G-CSF þ ICE (n ¼ 19), G-CSF alone (n ¼ 5). In addition, plerixafor was used in 21 patients. 90 patients were mobilized after first-line treatment (41%). Effective mobilization was defined as a count of 2.0x10^6/kg CD34 cells. Results: Overall, 69% of patients had a peak of 410/ml CD 34 cells in blood and 29% had 430/ml. Sufficient number of CD 34 cells were collected in 76% of patients, including 39% in first apheresis. Majority of CD34 cells were collected in the first (43%) and in the second (39%) day of apheresis. There was no difference in successful mobilization between myeloma and lymphoma patients. The use of platinum based regimen in previous treatment, number of days on G-CSF more than 10, and CD34 cell count less than 5/ml on the first day of collection correlated to poor mobilization in univariate analysis. There was no difference in apheresis outcome between chemotherapy regimens used for mobilization. 18 of 48 poor mobilizers had a second mobilization. Eleven of them collected sufficient CD34 cell count. Only 11 of 21 patients who received plerixafor reached a threshold CD 34 cell harvest.
Conclusion: More than 24% of patients needed a second mobilization cycle. Number of G-CSF administration days, maximum peripheral CD34 cell count, and platinum treatment were predictive for poor harvest. Chemotherapy regimen selection was non-predictive for the harvest Disclosure of Interest: None declared. P330 Chemosensitivity and not histology determines outcome of allogeneic stem cell transplantation in patients with advanced-phase lymphoma J. Apostolidis1,*, F. Panitsas1, S. Gigantes1, I. Baltadakis1, K. Kaisari1, V. Pardalis1, I. Markou1, M. Gonianaki1, E. Xenou1, K. Xirokosta1, T. Loidoris1, M. Bouzani1, T. Karmiris1, D. Karakasis1, N. Harhalakis1 1 Department of Hematology, BMT Unit, Evangelismos Hospital, Athens, Greece Introduction: Patients with relapsed/refractory (R/R) Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL) have poor prognosis. Allogeneic stem cell transplantation (allo-SCT) is a potentially curative strategy, albeit at the cost of significant mortality. However, its application has been facilitated over the last 15 years with the use of reduced-intensity conditioning (RIC) regimens. The aim of this study is to evaluate the outcomes of allo-SCT in a recent cohort of patients with advanced phase HL/NHL from a single institution. Materials (or patients) and methods: Between 01/2001 and 10/2013, 73 consecutive patients, aged 21-65 (median: 41) years, received allo-SCT at our center. Diagnoses were aggressive NHL in 17 patients (23%), indolent NHL in 34 (47%) and HL in 22 (30%). Patients had previously received a median of 3 lines of chemotherapy (range: 1-8), while 30 (41%) had relapsed after autologous transplant (ASCT). At transplant, 18 (25%) were in complete remission (CR) and 35 (48%) had chemosensitive disease (1st/2nd partial remission, Z 3rd CR), whereas the disease was chemorefractory in 20 (27%). Donors were HLA identical siblings in 36 (49%) cases, unrelated in 35 (48%) and umbilical cord blood in 2 (3%). Reduced intensity conditioning (RIC) was used in 60 (82%) cases, mainly a Fludarabine/Melphalan regimen. Results: With a median follow-up of 38 months (range: 4-118), 3-year overall survival (OS), 3-year progression-free survival (PFS) and 3-year relapse rate (RR) were 47% [95% CI: 34-58%], 41% [95% CI: 29-52%], and 32% [95% CI: 21-43%], respectively. The 3-year OS for patients with aggressive NHL, indolent NHL and HL were 37%, 52%, and 46%, respectively. Non-relapse mortality (NRM) was 22% at 1 year. The incidence of acute graft-versus-host disease (GVHD) was 46% (grade II-IV: 32%), and of chronic GVHD 66% (extensive: 49%). In multivariate analysis, age, donor type, and histology did not have any prognostic impact on outcome. On the other hand, disease status at transplant (chemorefractory versus CR/chemosensitive disease) was the only significant factor influencing OS [HR: 1.77 (95% CI: 0.9-3.3), P ¼ 0.07] and disease-free survival [HR: 1.89 (95% CI: 1.0-3.5) P ¼ 0.04]. Conclusion: Allo-SCT may offer prolonged PFS in about 40% of patients with advanced-phase HL/NHL with an acceptable risk of NRM in the era of RIC regimens. Referral of patients prior to the development of chemoresistance is critical for the successful outcome of allo-SCT. Moreover, novel agents (brentuximab vedotin, ibrutinib, PD-1 inhibitors) may serve as a bridge to overcome chemoresistance and improve outcome in this patient population. Disclosure of Interest: None declared.
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P331 Simultaneous inhibition of CXCR4 and VLA-4 exhibits combinatorial effect in overcoming stroma-mediated chemotherapy resistance in mantle cell lymphoma cells K.-S. Eom1,2,*, Y.-R. Kim3, S. Lee1 1 Hematology, Catholic Blood & Marrow Transplantation Center, Seoul St. Mary’s Hospital, The Catholic University of Korea, 2 Cancer Research Institute in the Catholic University of Korea, 3 Cancer Research Institute in teh Catholic Univeristy of Korea, Seoul, Korea, Republic Of Introduction: Mantle cell lymphoma is a rare, aggressive and distinct subtype of non-Hodgkin’s lymphoma. Despite substantial efforts to improve clinical outcomes, DFS remains low due to MRD. Cells constuting MRD have capacity to find refuge in protective microenvironments and the selective pressure of chemotherapy promotes the outgrowth of MRD. CXCR4/ CXCL12 axis has been considered as a major contributor to environment-mediated drug resistance (EMDR). Cell adhesion mediated by VLA-4 confers protection against chemotherapy in various cancers. The interactions between CXCR4 and VLA-4 constitute a 2-way pathway where both receptors work cooperatively. Therefore, it is expected that the combined
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blockade of both CXCR4 and VLA-4 is more effective in overcoming EMDR than soley blocking the respective receptor. Here, the combined use of specific Abs for both receptors was tested to see the effect on EMDR. Materials (or patients) and methods: MCL cell line Jeko-1 and the murine stromal cell line M2-10B4 were used. Chemotaxis and migration inhibition assay were performed using Transwell inserts. Purified NA/LE mouse anti-human CD184, FITC anti-human CD49d and FITC mouse anti-human CD45. For migration inhibition experiments, anti-CXCR4 Ab and anti-integrin alpha 4 Ab azide free for VLA-4 were purchased. Results: Strong expression of both CXCR4 and VLA-4 were observed. Chemotaxis was significantly inhibited by antiCXCR4 Ab. Pseudo-emperipolesis of Jeko-1 pre-treated with anti-VLA4 antibody was markedly reduced. Combined use of both antibodies (anti-CXCR4 and anti-VLA-4 antibody) reduces migration of MCL cells in a combinatorial manner. Blocking antibodies could reverse the protective effect of MSC on MCL cells. These effect was most prominent when Abs were used simultaneously, demonstrating a combinatorial effect on inducing apoptosis (Figure 1). Both antibodies resulted in decreased phosphorylation of downstream signaling proteins
(ERK1/2, AKT, NF-kB). The reduction in phosphorylation of the proteins was most prominent when the cells were treated simultaneously with both Abs. Combined use of the Abs induces a combinatorial increase in apoptosis in the upper chamber of Transwell insert, while the migrated cells in the lower chamber, and in turn, adhered to MSCs or underwent pseudoemperipolesis demonstrated marked decrease in apoptosis. Conclusion: The combined blockade of CXCR4 and VLA-4 resulted in combinatorial effects in reducing migration and inducing apoptosis of MCL cells. These findings are associated with reduced phosphorylation of downstream signaling proteins. The result of our study may provide a basis for future development of therapeutic tools targeting both CXCR4 and VLA-4 such as bispecific antibody to improve treatment outcome of MCL. Disclosure of Interest: None declared. P332 Radioimmunotherapy and BEAM chemotherapy versus BEAM as the conditioning regimen for autologous stem cell transplantation (ASCT) in relapsed follicular lymphoma (FL): a retrospective matched-control study of the LWP-EBMT L. Bento1,*, A. Boumendil1, H. Finel1, P. Chevallier1, S. Amorim1, H. Monjanel1, D. Blaise1, J.-O. Bay1, E. Nicolas-Virelizier1, G. McQuaker1, G. Rossi1, R. Johnson1, A. Huynh1, N. Fegueux1, A. Rambaldi1, G. Salles1, C. Craddock1, K. Orchard1, D. Pohlreich1, H. Tilly1, G. Bandini1, X. Poire´1, F. Guilhot1, A. Haenel1, C. Crawley1, B. Metzner1, J. Gribben1, N. H. Russell1, G. Damaj1, K. Thomson1, P. Dreger1, S. Montoto1 on behalf of EBMT Lymphoma Working Party, Paris, France 1 EBMT, Paris, France Introduction: Relapse after transplant remains the most common cause of treatment failure in patients receiving an autologous stem cell transplant (ASCT) for recurrent follicular lymphoma (FL). We hypothesized that the incorporation of radioimmunotherapy (RIT) in the conditioning regimen of ASCT might reduce the relapse rate and improve the outcome in patients with FL. Materials (or patients) and methods: Between 2004 and 2012, 2359 patients (57% male, median age at ASCT: 56– range:19-77) who had an ASCT for relapsed FL with BEAM conditioning regimen with or without RIT (Ibritumomabtiuxetan: Zevalins) were reported to the EBMT and included in this study. Z-BEAM (n ¼ 207), R-BEAM (n ¼ 179) and BEAM
(n ¼ 1973) were analysed excluding addition of other drugs. We conducted a matched-cohort analysis of BEAM versus Zevalins-BEAM (Z-BEAM) including 282 and 154 patients, respectively. R-BEAM cases were not considered for this analysis. The variables used to match the pairs were age, time from diagnosis to transplant, gender, disease status at ASCT, performance status (PS) and year of transplant. Overall survival (OS) and event-free survival (EFS) were determined using the Kaplan-Meier method, and curves were compared by log-rank test.In the matched sample, outcome in BEAM vs Z-BEAM were compared using a Cox model stratified on matched pairs. Results: The matched-cohort analysis of BEAM versus Z-BEAM was described. The majority of the patients had a good PS at transplant (KarnofskyZ80% in 96% and 94% of patients receiving BEAM and Z-BEAM, respectively) and were transplanted in good response (at least sensitive relapse in 96% of patients receiving BEAM and in 94% of the Z-BEAM cohort). After a median follow-up of 18.7 months (range: 1-85.7), 25% of patients in the BEAM group relapsed, in comparison with 34% in the Z-BEAM cohort. Forty (14%) and 20 (13%) patients died in the BEAM and Z-BEAM cohorts, respectively. There were no significant differences in non-relapse mortality (HR: 0.5 [95%CI: 0.13-1.93]; P ¼ 0.3), nor on cumulative incidence of relapse (HR ¼ 1.55 [0.96-2.51]; P ¼ 0.07). Similarly, no significant differences between BEAM and Z- BEAM cohorts were found in OS (HR ¼ 0.77 [CI95%: 0.41-1.46]; P ¼ 0.43) or EFS (HR ¼ 1.34 [0.86-2.08]; P ¼ 0.2). Conclusion: Based on the matching factors used here, this study failed to show a benefit of adding RIT to BEAM in ASCT for relapsed FL. However, the limitations of registry analyses have to be taken into account, and prospective trials are needed for definite information. Disclosure of Interest: L. Bento Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Boumendil Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, H. Finel Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, P. Chevallier Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, S. Amorim Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, H. Monjanel Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, D. Blaise Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, J.-O. Bay Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, E. Nicolas-Virelizier Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany,
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G. McQuaker Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Rossi Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, R. Johnson Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Huynh Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, N. Fegueux Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Rambaldi Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Salles Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, C. Craddock Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, K. Orchard Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, D. Pohlreich Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, H. Tilly Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Bandini Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, X. Poire´ Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, F. Guilhot Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, A. Haenel Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, C. Crawley Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, B. Metzner Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, J. Gribben Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, N. H. Russell Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, G. Damaj Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, K. Thomson Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, P. Dreger Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany, S. Montoto Conflict with: Supported by an unrestricted grant by Sepropharm GmbH, Munich, Germany. P333 Autologous-stem cell transplantation in diffuse-large B cell lymphoma: the Tuscany experience L. Iovino1,*, I. Donnini2, L. Rigacci2, B. Puccini2, S. Kovalchuk2, E. Orciuolo1, F. Mazziotta1, A. Fabbri3, E. Cencini3, M. Bocchia3, M. Petrini1, A. Bosi2 1 Hematology, University of Pisa, Pisa, 2Hematology, University of Florence, Florence, 3Hematology, University of Siena, Siena, Italy Introduction: Patients with diffuse-large B cell lymphoma (DLBCL, NOS) who doesn’t achieve a complete remission (CR) after a first-line chemotherapy regimen, young high-risk
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patients, and patients who relapsed after CR, are candidate to high-dose therapy and autologous stem cell transplantation (ASCT). The objective of the present study was to investigate the results of ASCT for DLBCL during recent years in Tuscany network. Materials (or patients) and methods: For this retrospective study, patients aged between 18 and 70 who were treated with a first ASCT between 2009 and 2014 in three Tuscan Institutions (Firenze, Pisa, Siena) with complete follow-up data were eligible. Baseline patient’s, disease’s, and transplant’s data were collected from MED-A forms. Statistical univariate analysis used log rank’s test to assess the impact of baseline characteristics on survival endpoints. Results: thirty patients with DLBCL were eligible for this analysis. 57% were male, median age was 41 years (range 2161). Forty-seven percent of the patients presented an advanced Ann-Arbor stage at diagnosis, and 27% had B symptoms. 27% of the patients received 3 or more treatment lines prior to transplant. Remission status at ASCT was CR in 43% of the patients, PR in 50%, and refractory disease in 7%. Second line therapy was a high-dose of ARA-C with a platinum-based drug in 66% of patients. Response after ASCT was CR in 22 patients (73%). Two patient achieved a PR, and two patients did not response. Three patients (10%) died in first three months for transplant-related mortality (TRM), and were not included in survival analyses. At the last follow-up, 23 patients (77%) were alive. Two of them (7%) relapsed. With a mean follow-up of 81 months, the 5-years overall survival (OS) and progression-free survival (PFS) for the whole series were 75% and 68%, respectively. Five-year OS and PFS was significant better in patients who achieved a CR after ASCT (p 0,005) (Figure 1). The type of second line did not impact on OS and PFS. Univariate analysis showed a significant trend (p 0,026) in PFS in favor of patients who achieved at least a PR pre-ASCT, whereas no differences were observed in OS for this variable. Other variables did not show a significant prognostic value. Conclusion: this study confirms the role of ASCT in DLBCL, and his benefit as salvage therapy, with a role in prevention of relapse and an high percentage of conversion in CR since PR. High-dose therapy is effective when the disease still chemosensitive. Disclosure of Interest: None declared. P334 Allogeneic stem cell transplantation for relapsed or refractory Hodgkin lymphoma: a better therapeutical perspective M. M. Greco1,*, A. M. carella1, E. Merla1, N. Cascavilla1 1 Hematopoietic Stem Cell Transplantation Unit, IRCSS Casa Sollievo Della Sofferenza, San Giovanni Rotondo (FG), Italy Introduction: The current standard of care of relapsed or resistant Hodgkin lymphoma is a salvage chemotherapy
consisting of a conclusive myeloablative chemotherapic regimen with autologous stem cells transplantation (Auto-SCT), if indicated followed by radiotherapy. Patients with relapsed or refractory disease after autologous bone marrow transplantation have a poor survival and the management of the disease is challenging. In the last decade, several factors have refined the role of allogeneic transplantation such as reduced intensity conditioning (RIC) transplantation, donor lymphocytes infusion, biologically based treatments, and monoclonal antibody drug-coniugate anti-CD30 Bretuximab Vedotin. Materials (or patients) and methods: Between 2007 and 2014 14 patients whose disease progressed or relapsed after Auto-SCT underwent an allogeneic RIC stem cell transplantation (RIC Allo-SCT). Four patients were male and ten female, median age was 29 years and age range was 25-40 years. Disease status at SCT was complete remission (CR) (n ¼ 5; 35%), partial remission (n ¼ 6; 45%) and progression disease (PD) (n ¼ 3; 20%). The median time from Auto-SCT to Allo-SCT was 6 months (range 2-8). The conditioning regimen was FluMel (n ¼ 1), BU-Flu (n ¼ 4) and Thio-BU-Flu (n ¼ 9). The donor was an HLA-identical sibling (n ¼ 4), a matched unrelated donor (MUD) (n ¼ 4) or a familiar aploidentical (n ¼ 6). Graft versus host disease (GVHD) profilaxis consisted of Cyclosporin (CSA) and Methotrexate (MTX) short-course in simbling transplants, CSA þ MTX and rabbit ATG in MUD, Cyclofosphamide 50 mg/kg day þ 3, þ 4, associated to CSA þ Mycophenolate by day þ 5. The Bretuximab Vedotin was utilized in 5 patients from 2013. Results: All patients engrafted and the median of neutrophils recovery was 14 days. Acute GWHD Z2 was documented in 6/ 14 patients (43%). 7/14 patients dead, the cause of death was infectious disease in 2 patients, respectively located in lung and CNS. 2 patients affected by chronic GVHD dead because of congestive heart failure and obliterans bronchiolytis and 3 patients dead because of the progression of the disease. The last 3 patients were primary refractory with Bulky disease. 7 patients (50%) are alive in CR. Conclusion: We conclude that Bretuximab Vedotin can induce CR or high citoreduction promoting a favorable outcome of transplantation. The early and late TRM is similar to that one detected in other hematological malignancies. The Bulky and the primary refractory disease represent a biological barrier of the tumour and its microenvironment to allogeneic activity of transplantation. Disclosure of Interest: None declared. P335 Differences in the outcome of Hodgkin’s lymphoma treated with high dose chemotherapy and autologous stem cell transplantation M. R. Todorovic1,*, B. Balint2, D. Stanisavljevic3, B. Andjelic 4, J. Bila1, D. Antic4, D. Vujuic5, N. Kraguljac Kurtovic4, D. Sefer4, J. Jeleicic4, B. Mihaljevic1 1 Medical faculty University of Belgrade, Clinic for hematology CCS, 2Institute for transfusiology and Hemobiologyof MMA, 3 Institute for statistics, Medical faculty University of Belgrade, 4 Clinic for hematology CCS, 5Medical faculty University of Belgrade, Belgrade, Serbia
regimen in first ASCT was BEAM. Second ASCT performed with CBV regimen in five patients relapsed after first transplantation. Results: Data on 80 patients who received ASCT between Jan 2006 and Dec 2013 were reviewed. Median time from first treatment to ASCT was 2.6 years (0.8-8.4). Demography at ASCT was 62.5% stage III and IV, median age 29.8 (18-47), median prior treatment regimens 3 (2-7), median Hasenclever index 3 (0-6); Initially, 43 patients had responding disease [20 (25%) complete remission (CR), 23 (28.7%) partial remission (PR)], and 37(46.3%) patients had stable disease/progressive disease. Post-first ASCT, an overall response rate (CR þ PR) was 76.2% with CR (38.7%) and PR (37.5%). Mean time to second ASCT was 18.6 (3-39) months. Post-second ASCT all 5 patients upgraded their response, with two consecutive CR after brentuximab vedotin, one CR after radiotherapy, and two after chemotherapy. Median overall survival (OS)/progressionfree survival (PFS) from first ASCT was 105 months/51months. Nineteen patients died due to progressive disease at the end of follow up period. Patients with initial CR 4 12 months had double longer OS, than patients with CRo12 months (110 months vs. 60 monts). Positive impact on OS had initial Hasenclever indexo3, CR after first line therapy, attaining CR at D þ 100 after ASCT, while number of CD34 þ peripheral blood stem cells 4 5.5x10e9/L had positive impact on PFS after ASCT. Multivariate Cox regression defined that independent prognostic factor for OS was favorable therapeutic outcome at D þ 100 (P ¼ 0.001). Conclusion: Although, the results of ASCT vary significantly depending on a number of prognostic factors, it is the standard of care for patients with relapsed HL and leads to a better outcome improving overall response rate for 22.5%. Disclosure of Interest: None declared. P336 Outcome of autologous stem-cell transplantation in Hodgkin and non hodgkin lymphoma patients N. Soyer1,*, C. Atmaca Mutlu2, F. Vural1, M. Tombuloglu1, M. Tobu1, F. Sahin1, G. Saydam1 1 Hematology, 2Internal Medicine, Ege University Medical Faculty, IZMIR, Turkey Introduction: Autologous hemopoetic stem cell transplantation (aHSCT) can offer potential long-term remission or cure in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Materials (or patients) and methods: One hundred eight NHL and 56 HL patients treated with autologous stem-cell transplantation in Ege University Hospital from January 2008 through December 2014 for relapsed or refractory HL and NHL were analyzed retrospectively.
