Cancer Immunol Immunother (2013) 62:1851–1858 DOI 10.1007/s00262-013-1496-5
Meeting Report
Tenth annual meeting of the Italian Network for Tumor Biotherapy (NIBIT), SIENA, Italy, November 5–7, 2012 Michele Maio · Hugues Jean Marie Nicolay · Paolo Antonio Ascierto · Filippo Belardelli · Roberto Camerini · Mario Paolo Colombo · Paola Queirolo · Ruggero Ridolfi · Vincenzo Russo · Giulia Parisi · Ornella Cutaia · Ester Fonsatti · Giorgio Parmiani
Received: 17 May 2013 / Accepted: 24 October 2013 / Published online: 5 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Keywords Cancer · Immunology · Immunotherapy · Networks · NIBIT Abbreviations 1-MT 1-Methyl tryptophan AML Acute myeloid leukemia ACT Adoptive cell therapy AEs Adverse events Haplo-HSCT Haploidentical hematopoietic stem cell transplantation
This study was conducted on behalf of Italian Network for Tumor Biotherapy (NIBIT), Siena, Italy. M. Maio (*) · H. J. M. Nicolay · G. Parisi · O. Cutaia · E. Fonsatti Division of Medical Oncology and Immunotherapy, Department of Oncology, University Hospital of Siena, Viale Mario Bracci n. 16, 53100 Siena, Italy e-mail: [email protected] P. A. Ascierto Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, Istituto Nazionale Tumori Fondazione “G. Pascale”, Naples, Italy F. Belardelli Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy R. Camerini Clinical Research, Sigma Tau S.p.A., Pomezia, Italy
APC Antigen-presenting cells APM Antigen processing and presentation machinery B-NHLs B cell non-Hodgkin lymphoma BLI Bioluminescence imaging BM Bone marrow CIC Cancer initiating cells CSC Cancer stem cells CTA Cancer–testis antigen CEA Carcinoembryonic antigen CCL Chemokine (C–C motif) ligand CAR Chimeric antigen receptors CLL Chronic lymphocytic leukemia CRC Colorectal cancer cells CREB cAMP response element-binding protein P. Queirolo Oncologia Medica A, IRCCS Ospedale San Martino-Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy R. Ridolfi Immunotherapy and Somatic Cell Therapy Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Forlì, Italy V. Russo Cancer Gene Therapy Unit, Division of Molecular Oncology, San Raffaele Foundation Center, Milan, Italy G. Parmiani Unit of Immunobiotherapy of Melanoma and Solid Tumors, Division of Molecular Oncology, San Raffaele Foundation Centre, Milan, Italy
M. P. Colombo Molecular Immunology Unit, Department of Experimental Oncology, Fondazione Istituto Ricovero e Cura a Carattere Scientifico, Istituto Nazionale dei Tumori, Milan, Italy
13
1852
CIK Cytokine-induced killer CTLA-4 Cytotoxic T-lymphocyte antigen 4 DC Dendritic cells T-bodies Engineered T cells EAP Expanded access program GVHD Graft versus host disease VH CDR3 Heavy complementarity-determining region 3 HLA Human leukocyte antigen hPSMA Human prostate-specific membrane antigen HAS Human serum albumin Ig Immunoglobulin IDO Indoleamine 2,3-dioxygenase isoDGR IsoAsp-Gly-Arg KIR Killer cell immunoglobulin-like receptor LAG-3 Lymphocyte activation gene-3 LN Lymph node MHC Major histocompatibility complex MC Mast cells MMP-9 Metalloproteinase-9 mAb Monoclonal antibody MM Multiple myeloma MDSC Myeloid-derived suppressor cells NK Natural killer NSCLC Non-small-cell lung cancer OS Overall survival PBMC Peripheral blood mononuclear cell PD-1 Programmed cell death-1 PD-L1 Programmed cell death-1 ligand 1 TGF Transforming growth factor TRAMP Transgenic adenocarcinoma of the mouse prostate TAA Tumor-associated antigen TNF-alpha Tumor necrosis factor alpha TCR T cell receptor Treg Regulatory T cells CD44v6 Variant 6 of the adhesion receptor CD44 VEGF Vascular endothelial growth factor
Introduction NIBIT (acronym for the Network Italiano per la Bioterapia dei Tumori-Italian Network for Tumor Biotherapy) is a non-profit association created in 2004 to promote and foster scientific interactions among Italian and international professionals working in the field of cancer immunotherapy. The aim of the NIBIT annual meeting is to share and discuss the more recent preclinical and clinical results among researchers engaged in the field of cancer immunology and biotherapy. The meeting included more than 40 Italian and international leading groups representing academia, biotechnology
13
Cancer Immunol Immunother (2013) 62:1851–1858
and pharmaceutical industry. It covered the following topics related to immunology and immunotherapy of cancer: (1) gene mutations as targets of immunobiotherapy, (2) recent insights from preclinical models of cancer immunotherapy, (3) shaping the tumor microenvironment by inflammation and altered metabolism, (4) immunomodulation in the course of CTLA-4 blockade, (5) immunoengineering for cancer therapy and (6) translation in cancer immunotherapy.
