Pediatr Transplantation 2015: 19: 422–427

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Pediatric Transplantation DOI: 10.1111/petr.12476

Non-total body irradiation myeloablative conditioning with intravenous busulfan and cyclophosphamide in hematopoietic stem cell transplantation for malignant infantile osteopetrosis Behfar M, Dehghani SS, Hosseini AS, Jalali A, Hamidieh AA, Ghavamzadeh A. (2015) Non-total body irradiation myeloablative conditioning with intravenous busulfan and cyclophosphamide in hematopoietic stem cell transplantation for malignant infantile osteopetrosis. Pediatr Transplant, 19: 422–427. DOI: 10.1111/ petr.12476. Abstract: HSCT is the only curative treatment for MIOP. We prospectively investigated the outcome of HSCT using intravenous busulfan-based conditioning regimen from 2008 to 2013. Nineteen patients (median age = 17 months) underwent transplantation from HLA-matched related donors (n = 14), HLA-haploidentical related donors (n = 2), partially matched cord blood donors (n = 2), and HLA-matched unrelated donor (n = 1). Bone marrow (n = 9), peripheral blood (n = 8), and cord blood (n = 2) were used as stem cell sources. All but one patient demonstrated primary engraftment. Two patients experienced secondary graft failure. During the follow-up period, three patients showed mixed chimerism (45%, 45%, and 70% of donor cells were engrafted in each one of these patients) but are disease free. Two-yr OS and DFS were 84.2% and 73.7%, respectively. Improvement of visual acuity and partial reversal of mild conductive hearing loss occurred in two and four patients, respectively. The causes of death among three patients were infection, GvHD, and disease progression. In conclusion, due to major side effects of MIOP such as visual and hearing loss, early treatment using myeloablative conditioning without irradiation HSCT is suggested. The use of an HLA-matched related donor seems to be highly successful in this regard. Also, according to results of our study, mixed chimerism may be sufficient to resolve symptoms of disease.

Abbreviations: ABR, auditory brainstem response; aGvHD, acute graft versus host disease; CBC, complete blood count; CMV, cytomegalovirus; CsA, cyclosporine A; DFS, disease-free survival; EBMT, European Society for Blood and Marrow Transplantation; G-CSF, granulocyte colonystimulating factor; GvHD, graft versus host disease; HLA, human leukocyte antigen; HSCT, hematopoietic stem cell transplantation; i.v., intravenous; MIOP, malignant infantile osteopetrosis; OS, overall survival; PBSCs, peripheral blood stem cells; TBI, total body irradiation; VEP, visual evoked potential.

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Maryam Behfar, S. Sharareh Dehghani, Ashraf Sadat Hosseini, Arash Jalali, Amir Ali Hamidieh and Ardeshir Ghavamzadeh Hematology-Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, Iran

Key words: malignant infantile osteopetrosis – hematopoietic stem cell transplantation – myeloablative conditioning regimen Amir Ali Hamidieh, Hematology-Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Shariati Hospital, Karghar Avenue, Tehran 14114, Iran Tel.: +98 912 159 3270 Fax: +98 21 8800 4140 E-mail: [email protected] Accepted for publication 17 March 2015

MIOP is a rare autosomal recessive inherited disorder in which the failure of osteoclasts to resorb the bone results in increased bone density in childhood (1–3). MIOP has an incidence of one in 250 000 births (4). To this date, mutations in nine genes have been discovered for this disease that could determine the severity of presentations (5). In most types of the disease, the number of osteoclasts is in normal range or higher than normal, but they are dysfunctional. It has been shown that mutation in TCIRG1 is responsible for over 50% of all cases of MIOP (6). Mutations