Introduction: Hodgkin’s lymphoma (HL) is highly curable disease to conventional chemotherapy. Even though, some patients are primary refractory or relapsed after first-line chemotherapy. For them autologous stem cell transplantation (ASCT) is the standard of care. The results of ASCT vary significantly depending on a number of prognostic factors. The purpose of this study was to assess prognostic factors and outcome of patients with relapsed/refractory HL who received high-dose chemotherapy and ASCT. Materials (or patients) and methods: Patients with relapsed or refractory HL who were initially treated with ABVD therapy, were analyzed. In the course of relapse or refractory disease, all patients received DHAP as first salvage, six patients received ICE as second salvage therapy, while conditioning
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Results: Data of the 164 lymphoma patients treated with aHSCT were evaluated retrospectively. Median age of HL and NHL was 40 years (22-75 years) and 53 years (19-76 years) respectively. Forty one (%38) patients were female, 67 (62%) were male from all of 108 NHL patients. 20 (35%) were female, 36 (65%) were male from all of 56 HL patients. Transplantation related mortality was 0.8% (13 of 164 patients;1 HL, 12 NHL). Complete remission and partial remission was obtained in 37 (67%) and 9 (16%) of HL; 52 (48%) and 25(23%) of NHL. Refractory disease was in 9 (16%) in HL and 19 (17%) in NHL. After a median follow-up of 13.5 months (range, 3- 110 months), 55 of 108 (50.9%) patients with NHL were alive. The estimated 5 year OS was % 50 for NHL patients. After a median follow-up of 62 months (range, 12180 months), 46 of 56 (82.1%) patients with HL were still alive. The 5-year overall survival (OS) were 86% for HL patients. Progression free survival of HL and NHL patients was shown Figure 1. Conclusion: Autologous stem-cell transplantation remains to be the standard treatment of relapsed or refractory HL and NHL patients. Prognosis of relapsed/refractory HL and NHL patients ineligible for autologous stem-cell transplantation and treated with several lines of standard chemotherapy ± radiotherapy is poor. Improving the treatment of patients at high risk of failure at diagnosis, evaluation of new chemotherapeutic agents, further development of immunotherapy and radioimmunoconjugates require further study. Disclosure of Interest: None declared. P337 Results of 10 years high dose chemotherapy with Tutshka protocol followed by autolougous stem cell transplantation in diffuse large B cell lymphoma R.-M. Hamladji1,*, N. Ait Amer1, M. Benakli1, R. Ahmed Nacer1, A. Talbi1, F. Harieche1, F. Mehdid1, R. Belhadj1 1 Hematology-Bone Marrow Transplantation Department. Pierre and Marie Curie Center, Algiers, Algeria Introduction: Despite numerous studies on the long term outcome of patients (pts) with diffuse large B cell lymphoma (DLBCL) treated with high dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) choosing the best effective HDT protocol is not yet known. This study reports the results of 10 years Tutshka protol applied to pts with DLBCL. Materials (or patients) and methods: Between January 2004 and December 2013, 94 DLBCL pts including 28 mediastinal large cell lymphoma pts (MLCL) underwent, before ASCT, HDT with Tutshka protocol that combines oral Busulfan (16 mg/Kg) and intra-venous cyclophosphamide (120 mg/Kg), intra venous Etoposide (30 mg/Kg) have been added since 2012. Median age is 37 years ( 17 to 65) and sex ratio (M/F) 1.2. For the 28 pts with MLCL median age is 31 years ( 17 to 56), sex ratio: 0.6 and their pre transplant disease status as follow: 14 pts in complete remission (CR) and 14 pts in partial remission (PR) after 2 to 3 lines of treatment. For the others 66 DLBCL pts the median age is 45 years (17 to 65), sex ratio:2.7 and their pre transplant disease status: 44 pts in CR within 10 pts after 2 to 3 lines of treatment and 22 pts in PR. All pts receive, after thawing, mobilized peripheral blood stem cells obtained by G-CSF mobilization (15mg/Kg/d, 5 days) alone and froze in liquid nitrogen. The median rate CD34 þ cells infused is 3.14 x 106/Kg (0,87-17,36). The median follow-up at 10/30/ 2014 is 48 months (4-120). Results: The median time to graft (PNC 4 0.5 x 109/l) was 12 days (10- 60). Five early deaths were observed including 4 infection (TRM: 4%) and one in disease progression at 3 months. After 3 months of 89 evaluable pts (27 MLCL and 62 others DLBCL) relapse was observed in 28/89 pts (22.5%) and it was earlier relapse in a period of 24 months in 15 pts (75%).
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Deaths were among 23/94 pts (24.5%): MLCL 10/28 (36%) and others DLBCL 13/66 (20%) with one very late death after second relapse at 101 months. Persistent CR was achieved in 66/89 pts (74%) including 16/27 (59%) MLCL and 50/66 (75.5%) others DLBCL. The overall survival (OS) and event free survival (EFS) at 10 years are respectively 66% and 59.5%. In terms of OS and EFS there is no difference between MLCL (60% and 57.5% respectively) and others DLBCL (66% and 72% respectively). Conclusion: Our results show that Tutshka protocol is well tolerated and is an alternative in DLBCL including MLCL. Disclosure of Interest: None declared. P338 Hodgkin lymphoma with early relapse (3-12 months) behaving clinically similar to primary refractory Hodgkin lymphoma (primary progressive: partial response: relapse within three months). Single institution analysis of 201 patients S. Akhtar1,*, M. S. Rauf1, T. A. Elhassan1, I. Maghfoor1 1 Oncology Center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia Introduction: The outcome of high dose chemotherapy (HDC) and auto-SCT in primary refractory Hodgkin lymphoma (PR-HL) is not as encouraging as in relapsed HL. Patient relapsing between 3-12 months (relapse 3-12) also have worse outcome. We compared HDC auto-SCT in PR-HL against relapse 3-12 group. Materials (or patients) and methods: From 1996 to 2013, patients with HL who received HDC auto-SCT for PR-HL and for relapse 3-12 were identified. PR-HL is defined as partial response (PR), no response (NR), stable disease (SD), progressive disease (PD), relapsing within 3 months (relapse 0-3) of finishing the planned treatment. Competing risk analysis method was used to estimate disease specific death (DSdeath) and disease specific event (DS-event) from the day 0 of auto-SCT. Results: Male 100 (50%), female 101 (50 %), Median age at diagnosis was 22.2 years (12-61 years) and at ASCT was 24 years (13.8-62 years). Initial chemotherapy was ABVD in 168 (84%), MOPP/COPP alternating with ABV or ABVD in 17 (8%), median chemotherapy cycles were 6. 58 patients (29%) also had radiation therapy (XRT) after initial chemotherapy. Response to chemo þ XRT was PR in 78 (39%), PD in 48 (24%), relapse 0-3 in 25 (12%) and relapse 3-12 in 50 (25%). ESHAP as first line salvage in 178 (89%); median cycles given were 3. Post salvage / prior to HDC auto-SCT disease status was PR in 126 (63%), CR in 61 (30%) and stable disease/no response in 14 (7%). BEAM was used as HDC. Median follow-up is 64 months from auto-SCT. Post auto-SCT overall response in 140 patients (70%); CR in 123 (61 %), PR in 17 (8.5 %), NR / SD in 1 (0.5%) and PD in 50 (25%) patients, unknown in 9 (4.5%). 66 patients had XRT post auto-SCT. Type of first event was no event in 94 (47%), persistent disease in 18 (9%), progressive disease in 50 (25%), relapsed disease in 28 (14%), treatment related mortality in 7 (3.5%) and died of other cause 4 (2%). At last follow-up in December 2014, 102 patients (51%) are alive with no disease, 14 (7%) alive with disease, 71 (35%) died of disease and 14 (7%) died of treatment related mortality or other causes. For whole group estimated cumulative risk of DS-death (36%) and DS-event (47%) is shown in Table and Figure. Factors Whole group Progressive disease Partial response Relapse 0-3 months Relapse 3-12 months
Total N DS-Death % P-value DS-Event % P-value 201 48 78 25 50
36 40 36 34 32
x 0.4
47 52 51 44 36
x 0.1
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Conclusion: In patients with all types of PR-HL and those with relapse 3-12, ESHAP þ BEAM combination results in high CR rate. Despite this high CR rate, one third of patients died of disease post auto-SCT. There is no statistically significant difference in various PR-HL types and relapse 3-12 group. Relapse 3-12 has high incidence of disease specific deaths and events. This cohort is behaving more like a PR-HL and would be more appropriate to be referred as refractory HL in future studies for better management and outcome. Disclosure of Interest: None declared. P339 Outcome of HDC auto-SCT in adolescent and young adults (AYA) patients with Hodgkin lymphoma: Primary refractory vs early relapse (3-12 months) vs relapse after 12 months: A single institution result of 216 patients S. Akhtar1,*, M. S. Rauf1, T. A. Elhassan1, I. Maghfoor1 1 Oncology Center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia Introduction: The outcome of high dose chemotherapy (HDC) and auto-SCT in adolescent and young adults (AYA) is not commonly reported separately. Outcome in primary refractory Hodgkin lymphoma (PR-HL) and relapsing between 3-12 months (relapse 3-12) is not as encouraging as in relapsed HL after 12 months (relapse 412). We are reporting HDC auto-SCT results in PR-HL vs relapse 3-12 vs relapse 412 in AYA. Materials (or patients) and methods: From 1996 to 2013, all adolescent (14-21 years) and young adults (421 to 30 years) (at the time of auto-SCT) patients with HL and HDC auto-SCT were identified. PR-HL is defined as partial response (PR), no response (NR), stable disease (SD), progressive disease (PD), relapsing within 3 months (relapse 0-3) of finishing the planned treatment. Competing risk analysis method was used to estimate disease specific death (DS-death) and disease specific event (DS-event) from the day 0. Results: Male 107 (49.5%), female 109 (50.5 %), Median age at diagnosis 19 years (5-29) and at ASCT 22.4 years (13.8-30). Adolescent 94 (43.5%), young adults 122 (56.5%). Initial chemotherapy was ABVD in 176 (81%), MOPP/COPP alternating with ABV or ABVD in 20 (9%), median chemotherapy cycles were 6. Sixty-eight patients (31%) also had XRT after initial chemotherapy. Reason for HDC auto-SCT: refractory disease in 129 (60%), relapse 3-12 in 33 (15%) and relapse 412 in 54
(25%). ESHAP was used as first line salvage in 181 patients (84%) and in another 20 (9%) as 2nd line salvage; median 3 cycles (3 in 63% and 4 in 14%). Post salvage / prior to HDC auto-SCT disease status was PR in 132 (61%), CR in 73 (34%) and stable disease/no response in 11 (5%). BEAM was used as HDC. After auto-SCT, overall response in 157 patients (73%) (CR in 140 (65 %), PR in 17 (8%)), NR/SD in 2 (1%) and PD in 48 (22%) patients, unknown in 9 (4%). 71 (33%) patients had XRT after auto-SCT. After auto-SCT in 26 (12%) patients had relapsed disease, treatment related mortality in 5 (2%) and died of other cause 2 (1%). Median post auto-SCT follow-up is 63 months. Currently 123 patients (57%) are in CR, 14 (7%) alive with disease, 70 (32%) died of disease. For whole group estimated risk of DS-death (33%) and DS-event (45%) and according to PR-HL vs relapse 3-12 vs relapse 412 is shown in Table and Figure. Factors
Total N
DS-Death %
Whole group Adolescent (14-21 yrs) Young adults (421-30 yrs) Primary refractory Relapse 3-12 months Relapse after 12 months
216 94 122
33 32 34
119 37 60
43 32 12
Pvalue 0.7 0.0001
DS-Event % 45 49 41 57 35 27
P-value
0.4 0.00003
Conclusion: In this relatively high risk group of AYA, ESHAP þ BEAM combination resulted in high CR rate. Even PR-HL and relapse 3-12 have encouraging survival. There is no difference in the outcome of adolescent vs young adults. Relapse 3-12 has high incidence of disease specific deaths and events. This cohort is behaving more like a PR-HL and would be more appropriate to be referred as refractory HL in future studies for better management and outcome. Disclosure of Interest: None declared.
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P340 High dose chemotherapy and auto-SCT for nodular lymphocyte-predominant Hodgkin lymphoma: Single institution results of sixteen patients treated with and without rituximab containing salvage chemotherapy S. Akhtar1,*, M. S. Rauf1, T. A. M. Elhassan1, I. Maghfoor1 1 Oncology Center, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia Introduction: Nodular lymphocyte-predominant Hodgkin lymphoma (NLP-HL) is a distinct subtype of Hodgkin lymphoma with unique clinical and histological features and indolent behavior with frequent recurrences. We are reporting our experience of NLP-HL patients who underwent High dose chemotherapy (HDC) and auto-SCT for refractory or relapsed disease. Materials (or patients) and methods: From 1996 to 2013, 290 patients with relapsed / refractory HL received HDC autoSCT and 16 were identified as NLP-HL. Refractory disease is defined as partial response (PR), no response (NR), stable disease (SD), progressive disease (PD) or relapsing within 3 months (relapse 0-3) of finishing the planned treatment. Kaplan-Meir method was used for survival analysis from day 0. Results: Sixteen patients underwent HDC auto-SCT for NLP-HL. At the time of auto-SCT, 13 (81%) had NLP-HL and 3 (19%) had transformed diffuse large B cell lymphoma (DLBCL) (1 had
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DLBCL and 2 had T cell histiocytic rich B cell lymphoma). Male 14 (88%), female 2 (12 %), Median age at diagnosis 22 years (10-54 yrs) and at auto-SCT 27 years (15-55 yrs). At initial presentation patients characteristics were stages I:II:III:IV, 2:6:4:4 respectively. Primary treatment was ABVD in 13 (81%) and 3 (19%) received 1 of each MOPP, R-CHOP and radiation alone. 2 patients also had radiation therapy (XRT) after chemo. 12 (75%) had tissue confirmation for disease prior to salvage chemo. Reason for HDC auto-SCT was refractory NLP-HL in 10 patients (PD in 4, PR in 4, and relapse within 3 months in 2) and relapsed disease in 6. Prior to salvage chemo, stages I:II:III:IV were 2:4:5:5 respectively. Bulky disease in 2 (12%), spleen involvement in 8 (50%) and extranodal-site involvement in 5 (31%), mediastinal involvement in 6 (38%) patients and positive bone marrow in 2 (12%). ESHAP was used in 15/ 16 patients: as first line salvage in 10 (63%), 2nd and 3rd line 3 (19%); median number of cycles given were 3. 5 patients also had rituximab with ESHAP. Response to salvage was PR in 7 (44%), CR in 9 (56%). All patients had BEAM as HDC. Median follow-up is 65 months from auto-SCT. Post auto-SCT overall response was observed in 14 patients (88%); CR / CRunconfirmed in 13 (81%) and PR in 1 (6%). 2 (13%) patients had PD patients. 6 (38%) patients had XRT post auto-SCT; for consolidation post CR in 4 patients and 1 each for eradication of residual and progressive disease. 11 patients who received ESHAP only, 4 of them had post auto-SCT disease specific events (persistent disease in 1, relapsed in 1 and PD in 2
patients) as compared to no event was observed in 5 patients who received rituximab þ ESHAP. Currently 15 (94%) patients are alive, 14 with no evidence of disease and only 1 with disease (on observation for slowly progressive disease for 7 years). Five year event free survival is 75% and overall survival is 94%. Conclusion: In patients with NLP-HL, even in refractory disease, attainment of long term disease free survival is possible after HDC auto-SCT. Except one, all patients are alive. Post auto-SCT relapse or progression can still be managed with chemo / chemo þ immunotherapy. Although limited number of patients received rituximab, results are very encouraging. Integration of rituximab in salvage setting should be explored further in this unique set of patient population. Disclosure of Interest: None declared. P341 In vivo purging may not be required in the era of universal use of rituximab containing chemo-immunotherapy in patients with follicular and mantle cell lymphoma: A single center experience A. Singh1, C. Divine1, O. Aljitawi1, S. Abhyankar1, J. McGuirk1, S. Ganguly1,* 1 Hematology/Oncology, UNIVERSITY OF KANSAS MEDICAL CENTER, Westwood, United States Introduction: Although, rituximab containing chemotherapy regimens have improved response rates in B-cell lymphomas, a significant percentage of patients will eventually relapse. Autologous stem cell transplantation (Auto SCT) may provide long term remission in some of these patients. Tumor contamination of harvested stem cell graft has been postulated as a reason for relapse after Auto SCT. Single agent rituximab has been used for in-vivo purging of CD20 þ tumor cells with positive results. In this study, we aimed to evaluate whether use of rituximab based in-vivo purging is still beneficial in the era of universal use of rituximab containing chemotherapy in patients with follicular (FL) or mantle cell lymphoma (MCL). Materials (or patients) and methods: Twenty consecutive patients with a diagnosis of relapsed CD20 þ FL or transplant eligible MCL were included in this study. All patients received rituximab containing chemotherapy. Restaging was done after completion of chemotherapy and those with chemo-sensitive disease (n ¼ 14) were considered for Auto SCT. Polymerase Chain Reaction (PCR) analysis for bcl2 and bcl1 were done on apheresis products from patients with FL and MCL respectively. In-vivo purging with single agent rituximab was planned if any stem cell product were to be found PCR positive prior to Auto SCT. Results: A total of 19 patients were eligible for analysis. Patient characteristics are shown in the accompanying table. The bcl-2/bcl-1 status of the graft was available in 14 patients (FL; n ¼ 10 and MCL; n ¼ 4). The stem cell products in all 14 patients tested negative by PCR for both bcl2 (FL) and bcl1(MCL). None of these patients required additional rituximab treatment for in-vivo purging. Eleven patients (FL; n ¼ 7 and MCL; n ¼ 4) underwent Auto SCT. Three Patients with FL opted for ‘Harvest and Hold’ approach and delayed transplantation. Median (range) PFS and OS of patients with FL undergoing Auto SCT were 2 yrs. (9 mo-5 y) and 3 yrs. (2-5 y) and those with MCL were 3 yrs. (2-4y) and 4 yrs. (2-5 y) respectively (median duration of follow up-3yrs.). Conclusion: In this small cohort of patients with FL and MCL treated by R-chemotherapy, 100% of the harvested stem cell products were tumor free by PCR analysis. Our findings suggest that with universal use of rituximab containing chemo-immunotherapy in patients with FL and MCL, additional in-vivo purging may not be required. We may also conclude that persistence of residual/refractory disease after
auto SCT rather than tumor contamination of the graft was the most likely cause of relapse in this cohort. Disclosure of Interest: None declared. P342 Adequacy of peripheral blood stem cell mobilizatıon in patients with relapsed B-cell non-Hodgkin’s lymphoma (NHL) treated wıth bendamustine: a pilot project and a proof of concept study A. Iliff1, C. Divine1, F. Diaz2, O. Aljitawi1, S. Abhyankar1, J. McGuirk1, S. Ganguly1,* 1 Hematology/Oncology, 2Biostatistics, UNIVERSITY OF KANSAS MEDICAL CENTER, Westwood, United States Introduction: Although rituximab containing chemo-immunotherapy regimens have improved response rates in B-cell non Hodgkin lymphomas (B-NHL), a significant percentage of patients will eventually relapse. Autologous stem cell transplantation (auto SCT) may provide long term remission in some of these patients. A pre-requisite for successful transplantation is the ability to collect adequate number of stem cells. Use of alkylating agents has been historically thought to be stem cell toxic. Repeated or prolonged exposure to such chemotherapy may lead to poor mobilization of peripheral blood stem cells, adversely affecting outcome after auto SCT. Bendamustine, an alkylating agent, with or without rituximab have been used in the treatment of various lymphomas in both upfront and salvage settings. Materials (or patients) and methods: The primary objective of this study was to determine if use of bendamustine in combination with rituximab (BR) in patients with relapsed B-NHL, leads to adequate response and collection of stem cells prior to proceeding with high dose chemotherapy and auto SCT. Patients with relapsed or refractory CD20 þ B-NHL, eligible for high dose chemotherapy and auto SCT were enrolled for this study. Enrolled patients received salvage regimen with BR for two to three cycles, depending on the disease response. In evaluable, chemo-sensitive patients, G-CSF was used for stem cell mobilization. A minimum of 2 million CD34 þ cells per Kg of body weight was accepted as adequate collection. All eligible patients underwent high dose chemotherapy followed by auto SCT. Results: Three patients with relapsed diffuse large B cell lymphoma (DLBCL) and 15 patients with relapsed follicular lymphoma (FL) were enrolled and participated in the study. None of the 3 patients with DLBCL responded to BR salvage. Twelve out of 15 patients (83%) with FL had response (9 CR and 3 PR). Ten patients (All with Follicular Lymphoma) underwent stem cell harvest followed by auto SCT. Of the 10 patients, who proceeded onto auto SCT, we were able to collect 42 million CD34 þ stem cells per Kg of body weight in all patients. Only 2 patients had to use plerixafor according to institutional guidelines. All 10 patients engrafted (median neutrophil recovery-11 days; range 10-16 days and median platelet recovery-15.5 days; range 0-21 days). BR was well tolerated with most common AE being GI toxicity followed by neutropenia. No patient died from salvage BR related toxicity. With a median duration of follow up of 2 years, 100% and 42% overall and disease free survival were noted at 3 years post transplantation, respectively. Conclusion: This study shows that combined chemotherapy with BR had a high efficacy in the treatment of relapsed or progressive FL with 12 out of 15 patients (83%) demonstrating response to therapy. Additionally we were able to harvest sufficient stem cells for all patients that proceeded onto auto SCT. In future, we propose to use Bendamustine based salvage chemotherapy followed by Bendamustine containing high dose chemotherapy and auto SCT in the management of relapsed/refractory FL. In this small
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study, we did not see any response to BR salvage in patients with relapsed DLBCL. Disclosure of Interest: A. Iliff: None declared, C. Divine: None declared, F. Diaz: None declared, O. Aljitawi: None declared, S. Abhyankar: None declared, J. McGuirk: None declared, S. Ganguly Funding from: Pharmaceutical Funded Project. P343 Clinical outcome of a consecutive series of lymphoma patients fulfilling indication to allogenic stem cell transplantation S. Bramanti1,*, R. Crocchiolo2, B. Sarina1, L. Morabito1, A. Vai1, C. Carlo Stella1, A. Santoro1, L. Castagna1 1 IST. CLINICO HUMANITAS, rozzano, Italy, 2ISTITUTO CLINICO HUMANITAS, IST. CLINICO HUMANITAS, rozzano, Italy Introduction: Allogeneic stem cell transplantation (allo-SCT) is a curative salvage options for relapse/refractory lymphoma patients with a dismal prognosis. Materials (or patients) and methods: From 2009 to 2013, we searched for all lymphoma in our center, with an indication to proceed to allo-SCT. Indications to allo-SCT were: relapse after high dose chemotherapy and refractoriness to salvage chemotherapy or to more CT lines. The aim of this monocentric retrospective study was to analyze the clinical outcome of an unselected consecutive lymphoma patients for whom an allogeneic transplant was planned. Results: 120 patients were retrieved and 117 were evaluable. 79 patients (67%) underwent to allo-SCT and 41 (35%) did not. The median follow up was 42 months (range 4-85). Patient characteristics were reported in Table 1. Main causes to inelegibility to allo-SCT were: disease progression (56%) and patient refusal (35%). In most of the patients (65%) the donor was haploidentical. The 4 years OS (Figure 1) of the allo group and no allo group were 46% and 20% respectively (P ¼ 0.0001). 4-year NRM in the allo group was 29%. Overall 52% (41) of the patients in allo group is still alive in complete remission. Only 1 patient is alive with active disease. In the no allo group, 9(23%) patients are alive, 5 (13%) in complete remission, 4 with active disease.