Session 1: Gene mutations as targets of immunobiotherapy Unique tumor-associated antigens (TAAs), encoded by somatically mutated cancer genes, generate strong immunogenic epitopes, and cancer vaccination with mutated TAAs can result in increased clinical benefit. Daniele Mennonna (San Raffaele Scientific Institute, Milan, Italy) focused on the identification of unique colorectal cancer antigens by next generation sequencing of somatically mutated genes. Preliminary results obtained in colorectal cancer cells (CRC) show that the frame shift mutation, occurring in the antigen-presenting cell (APC) genes, generates a neoepitope that is presented by human leukocyte antigen (HLA)–DR molecules, and it is recognized by specific CD4+ T cells. Peptides corresponding to the non-synonymous point mutations in TP53 and SMAD4 genes elicit CD8+ and CD4+ T cells that specifically react against CRC cells. These results support the feasibility of this approach to identify unique TAAs in CRC by massive sequencing and reverse immunology. Cristina Maccalli (San Raffaele Scientific Institute, Milan, Italy) discussed about the immunoglobulin gene repertoire in chronic lymphocytic leukemia (CLL). She reported that this repertoire is remarkably restricted with >30 % of cases carrying quasi-identical (stereotyped) heavy complementarity-determining region 3 (VH CDR3) sequences. She suggested that peptides from stereotyped VH CDR3 sequences can represent candidate antigens to elicit anti-CLL T cell responses, especially in poor prognosis, where novel therapeutic interventions are needed. Although preliminary, the presented results may provide the proof of principle for the design of new immunotherapeutic protocols for CLL patients, including both active vaccination and adoptive cell therapy. Franco Locatelli (Bambino Gesù Children’s Hospital, Rome, Italy) presented data on haploidentical hematopoietic stem cell transplantation (haplo-HSCT) and innate immunity. He showed that in haplo-HSCT, alloreactive natural killer (NK) cells mediate potent anti-leukemia effects, which alloreactivity can be predicted by killer cell immunoglobulin-like receptor (KIR)/KIR-ligand genotypic analysis of donor/recipient cells. Also, the identification, the size
Cancer Immunol Immunother (2013) 62:1851–1858
of the NK alloreactive subset and the presence of activating KIR are important for the optimal donor selection. He finally discussed as failure to reconstitute the invariant NK T cell repertoire after haplo-HSCT is associated with high risk of leukemia relapse. By using proteome-based analyses and/or cDNA microarrays of RAS and/or HER-2/neu transformants candidate genes/proteins, Barbara Seliger (Martin-Luther University, Halle, Germany) showed that the overexpression of biglycan in oncogene-transformed cells leads to a transcriptional up-regulation of antigen processing machinery (APM) components associated with an increased major histocompatibility complex (MHC) class I surface expression. These data suggest that deficient biglycan expression in neoplastic cells may promote MHC class I down-regulation and allow their escape from immune surveillance. Finally, both inhibition of cAMP response element-binding protein (CREB) and biglycan overexpression lead to reduced tumor formation in oncogene-transformed cells. Silvia Carluccio (San Raffaele Scientific Institute, Milan, Italy) showed that tumor cell lines and dendritic cells (DC) were successfully obtained from CRC patients to stimulate T cells isolated from autologous PBMC. To date, ELISPOT was performed for two patients: Statistically significant INF-gamma secretion was detected at third, fourth and fifth stimulation for one patient, while no relevant cytokine secretion was found for the other patient. These data demonstrate the feasibility to generate tumorspecific CTL from peripheral blood to be useful for supporting adoptive cell therapy in patients with CRC. Peptide-based vaccines, although safe in patients, have problems related to HLA restriction and rapid clearance. To overcome these issues, Gilberto Filaci (University of Genoa, Genoa, Italy) set a telomerase-based vaccine, named GX301, constituted by four different peptides (i.e., hTERT540–548, hTERT611–626, hTERT672–686 and hTERT766–780) and two adjuvants (i.e., Montanide ISA51 and Imiquimod). Preliminary results, from a phase I/II clinical trial enrolling stage IV prostate and renal cancer patients resistant to conventional therapies, indicate that this therapeutic strategy is safe and induces efficient immune responses against potentially weak immunogenic antigens such as self-tumor antigens.