HSCT using non-TBI conditioning for osteopetrosis

in CLCN7 are the second most determining factor in the pathogenesis of osteopetrosis that involves acidification of extracellular lacunae (7). Other mutations are less frequent, and in 25% of cases, the disease causing gene is unknown (8). MIOP affects both medullary and cortical bone. Bone fractures, visual impairment (due to compression of optic nerve), and bone marrow failure are the classic features of the disease (9, 10). Also deafness, hepatosplenomegaly, hypocalcemia, growth failure, delayed tooth eruption, and change of facial appearance are the other manifestations due to the primary disease (11– 13). Without effective treatment, all patients with MIOP will die within the first decade of life due to consequences of bone marrow failure and pancytopenia (14). At present, there is no effective medical treatment and medications such as vitamin D, calcitriol, parathyroid hormone, erythropoietin, and corticosteroids are used only as supportive and symptom management drugs (15–17). Interferon gamma is another drug that has been used in MIOP settings (18). Orthopedic surgery is sometimes used for fractures and other skeletal complications (19). However, HSCT is the only effective and curative therapy for most types of MIOP (20, 21). Due to the rarity of the disease, very few studies have been conducted on disease treatment in all regions, especially in developing countries. With a view to assess the effects of intravenous busulfan-based conditioning regimen on engraftment and mixed chimerism on patients’ outcome, this prospective survey was carried out on patients with MIOP.

Donor candidates were examined for HLA matching, with siblings being the preferred choice. Because of consanguineous marriages, we also examined parents and other relatives for patients without any HLA-matched sibling. We performed a low-resolution molecular typing of HLA-A, -B, and -DRB1 in sibling donors and recipients and a high-resolution typing for other relatives. We considered unrelated donors as the last choice for transplant. Patients received stem cells from bone marrow, peripheral blood, or cord blood. PBSCs were mobilized by injection of G-CSF. All patients received the same conditioning regimen consisting of busulfan plus cyclophosphamide, with or without antithymocyte globulin. Busulfan (BusilvexÒ; Pierre Fabre Medicament, Boulogne, France) was administered i.v., and the dosage was adjusted according to weight; 4 mg/kg/day for under 9 kg patients, 4.8 mg/kg/day for 9–15 kg patients, and 4.4 mg/kg/day for patients between 15 and 23 kg for four consecutive days (days 8 to 5). Cyclophosphamide 50 mg/kg/day i.v. was also administered for four consecutive days (days 4 to 1). For the patients without identical sibling donors, we started rabbit antithymocyte globulin (Thymoglobulin; Sanofi Inc., Laval, QC, Canada) 2.5 mg/ kg/day i.v. for four consecutive days (days 4 to 1). No patient received TBI. All patients were protected against GvHD by CsA 1.5 mg/kg/day i.v., starting on day 2 and then abruptly titrated up to 3 mg/kg/day from day +7 in patients receiving PBSCs and day +11 in patients receiving BMSCs and cord blood. Methotrexate (10 mg/m2 on day +1 and 6 mg/m2 on days +3, +6) was used for all the patients except those who received stem cells from the source of cord blood. CsA level was monitored twice in a week (therapeutic range 100–250 mg/mL).

Patients and methods

Supportive care

Patient characteristics

During hospitalization, all patients were isolated in rooms with high-efficiency particulate arresting filters. They all received acyclovir 15 mg/kg/day i.v. in divided doses from day 2 to +3 then 15 mg/kg/day orally as prophylaxis for herpes simplex and varicella–zoster virus. Trimethoprim-sulfamethoxazole was the choice of prophylaxis for Pneumocystis jirovecii; 10 mg/kg/day (trimethoprim-based) orally which was given from day 9 to day 1 and then resumed from day +36 to two yr after transplant. Fluconazole was used as prophylaxis for fungal infections. It was started at 3–6 mg/kg/day orally from the first day of implementing conditioning regimen until day +90. Phenytoin was administered to prevent seizures due to the busulfan treatment. We also performed CMV PP65 antigen assay or CMV DNA PCR tests for diagnosis of CMV, twice a week. Positive cases were treated by gancyclovir for at least 21 days or until the tests proved negative. Irradiated blood products were used as needed. Empiric broad-spectrum antibiotics were administered to individuals with febrile neutropenia.