Table 1.
Patient characteristics
Allo N 78 (%)
No allo N 39 (%)
P
LNH HD
40 (51%) 38 (49%)
24 (61%) 15 (38%)
0.30
Indication for allo Relapse after auto Relapsed/Refractory High risk Relapse after allo
43 31 3 1
12 26 1 1
0.001
(55%) (39%) (4%) (1%)
(31%) (66%) (2%) (2%)
Donor availability at the time of indication no donor / 11 (28%) Haplo 51 (65%) 18 (46%) HLA id sib 18 (23%) 7 (18%) HLA id MUD 7 (9%) 3 (8%) mmMUD 2 (2%) / Unrelated donor 32 (41%) 7 (18%) search activation Median time from 23 18 diagnosis to indication months months to allo-SCT (4-147 ) (2-234)
0.01 0.12
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Conclusion: Even though allo-SCT is an effective approach in poor prognosis lymphomas, it is still difficult to fully appreciate the real impact on survival because of the lack of prospective randomized study. Furthermore, in published studies, it was reported only on the outcome of patients receiving allotransplanted, and few informations were available of patients potentially candidate to transplant. Similarly to donor vs no donor analysis, this retrospective study suggests that receiving allo-SCT conferred survival advantage in refractory lymphoma patients. Disclosure of Interest: None declared. P344 Prognostic factors and long term outcome after autologous hematopoietic stem cell transplantation in refractory or recurrent Hodgkin’s lymphoma of children and adolescents: a multicenter survey of Turkish Pediatric BMT Study Group V. Kesik1,*, E. Atas1, M. Karakukcu2, S. Aksoylar3, F. Erbey4, N. Tacyildiz5, A. Kupesiz6, H. Oniz7, E. Unal2, S. Kansoy3, G. Ozturk4, M. Elli8, Z. Kaya9, E. Unal5, V. Hazar6, S. Yilmaz Bengoa10, G. Karasu11, D. Atay4, A. Dagdemir8, H. Oren10, U¨. Kocak9, A. Yesilipek11 1 Department of Pediatric Oncology, Gulhane Military Medical Academy, Ankara, 2Department of Pediatric Hematology&Oncology and BMT Unit, Erciyes University Faculty of Medicine, Kayseri, 3 Department of Pediatric Oncology and BMT Unit, Ege University Faculty of Medicine, Izmir, 4Pediatric BMT Unit, Medical Park Bahc¸elievler Hospital, Istanbul, 5Department of Pediatric Oncology and BMT Unit, Ankara University Faculty of Medicine, Ankara, 6Department of Pediatric Hematology&Oncology and BMT Unit, Akdeniz University Faculty of Medicine, Antalya, 7 Department of Pediatric Oncology and BMT Unit, Tepecik Education and Research Hospital, Izmir, 8Department of Pediatric Oncology and BMT Unit, On Dokuz Mayıs University Faculty of Medicine, Samsun, 9Department of Pediatric Hematology and BMT Unit, Gazi University Faculty of Medicine, Ankara, 10Department of Pediatric Hematology and BMT Unit, Dokuz Eylu¨l University Faculty of Medicine, Izmir, 11Pediatric BMT Unit, Medical Park Go¨ztepe Hospital, Istanbul, Turkey Introduction: We examined the effect of some factors and age-adjusted international prognostic index (AAIPI) on relapse and survival of the children with Hodgkin’s lymphoma (HL) undergone autologous stem cell transplantation (ASCT) as a multicenter study in Turkey. Materials (or patients) and methods: Sixty-one children from 10 centers in Turkey who underwent autologous ASCT because of refractory or recurrent HL were retrospectively
P345 Autologous/Allogeneic Stem Cell Transplantation following High-dose Sequential Chemotherapy in Patients with Stage III-IV Burkitt and Lymphoblastic Lymphoma : Long term survival data from LLBL2001 protocol B. Gungor1, C. Unsal1, I. Barista2, M. Adam1, Y. Koc1,* 1 SCT Unit, Medical Park, Antalya, 2Oncology, Hacettepe University, Ankara, Turkey
analyzed. The evaluated possible prognostic factors were age, relapse number, stage, Hodgkin type, bulky tumor, spleen, extranodal bone and bone marrow involvement, B symptoms, involvement, hemoglobin, WBC, lymphocyte, monocyte, MPV, ferritin, albumin, LDH, sedimentation at diagnosis, pretransplant Karnofsky, PET positivity, number of chemotherapy regimens, radiotherapy, postransplant day 15 and 100 lymphocyte, neutrophil /lymphocyte ratio, MPV, and day 100 PET positivity. Risk factors affecting relapse free survival (RFS) were evaluated using stratified Cox regression. Results: At the time of study, 44 of 61 patients were alive, 15 patients were expired, and 2 patients were lost. Nine patients due to progressive disease, 2 patients due to infection, 4 patients due to other reasons except relapse were dead. Nonrelapse mortality rate was 40%. Median relapse time was 11 months (range, 1-105). Three-year overall survival (OS)/RFS were 77.3% and 68.5% with a median follow-up of 27 months (range, 1-114 months) for all patients, respectively. Subtype of HL (P ¼ 0.042, HR ¼ 1.6), WBC 410000/mm3 at postHSCT day100 (P ¼ 0.009, HR ¼ 5.4) no RT before HSCT (P ¼ 0.019, HR ¼ 3.1), no remission at HSCT (P ¼ 0.012, HR ¼ 3.2), refractory disease (P ¼ 0.003, HR ¼ 4.1), type of relapse (P ¼ 0.003, HR ¼ 0.17), PET-CT positivity at post-HSCT day 100 (P ¼ 0.009, HR ¼ 4.4), serum albumin under 2.5 mg/dl (P ¼ 0.035, HR ¼ 13.1) were predictive of relapse free survival (RFS). The factors effecting survival were represented on Table 1. When AAIPI was adapted to children according to LDH 500 IU/L instead of 250 IU/L, we called this new term as CAAIPI. Threeyear DFS rates were 100% for Score 0, 61.3 % for Score 1, 52.7 % for Score 2 (P ¼ 0.26) and OS rates were 80.0% for Score 0 (exitus due to infection), 93.3% for Score 1, 77.0% for Score 2 (P ¼ 0.50) according to CAAIPI. Conclusion: Although ASCT increase the survival rate, survival is worse among patients that relapse after HSCT, with no remission at the time of HSCT, and bone marrow positivity at diagnosis. Therefore, we suggest more aggressive therapies for children with bone marrow involvement at diagnosis and early ASCT at the first remission. We also suggest different treatment approaches for the latter. The ones with high IPI or AAIPI score can also be treated in this manner. We suggest that CAAIPI score that use LDH level as 500 IU/L is more useful in childhood. In addition, the characteristics that showed up significance in univariate but not in multivariate analysis appears to have an influence as well and might show a stronger correlation in larger trials. Disclosure of Interest: None declared.
Introduction: In patients with advanced stage highly-agressive NHL, a satisfying response rate and long-term survival cannot be achieved by conventional standard lymphoma chemotherapy regimens. Despite reports of high-dose chemotherapy regimens coupled with or without stem cell transplantation (SCT), medical literature lacks a standard treatment protocol in the management of patients with stage III-IV Burkitt (BL) and Lymphoblastic Lymphoma (LL). In this study, we report disease-free (DFS) and long-term overall survival (OS) of patients treated with LLBL2001 treatment protocol consisting of high-dose chemotherapy and autologous or allogeneic SCT in the past 12 years. Materials (or patients) and methods: Thirty newly diagnosed patients with LL (n ¼ 18) and BL (n ¼ 12) were treated with the LLBL2001 protocol consisting of induction (CTX þ DOX þ VCR þ PRED), consolidation with high-dose chemotherapy (2 cycles of HIDAC þ HDMTX, 3g/m2 each) and pegylated L-asparaginase, prophylactic intrathecal MTX þ Ara-C, pre-transplant conditioning with IIVP and HD-VP16 chemotherapy (2 g/m2), followed by autologous SCT with HD-MTZ (60 mg/m2) and HD-MEL (180 mg/ m2) conditioning. Patients with B-cell phenotype received invivo purging with rituximab prior to Auto-SCT. Protocol did not include prophylactic cranial radiotherapy. In cases with relapse or CNS involvement during administration of the LLBL2001 protocol, patients proceeded with allogeneic SCT after CR was achieved. Median age of the patient group was 30 (17-48) and 86% had bulky disease Z 6 cm. Forty-seven percent of patients had bone marrow involvement at diagnosis. Results: Following induction phase, 87% patient achieved CR, and transplantation could not be performed in 4 patients due to refractory lymphoma or social reasons. Twenty-six patients completed the treatment protocol (23 Auto-SCT, 3 Allo-SCT). Eight patients died due to relapsed/refractory disease (n ¼ 5), sepsis (n ¼ 2), and cGVHD (n ¼ 1). Significant complications observed in the patient cohort are as follows; neutropenic fever (25), CNS recurrence (4), sepsis (3), VZV reactivation (3), acute GVHD (3), chronic GVHD (3), aseptic femoral head necrosis (3), CMV reactivation (2), tumor lysis (2), BK virus cystitis (1), fungal pneumoniae (1), HSV keratitis (1), IVC thrombosis (1). When analysed by intent-to-treat principle, 12 year DFS and OS were 81% (88% in BL, 78% in LL, P ¼ 0.56) and 73% (88% in BL, 67% in LL, P ¼ 0.32), respectively. No relapse was observed beyond 3 years.
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Conclusion: LLBL2001 protocol is capable of inducing high long-term disease-free and overall survival rates and provides a curative option for patients with advanced stage highlyaggressive bulky NHL. Disclosure of Interest: None declared. P346 Allogeneic hematopoietic stem cell transplantation in patients with T cell lymphoma is prefer to chemotherapy Y. Luo1,*, Y. Wu2, Y. Tan2, H. Huang2 1 Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, 2Bone Marrow Transplantation Center, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Introduction: To compare the efficiency and safety of allogeneic hematopoietic stem cell transplantation versus chemotherapy in patients with T cell lymphoma. To analyze the clinical features and prognostic factors of these patients. Materials (or patients) and methods: 59 patients who were diagnosed as T cell lymphoma by pathology between Jan 1st 2007 to Dec 31 2012 in the First Affiliated Hospital of Zhejiang University School of Medicine were enrolled and analyzed retrospectively. All of the cases were determined according to WHO 2008 classification and classified by Ann Arbor staging system. These patients were all younger than 60 years old at clinical stage III/IV period and have completed two or more courses chemotherapy. 19 patients finally received allogeneic stem cell transplantation and 40 patients still received chemotherapy, followed up by telephone to Dec 31 2013. We evaluate the PFS and OS between two groups. At the same time, some clinical factors such as the level of serum lactate dehydrogenase (LDH) , B symptoms, and international prognostic index (IPI) were collected to analyze the relation between these parameters and survival. Results: After a median follow-up of 21 months, one-year overall survival(OS) of SCT and CTx were 94.4% and 65.0% (Po0.05). Three-year OS of SCT and CTx were 57.2% and 39.1% (Po0.05). Five-year OS of SCT and CTx were 45.8% and 23.1% (Po0.05). One-year progression-free survival (PFS) of SCT and CTx were 88.5% and 47.5% (Po0.001). Three-year PFS of SCT and CTx were 51.5% and 22.5% (Po0.001). Five-year PFS of SCT and CTx were 41.2% and 7.5% (Po0.001). The treatment related mortality (TRM) rate has no statistical significance between the two groups. 100-day TRM of SCT and CTx were 2.5% and 10.5% (P ¼ 0.190). One-year TRM of SCT and CTx were 17.5% and 15.8% (P ¼ 0.870). Three-year TRM of SCT and CTx were 27.5% and 21.1% (P ¼ 0.595). Five-year TRM of SCT and CTx were 30.0% and 26.3% (P ¼ 0.770). The level of serum LDH, extranodal violations, Ann Arbor stage and international prognostic index (IPI) were statistically relevant in predicting the prognosis of the overall survival (OS). Multivariate analysis showed that only treatment is an independent prognostic factor. Conclusion: Allogeneic hematopoietic stem cell transplantation is safe and effective for T cell lymphoma patients and can result in long-term disease control. Disclosure of Interest: None declared.
Multiple myeloma P347 The impact of autologous stem cell transplantation (ASCT) on survival of patients with multiple myeloma (MM) aged under 65 years in compliance with risk stratification on the modified scales mSMART 1.0 and mSMART 2.0 A. Garifullin1,*, I. Martynkevich1, S. Voloshin1, L. Martynenko1, A. Kuvshinov1, L. Stelmashenko1, E. Kleina1, G. Salogub2, E. Karyagina3, A. Schmidt1, A. Kuzyaeva1, K. Abdulkadyrov1 1 Russian Research Institute of Hematology and Transfusiology, 2 Saint-Petersburg Pavlov State Medical University, 3City Hospital N15, Saint-Petersburg, Russian Federation Introduction: Seventy percent of patients with MM have chromosomal aberrations. Part of them have prognostic value. That allows to stratify patients in separate groups of risk and select optimal strategy of treatment. Autologous stem cells transplantation is one of the effective methods for patients aged under 65 years with MM. Materials (or patients) and methods: We retrospectively analyzed 121 patientsr65 years (median age 56 years, range 26 - 65; male/female – 1:1.24). The incidences of genetic abnormalities were determined in all cases. Cytogenetic studies were performed on bone marrow samples using standard GTD-method. Metaphase FISH analyses were performed according to the manufacturer’s protocol using DNA probes: LSI 13(RB1)13q14, IGH/CCND1, IGH/FGFR3, LSI TP53 (17q13.1) (Abbott). Stratification of patients was carried in groups of risk according to the modified molecular classification mSMART 1.0 and mSMART 2.0. Patients with complex karyotype (3 and more chromosomal aberrations) were additionally entered in high risk group of both systems. Results: In group of patients after ASCT (single and tandem) (n ¼ 45) 5-years OS was 92% vs 86% (P ¼ .14) in group without ASCT (n ¼ 76). 5-years OS (mSMARTmod 1.0) in standard risk groups (mSMARTmod 1.0 and mSMARTmod 2.0) of patients with ASCT (n ¼ 34) and without ASCT (n ¼ 65) was 87% and 83%, respectively (P ¼ .68). In high risk group (mSMARTmod 1.0) 5years OS of patients with ASCT (n ¼ 11) was 100%, without ASCT (n ¼ 11) – 55% (P ¼ .04). Results 5-years OS (mSMARTmod 2.0) in intermediate risk group in patients with ASCT (n ¼ 9) was 100%, without ASCT (n ¼ 7) – 64% (P ¼ .12). In high risk group (mSMARTmod 2.0) was reached only 2-years OS of patients with and without ASCT which made 100% and 50%, respectively (P ¼ .36). The median of OS wasn’t reached in all groups of patients. Conclusion: ASCT improves treatment outcome in young MM high risk patients. Disclosure of Interest: None declared. P348 Outcome improvement in newly diagnosed Multiple Myeloma patients elegibile for Autologous Stem Cell Transplant: a single centre retrospective analysis A. Natale1,*, S. Pulini1, L. Torti1, A. M. Morelli1, P. Ranalli1, S. Angelini2, A. Spadano1, P. Di Bartolomeo1 1 Clinical Hematology, Department of Hematology, Transfusion Medicine and Biotechnology, ‘‘Spirito Santo’’ Civic Hospital, Pescara, 2Hematology, Mazzoni Hospital, Ascoli Piceno, Italy Introduction: Overall survival (OS) in Multiple Myeloma (MM) patients (pts) significantly improved during the past decade after the introduction of new drugs (proteasome inhibitors and immunomodulators). Materials (or patients) and methods: We retrospectively analyzed the outcome of 165 newly diagnosed MM pts transplanted in our institution. Various induction regimens were administered during different time periods: conventional chemotherapy-based (VAD 27 pts, 17% of total; TT2-like 37,
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22%; EDAP 1, 1%), thalidomide-based (TD 15, 9%), bortezomibbased (VTD 62, 37%; VD/VCD 11, 7%; PAD 3, 2%), lenalidomide-based (RD 9, 5%). We analyzed data of the 4 more representative regimens (VAD, TT2-like, TD and VTD) considering the number of the pts treated. Results: From November 1999 to December 2013 165 MM pts received at least one autologous stem cell transplant (ASCT) after induction. Median age at start therapy was 58 years (range 36-71) and median follow up time 52 months (range 7-179). 66% of the pts achieved at least very good partial response (VGPR) after induction therapy: sCR (stringent Complete Response) was achieved in 1%, CR in 26%, VGPR in 39%. After VTD induction regimen pts obtained the overall best quality of response (86% of them at least in VGPR) and the highest percentage of sCR (2%). 22 pts (13%) received a salvage therapy before ASCT: 13 pts experienced PD (Progressive Disease), 9 pts were considered in suboptimal or unsatisfactory response (4 of them were in Stable Disease, SD and 5 in Partial Remission, PR). After induction therapy 67 pts received a single ASCT, 98 pts double ASCT; 11 patients received tandem autologous and allogeneic SCT. ASCT improved quality of response in all patients, irrespective of the induction regimen received. None of the pts examined underwent PD after ASCT. After ASCT 87% of the pts were at least in VGPR: sCR was achieved in 5%, CR in 48%, VGPR in 34%. Also after transplant VTD induction confirmed to be the regimen with the best quality of response (93% were at least in VGPR) and the highest percentage of sCR (11%). MM relapsed after ASCT in 86 pts, median time from ASCT to relapse was 24 months (range 4-113). At last follow-up 113 (69%) pts were alive and 52 (31%) pts died, the main cause of death was PD (85%). The median OS was 9 years and 11 months, after 15 years the probability of survival was 35%. Considering the different induction regimen the best probability of survival (77% after 7 years and 6 month from start therapy) was achieved after VTD (median OS not reached, more than 7.5 years), followed by VAD (probability of survival 35% at 14 years and 11 months from start therapy, median OS 11 years), TT2-like (probability of survival 45% at 14 years and 8 months, median OS 9 years and 11 months) and TD (probability of survival 38% at 7 years and 8 months, median OS 6 years and 11 months). Conclusion: The introduction of novel agents improved outcome of MM pts transplanted in our center, according with published data. The improvement of quality of response after induction therapy results in better long term control of the disease. Moreover the avaibility of new drugs in relapse post transplant for patient treated with first line chemotherapy regimen improved OS also in this setting. ‘‘Does the dogma of multiple myeloma is incurable still hold?’’, Dr. Bart Barlogie recently asked (Blood, 2014). We are moving forward. Disclosure of Interest: None declared. P349 Polyclonal activation of the immunoglobulin determined by free-light assay after lenalidomide-treatment ın patients wıth multiple myeloma prolongs time to treatment failure A. L. Kluger1,*, G. Scho¨n2, A. Gerritzen3, U.-M. von Pein1, F. Ayuk1, C. Wolschke1, N. Kro¨ger1 1 Department of Stem Cell Transplantation, 2Institute of Medical Biometry, University Medical Center Hamburg, Hamburg, 3 Medical Laboratory Bremen, Bremen, Germany Introduction: Lenalidomide is an immunomodulatory drug, which induced T-cell and NK-cell activation and is approved for treatment of patients with multiple myeloma. Here we investigated the effect of lenalidomide on polyclonal activation of immunoglobuline detected by free-light assay and on its impact on outcome in patients with multiple myeloma.
Materials (or patients) and methods: In this study, we included 61 patients with multiple myeloma (male ¼ 42, female ¼ 18) with a median age of 61 years (range 39-77 years), who received lenalidomide at a median dose of 15 mg either as maintenance (n ¼ 28) or as salvage therapy (n ¼ 33) from 2006 to 2013 in our institution. The median duration of lenalidomide therapy was 14 months (range 2-69 months). 29.51% (n ¼ 18) received lenalidomide as single agent and 70% (n ¼ 43) in combination with Dexamethasone. All patients were inspected for the presence of a polyclonal activation on the basis of free-light chain serum levels. "Polyclonal activation" was defined as an increase of both free-light chain serum levels out of normal range (range k: 3.3mg/l-19,4mg/l; l: 5.7mg/l-26.3mg/l). Results: A total of 23 patients (37,70%) had shown a polyclonal activation during lenalidomide-treatment as described above. Polyclonal activation was seen in 11/23 patients with partial response, in 9/23 with complete response and in 2/23 with stable disease. One response-level was not specified. Comparing the time to treatment failure of the polyclonal activation population (n ¼ 23) and the non-polyclonal activation population (n ¼ 38), the patients with polyclonal activation had a significant higher median time to treatment failure (41.03 vs.16.9 months, P ¼ 0.049). The significance was meanly seen in patients with relapsed myeloma (58.0 vs. 12.94 months, P ¼ 0.043) while in maintenance the difference (41.03 vs. 26.0 months) was not significant (P ¼ 0.40). Conclusion: Our study suggests that a polyclonal activation measured by free-light chain assay after lenalidomide therapy resulted in a prolonged time to treatment failure, especially for the relapse therapy population. Disclosure of Interest: None declared. P350 Albumin is the main predıctor of survival after Autologous Stem Cell Transplantation in Multiple Myeloma patients E. Birtas Atesoglu1,*, A. Hacihanefioglu1, Z. Gulbas2 1 Department of Hematology, Kocaeli University, 2Bone Marrow Transplantation Center, Anadolu Medical Center Hospital, Kocaeli, Turkey Introduction: Multiple myeloma(MM) remains a rarely curable disease. However, studies have shown benefit for overall survival (OS) and progression free survival (PFS) by the use of high-dose chemotherapy with subsequent autologous stem cell transplantation (ASCT). The aim of the present study was to retrospectively analyze the impact on outcomes of various factors especially albumin levels at the time of ASCT in MM patients. Materials (or patients) and methods: We analyzed retrospectively 137 multiple myeloma(MM) patients who had undergone autologous stem cell transplantation from August 2010 to January 2013. Detailed information concerning patients presentation at diagnosis as well as the disease status and other variables of known prognostic importance were evaluated before the initiation of conditioning chemotherapy for AHSCT. Results: Median follow-up time is 22 (1-46) months. When we analyze, the impact of gender, b2microglobulin levels (o3,5 vs Z3,5), ISS stage, LDH (normal limit vs Z upper normal limit), ferritin (o1000 vs Z1000), hemoglobin level (o12 vs Z12), age (r65 vs 465), remission status before transplantation, tandem vs single transplantation on overall survival (OS) and progression free survival (PFS), none of them had an impact on OS or PFS (P40,05). Only patients with albumin levels 43,5 had significantly better OS and PFS in comparison to patients with albumin levels o3,5 (P ¼ 0,02, P ¼ 0,04, respectively) (Figure 1). Conclusion: We conclude that albumin level measured before ASCT is a useful predictor for both OS and PFS in MM patients. Disclosure of Interest: None declared.
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P352 Minimal residual disease by multiparameter flow cytometry in Mieloma patients undergoing an up-front tandem ASCT: a real life single-institution study I. Cordone1, F. Marchesi2,*, S. Masi1, V. Summa1, M. L. Dessanti2, S. Gumenyuk2, R. Merola1, G. Cigliana1, G. Orlandi1, F. Palombi2, F. Pisani2, A. Romano2, A. Spadea2, L. Conti1, M. C. Petti2, A. Mengarelli2 1 Clinical Pathology Unit, 2Hematology and Stem Cell Transplantation Unit, Regina Elena National Cancer Insitute, Rome, Italy Introduction: Multiparameter Flow Cytometry (MFC) is a powerful technique in both diagnosis and Minimal Residual Disease (MRD) detection in multiple myeloma (MM) patients. We performed a prospective single center study on real-life MM patients who underwent up-front tandem autologous stem cell transplantation (ASCT) utilizing an innovative MFC MRD monitoring. The aims of the study were 1) to establish whether the 2nd ASCT allows a better quality of response in terms of MRD assessment; 2) to evaluate patients’ clinical outcome after 2nd ASCT according to MRD status and to postASCT treatment. Materials (or patients) and methods: From January 2006 to January 2012, 50 consecutive MM patients, median age 56 years (41-68) treated with a program containing an up-front tandem ASCT (Melphalan 200 mg/m2) entered the study. As induction, 23 patients received 3 courses of VAD chemotherapy and 27 received 3 courses of novel agents (Bortezomibbased n ¼ 21; immunomodulators only n ¼ 6). After the 2nd ASCT 28 patients underwent a consolidation/maintenance treatment based on Bortezomib (n ¼ 14), immunomodulators only (n ¼ 12) or others (n ¼ 2). Plasma-cells (PC) MFC characterization was performed on bone marrow (BM) samples at diagnosis utilizing the antibody combinations (Fitc/PE/PerCP/PE-Cy7/APC/APC-CY7): I)CD28/CD138/CD45/ CD38/CD33/CD20; II)CD38/CD138/CD45/CD56/CD117/CD19. The PC phenotypic aberrancies detected in the surface characterization were used as patient-specific disease markers (PSDM) to document intra-cytoplasmic immunoglobulin (cy-Ig) light chains restriction: III)cy-Ig Lambda/cy-Ig Kappa/CD19/ CD38/anti-PSDM/CD45 at diagnosis, after induction and at day þ 100 approximately after 1st and 2nd ASCT. Patients were
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considered to be in immunophenotypic complete response (iCR) when pathological PC were undetected at the MFC sensitivity limit of 10-4 cells, allowing a maximum sensitivity of detection of 0.001% in all cases. Treatment response by MFC criteria and progression-free survival (PFS) according to MRD status after 2nd ASCT were evaluated. Results: A statistically significant decrease of the mean (±SD) percentage of residual pathologic PC (2.6% [±6.3] vs 0.2% [±0.3]; P ¼ 0.01) and a significant increase of the proportion of patients in iCR (13 [26%] vs 29 [58%]; P ¼ 0.001) were observed comparing induction and 1st ASCT but not comparing 1st and 2st ASCT (0.2% [±0.3] vs 0.1% [±0.8]; P ¼ 0.764 and 29 [58%] vs 36 [72%]; P ¼ 0.236). The 4 years-PFS (median follow-up from diagnosis) of patients in iCR after the 2nd ASCT was significantly better than that of MFC-MRD positive patients (72% vs 23%; P ¼ 0.002). In 36 patients in iCR and 14 patients MFC-MRD positive after 2nd ASCT, no significant differences were observed in terms of PFS between those receiving (19 vs 9) or not (17 vs 5) a consolidation/maintenance treatment (4yPFS: 73% vs 57% [P ¼ 0.336] and 47% vs 20% [P ¼ 0.703], respectively). Conclusion: The cy-Ig light restriction on PSDM is a powerful tool of clinical relevance in MM MRD monitoring. In MM patients undergoing an up-front tandem ASCT, the 2nd ASCT doesn’t seem to add significant advantages in terms of quality of response assessed by MFC compared with the first one. With the caution due to the low number of patients, our data seem also to suggest that a consolidation/maintenance treatment in patients in iCR after 2nd ASCT may not be translated into significantly better PFS. Disclosure of Interest: None declared. P353 23 year Iranian experience in HSCT in multiple myeloma H. Kamranzadehfumani1,*, K. Alimoghadam1, M. Jahani1, M. Vaezi1, B. Bahar2, A. Mousavi1, A. Ghavamzadeh1 1 HORCSCT, 2Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic Of Introduction: Aim: Autologos HSCT is standard of care for eligible myeloma patients and allogenic HSCT may be the only curable treatment. We began this procedure in 1991 and later we performed allogenic HSCT in some cases. Materials (or patients) and methods: Methods: This retrospective study is a report of 557 patients who underwent HSCT in our center. The conditioning regimen was melphalan for autologos and melphalan þ fludarabine for allogenic
HSCT.MTX þ Cyclosporine A were used as GVHD prophylaxis. All cases of allogenic transplantation, were performed from match sibling donor. Results: Results: 557 patients ( 348 male,209 female) from 1991 to September 2014 resived HSCT. Mean age at transplant is 51 years (range 24-72). 489 autologos and 68 allogenic HSCT performed. Median follow up time is 27.7 months in autologos and 27.2 months in allogenic group. 97 patients (17.4%) died (1.9%in allogenic, 15.5% in autologos). The major cause of death in two group was relapse. 168 relapses ocured (31.3% of autologos, 10.2% of allogenic). Mean time to relapse is 24.7 months. In allogenic group 36% had acute and 31.34% had chronic GVHD. Most of them were limited. 2 patients died from GVHD(acute and chronic) and one patient die from CMV infection and one from DVT. Conclusion: Conclusion: We found relapse rate is lower in allogenic HSCT than autologos (P ¼ 0.01), but there is no difference in overall mortality rate. Acute or chronic GVHD had no relationship with relapse rate and it may be due to short time of follow up and few patients number in allogenic group. we found no relationship with sex, mortality and relapse. Disclosure of Interest: None declared. P354 The impact of pre-transplant remission status on posttransplant disease outcomes in patients with multiple myeloma T. Toptas1, I. Kaygusuz–Atagunduz2,*, F. Pepedil1, A. Eser1, O. Kara1, A. Sezgin1, T. Ozgumus1, C. Adiguzel1, T. Firatli–Tuglular1 1 Marmara University Hospital, Istanbul, Turkey, 2Department of Hematology, Marmara University Hospital, Istanbul, Turkey Introduction: We aimed to investigate the impact of pretransplant response depth on myeloma outcomes. Materials (or patients) and methods: We retrospectively analyzed the data of a total of 56 patients with multiple myeloma who underwent autologous stem cell transplantation (SCT) between 2000 and 2010. Response status was defined according to IMWG criteria. Post-hoc power analysis revealed that study show a hazard ratio (HR) of 0.5 with a power of 72% and an overall two-sided type I error of 5%, with use of a two-sided log-rank test. Results: Time-to-transplantation, median follow-up, and treatment related mortality were 12 (6-36) months, 32 months, and 3.6%, respectively. For all cohort, median PFS, TTP, TNT, and OS were 25, 32, 47, and 84 months respectively. 5- and 10-year OS were 60.9%, and 26.6%. The proportion of patients who achieved Z very good partial response (VGPR) before transplantation was 41.9%. Post-transplant ratio of patients with ZVGPR estimated to be 75.8%. There were no significant differences between patients with a pre-transplant response ZVGPR and partial response (PR) in terms of PFS (median 33 vs 24 months, P ¼ 0.3), TTP (not reached vs 25 months, P ¼ 0.2), TNT (not reached vs 33 months, P ¼ 0.09), and OS (not reached vs 71 months, P ¼ 0.8). Conclusion: Patients with multiple myeloma who achieve PR or ZVGPR before transplantation have similar disease outcomes. Since only 30% of patients will have a ZVGPR with novel agents, at least PR is a reasonable pre-transplant treatment goal. Disclosure of Interest: None declared. P355 bortezomib targets side population of human myeloma cells enriched clonogenic tumor initiation subpopulations J.-H. Won1,*, D. S. Hong2, K. H. Kim1, J. Yun2, H. J. Kim3, S. Kim2, H. J. Kim2, S.-C. Lee3, S. B. Bae3, N. S. Lee1, K.-T. Lee3, S. K. Park2 1 Soonchunhyang University Hospital, Seoul, 2Soonchunhyang University Hospital, Bucheon, 3Soonchunhyang University Hospital, Cheonan, Korea, Republic Of Introduction: Cytotoxic chemotherapy based treatment of multiple myeloma (MM) is not curative and the disease eventually recurs. This is in part because although currently
available antiMM strategies have been effective in targeting the bulk of tumor cells, they do not target the tumor initiating subpopulation or cancer stem cells. Materials (or patients) and methods: In our study, we performed flow cytometry based Hoechst 33342 staining to evaluate the existence of side population (SP) cells and investigated the prevalence and biologic function of SP cells in multiple myeloma cell lines. Results: Frequency of SP cells was heterogenous: most cell lines (ARH77, IM9, and MM.1R) had a less than 1% of SP cells but RPMI8226 had around 10% of SP cells. Sorted SP cells showed a capacity of higher proliferation abilities and clonogenecity than main population (MP) in RPMI8226 myeloma cell line and also observed the ability of SP cells to regenerate original population. We detected higher activity of ABCG2 in SP cells which associated with high rates of proliferation. But we could not found the difference of expression of specific surface markers between SP and MP, such as expression of CD138, CD34, CD38, CD19, CD20, and CD27. We compared relative sensitivity of SP and MP to clinical anti-myeloma agents (bortezomib, dexamethasone, doxorubicin, and melphalan), bortezomib only exhibit substantial activity to SP cells. Conclusion: Our studies demonstrate SP fraction of myeloma cells contained clonogenic tumor initiating potential and provide new mechanisms of action for bortezomib to target the SP cells. Further studies to define the myeloma initiating cell markers leave in abeyance. Disclosure of Interest: None declared. P356 Additional therapy before autologous transplantation improves the quality of response but not the outcome of Multiple Myeloma patients: a single centre experience L. Torti1,*, S. Pulini1, A. Natale1, A. M. Morelli1, R. Spadano1, S. Angelini2, A. Spadano1, P. Di Bartolomeo1 1 Clinical Hematology, Department of Hematology, Transfusion Medicine and Biotechnology, ‘‘Spirito Santo’’ Civic Hospital, Pescara, 2Hematology, Mazzoni Hospital, Ascoli Piceno, Italy Introduction: Newly diagnosed Multiple Myeloma (MM) patients (pts) who are eligible for high dose chemotherapy (CHT) and autologous stem cell transplant (ASCT) receive induction therapy before ASCT. Results from randomized clinical trials incorporating novel agents suggest that the quality of response after induction correlates with a longer control of MM. However, data published in the literature show that ASCT may improve the quality of response even in pts refractory to induction. It is not clear if pts with suboptimal response really benefit from additional treatment before ASCT. Materials (or patients) and methods: We retrospectively analyzed 165 newly diagnosed MM pts transplanted in our institution from November 1999 to December 2013. After different induction regimen (CHT-based, thalidomide-based,