In this prospective trial, 19 patients with MIOP were treated with HSCT in the hematology–oncology and stem cell transplantation research center and followed up afterward. All patients referred to our center were primarily diagnosed with MIOP (i.e., manifesting features such as pathologic features, bone marrow failure, and visual and auditory impairments) in the referral institutes. The institutional review board and ethics committee approved the study protocol. Signed informed consents were obtained from the patients’ parents due to ethical obligation. To confirm the diagnosis, biopsy of bone and whole body bone survey were performed which were compatible with the diagnosis. Also, VEP and ABR were conducted to assess the level of auditory and visual impairments. Abiding by the Osteopetrosis Consensus Guideline of the European Society for Immunodeficiencies and the EBMT (22), patients who simultaneously suffered from both complete blindness and deafness were excluded from the study. Before the

transplant, routine laboratory tests including CBC, lactate dehydrogenase, alkaline phosphatase, calcium and phosphate levels, and venous blood gas measurement were conducted. We also consulted with a neurologist for detailed evaluation of the patients.

Transplant preparation and procedure

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Behfar et al. Follow-up All patients were followed up through regular medical visits; every week for the first month, every two wk to day +100, and then, each case was followed up differently according to his or her condition. We assessed the engraftment by chimerism ratio of bone marrow which was obtained by short tandem repeat or fluorescence in situ hybridization techniques on the days +15 and +30 and then assessed the same ratio of peripheral blood on the days +60, +90, +180, and +365. Acute and chronic GvHD were observed and documented based on standard criteria (23, 24). Evaluation of hearing and visual development was conducted by ABR and VEP at six and 12 months after transplantation. Other routine tests including CBC and biochemistry of blood were performed in every medical visit, and radiographic studies and whole body bone survey were performed at six and 12 months post-transplant to assess the effect of transplant on patients.

Definitions Neutrophil engraftment was defined as a neutrophil count >0.5 9 109/L for at least three consecutive days without infusion of G-CSF, and platelet engraftment was defined as a platelet number of more than 20 9 109/L for seven consecutive days, unsupported by platelet transfusion for seven days. When more than 95% of the cells in peripheral blood or bone marrow of the patient belonged to the donor, the situation was qualified for full chimerism and was labeled mixed chimerism when a percent between 5% and 95% of donor cells existed and termed rejection when 0.5 9 109/L for three consecutive days after HSCT. Secondary graft failure was defined as failure to sustain an absolute neutrophil count >0.5 9 109/L after achievement of primary engraftment (27).

OS was defined as the time elapsed between HSCT and time of death or last recorded contact, and DFS was estimated as the time period between HSCT and recurrence of primary disease or death or the last contact.

Results

This study included 19 patients: eight girls and 11 boys. The median age of patients at diagnosis and transplant was six months (range 0.5– 27 months) and 17 months (range 5–54 months), respectively. The median age of donors was 25 yr (range 3–40 yr), 11 of whom were female. All parents of patients were consanguineous. Most patients underwent HSCT from an HLA-identical sibling, and other related donors were our second priority for transplantation. The characteristics of patients before the transplant are shown in Table 1. The patients received cell dose with median mononuclear cells of 7.49 9 108/kg (range 4.0– 8.48 9 108) and median CD34+ cells of 3.62 9 108/kg (range 1.48–8 9 106). For recipients of cord blood, the mean number of infused total nucleated cells and CD34+ cells was 11.73 9 108/kg and 8.84 9 105/kg, respectively. Primary engraftment was achieved in all but one patient, while secondary graft failure occurred in two patients. Two of these three patients had a full-matched donor, while one of them had a two locus mismatched cord blood donor. Characteristics of patients after transplantation are shown in Table 2. The two patients who developed secondary graft failure

Table 1. Characteristics of patients and donors Donors’ characteristics Patient no.