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bortezomib-based or lenalidomide-based) 143 pts underwent ASCT; 22 pts (13%) received additional therapy before ASCT. Results: Among the 22 pts analyzed median age at start therapy was 58 years (range 36-66 years) and median follow up time 47 months (range 18-144 months). As first line therapy 6 pts received conventional CHT, 7 bortezomib basedregimen, 4 thalidomide-based and 5 lenalidomide-based. After induction 13 pts were in PD (Progressive Disease), 9 pts were considered in suboptimal/unsatisfactory response (4 in Stable Disease, SD and 5 in Partial Response, PR). They therefore underwent additional therapy: 15 pts were treated according bortezomib-based regimen, 4 lenalidomide-based, 3 conventional CHT. 18% of the pts achieved Complete Response (CR), 45% Very Good Partial Response (VGPR), 32% PR and 5% SD. All of the 22 pts received at least one ASCT, 11 pts double ASCT, 5 tandem autologous and allogenic SCT. ASCT improved quality of response in all patients and none of them underwent PD after transplant. After ASCT 23% patients were in CR, 41% in VGPR and 36% in PR. 15 pts experienced at least one MM relapse with a median time from ASCT of 16 months (range 4-51). At last follow up 10 pts were alive and 12 were dead, 11 for PD and 1 patient for complications related to Allogenic BMT (acute GvHD and mycotic pneumonia). The median Overall Survival (OS) for pts who received addition therapy before ASCT was 4 years and 4 months and the probability of survival was 23% at 12 years, significantly inferior respect to pts that did not receive additional therapy (median OS 11 years, 38% at 14 years and 11 months), p-value ¼ 0.0009. Conclusion: There is no agreement if MM patients with suboptimal response to induction may benefit from additional alternative therapy with the intent to maximize pretransplant response and therefore the control of MM. In our retrospective analysis pts that received additional therapy before ASCT improved depth of response but this result did not translate in improvement of OS. Our study has some limitations (retrospective study, number of pts, heterogeneous therapy administered, lack of biological data) but it addresses a significant clinical issue. In the era of the new drugs we aim to achieve the best response before ASCT, but if the quality of the response does not correlate with outcome do we need to postpone ASCT considering that high-dose melphalan remains one of the most active treatment in MM? We hope that prospective, randomized clinical trials will clarify the importance of depth or response before and after ASCT. Disclosure of Interest: None declared. P357 Tandem autologous plus reduced-intensity allogeneic stem cell transplantation without standard immunosuppression in young patients with relapsed and refractory multiple myeloma O. Pokrovskaya1,*, L. Mendeleeva1, E. Makunina1, L. Kuzmina1, M. Firsova1, M. Nareyko1, E. Urnova1, I. Galtseva1, T. Sorokina1, E. Parovichnikova1, V. Savchenko1 1 Bone Marrow Transplant Department, National Research Center for Hematology, Moscow, Russian Federation Introduction: Multiple myeloma (MM) remains an incurable disease and almost all patients (pts) will relapse or develop refractory disease even after autologous stem cell transplantation (autoSCT). Allogenic bone marrow transplantation (alloBMT) carries the benefit of graft versus myeloma effect but also high risk of treatment related mortality. Combining benefits of cytoreductive-therapy with adoptive immunotherapy forms the basis of a tandem auto-allo SCT treatment strategy which is very actual, especially in young pts with MM. Materials (or patients) and methods: Since April 2012 to December 2014 four young pts (three men and one woman, 26-40 year old) with advanced multiple myeloma underwent tandem autoSCT and alloBMT in the department of Bone Marrow Transplantation. 3 of them had myeloma G and one revealed secretion of immunoglobulin light chains only. At the
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time of diagnosis all pts had negative prognostic factors, such as unfavourable cytogenetics, extramedullary disease, high LDH level, ISS stage III. All patients received first-line induction chemotherapy with bortezomib þ dexamethasone þ /cyclophosphamide þ /- adriamicin. 3 pts progressed during induction and received 2 to 4 lines of therapy containing lenalidomide, bendamustine and cisplatin. One patients underwent irradiation of extramedullary lesion. The mobilization regimen was cyclophosphamide (4 g/m2) followed by daily administration of G-CSF. AutoSCT entailed conditioning with melphalan 200 mg/m2. Conditioning regimen before alloBMT was composed of fludarabine, busulfan and horse anti-thymocyte immunoglobuline. There was no standard acute graft-versus-host disease (GVHD) prophylaxis. For this purpose cyclophosphamide at dose 50 mg/kg at day þ 3, þ 4 was used. Also all pts received mesenchymal stem cells for acute GVHD prophylaxis at the day of WBC recovery 41*10^9/l. Response was defined according to IMWG criteria. Results: After induction only 1 pt achieved partial response, 1 had minimal response and 2 pts had refractory disease. No increase of antimyeloma effect was seen after autoSCT. The median time between auto-SCT and allo-BMT was 5 months (2-5). Neutrophil engraftment was achieved at a median day þ 17. Late acute GVHD had occurred in 1 case. This pt responded to initial treatment with prednisone 1 mg/kg. Three pts achieved complete response (CR) in 2-4 months after alloBMT, (with stringent CR in one case) and 1 pt remained in very good partial response after 4 months after alloBMT. All patients are alive. The median time of follow-up after alloBMT was 7 months (4-18,5). Conclusion: Despite high risk of treatment related mortality, even performed in advanced stage of MM, tandem autoSCT plus alloBMT without standard acute GVHD prophylaxis may result in high CR rate and long-term overall and progressionfree survival. Disclosure of Interest: None declared. P358 The efficacy and tolerability of pomalidomide treatment for multiple myeloma in patients after allogeneic hematopoietic stem cell transplantation S. Janjetovic1,*, F. Ayuk1, M. Christopeit1, C. Wolschke1, U.-M. von Pein1, T. Stuebig1, N. Kroeger1 1 University Medical Center Hamburg-Eppendorf, Hamburg, Germany Introduction: Disease outcome in patients suffering from multiple myeloma (MM) has dramatically improved with the introduction of novel agents, including immunomodulators and proteasome inhibitors. Increased rates of complete remission translated into increased overall survival. Pomalidomide is one of a new immunomodulatory agent approved for the treatment of advanced MM patients. The goal of our study was to determine the tolerability and efficacy of pomalidomide in the therapy of MM patients after allogeneic hematopoietic stem cell transplantation (HSCT). Materials (or patients) and methods: Our study included nine patients, who received pomalidomide therapy (2 to 4 mg) in combination with (n ¼ 7) or without (n ¼ 2) low dose dexamethasone (median dose 20 mg) as salvage (n ¼ 8) or due to persistence of disease (n ¼ 1) after allogeneic HSCT. Results: Pomalidomide therapy started after a median of 13 months (range 4 to 19 months) after allogeneic HSCT. Overall, pomalidomine therapy was well tolerated. In three patients infectious complications like pneumonia and gastroenteritis were observed leading to a temporary discontinuation of the drug. Four patients developed neutropenia (grade I) while one patient developed thrombocytopenia (grade II), which led to a dose reduction of pomalidomide. One patient developed a mild cutaneous Graft-versus-Host-Disease (grade I). Patients, who received 4 mg of pomalidomide, complained more about weakness, when compared to those who received 2 mg. Three out of nine patients showed a very good partial response, four
a partial response, and two stable disease during the therapy. The median duration of response was 8 months (range 4 to 13 months). Conclusion: Although our study group was small, we observed that the therapy with pomalidomide was effective and generally well tolerated after allogeneic HSCT. However, we observed hematologic toxicities and infectious complications requiring temporary discontinuation of the treatment. The incidence of Graft-versus-Host-Disease was not increased. Therefore, we conclude that pomalidomide can be applied safely as salvage treatment in MM patients after allogeneic HSCT. Disclosure of Interest: None declared. P359 Prognostic impact of STAT3 expression in patients with multiple myeloma treated with front-line autologous stem cell transplantation S.-H. Jung1,*, H.-W. Choi2, M.-G. Shin2, D.-H. Yang1, J.-S. Ahn1, Y.-K. Kim1, H.-J. Kim1, J.-J. Lee1 1 Department of Hematology-Oncology, 2Department of Laboratory medicine, Chonnam National University Hwasun Hospital, Hwasun, Korea, Republic Of Introduction: The signal transduced and activator of transcription 3 (STAT3) is considered as prognostic marker in various type of cancer. In this study, we investigated the prognostic significance of STAT3 expression in patients with multiple myeloma (MM) that treated with front-line high-dose therapy and autologous stem cell transplantation (HDT/ASCT). Materials (or patients) and methods: Sixty-nine patients with MM treated with front-line HDT/ASCT were enrolled between June 2005 and March 2013. Dual immunochemical staining (IHC) for phosphotyrosine-STAT3 (PY-STAT3) and CD138 on paraffin-embedded bone marrow sections at diagnosis was performed to evaluate tumor cell-specific PYSTAT3 expression. Results: PY-STAT3 was detected in 7 patients (10.1%) at diagnosis, and there were no significant differences of baseline clinical characteristics according to PY-STAT3 expression. After completion of induction chemotherapy, HDT/ASCT was performed at median 6.3 months. With a median follow-up of 37.7 months, the median progression-free survival (PFS) and
overall survival (OS) were 24.6 months and 76.0 months, respectively. Patients with PY-STAT3 expression had significantly shorter PFS compared to patients without PY-STAT3 expression (10.8 vs. 26.8 months, P ¼ 0.012). In addition, patients with PY-STAT3 expression had poor OS compared to patients without PY-STAT3 expression (57.1 vs. 76.0 months, P ¼ 0.018). Conclusion: This data suggest that PY-STAT3 expression using on dual IHC method is associated with poor survival outcomes in patients with MM treated with upfront HDT/ASCT. Disclosure of Interest: None declared. P360 Successful Stem Cell Mobilization and Autologous Stem Cell Transplantation after Pretreatment consisting of Bendamustine, Prednisone and Bortezomib (BPV) in 35 Patients with newly diagnosed/untreated Multiple Myeloma W. Poenisch1,*, M. Ploetze1, B. Holzvogt1, M. Andrea1, T. Schliwa1, M. Bourgeois1, S. Heyn1, G. N. Franke1, M. Jentzsch1, S. Leiblein1, R. Krahl1, S. Schwind1, V. Vucinic1, H. K. Al-Ali1, D. W. Niederwieser1 on behalf of OSHO (East German Study Group of Hematology and Oncology) 1 Department of Hematology and Oncology, University of Leipzig, Leipzig, Germany Introduction: Bendamustine is a bifunctional alkylating agent with low toxicity that produces both single- and double-strand breaks in DNA, and shows only partial cross resistance with other alkylating drugs. Treatment of patients with newly diagnosed multiple myeloma (MM) using bendamustine and prednisone in comparison to melphalan and prednisone results in superior complete response rate and prolonged time to treatment failure (Poenisch et al, Res Clin Oncol 132: 205-212;2006). So far, however, reliable information on stem cell toxicity and mobilization of stem cells for autologous stem cell transplantation (SCT) after induction treatment with a combination of bendamustine, prednisone and bortezomib (BPV) is missing. Materials (or patients) and methods: A retrospective analysis of peripheral blood stem cell mobilization and autologous SCT was performed in 35 patients with MM who had received at least one cycle of a BPV induction therapy consisting of
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bendamustine 60mg/m2 on days 1 and 2, bortezomib 1.3mg/ m2 on days 1, 4, 8 and 11, and prednisone 100mg on days 1, 2, 4, 8 and 11 between October 2008 and May 2014. The mobilization regimen consisted of cyclophosphamide 4 g/m2 and G-CSF (2x5mg/kg). Apheresis was started as soon as peripheral blood CD34 þ counts exceeded 20x106/L with a harvest target of 8x106 CD34 þ /kg. The minimal accepted target was 2x106 CD34 þ /kg. The pre-transplantation conditioning therapy consisted of melphalan 200mg/m2. Results: A median number of two (range 1–5) BPV treatment cycles were given to the patients. The majority of the patients (n ¼ 31, 89%) responded with 2 sCR, 5 nCR, 11 VGPR, and 13 PR. Three patients had MR, and 1 SD. Stem cell mobilization and harvest was successful in all patients. In 19 of 35 patients (54%) a single apharesis was sufficient to reach the target. The median number of aphareses was one (range 1–4) and the median CD34 þ cell-count/kg was 13.5 (range 3.2–33.1) x106. All patients received an autologous SCT. Engraftment was successful in 34 of 35 patients. The median time to leukocytes count 4lx109/L was reached after 11 days and the time to untransfused platelet count of 450x109/L was 13 days. 34 patients (97%) responded after the autologous SCT with 11 sCR, 2 CR, 7 nCR, 7 VGPR, and 7 PR. The progression free survival at 18 months was 87% and overall survival was 92%. Conclusion: The stem cell mobilization and autologous SCT is feasible in MM patients who have received BPV induction therapy. Disclosure of Interest: None declared.
Myelodysplastic syndromes P361 Prospective analysis of prognostic pre-transplant factors in MDS primarily treated by allogeneic hematopoietic stem cell transplantation: a study on behalf of the MDS subcommittee of the Chronic Malignancies Working Party of the EBMT E. M. Cremers1,*, T. de Witte1, A. van Biezen2, E. Kno¨dler2, J. Finke3, D. Beelen4, G. Socie´5, A. Nagler6, G. Kobbe7, L. Volin8, T. Gedde-Dahl9, J. Sierra10, Y. Beguin11, G. Sucak12, P. Ljungman13, A. Anagnostopoulos14, M. Robin5, N. Kro¨ger15 on behalf of Chronic Malignancies Working Party 1 Hematology, Radboud University Medical Center, Nijmegen, 2 Leiden University Medical Center, Leiden, Netherlands, 3University of Freiburg, Freiburg, 4University Hospital, Essen, Germany, 5 Hopital St. Louis, Paris, France, 6Chaim Sheba Medical Center, Tel-Hashomer, Israel, 7Heinrich Heine Universita¨t, Du¨sseldorf, Germany, 8HUCH Comprehensive Cancer Center, Helsinki, Finland, 9Rikshospitalet, Oslo, Norway, 10Hospital Santa Creu i Sant Pau, Barcelona, Spain, 11University of Liege, Liege, Belgium, 12 Gazi Universitesi Tip Faku¨ltesi Hastanesi, Ankara, Turkey, 13 Karolinska University Hospital, Stockholm, Sweden, 14George Papanicolaou General Hospital, Thessaloniki, Greece, 15University Hospital Eppendorf, Hamburg, Germany Introduction: Allogeneic HSCT is the most potent curative modality in MDS. Major cause of death after HSCT is NRM. NRM has decreased after introduction of reduced intensity regimens (RIC) and has allowed HSCT in more advanced age groups. In this large prospective analysis, performed by the Chronic Myeloid Malignancies Party (CMWP) of the EBMT, prognostic pre-transplant factors in MDS have been analyzed with the focus on the impact of red blood cell transfusions (RBCT) on outcome. Materials (or patients) and methods: All 225 patients in this study received upfront allogeneic HSCT after bone marrow ablative or RIC regimens, between January 2009 and January 2014. Data were collected at diagnosis, at transplantation and 6 weeks, 100 days, 1 year and 2 years after SCT. We analyzed
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the impact of WHO classification, number of transfusions, iron parameters, comorbidities, (non-)infectious complications on the occurrence of non-relapse mortality and overall survival. Results: 48% of the patients showed unilineage or multilineage MDS with o5% marrow blasts, 62% had 45% marrow blasts. According to IPSS 48% had lower risk MDS and 52% had higher risk MDS, at HSCT. 61 Patients have received RBCT before HSCT. 45% of patients received HSCT after standard intensity conditioning regimens and 55% after RIC regimens. Median interval between diagnosis and SCT was 10 months (range 1-128 months). Analyzed pre-transplant factors were: transplant and donor characteristics, comorbidities (present in 41% of the patients), median number of RBCT in 139 patients treated with RBCT before HSCT: 13 units (range: 1-146), ferritin levels (median 715 ng/ml; range 10-9033), median transferrin levels: 201 mg/dL (range 2-1946), unbound serum iron (median 116ug/dL; range 0-463). Twenty-five patients have been treated iron chelation prior to HSCT, and 25 patients at 2 years after SCT. 6 Patients received chelation both prior to and after HSCT. Nine patients underwent phlebotomies after HSCT. Two years OS was 64%, NRM 22% and RI 25%; 14% of patients died after relapse or progression of disease. Within this upfront allografted MDS cohort WHO-classification and cytogenetics had only minimal impact on treatment outcome (data not shown). Age had a significant impact on OS (HR 2.22; P ¼ 0.008), but not on NRM or RI. This might be explained by the influence of aging and increased comorbidities at higher age. Patients transplanted with peripheral blood as stem cell source had a significantly lower risk of death, OS (HR 0.46; P ¼ 0.049) and NRM (HR 0.32; P ¼ 0.017), but impact on RI was absent. We found a significant impact of transfusion burden (o20 RBCT versus 420 RBCT) on NRM (HR 1.89; Po0.05), without a significant effect on OS (HR 1.48 P ¼ 0.116). Ferritin levels (41000 ng/ml) had nonsignificant (P ¼ 0.06) negative impact on survival after SCT. Conclusion: This large observational study is the first prospective study in HSCT for MDS with detailed information of several important prognostic factors including disease modalities, transfusional data and iron parameters. Diseasespecific modalities had only minor impact on treatment outcome. However, age and transfusion burden predicted outcome after allogeneic HSCT. The impact of iron chelation before and after HSCT will be analyzed in mi ¼ ore detail in subsequent analyses. Disclosure of Interest: None declared. P362 Characterization and MicroRNA Expression Profile Analysis of Mesenchymal Stem Cells Derived From Myelodysplastic Syndrome and Acute myeloid leukemia (AML) Patients and Comparison with Healthy Controls H. Ozdogan1,*, B. Gur Dedeoglu1, A. Atalay1, S. Kose2, Z. A. Yegin3, F. Avcu4, D. Uckan Cetinkaya2, O. Ilhan5 1 Biotechnology Institute, Ankara University, 2Stem Cell Research and Application Center, Hacettepe University, 3Hematology, Gazi University, 4Hematology, Gulhane Military Medical Academy, 5 Hematology, Ankara University, Ankara, Turkey Introduction: Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) represent the heterogenous groups of clonal diseases that affect the stem cells in bone marrow. Their pathogenesis is complex and likely depends on interplay between aberrant hematopoietic cells and hematopoietic microenvironment (HM). The key component of the HM is mesenchymal stem cells (MSC). Only a few studies characterized abnormalities in the HM in MDS and AML. Recent findings suggest that a specific deletion of Dicer1 in MSC-derived osteoprogenitors triggers several features of MDS and AML in a murine model. Dicer1 is an RNase III endonuclease essential for microRNA (miRNA) biogenesis and RNA processing. miRNAs are 18 to 25 nucleotide non-coding RNAs regulating gene expression. In recent years, several studies have reported that the miRNAs play important roles in the regulation of
expression of the cytokine molecules as well as in the regulation of the differentiation of MSC and hematopoietic stem cells (HSC). These data suggest that there may be functional roles of miRNAs in the regulation of HM and miRNA deregulation in MSC may play a role in the pathology of MDS and AML. The aim of the present study is to analyze miRNA expression in MSC obtained from bone marrow (BM) of MDS and AML patients. Materials (or patients) and methods: In this study miRNA expression profiles of MSCs from BM samples of MDS (n:3 RAEB and RA)/AML patients (n:3 M1, M4) and healty donors (n:2) were evaluated. BM aspirates were collected from MDS and AML patients and healty donors. MSCs from MDS and AML were expanded in vitro and characterized. RNA isolation was performed from MSC cultures at passage 3. miRNA expression profiles were identified by miRNA microarray analysis (Agilent). miRNA microarray data analysis was performed by BRB Array Tools. Statistical methods such as ANOVA and t-test were used for the determination of miRNAs differentially expressed in MDS and AML patients compared to that of healty donors. The targets of differentially expressed miRNAs were identified by using bioinformatics tools and pathway enrichment analysis was performed with these target genes. Results: Three sets of differentially expressed miRNAs were obtained at the end of microarray experiments. To find out the miRNAs of MSC specific to AML and MDS, the two former group was compared to heathy donors and 287 miRNAs were found to be differentially expressed in MDS while it was 87 for AML patients compared to healthy donors. Additionally the comparison of MDS and AML resulted 34 differentially expressd miRNAs (Po0,05 and fold change 42). The pathway enrichment analysis was conducted with the target genes of top 3 miRNAs from each group. The target genes were found to be enriched in the pathways, which are important in the regulation of HM and cancer development and progression; MAPK, chemokine signaling pathway, cytokine-cytokine receptor interaction, hedgehog signaling pathway, wnt signaling pathway, VEGF signaling pathway, notch signaling pathway and TGF-beta signaling pathway. Conclusion: With the preliminary findings obtained from this study it could be suggested that miRNA exspression changes in MSCs might be involved in MDS and AML pathogenesis and miRNAs might be new players in the regulation of HM by targeting the genes taking part in pathways important in HM. Disclosure of Interest: H. Ozdogan Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest , Personal Interest: There is no any conflict of interest , Conflict with: There is no any conflict of interest, B. Gur Dedeoglu Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, A. Atalay Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, S. Kose Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, Z. A. Yegin Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, F. Avcu Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, D. Uckan Cetinkaya Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest, O. Ilhan Funding from: There is no any conflict of interest, Employee of: There is no any conflict of interest, Personal Interest: There is no any conflict of interest, Conflict with: There is no any conflict of interest
P363 Superior long-term outcomes of allogeneic hematopoietic cell transplantation when transplanted in response compared to relapsed or refractory after hypomethylating agents for patients with lower risk myelodysplastic syndrome J. H. Moon1,*, S. K. Sohn1, J. S. Ahn2, H. J. Kim2, Y.-R. Do3, M. K. Song4, H. J. Shin4, W. S. Lee5, S. M. Lee5, Y. D. Joo6, H. Kim7, H. S. Lee8, Y. S. Kim8, Y. Y. Cho9, S. H. Bae9 1 Hematology/Oncology, KYUNGPOOK NATIONAL UNIVERSITY HOSPITAL, Deagu, 2Hematology/Oncology, Chonnam National University Hwasun Hospital, Hwasun , 3Hematology/Oncology, Keimyung University Dongsan Medical Center, Deagu, 4Hematology/Oncology, Busan National University Hospital, 5Hematology/ Oncology, Inje University Busan Baik Hospital, 6Hematology/ Oncology, Inje University Haewoondae Baik Hospital, Busan, 7 Hematology/Oncology, Ulsan University Hospital, Ulsan, 8Hematology/Oncology, Kosin University Gosper Hospital, Busan, 9 Hematology/Oncology, Daegu Catholic University Medical Center, Deagu, Korea, Republic Of Introduction: Allogeneic hematopoieic cell transplantation (Allo-HCT) is generally recommended for lower-risk myelodysplastic syndrome (LR-MDS) patients with relapsed or refractory to HMA treatment. It is still under debate when LR-MDS patients should be transplanted in response or at the time of loss of response. This study retrospectively evaluated the outcomes of allo-HCT who transplanted in response or relapsed/refractory after HMA treatment. Materials (or patients) and methods: The data of 307 patients diagnosed with LR-MDS from Oct 1992 to Jul 2013 were retrospectively evaluated. To address the time dependence of allo-HCT, the methods of Simon and Makuch and the Mantel-Byar test was used. Results: Among 307 patients with LR-MDS, 200 patients were treated with HMA. Sixty-three (31.5%) patients showed response (CR/PR/HI) to HMA and 119 (59.5%) were relapsed/ refractory (SD/PD). Prior to Allo-HCT, 7 patients were in response and 26 patients in relapsed/refractory. Median time to allo-HCT were 2.8 years in responders and 1.9 years for patients with relapsed/refractory. Among HMA responders, the 5-year overall survival (OS) rate were 83.3% with allo-HCT (n ¼ 7) and 34.2% with continued HMA treatment (n ¼ 53) (P ¼ 0.07). Among patients with relapsed/refractory, the 5-year OS rate were 47.0% with allo-HCT (n ¼ 26) and 29.4% with BSC after HMA treatment (n ¼ 93) (P ¼ 0.45). There showed a trend in the benefits of OS for patients treated with allo-HCT in response compared to allo-HCT in SD/PD (P ¼ 0.17). In the multivariate analysis, ECOG-PS 2-3 (HR 3.030, P ¼ 0.006), IPSS blast Z0.5% (HR 2.003, P ¼ 0.018), poor cytogenetic risk group
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(HR 12.362, Po0.001), and non-response to HMA (HR 2.487, P ¼ 0.001) were unfavorable factors for OS among LR-MDS patients. Conclusion: Allo-HCT in response to HMA showed superior OS compared to allo-HCT in relapsed/refractory after HMA for patient with LR-MDS. To elucidate best time points of allo-HCT for LR-MDS patients, a large scale study will be needed. Disclosure of Interest: None declared. P364 The outcome of allogeneic stem cell transplantation in myelodysplastic syndrome is related to disease burden at transplant: a multicenter study of the Polish Adult Leukemia Group L. A. Gil1,*, J. Dwilewicz-Trojaczek2, M. Komarnicki1, S. Kyrcz-Krzemien3, M. Markiewicz3, M. Dzierzak-Mietla3, P. Zielinska3, J. Styczynski4, M. Borowczyk1, A. Nowicki1, M. Baranska1, M. Bieniaszewska5, A. Hellmann5, G. Basak6, W. W. Jedrzejczak6, K. Madry6, A. Lange7, K. Halaburda8, B. Jakubas9, P. Mensah-Glanowska9, P. Rzepecki10, A. Wierzbowska11, M. Ussowicz12, J. Dybko12, T. Wrobel12, T. Czerw13, S. Giebel13 1 Department of Hematology, Poznan University of Medical Sciences, Poznan, 2Department of Hematology, Oncology and Internal Diseases, Medical University of Warsaw, Warszawa, 3 Depatment of Hematology and BMT, Medical University of Silesia, Katowice, 4Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, 5Department of Hematology, Medical University of Gdansk, Gdansk, 6Department of Hematology, Oncology and Internal Diseases, Medical University of Warsaw, Warszawa, 7 Department of Hematology, Lower Silesian Center for Cellular Transplantation, Wroclaw, 8Department of Hematology, Institute of Hematology and Transfusion Medicine, Warszawa, 9Department of Hematology, Collegium Medicum, Jagiellonian University, Krakow, 10Department of Hematology, Military Institute of Medicine, Warszawa, 11Department of Hematology, Medical University of Lodz, Lodz, 12Department of Hematology, Wroclaw Medical University, Wroclaw, 13Department of Hematology and BMT, Cancer Center and Institute of Oncology, Gliwice, Poland Introduction: Allogeneic stem cell transplantation (alloSCT) is the only curative strategy for patients (pts) with myelodysplastic syndrome (MDS), however numerous controversies exist regarding timing of transplant, disease burden at transplant, the need for pretransplant therapy, conditioning intensity and stem cell source. Therefore, the Polish Adult Leukemia Group (PALG) analyzed the outcomes of MDS pts transplanted between 2000 and 2013 with respect to pretransplant factors. Materials (or patients) and methods: Two hundred and sixteen pts with median age of 47 years (range: 18-69) were retrospectively analyzed. The study cohort included pts with intermediate-2/high risk according to IPSS (101), unfavorable cytogenetics (70), secAML (34) and therapy related disease (tMDS) (26). Seventy four (34%) pts were older than 50. Before transplantation 90 pts had o5% bone marrow blasts, while 43 (20%) pts underwent alloSCT with blasts 410%. Ninety three pts were reported to be dependent on blood transfusion. Ferritin level was available for 48 pts, with elevated level 41000 ng/mL found in 28. In 68 (31%) pts pretransplant intensive therapy was applied. For conditioning, RIC protocols based on fludarabine were used in 113 (53%) pts and 103 pts were treated with MAC, mainly based on busulfan (66) or treosulfan (34). Stem cells from related (113) or unrelated (103) donors were grafted in median dose 4.6 (0.5-12.5)x106 of CD34 þ cells/kg. PB stem cells were transplanted in 168 (78%) pts. Twenty five pts received allograft from HLA mismatched donors. Results: Engraftment occurred in 206 (95%) pts and 6 pts died before þ 21 day after alloSCT. Infectious complications were observed in 189 (87%) pts during neutropenia (FUO 120, bacterial 45, fungal 17, bacterial and fungal 7). Late infections
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occurred in 106 pts, and were related mainly to CMV infection. Toxic complications (CTCAE grade 3-4) developed in 42 (19%) pts. Acute GVHD grade 2-4 occurred in 85 (36%) pts, while chronic extensive GVHD in 32 (15%) pts. Forty two pts experienced disease relapse after treatment. Over a median follow-up of 43 months (range: 15-70) 117 (54%) pts were alive. In 29 cases deaths were related to relapse, 20 pts died due to GVHD, 36 due to infection and 13 because of organ toxicity. Probability of 3-years overall survival (OS), disease free survival (DFS), relapse incidence (RI) and treatment related mortality (TRM) were 0.396±0.051, 0.384±0.05, 0.28±0.042 and 0.421±0.056, respectively. Among factors studied in univariate analysis those associated with lower OS were: tMDS (P ¼ 0.04; HR 2.2), bone marrow blasts 4 10% before alloSCT (P ¼ 0.04, HR 1.7) and PB as a source of stem cells (P ¼ 0.05; HR 1.7). In multivariate analysis the only factor associated with lower OS was bone marrow blasts 410% (P ¼ 0.023, HR 1.7). In subgroup analysis ferritin level 41000 ng/mL was significantly related to lower OS (P ¼ 0.001; HR 4.6). There were no statistically significant differences in OS, DFS, RI and TRM with respect to age, IPSS risk group, cytogenetics, dependence on transfusion, pretransplant therapy, intensity and type of conditioning, donor choice and HLA compatibility. Conclusion: Allogeneic SCT is an option for MDS patients in different age groups. Disease status at time of transplant is the most important determinant for the outcome. High rate of TRM remains a concern. Disclosure of Interest: None declared. P365 Next generation sequencing – based delineation of mutational spectrum in 40 patients with MDS uniformly treated with sequential FLAMSA-RIC-conditioning and allogeneic hematopoietic stem cell transplantation M. Christopeit1,*, A. Badbaran1, T. Zabelina1, N. Kro¨ger1 1 Department of Stem Cell Transplantation, University Hospital Eppendorf, Hamburg, Germany Introduction: Prognosis of patients with advanced MDS is poor. Allogeneic stem cell transplantation is a curative treatment. Current prognostic schemes take, amongst other parameters, cytogenetics into account. A classification that relies on the mutational status of recurrently mutated genes in MDS has not been developed. To correct for potential bias, correlation of mutational status with outcome best is performed in uniformly treated groups of patients. Materials (or patients) and methods: To this end, the mutational status of 40 patients suffering MDS was evaluated and correlated with overall survival (OS). Conditioning consisted of sequential chemotherapy (FLAMSA scheme) and busulfan-based reduced-intensity conditioning. Patients suffered RAEB-I (n ¼ 6), RAEB-II (n ¼ 18), or other forms of MDS (n ¼ 16). Median age was 62 years (range 39-73). Patients were transplanted from a matched related donor (n ¼ 4) or an unrelated donor (n ¼ 36). Results: DNA was isolated following standard procedures. Semiconductor based Next Generation Amplicon Sequencing (NGS) was carried out using an Ion Torrent Personal Genome Machine (PGMTM, Thermofischer). A mutational panel was constructed and consisted of ASXL1 (exon 12), BRAF COSMIC mutation (COSM476), CBL (exon 8, 9), CEBPA (all coding exons), DNMT3A (all coding exons), FLT3 (codons 676, 830850), GATA2 (all coding exons), IDH1 (exon 4), IDH2 (exon 4), JAK2 (exon 14), KIT (exons 8, 10, 11, 17), KRAS (exons 2, 3, 4), NPM1 (exon 12), NRAS (exons 2, 3, 4), PTPN11 (exons 3, 7, 8, 13), RUNX1 (exons 3-8), TET2 (all coding exons), TP53 (all coding exons), WT1 (exons 7, 9). One run was finished within two days. Mutations were found in 38/40 patients (95%, median number of mutations 2, range 0-6, exactly 1 mutation in 15 patients, 2 mutations in 13 patients, 3 mutations in 4 patients, 4 mutations in 3 patients, 5 mutations in 2 patients, 6 mutations in 1 patient). Mutated genes were ASXL1 (6 pts; missense, ms, 2, frameshift, fs, 3, ms þ fs 1), BRAF (ms 1),
CEBPA (8 pts; ms 5, fs 2, silent, sil, 1), DNMT3A (8 pts; ms 6, fs 2), FLT3 (ms 2), GATA2 (12 pts; ms 10, fs 2), IDH2 (ms 4), KIT (ms 1), KRAS (4 pts; ms 1, sil 1), NPM1 (fs 2), NRAS (ms 5), RUNX1 (12 pts; ms 2, fs 6, stop codon 4), TET2 (13 pts; ms 3, fs 2, stop codon 1, 2xms 1, ms þ sil 1, ms þ stop 1, 2xms þ stop 1, 2xms þ sil 1, 2xms þ stop 1, 2xstop 1), TP53 (5 pts; ms 4, 2xms 1). No significant correlation between the number of mutations and OS was found (P ¼ 0.672, log rank test). In multivariate analysis including all mutations discovered as covariates, FLT3 mutations were a significant covariate for OS (Po0.001) with an adverse prognostic impact. Conclusion: In conclusion, semiconductor based Next Generation Sequencing analysis of amplicon mutations in patients with MDS before allogeneic HSCT might prove a feasible means to a timely risk stratification. Further follow up analysis should cover the mutational status of patients missing complete hematologic remission in order to unravel treatable remaining malignant clones and the prognostic power of minimal residual disease. Unbiased analysis of mutations by whole genome sequencing might lead to further marker mutations and mechanistic insights. MC and AB contributed equally to this work. Disclosure of Interest: None declared. P366 Use of azacytidine as a bridge to allogeneic stem cell transplantation in MDS R. R. Pol1,*, K. Raj2, I. Lazana3, N. Russell1, P. Ferguson4, C. Craddock4, P. Mehta5, S. Killick6, E. Das-gupta1 1 Nottingham University hospital, U.K., Nottingham, 2Kings College hospital, Haematology, 3Kings College hospital, Kings College hospital, London, 4Queen Elizabeth Hospital Birmingham, Birmingham, 5University Hospitals Bristol NHS Foundation Trust, Bristol, 6Royal Bournemouth General Hospital, Bourmouth, United Kingdom Introduction: Allogeneic stem cell transplantation (alloSCT) is the only potential curative therapeutic approach in myelodysplastic syndrome (MDS). AML induction chemotherapies are associated with toxicities which could prohibit proceeding to alloSCT. Hence we investigated the impact of prior-totransplantation azacytidine (AZA) on patient outcome for MDS. Materials (or patients) and methods: We included 58 consecutive patients from 5 centres who underwent alloSCT for MDS between November 2006 and July 2014. They were male-37, female-21. The median age was 57 (range 24-75). WHO diagnosis were RCMD-3, RAEB1-19, RAEB2 -22, CMML-6, Fibrotic MDS-3, MDS/MPN – 2 and AML – 3. At diagnosis of MDS, 46/52 (86.5%) had intermediate-2 or high risk MDS as per IPSS and 7/52 (13.4%) had IPSS-1. Median number of days from diagnosis to HSCT was 414 (range 120-2920). Median Aza cycles to best response was 4 (range 1-9). Single agent azacytidine was used as a bridge to alloSCT in 46/58 patients. Additional chemotherapy was required in the remaining 12/58
patients post azacytidine but prior to HSCT to achieve remission (AML-8, SD-2, PR-1, CR-1). Additional treatment was FLAG-2, Flag-Ida -2, DA-3, DA þ Myelotarg-4, Clofarabine 1). Conditioning was myeloablative in 4 and RIC in 54. Graft was T replete in 5 (FLAG - 3, Flu-Cy-TBI-2) and T cell deplete in 50 (ATG/Alemtuzumab). The donor source was from siblings – 15, Cord-3, Haploidentical -2, and MUD-38. Results: Response to azacytidine before alloSCT was CR-30 (51.7%), PR-10 (17.2%), SD-10 (17.2%), and failure-8 (13.7%). Additional chemotherapy achieved CR in 11 and SD in 1. All patients who received azacytidine only (46) and additional chemotherapy (12) successfully received HSCT. After a median follow-up of 405 days (range 38-1882), the median survival was 480 days (1 year 3 months). OS was 64.7% at 1 year, 38% at 2 years and 34.7% at 3 years. Relapse at 1 year was seen in 16/58 patients (27.5%). Nonrelapse mortality was seen in 9/58 (15.5%) at 1 year. Mortality due to disease relapse/progression was seen in 9/58 (15.5%). DFS was 56% at 1 year. The median time to neutrophil engraftment (4 0.5) was 12 days (range 623) and median time to platelets engraftment (4 50) was 14 days (range 1-41). 56 patients were evaluable for aGVHD and this was seen in 34(60.7%). Grade I and II in 22(39.2%), Grade III and IV in 12(21.4%). Out of the 54 evaluable patients, 23 (42.5%) developed cGvHD (extensive in 11 (20.3%) and limited in 12 (22.2%). Conclusion: Single agent Aza is of value in stabilizing, achieving remission and allowing time for patients to reach transplantation. This is with less toxicity than conventional intensive chemotherapy and is useful as a bridge to alloSCT without affecting transplant outcomes. Disclosure of Interest: None declared. P367 Allogeneic stem cell transplantation for MDS patients more than 70 years of age. A report of MDS subcommittee of CMWP of EBMT S. Heidenreich1,*, T. Zabelina1, A. van Biezen2, J. Finke3, U. Platzbecker4, D. Niederwieser5, H. Einsele6, W. Bethge7, R. Schwerdtfeger8, D. Beelen9, J. Tischer10, A. Nagler11, P. Zache´e12, C. Scheid13, T. de Witte14, M. Robin15, N. Kro¨ger1 1 University Hospital Eppendorf, Hamburg, Germany, 2EBMT Data Office, Leiden, Netherlands, 3University of Freiburg, Freiburg, 4 University Hospital, Dresden, 5University Hospital, Leipzig, 6 University Hospital, Wu¨rzburg, 7University of Tu¨bingen, Tu¨bingen, 8Deutsche Klinik fu¨r Diagnostik, Wiesbaden, 9University Hospital, Essen, 10Klinikum Grosshadern, Munich, Germany, 11 Chaim Sheba Medical Center, Tel-Hashomer, Israel, 12ZNA, Antwerp, Belgium, 13University of Cologne, Cologne, Germany, 14 Radboud University Medical Centre, Nijmegen, Netherlands, 15 Hopital St. Louis, Paris, France Introduction: Myelodysplastic syndromes (MDS) are diagnosed at median age of 75 years. Allogeneic stem cell transplantation (HSCT) is the only curative treatment option, but with an increasing age, the number of comorbidities is escalating. Treatment guidelines suggest HSCT for intermediate-II and high risk constellations up to the age of 65 years, and reduced intensity conditioning (RIC) regimens are commonly used up to 70 years of age. However, increasing life expectancy, availability of RIC regimens and good Karnofsky performance status (KPS) of MDS patients more than 70 years of age, has led to an increased use of HSCT. We performed a retrospective analysis to investigate results after HSCT for those patients and influence of KPS on outcome. Materials (or patients) and methods: We analyzed data of 345 patients in the EMBT database older than 70 years with MDS (n ¼ 238) and secondary acute leukemia (sAL, n ¼ 107) which have undergone HSCT from related (n ¼ 89) and unrelated (n ¼ 256) donors between 2000 and 2014. Median follow up was 13 months (4-37 months). Median age at transplantation was 72 years (70-79 years) with male:female ratio of approximately 2:1. KPS was defined in 295 cases.