Sex

Age at diagnosis (months)

Age at transplant (months)

Stem cell source

Relationship

Sex

Age (yr)

Compatibility

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

F M M F M F M M M F M F F F M F M M M

4 4 9 2 8 27 2 2.5 6 5 15 6 24 24 2 4 6 0.5 6

15 10 22 36 13 30 13 6 9 9 18 54 30 30 6 18 17 5 18

PB BM PB CB BM PB BM BM PB PB PB PB BM BM BM CB BM BM PB

Sibling Other relative Sibling Unrelated Other relative Sibling Other relative Other relative Sibling Sibling Sibling Sibling Other relative Unrelated Other relative Unrelated Other relative Other relative Other relative

M F M F M F M M F F F M M F M F F F F

3 26 11 – 36 11 27 36 7 4 6 9 40 31 25 – 32 26 9

Full match Full match Full match 4/6 Full match Full match Haploidentical Full match Full match Full match Full match Full match Full match Full match Full match 4/6 Full match Haploidentical Full match

F, female; M, male; BM, bone marrow; PB, peripheral blood; CB, cord blood.

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HSCT using non-TBI conditioning for osteopetrosis

are still alive and are currently being treated with transfusion of blood products and supportive care. They are considered candidates for second transplantation. aGvHD occurred in 12 patients. Eight patients had grade I–II aGvHD and four patients had grade III–IV aGvHD. They showed a good response to standard treatment except one patient who expired due to this complication. Only one patient developed limited chronic GvHD after transplantation. The median duration of follow-up for survived patients was 24 months. The 24-month OS and DFS was 84.2% (s.e. = 8.4%) and 73.7% (s.e. = 10.1%), respectively. Eleven patients have full chimerism, and three patients have a mixed chimerism status, that is, 45%, 45%, and 75% of the donor cells transplanted into each one of them have engrafted, in order, and have no clinical complaints. Only one patient who developed primary graft failure expired because of progression of the primary disease, and two patients died because of CMV infection and GvHD after transplantation. We evaluated the hearing and visual level in patients who were alive and disease free at six and 12 months after transplantation. Two patients demonstrated vision improvement in

VEP test, one developed visual deterioration and the rest remained at the same level. Our study showed partial reversal of mild conductive hearing loss in four patients versus remaining mild or moderate sensory neural hearing loss in two patients. Other patients had no change in their follow-up ABRs. Serial clinical radiographs of patients showed dramatic improvement in bone density at months six and 12 after transplantation. Complete recovery is evident in all patients who are alive and disease free. Discussion

MIOP is a rare autosomal recessive disease, and HSCT is the only potential curative treatment for these patients, mainly in younger patients, to prevent bone marrow failure and preserve the hearing and visual ability. Hearing loss can be reversible, provided that it is conductive and due to stenosis of the auditory canal (28). Furthermore, the risk of hypercalcemia is much lower in infants after transplantation than in elder children (12). In response to the need of diagnosis in infants, it seems that prenatal screening in families with a history of osteopetrosis is a useful way to detect the disease and subsequently perform HSCT in infants in the early months (28).

Table 2. Outcome of HSCT in patients with MIOP Patient no. 1 2 3

Engraftment

Day of neutrophil engraftment

Day of platelet engraftment

aGvHD (grade)

Chronic GvHD

Follow-up (months)

Last chimerism

Outcome

Other complications

+16 +17 +12

+44 +17 +12

1 1 1

– – –

35.5 36.8 20.5

>95 >95 95 >95 45 >95 >95 >95 >95

Alive Alive Alive Alive Alive Alive Alive Alive

13 14

Yes Yes

+20 +21

+34 +58

2 2

– –

17.5 18.1

45 >95

Alive Alive

15 16 17 18 19

Yes Yes Yes Yes Primary failure

+17 +25 +16 – –

+40 – +25 – –

– 3 1 2 –

– – – – –

16.3 1 24.1 0.5 3

70 >95 >95 >95

Non-total body irradiation myeloablative conditioning with intravenous busulfan and cyclophosphamide in hematopoietic stem cell transplantation for malignant infantile osteopetrosis.

HSCT is the only curative treatment for MIOP. We prospectively investigated the outcome of HSCT using intravenous busulfan-based conditioning regimen ...
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