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Presence of acute graft-versus-host disease (aGVHD) (n ¼ 129) was documented for 320 patients. Univariate (Kaplan-Meier) and multivariate analysis (Cox regression test, competing risks - Fine & Gray test) were used to calculate overall survival (OS), transplantation related mortality (TRM), Relapse rates (REL) in relation to RIC (n ¼ 261) versus MAC (n ¼ 84) regimes, related or unrelated donor, KPS 90-100% versus r80% and presence of CMV antibodies. Due to missing data, FAB or WHO subclassification, IPSS scores, cytogenetics and information about HLA-match of the graft, as well as occurrence of chronic GVHD could not further be considered. Results: The number of HSCT for MDS patients of 70 years or more has increased over time. While 2000-2004 only 19 patients received transplantation, the following 3-year periods included 28 (2005-2007), 97 (2008-2010) and 200 (2011-2013) patients, respectively. 3-year estimated OS and REL were 34% (28-40%) and 44% (34-54%), respectively. TRM after 1 year was 40% (34-46%). KPS of 90-100% had a significant benefit for OS (P ¼ 0.037) and TRM (P ¼ 0.129) but not for REL. These results were confirmed in the multivariate analysis. Use of RIC regimens was superior in OS (P ¼ 0.037) and TRM (P ¼ 0.016) but did not result in higher REL. Patients with related donors had more REL (P ¼ 0.0008) without influence on OS. The presence of CMV antibodies in the patient improved OS (P ¼ 0.015). No significant influence on OS was seen for diagnosis at transplantation (MDS/sAL), complete remission before transplantation, use of TBI, in vivo t-cell depletion and use of bone marrow versus peripheral blood stem cells. Conclusion: HSCT for MDS patients 70-years of age or older is a possible choice of treatment with a 3-year OS of 34%. Good performance, determined by KPS, increases the chance of survival as well as the use of RIC regimens, which is not compromised by higher relapse rates. According to these data, MAC regimens are associated with high risk for TRM. Relevance of IPSS, cytogenetics, MDS subclassification and HLA-match are unclear due to a lack of data and should be considered in subsequent investigations. Disclosure of Interest: None declared. P368 Abstract Withdrawn
Solid tumours P369 Peripheral blood stem cell autografts for the treatment of children neuroblastoma A. Hedayati Asl1,* on behalf of Tashvighi M, Zangooei R, Mehrvar A, Iravani N, Astaraki S, Odameh Sh,Fallah V 1 Pediatric Stem cell transplantation, MAHAK, Tehran, Iran, Islamic Republic Of Introduction: The long-term survival probability for children with high-risk neuroblastoma was less than 15 percent. Better results have been achieved using an aggressive multimodality approach that includes chemotherapy, surgical resection, high-dose chemotherapy with hematopoietic stem-cell rescue, and radiation therapy. The prognosis of high-risk neuroblastoma after conventional chemotherapy is generally poor. The use of high-dose chemotherapy with autologous hematopoietic stem cell rescue in consolidation has resulted in improvements in survival, although further advances are still needed. Materials (or patients) and methods: seventeen patients age 5 to 20 years (median 4 years, M/F ¼ 13/4), with high risk, relapsed or metastatic underwent ASCT in our hospital (from 2012-2014). 15 of the 17 patients underwent a first HDCT/ASCR
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and 2 patients underwent a second HDCT/ASCR. 7 of the 17 patients who were evaluated for N-myc amplification were positive. Status at transplant was: second complete remission (CR2): n ¼ 11; CR3: n ¼ 3, (CR 43) n ¼ 3. All patients received chemotherapy- based conditioning regimens: MEC regimen consisting of carboplatin for 3 days, VP-16 for 3 days and melphalan in 2 days was the primary cytoreductive regimen. Peripheral blood (PB) was the source of progenitor cells in 17 patients. All patients engrafted. Results: The median mononuclear cell dose was 4.8 108/ kg. The median time to absolute neutrophil count 4 0.5 109/L was 11 days, and the median time to platelet count 4 20 109 was 14 days. One patient out of 17 (5.8%) experienced transplant-related mortality. Three patients relapsed after transplant (mean 8m.). With a median followup of 11 months (3-20 months) after transplant the event-free survival (95% confidence interval) was 0.64. 14 patients remain alive. Conclusion: Longer follow-up is required to evaluate fully efficacy and long term survival of our patients. However, further studies will be needed to decrease the toxic death rate in the transplantation. Disclosure of Interest: None declared. P370 Potential use of cytokine activated patients’ NK cells in metastatic colorectal cancer refractory to conventional therapies D. Montagna1,*, F. Ferulli2, S. Brugnatelli2, I. Turin2, M. Tanzi2, S. Delfanti2, C. Klersy2, D. Lisini3, E. Todisco4, D. Pende5, R. Maccario2, P. Pedrazzoli2 1 Fondazione IRCCS Pol. San Matteo, Universita` di Pavia, 2 Fondazione IRCCS Pol San Matteo, Pavia, 3Fond. IRCCS Istituto Neurologico C. Besta, Milano, 4Humanitas Cancer Center, Rozzano (MI), 5AOU San Martino-IST Genoa, Genova, Italy Introduction: The failure of conventional treatment to significantly improve outcomes in mCRC has prompted the development of immune-based therapies, including natural killer (NK) cells-based strategies. We investigate the capacity of patients-derived NK cells, either resting or after cytokine activation, to lyse autologous metastatic colorectal cancer (mCRC) cells which have been analyzed for expression of HLA Class I and of ligands for adhesion and triggering NK receptors involved in their recognition and killing. We also evaluated whether KRAS mutated mCRC cells were susceptible to antiEGFR-induced ADCC mediated by NK cells. Materials (or patients) and methods: After obtaining signed informed consent, 25 mCRC patients have been enrolled. mCRC cells, from primary or metastatic tissue, were expanded in vitro and analyzed to confirm their neoplastic origin. Ligand expression was evaluated by cytofluorimetric analysis and gene expression on mCRC cells. Purified NK cells were activated overnight (ON) and for five days with IL-2 or IL-15 and were analyzed for expression of triggering and inhibitory receptors and for their ability to kill autologous mCRC cells alone or previously coated with antiEGFR mAbs. Results: mCRC were successfully expanded from 21 of 25 samples. Experiments from 10 patients showed a substantial inability of patients resting NK cells to lyse mCRC cells. ON activation resulted in greatly increased NK cytotoxic potential (IL-2: mean 28%; range:10-68; and IL-15: mean 38%; range 1881 at E:T ratio of 40:1). Five days of NK cell activation were able to further enhance their lytic capability and to reduce the variability of the results obtained in ON condition(IL-2: mean 43%; range: 33-56; and IL-15: mean 58%; range 36-87 at E:T ratio of 40:1). Experiments performed in 6 patients (3 wild type and 3 KRAS mutated) demonstrated that the incubation of mCRC cells with anti-EGFR mAbs increase their susceptibility to NK-mediated lysis irrespective of KRAS status of tumor cells (mCRC alone: IL-2: mean 22%; range:17-39; and IL-15: mean 41%; range 18-87; mCRC EGFR-coated: IL-2: mean
32%; range: 22-48; and IL-15: mean 53%; range 44-84 at E:T ratio of 20:1). NKp30 and NKp46 were variably expressed by patient NK cells and could be up-regulated after cytokine activation, while mCRC expressed different levels of most NK ligands. Conclusion: The demonstration that mCRC cells can be efficiently lysed after in vitro NK cells activation support the ongoing design of immunotherapy with autologous NK cells for poor prognosis mCRC patients. References: Disclosure of Interest: None declared. P371 Pediatric Solid Tumors: an in vitro model to improve the efficacy of haploidentical stem cell transplantation M. Comini1, F. Bolda1,*, A. Beghin1, C. D’Ippolito2, D. Alberti3, L. Ruggeri4, L. Bercich5, G. Carella6, F. Porta2, A. Caruso7, A. Lanfranchi1 1 Stem Cell Lab, Children’s Hospital, 2Pediatric Oncohaematology and BMT Unit, Children’s Hospital, 3Pediatric Surgery Unit, Children’s Hospital, Spedali Civili di Brescia, Brescia, 4Division of Hematology and Clinical Immunology, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, 5 Department of Pathology I, University of Brescia, 6Reumatology and Clinical Immunology Lab, Spedali Civili di Brescia, 7Section of Microbiology, University of Brescia, Brescia, Italy Introduction: Pediatric solid tumors are often characterized by a poor prognosis. Some of these, such as Rhabdomyosarcoma (RMS), Neuroblastoma(NB), Ewing Sarcoma(ES), are defined high-risk and disease free survival is less than 20%. In these patients a standard approach with chemotherapy and/or radiotherapy is not sufficient to allow a good prognosis. This fact highlights the need for new therapeutic strategies as the haploidentical hematopoietic stem cell transplantation (HSCT) that could represent a therapeutic immunological approach thanks to GvL and GvT. The aim of the study is to design an in vitro model to select the best donor to use in haploidentical HSCT in a solid tumor setting. Materials (or patients) and methods: We enrolled 29 patients with age between 0 and 16 years (mean: 5.4, median: 4). There were 8 RMS, 12 NB, 3 ES and 6 Wilms Tumor (WT). We decided to introduce in this study also WT to evaluate a possible immunological approach in an intermediate-risk solid tumor setting. We set up primary tumor cell lines and we characterized them first immunohistochemically, with specific tumor markers to evaluate the % of neoplastic cells, and phenotypically by flow cytometry. We created a specific panel of 22 markers: 5 hematopoietic markers as negative controls (CD45, CD14, CD34, HLA-DR, CD56), 7 mesenchymal (CD133, CD44, CD29, CD73, CD105, CD90, HLA-ABC), 1 endothelial (CD31) and 9 tumoral (CD271, CD71, MIC-A/B, CD155, CD112, CD9, CD58, CD99, Myogenin). Finally, we analyzed the HLA of patients whose culture was evaluated with good % of neoplastic cells and the alloreactivity of donor NK cells against patient’s blasts and primary neoplastic cell lines. We performed 2 HSCT by selection of
CD34 þ cells and we studied the patient’s post-transplantation follow up. Results: We obtained about 60% of primary culture evaluated as neoplastic with immunoistochemical analysis. The phenotypic evaluation analysis shows high expression of mesenchymal markers, particularly CD44, CD105 and CD90, while CD73 has an heterogeneous expression, and a low expression of CD45 (always less than 1%). Tumor markers, such as CD10, CD58, CD9, CD271, have a variable expression. The evaluation of HLA allowed us to find 3 patients missing of a receptor: 2 missing Bw4 and 1 missing C1. 2 patients underwent haploidentical HSCT (Table). Conclusion: The high expression of mesenchymal markers confirms what reported in literature: mesenchymal markers are often adhesion molecules physiologically expressed on the membrane of normal cells and involved in processes such as migration or cell-cell interaction, proliferation or differentiation, but on tumor cells can be up-regulated and act as positive regulators for tumor progression and metastasis processes. Solid tumors haven’t hematopoietic origin, so the negativity for the hematopoietic markers makes the great first screening of hematopoietic tumors and not hematopoietic ones. We found heterogeneity in the expression of tumoral markers; this can be explained by the fact that the expression of some cell surface receptors may represent different stages of maturation of tumor as well as different expressions related to different tissues and organs. Thanks to our work we document the importance of have an in vitro model that better mimics what occurs in vivo and consequently improves the opportunities and criteria for the selection of the best potential haploidentical alloreactive donor. Disclosure of Interest: None declared. P372 Biosimilar filgrastim in the treatment and prevention of chemotherapy-induced neutropenia in patients with solid tumours: a sub-analysis of the NEXT study F. Maloisel1,*, S. Lepreˆtre2, D. Kamioner3, C. Berthou4, H. Albrand5 1 Sainte-Anne Clinic, Strasbourg, 2Henri Becquerel Hospital, Rouen, 3Hoˆpital Prive´ de l’Ouest Parisien, Trappes, 4Hoˆpital Morvan, Brest, 5Hospira France, Meudon La Foret, France Introduction: The safety and efficacy of biosimilar filgrastim (Nivestimt, Hospira Ltd), a granulocyte-colony stimulating factor licensed for the treatment of chemotherapy (CT)induced febrile neutropenia (FN), were assessed post-market in the NEXT study. Here we present the results for patients (pts) receiving CT for solid tumours. Materials (or patients) and methods: The NEXT study was a prospective, observational, non-interventional, longitudinal, national, multicentre study, which recruited 2114 adult pts undergoing cytotoxic CT and receiving biosimilar filgrastim as primary or secondary prophylaxis, or as curative treatment. The primary objective was to evaluate the safety of biosimilar filgrastim. Secondary objectives were to evaluate the efficacy of biosimilar filgrastim, to describe characteristics of patients treated with biosimilar filgrastim, and patterns of use of biosimilar filgrastim.
[P371]
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Data collected included pt characteristics, biosimilar filgrastim treatment-related data and treatment emergent AEs. Pts were followed up for a maximum of six CT cycles at three visits: inclusion, during treatment, and following CT. The current subanalysis includes pts receiving CT for a solid tumour. Results: Of 2114 pts enrolled in the NEXT study, 2102 were included in the overall analysis. Mean age ± standard deviation (SD) was 63.5±12.7 years. On inclusion, 90% of the pts had a performance status of 0 or 1. The majority (98.2%) of pts received prophylactic biosimilar filgrastim (primary prophylaxis: 91.0%; secondary prophylaxis: 7.3%). Of the pts receiving prophylactic biosimilar filgrastim, 79.9% received a dose of 30 MIU and therapy was administered subcutaneously in 99.4% of these. In total, 1579 (75.1%) pts had solid tumours. Of these, 29.3% had breast cancer, 18.8% had lung cancer, 12.5% had colorectal cancer (colon-rectum-anal canal), 9.6% had urological cancer, 7.9% had a gynaecological cancer, 6.8% had pancreatic cancer, and 6.4% had ear, nose and throat cancer. 50.6% of pts were receiving metastatic CT (44.4% adjunctive; 5.1% other). The majority of pts were receiving first-line CT for their solid tumour (89.0% of pts with non-metastatic disease and 62.7% of pts with metastastic disease). During the study, 21.1% of pts experienced Z1 AE. 12.4% of pts reported muscle or bone disorders. The other most common AEs (Z2.0% incidence) included gastrointestinal disorders (6.2%), nausea (3.6%), diarrhoea (2.8%), nervous system disorders (2.6%) and headache (2.0%). 76.3% of pts were treated with an adequate dose according to their weight. In total, 4.1% of pts with a solid tumour experienced FN. In addition, 2.2% were receiving concomitant anti-infective prophylaxis (compared with 51% of pts with haematological malignancies). Conclusion: Biosimilar filgrastim (Nivestimt) was effective and well-tolerated in pts undergoing cytotoxic CT for solid tumours. Muscular and chest pain were the main expected adverse events. Disclosure of Interest: F. Maloisel Funding from: Hospira, Sandoz, Pfizer, S. Lepreˆtre: None declared, D. Kamioner: None declared, C. Berthou: None declared, H. Albrand Employee of: Hospira SAS. P373 Busulfan and treosulfan have a comparable efficacy in patients with Ewing sarcoma – retrospective analysis of Polish pediatric centers K. A. Drabko1,*, A. Raciborska2, K. Bilska2, M. Ussowicz3, J. Styczynski4, A. Zaucha-Prazmo1, M. Pogorza"a4, R. Chaber3, E. Gorczynska3, J. Musia"3, J. Kowalczyk1 1 Department of Pediatric Hematology, Oncology and Transplantology, Medical University, Lublin, 2Department of Surgical Oncology for Children and Youth, Institute of Mother and Child, Warsaw, 3Department of Pediatric BMT, Hematology and Oncology, Medical University, Wroc"aw, 4Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland Introduction: Megachemotherapy (MCT) with autologous stem cell transplantation has been used in clinical practice in high risk Ewing sarcoma for 30 years. Recent registry study confirms efficacy of this procedure however suggest possible lower survival after treoseulfan based conditioning when compare to busulfan based regimen. Aim of the study: Comparison of busulfan and treosulfan based MCT results in patients treated in Polish pediatric centers for high risk Ewing sarcoma. Materials (or patients) and methods: Between 1999 and 2013 in 11 pediatric centers 207 patients with Ewing sarcoma were treated according to EuroEwing protocols. Among this group 49 (24%) children (24 females) underwent MCT after pre-operative chemotherapy (VIDE, median 6 cycles) and local therapy. Median age of the patients was 13 years (range 2.6-19.2). busulfan based MCT was administered to 34 and treosulfan based in 15 children. In busulfan group 23 patients had metastatic disease at presentation and 11 were transplanted due to poor histological
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response or non-resectable tumor. In treosulfan group 12 patients had metastases and 3 poor histology. Results: Median follow up of 49 transplanted patients was 39 months (range 11-176 months); 53 months in busulfan group and 31 in treosufan group P ¼ 0.08. 3-years overall survival (OS) in busulfan group was 67% as compare to 64% in treosulfan group and 5-years 63% and 52% respectively (P ¼ 0.31). Disease free survival (DFS) at 3 years was 56% in busulfan and 32% in treosulfan group while 5-years DFS was 53% and 32% respectively ( P ¼ 0.09). Median follow up in non-transplanted patients with high risk features (n ¼ 97) was 20 months (range 2141 months). OS all transplanted patients (n ¼ 49) was 66% at 3years and 58% at 5-years as compared to 30% and 19%, respectively, in non-transplanted patients (P ¼ 0.001). DFS was also significantly better in transplanted patients as compared to other with high risk factors (3-years 49% vs 30%; 5-years 46% vs 15% respectively (P ¼ 0.003). Conclusion: Our retrospective analysis does not confirm lower efficacy of treosulfan based MCT as compared to busulfan based in patients with high risk Ewing sarcoma. We concluded that patients with high risk features have significantly higher probability of survival, when treated with MCT (busulfan or treosulfan). Further studies are necessary to compare efficacy of busulfan and treosulfan in patients with high risk Ewing Sarcoma. Disclosure of Interest: None declared. P374 Retrospective comparison of treosulphan/melphalan versus carboplatin/etoposide/melphalan as preparative regimen for autologous transplantation in pediatric neuroblastoma R. Khismatullina1, M. Maschan2, D. Balashov 2, J. Skvortsova2, D. Shasheleva2, G. Novichkova2, D. Kachanov3, E. Skorobogatova4, T. Shamanskaya5, G. Muftakhova6, K. Kirgizov7, S. Varvolomeeva5, L. Shelikhova8,*, A. Maschan2, 1 hematopoietic stem cell transplantation, FEDERAL CENTER FOR PEDIATRIC HEMATOLOGY, 2Hematopoietic stem cell transplantation, 3Oncology, DMITRIY ROGACHEV CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, 4Hematopoietic stem cell transplantation, Russian chilren’s hospital, 5Oncology, FEDERAL CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, 6Oncology, FEDERAL CENTER FOE PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, 7Hematopoietic stem cell transplantation, Russian children’s hospital, 8 Hematopoietic stem cell transplantation, DMITRIY ROGACHEV FEDERAL CENTER FOR PEDIATRIC HEMATOLOGY, ONCOLOGY AND IMMUNOLOGY, Moscow, Russian Federation Introduction: High-dose chemotherapy with autologous stem cell transplantation is a standard component of multimodality treatment for high-risk neuroblastoma. Common high-dose preparative regimens for neuroblastoma include busulphan/ melphalan (Bu/Mel), carboplatin/etoposide/melphalan (CEM)
and TBI-based combinations. Definitive comparison of alternative preparative regimens has not been performed thus far. In the current study we analyse retrospectively the results of autotransplantation with two regimens, CEM and treosulfan/ melphalan (Treo/Mel), used consecutively for conditioning in patients with high-risk neuroblastoma. Materials (or patients) and methods: Fifty three patients with high-risk neuroblastoma were treated in the Federal Center for pediatric hematology and Russian children’s hospital since April 2012 till September 2014. All patients were treated according to the NB-2004 protocol, excluding MIBG therapy, which was not available. Two types of preparative regimens were used. Twenty nine (f-8, m-21, median age 2,3 years) recieved CEM regimen (since September 2012 till June 2013): Melphalan 45 mg/m2/day, day -8,-7,-6,-5, Etoposide 40 mg/kg, day -4, Carboplatin 500 mg/ m2/day, day -4,-3,-2, G-CSF 10 mcg/kg/day IV since day þ 5 till WBC 4 5 x 109/l. Twenty four patients (f-12, m-12, median age 2,8 years) recieved Treo/Mel regimen (since July 2013 till September 2014): Treosulfan 14 g/m2/day, day -5,-4,-3, Melphalan 140 mg/m2, day -1, G-CSF 10 mkg/kg/day since day þ 5 till WBC 4 5 x 109/l. The median dose of infused CD34 þ cells was 10 (3,5-26) x 106/kg and 8,2 (1,3-31) x 106/kg respectively. The groups were balanced for disease stage, response to induction therapy and bone marrow involvement. There was a trend towards overrepresentation of MYC-N amplification (58% vs. 29%, p - 0,073) in the CEM group. Results: Primary engraftment was achieved in 50 of 53 pts, the median time to neutrophil and platelet recovery was 10 and 15 days in the CEM group, 10,5 and 13,5 days in the Treo/Mel group, respectively. In the CEM group 3 pts died of bacterial sepsis before 100-day after SCT, 1y. pTRM - 10,6% (95%CI: 3,7– 30,1). None of the patients died of transplant complications in the Treo/Mel group, 1y. pTRM 0%. In CEM group (median followup 1,9y) at 1 year cumulative incidence of relapse is 41,4% (95%CI: 26,8-63,8), pEFS is 48,3% (95%CI: 30,1-66,5), pOS - 69% (95%CI: 52,1-85,8). In the Treo/Mel group (median follow-up 0,67y) 1 year cumulative incidence of relapse is 11,2% (95%CI: 341,5), pEFS is 88,8% (95%CI: 74,2-100), pOS - 93,8% (95%CI: 81,9100). There is a trend towards improved event-free (p - 0,035) and overall survival (p - 0,07) in the Treo/Mel group. Conclusion: In this retrospective analysis Treo/Mel preparative regimen was associated with improved short-term results in comparison to the more conventional CEM regimen in highrisk neuroblastoma. Further observation, matched pair analysis and prospective testing will be performed to confirm these results and account for potential bias. Disclosure of Interest: None declared. P375 High-dose chemotherapy and autologous hematopoietic stem cell transplantation as adjuvant treatment in highrisk breast cancer: data from the EBMT registry 1995-2005 M. Martino1,*, P. Pedrazzoli2, A. Zambelli3, U. de Giorgi4, F. Arpaci5, D. Blaise6, R. Leno Nu´n˜ez7, H. Schouten8, M. Badoglio9, M. Bregni10, C. Bengala11, F. Lanza12 on behalf of on behalf of the European Society for Blood and Marrow Transplantation, Solid Tumors Working Party (EBMT-STWP) 1 Azienda Ospedaliera BMM, Reggio Calabria, Hematology and Stem Cell Transplant Unit, Reggio Calabria, 2Dept. Onco Hematology, Fondazione IRCCS Policlinico S. Matteo - Pavia, 3 Fondazione S.Maugeri, Pavia, 4Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Mendola, Italy, 5GATA BMT Center, Gu¨lhane Military Medical Academy, Ankara Etlik, Turkey, 6Institut Paoli Calmettes, Inserm UMR 891, Marseille, France, 7Hospital Universitario de Salamanca, Servicio de Oncologı´a Me´dica, Salamanca, Spain, 8University Hospital Maastricht, Maastricht, Netherlands, 9Paris EBMT Study Office, Paris, France, 10Medical Oncology, di Circolo Hospital, Busto Arsizio, 11Medical Oncology, Misericordia Hospital, Grosseto, 12 Hematology Institute, Hospital of Cremona , Cremona, Italy Introduction: The efficacy of high-dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation
(AHSCT) for high-risk breast cancer (HRBC) patients has been an area of intense debate and controversy among the medical oncology community. Although limited informations are available on efficacy of HDC in subgroups of patients with different biologic breast cancer subtype, data from modern phase III studies suggest that HDC may still have a role in selected patients. Materials (or patients) and methods: The aim of this retrospective, multicentre study was to assess toxicity and efficacy of this procedure in a large cohort of operable HRBC treated with adjuvant HDC and transplanted between 1995 and 2005 in Europe. The outcomes that we analyzed were probabilities of DFS, OS, and TRM. Stratifying parameters included menopausal status, age, hormone receptor status (estrogen- or progesterone-receptor positive versus both negative), HER2 status (positive versus negative), number of positive lymph nodes, primary and grading tumor categories. Results: We have analyzed the EBMT registry and a total of 583 patients (43 positive nodes) were eligible for survival analysis. All patients received surgery before transplant and 55 (9.5%) patients received a neo-adjuvant treatment before surgery. The median age was 47 years, 66.9% of patients were premenopausal at treatment, 66.7% had endocrine-responsive tumors, 38% had a human epidermal growth factor receptor 2 (HER2) positive tumor and 72.7 % had 4 10 positive lymph nodes at surgery. Eighty percent received a single HDC procedure. The source of stem cells was peripheral blood in the vast majority of patients. Transplant-related mortality was 1.7%, whereas secondary tumor-related mortality was 0.91%. With a median follow-up of 120 months, OS and DFS at 5 and 10 years in the whole population were 75/61 and 64/44%, respectively. Subgroup analysis demonstrated that OS was significantly better in patients with endocrineresponsive tumors, less than 10 positive lymph nodes and smaller tumour size. HER2 status did not affect survival probability. Conclusion: Adjuvant HDC with AHSCT has a low mortality rate and provides impressive long-term survival rates in patients with HRBC. Our results suggest that this treatment modality should be proposed in selected HRBC patients and further investigated in clinical trials. Disclosure of Interest: None declared. P376 The efficacy of tandem stem cell transplantation in patients with high-risk neuroblastoma M. Sugiyama1,*, Y. Terashita1, J. Ohshima1, Y. Cho1,2, A. Iguchi1 1 pediatrics, Hokkaido University Graduate School of Medicine, 2 pediatrics, Hokkaido Cancer Center, Sapporo, Japan Introduction: Despite of high-dose chemotherapy (HDC), stem cell transplantation (SCT) and advanced therapies, such as 131I-MIBG, 13-cis-retinoic acid and anti-GD2 antibody, the prognosis of patients with high-risk neuroblastoma remains poor. Thus, we have analysed tandem SCT, including allogeneic SCT, in patients with high-risk neuroblastoma to improve their prognosis. Materials (or patients) and methods: We retrospectively reviewed medical records to evaluate the efficacy and safety of tandem SCT for patients with high-risk neuroblastoma between January 1998 and November 2013. We analysed 33 consecutive SCT for 27 patients (M/F ¼ 15/12) with advanced stage neuroblastoma (stage 4). Their median age was 3 years (range: 1-10 years). Twenty patients received SCT during first complete remission or very good partial response and 7 received SCT during second remission or non-remission: all of them were included in this study. Twenty-one patients received autologous peripheral blood SCT (PBSCT) or bone marrow transplantation (BMT), 8 received un-related cord blood transplantation (CBT) and 4 received related PBSCT or BMT. Five patients received tandem SCT: among them, 3 received auto-allo SCT: HDC and auto-PBSCT in addition to
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reduced-intensity conditioning (RIC) and allogenic CBT and 2 received tandem-auto-SCT: double HDC and double autoPBSCTs. HDC comprised melphalan (LPAM), etoposide (VP-16), and carboplatin in 8 patients, busulfan and LPAM in 7 patients and total body irradiation (TBI), LPAM/Cyclophosphamide, and VP-16 in 3 patients. RIC comprised of TBI, LPAM, fludarabine and anti-thymocyte globulin in 3 patients. Results: In all patients, the 4-year estimated overall survival (OS) and disease-free survival (DFS) rates were 54.5% ± 9.8% and 40.0% ± 9.6%, respectively. In patients receiving single HDC and SCT, OS and DFS were 41.6% ±11.0% and 28.6% ±9.9%100%, respectively. In contrast, in patients with tandem HDC and SCT, OS and DFS were 100% and 83.3% ± 15.2%, respectively (P ¼ 0.029 and P ¼ 0.034). There was a tendency that SCT during first remission or very good partial response had better outcome (P ¼ 0.059). There were no significant differences in age, sex, conditioning regimen and stem cell source for OS and DFS. Conclusion: Although it was a small scale analysis, significant favourable outcome has been achieved in patients receiving tandem SCT. Not only tandem auto SCT but also auto-allo SCT was effective. This suggests that auto-allo SCT is an alternative treatment to reduce late side-effects, and CBT as a second SCT could increase allo-reactive anti-tumour effects without severe graft-versus-host disease. Moreover, in advanced neuroblastoma, early tandem SCT should be performed because the prognosis of the patients with relapse or beyond second remission was extremely poor. Disclosure of Interest: None declared. P377 Autologous peripheral stem cell transplantation in testicular cancers: single institution experience N. Soyer1,*, A. F. Yilmaz1, R. Uslu2, A. P. Gorcegiz2, G. Saydam1, F. Sahin1, F. Vural1 1 Hematology, 2Medical Oncology, Ege University Medical Faculty, IZMIR, Turkey Introduction: Germ cell tumors are curable disease even in the presence of metastasis. Patients with relapsed or refractory tumors are candidates for salvage therapy. Salvage chemotherapy consisted of cisplatin, ifosfamide, vinblastine, paclitaxel or high-dose chemotherapy with autologous stemcell transplantation (ASCT) but the effectiveness of ASCT is not documented clearly. Materials (or patients) and methods: We retrospectively analyzed patients diagnosed as testicular cancer and treated with ASCT between January 2004 and November 2013. There were 28 ASCT transplantations of 19 patients, 9 of them were tandemly transplanted. Results: The characteristics of the patients and their response to chemotherapy before ASCT were shown at table 1. BEP regimen (bleomycin þ etoposide þ cisplatin) was given to patients with testicular cancer as first line treatment. Patients with severe lung involvement treated with BEP chemotherapy without bleomycin. Salvage chemotherapies were consisted of VIP- VeIP (etoposide þ ifosfamide þ cisplatin - vinblastin þ ifosfamide þ cisplatin) and TIP (paclitaxel þ ifosfamide þ cisplatin) for relapsed and refractory patients with testicular cancer. Median numbers of CD 34 ( þ ) stem cells infused to all transplanted patients were 5,71x106/kg (range; 2,5 – 6,83x106 / kg). Carboplatin and etoposide containing regimen were administered to 18 patients and one patient received ICE chemotherapy (ifosfamide- carboplatin- etoposide) as a conditioning regimen. 9 patients who underwent tandem transplantation received conditioning regimen consisted of carboplatin and etoposide in each transplantation. Complete response and partial response was documented in eleven of patients after ASCT. There was progressive or stabile disease in six patients. Two patients were died during transplantation procedure without response evaluation.Relapsed or progressive disease was documented in five patients with complete or partial response after median of 5 (range; 2- 13 months)
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months. Nine of 19 patients (%) were died because of progressive disease after transplantation. The median overall survival was 21 months (range; 4-264 months). N ¼ 19 Diagnosis Testicular cancer Site of diagnosis of testicular cancer Orchiectomy Liver biopsy Biopsy of mediastinal mass Biopsy of retroperitoneal mass Age (median, range, in years) Metastatic disease yes no Response to first line treatment Complete response Partial response Progressive disease Radiotherapy Yes No
19 16 1 1 1 31,5 (15-44) 18 1 4 8 7 2 17
Number of treatment before ASCT First line Second line Z 3 lines
1 13 5
Response before ASCT Complete/ partial response Progressive / stabile disease
6 13
Conclusion: ASCT transplantation is a treatment option in relapsed and refractory testicular cancers. It is a relatively safe procedure in terms of both mobilization and transplantation. Results of long term follow up were still not favorable. The effectiveness of ASCT should be evaluated in prospective randomized studies with large cohorts. Disclosure of Interest: None declared. P378 Cell therapy with EBV LMP2-specific cytotoxic T-lymphocytes for patients with nasopharingeal carcinoma P. Comoli1,*, S. Basso2, S. Secondino3, A. Gurrado2, P. Morbini 4, M. Benazzo5, G. Quartuccio2, I. Guido2, A. Pellerano2, C. Paglino3, M. Zecca2, P. Pedrazzoli3 on behalf of on behalf of The ‘‘San Matteo’’ Head and Neck Tumor Multidisciplinary Group 1 Pediatric Hematology/Oncology, Fondazione IRCCS Policlinico S. Matteo, 2Pediatric Hematology/Oncology, 3Oncology, Fondazione IRCCS Policlinico San Matteo, 4Pathology, 5Otolaryngology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia, Italy Introduction: Nasopharyngeal carcinoma (NPC) is an EpsteinBarr virus (EBV)-related malignancy expressing a restricted set of viral antigens. The standard of care for locally advanced head & neck cancer is concurrent platinum-based chemotherapy and radiotherapy, with overall response rates exceeding 90% in NPC. However, for patients with local-regional recurrent or metastatic disease, the benefits of combination chemotherapy are short-lived. Onset of these virus-related tumors is favored by immune surveillance defects, and expression of antigenic viral proteins by malignant cells constitutes a good target for immunotherapeutic strategies. Materials (or patients) and methods: Since 2001, we have implemented a T-cell therapy program for patients with NPC failing conventional treatment. Standard EBV-specific cytotoxic T-lymphocytes (EBV-CTLs) have been so far expanded for 28/35 patients, while LMP2-specific CTLs expanded with LMP2-derived 15mer peptide pools could be successfully generated from 15 patients. Eligible patients with stage IV disease in relapse were treated in two sequential trials of cell therapy with autologous EBV-CTLs. In the first set
of patients, 4 escalating doses to a maximum of 8x107 CTL/dose were administered, followed by maintenance infusions in case of response; in the second protocol, the patients received 2 CTL administrations with a median cell dose/infusion of 3.5 x 108 (range 2-4 x 108) preceded by lymphodepleting chemotherapy. Results: We have treated 27 patients with disease resistant/ relapsing after 42 lines of radio-chemotherapy. Overall, the objective control of disease was 57%, with 6/27 patients showing complete (n ¼ 1), partial (n ¼ 4) and minimal (n ¼ 1) responses, and 10/27 disease stabilization (median duration 7 months). No severe adverse events were observed after cell therapy; 4 patients showed an inflammatory reaction at the tumor site. The use of preparative chemotherapy and increased CTL dose did not influence outcome. Importantly, patients showing response to cell therapy showed the emergence of EBV LMP2 antigen-specific T-cells in their peripheral blood. As we did not observe an increase in cell therapy activity, measured as proportion of CR þ PR measured according to the RECIST criteria, we inferred that a more specific T cell product, administered in earlier disease stages, may yield better results. Thus, we started a new trial in patients treated for metastatic disease, with EBV- or LMP2-CTL administered as adjuvant therapy (at completion of first line treatment). So far, we treated 6 patients (3 in each arm). None of the patients treated with EBV-CTL þ LMP2-CTL experienced disease relapse at a median follow-up of 12 months, while 2 of the 3 patients enrolled in the EBV-CTL only arm showed disease relapse at months þ 1 and þ 8, respectively. Conclusion: EBV-specific CTL therapy is safe and associated with clinical benefit in patients with advanced NPC refractory to standard therapies. The use of CTL with higher specificity for the EBV subdominant antigens expressed by the tumor, such as LMP2, at an earlier stage of disease, could further implement the strategy and ameliorate the outcome of patients with relapsing/refractory NPC. Disclosure of Interest: None declared. P379 High dose chemotherapy supported with autologous hematopoietic stem cells transplantation in patients with advanced germ cell tumors who had failed to be cured by standard treatment. Warsaw experience T. Sarosiek1, B. Mlot1, C. Szczylik1, P. K. Rzepecki1,* 1 MILITARY INST. OF MEDICINE, Warsaw, Poland Introduction: About 5 to 50% of all patients with advanced germ cell cancer (depending on the IGCCCG prognostic group) will experience a relapse or a disease progression, despite undergoing standard treatment including chemotherapy and resection of residual lesions. According to the published papers, patients who have failed to be cured with the first line chemotherapy have rather poor prognosis with only 20 to 40% probability of long term survival using the standard rescue treatment. High dose chemotherapy (HDC) followed by autologous haematopoietic stem cell transplantation has already been studied for the last 20 years as a rescue treatment for such patients. To find factors influencing the efficacy and toxicity of the high dose chemotherapy (HDC) and autologous hematopoietic stem cell transplantation (BMT) in patients with relapsed or refractory germ cell tumors (GCT). Materials (or patients) and methods: We performed 65 procedures of HDC þ BMT in 51 patients. One (in 37 patients) or two sequential (14 patients) cycles of HDC were used. The median of 2 x 106/kg CD34 þ cells were infused after HDC. Results: Treatment toxicity increased with Beyer prognostic score, number of previous chemotherapy cycles and ECOG performance status. Complete remission has been achieved in 31,37%, partial in 39,22% and stable disease in 4% of patients. 12-month overall survival rate (OS) averaged 58,5% , 24-month
OS was 50,6%. 12- and 24-month progression free survivals (PFS) were 58% and 54,5%. Conclusion: Independent prognostic factors for OS included beta-HCG level above 1000 mIu/ml, elevated AFP level before transplantation, IGCCCG prognostic group at diagnosis. Beyer prognostic score was an independent prognostic factor for PFS. HDC can be curative for some relapsed or refractory GCT patients. Those with upfront poor prognosis (according to Beyer prognostic score), high beta-HCG levels or poor ECOG performance status do not benefit from this kind of treatment. Disclosure of Interest: None declared. P380 Hematopoietic Stem Cell Transplantation in Relapsed or Refractory Extracranial Primitive Neuro-Ectodermal Tumor of Children and Adolescents: a Multicenter Survey by the Turkish Pediatric Bone Marrow Transplantation Study Group (TPBMT-SG) V. Hazar1,*, M. Karakukcu2, A. Kupesiz3, V. Kesik4, E. Unal2, U. Kocak5, N. Eker3, E. Atas4, S. Aksoylar6, N. Tacyildiz7, M. Elli8, S. Yilmaz9, Z. Kaya5, S. Kansoy6, E. Unal7, F. Erbey10, S. Emir11, H. Oniz12, A. Yesilipek13 1 Pediatric Hematology/Oncology and BMT Unit, Medipol University Faculty of Medicine, Istanbul, 2Pediatric Hematology/ Oncology and BMT Unit, Erciyes University Faculty of Medicine, Kayseri, 3Pediatric Hematology/Oncology and BMT Unit, Akdeniz University Facuty of Medicine, Antalya, 4Pediatric Oncology and BMT Unit, Gulhane Military Medical Academy, 5Pediatric Hematologyand BMT Unit, Gazi University Faculty of Medicine, Ankara, 6Pediatric Oncology and BMT Unit, Ege University Faculty of Medicine, ˙Izmir, 7Pediatric Oncology and BMT Unit, Ankara University Faculty of Medicine, Ankara, 8Pediatric Oncology and BMT Unit, On Dokuz Mayis University Faculty of Medicine, Samsun, 9Pediatric Hematologyand BMT Unit, Dokuz Eylul University Faculty of Medicine, ˙Izmir, 10Pediatric Hematology/ Oncology and BMT Unit, Medical Park Bahcelievler Hospital, Istanbul, 11Pediatric Hematology/Oncology and BMT Unit, Childrens Hematology and Oncology Hospital, Ankara, 12Pediatric Oncology and BMT Unit, Tepecik Childrens Hospital, ˙Izmir, 13 Pediatric Hematology/Oncology and BMT Unit, Bahcesehir University Faculty of Medicine, Istanbul, Turkey Introduction: The objective of this study was to evaluate the outcomes experienced by children with relapsed or refractory extracranial Primitive Neuro-Ectodermal Tumor (rr-ePNET) following high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT). Materials (or patients) and methods: We retrospectively analyzed the outcomes of 44 patients with rr-ePNET. Complete remission could not be achieved in 7 patients prior to transplantation due to primary refractory disease. At the time of transplantation, only 66% of the patients were chemosensitive. The majority of patients received busulphan þ melphalan for conditioning (28/44), and peripheral blood (41/44) was used as a source of stem cells. Results: After a median follow-up period of 14 months, 20 patients were alive. At 2 years, the probabilities of overall survival (OS) and disease-free survival (DFS), the relapse rate (RR) and the non-relapse mortality (NRM) rate were 33.3%, 41.4%, 50.0% and 9.1%, respectively. The probability of DFS in chemosensitive and chemoresistant patients at 2 years was 63.8% and 13.3%, respectively (P ¼ 0.004). Patients with only lung metastases had better PFS than those with other or combined metastases (2-year DFS 63.2% vs 31.5%, P ¼ 0.102). Multivariate analysis showed that chemoresistant disease at the time of transplantation was the only factor predicting limited PFS (odds ¼ 2.739, P ¼ 0.026). Conclusion: HSCT increased PFS in a significant proportion of children with rr-ePNET. Survival rates were better for patients with chemosensitive disease at the time of HSCT. Disclosure of Interest: None declared.
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