Session 2: Recent insights from preclinical models of cancer immunotherapy Antonio Rosato (University of Padua, Padua, Italy) focused on the importance of tracking T cells in vivo to assess the persistence, expansion and homing properties of injected T cells in ACT protocols. He presented data on the development of a second generation chimeric antigen
1853
receptor (CAR) directed against the human prostate-specific membrane antigen (hPSMA), to engineer human peripheral blood mononuclear cell (PBMC) for immunotherapeutic strategies in prostate cancer. Lentiviral vectors were used as gene transfer method, designed with a bidirectional promoter for the expression of both CAR and firefly luciferase, enabling the in vivo monitoring of engineered T cells (T-bodies) with bioluminescence imaging (BLI). T-bodies expressed high levels of CAR, a memory phenotype and the ability to kill hPSMA-expressing cells. However, data from BLI showed limited in vivo persistence and homing properties of transferred T-bodies in mouse model. In the last decade, several types of vaccines for boosting the body’s immune response to cancer cells are being tested in clinical trials with limited therapeutic efficacy. Matteo Bellone (San Raffaele Scientific Institute, Milan, Italy) reported on different effects of booster vaccinations in prophylactic and therapeutic settings. In vivo data demonstrated that priming with DC-based vaccines and the correct timing of booster injections favor the persistence of antigen-specific memory T cells and correlate with tumor rejection, thus increasing anti-tumor immunity in prophylactic settings. In contrast, prime/boost vaccination proved to be of no advantage or even detrimental in therapeutic settings in B16F1 and transgenic adenocarcinoma of the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse models. Paola Pittoni (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) discussed the role of mast cells (MC) in mouse and human prostate cancer and the possible application of MC-targeted cancer therapy. She showed that MC display different functions according to tumor stage. In fact, MC support the growth of early-stage tumors, providing metalloproteinase-9 (MMP-9), while prostate tumors after epithelial-to-mesenchymal transition produce MMP-9 and become independent from MC. Moreover, results highlighted a new, opposite role of MC in suppressing the occurrence of aggressive c-Kit+ neuroendocrine tumor variants. These findings support the hypothesis that c-Kit tyrosine kinase inhibitors, as imatinib, could block both adenocarcinoma-promoting MC (stroma-targeting) and neuroendocrine tumor variants (tumor-targeting) in prostate cancer. Elena Quaglino (University of Turin, Turin, Italy) reported on the identification of oncoantigens with a critical role in mammary carcinogenesis in ErbB2+ transgenic (BALB-neuT) mice. The results obtained from the analysis of the oncoantigens ErbB2 (expressed on the tumor cell surface) and Amot (expressed by the endothelia of angiogenic tumor tissues) demonstrated that in BALBneuT mice: (1) DNA vaccination targeting ErbB2 persistently inhibits cell proliferation of early mammary lesions, (2) DNA vaccination targeting Amot induces an antibody
13
1854
response that significantly halts the progression of incipient carcinomas and alters tumor vessel structure and permeability. These findings suggest the importance to extend the investigation on cancer initiating cells (CIC), responsible for tumor progression, metastasis, resistance to therapy and tumor recurrence. Marcella Tazzari (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) discussed the immunomodulating potential of targeted therapies in patients with solitary fibrous tumor. The study revealed a reduction in the circulating immunosuppressive cells myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs) by treatment with sunitinib, a multi-targeted receptor tyrosine kinase inhibitor. This peripheral immune suppression was associated with the modulation of the immunological status at tumor site resulting in the acquisition of immunological traits compatible with the setting of an adaptive response.
Session 3: Shaping the tumor microenvironment by inflammation and altered metabolism In her keynote lecture, Licia Rivoltini (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) gave an overview of the role of MDSC in cancer. She showed that at least 2 MDSC subsets can be identified (monocytic and granulocytic), involved in different phases of tumor progression. In particular, MDSC accumulation was reported to be associated with bad prognosis in stage II–III melanoma patients and in kidney cancer patients. She illustrated a hypothesis on genesis of tumor-induced MDSC accumulation in peripheral blood, due to selective tumor factors delivered as embedded in nanovesicular structures (exosomes), selectively homing to the bone marrow (BM) to influence myelopoiesis. Finally, data were presented on the pharmacologic down-modulation of MDSC in cancer patients induced by sunitinib, esomeprazole and BRAF inhibitors, suggesting the use of these therapeutic agents in combination with immune-based strategies to achieve more effective immune responses and disease control. Andrea Anichini (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) provided evidence of presence of CD8+ FOXP3+ T cells, which do not present an “early effector” profile (CD127−, KLRG1−, HLADR+, CD38+, T-bet+, perforin+) within neoplastic tissues from primary and metastatic lesions derived from melanoma patients. This subset of CD8+ FOXP3+ T cells is neither exhausted nor senescent and do not exhibit a regulatory [cytotoxic T-lymphocyte antigen 4 (CTLA-4), chemokine (C–C motif) ligand (CCL)-4, IL-10, transforming growth factor (TGF)-beta-1 phenotype]. He suggested that blockade of CTLA-4 by the monoclonal antibody (mAb) ipilimumab promotes CD3+ T cell infiltration, the presence of CD1a+ DC and a lower frequency of FOXP3+ lymphocytes, mimicking the early
13
Cancer Immunol Immunother (2013) 62:1851–1858
stages of T cell-mediated responses at the tumor site of responding lesions. Rosa Maria Moresco (University of Milan Bicocca, Milan, Italy) discussed on the development of specific tracers for positron emission tomography-based molecular imaging of metabolism and inflammation in cancer. She underlined that [18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) tracer, largely used for patients staging and followup, may result in false-positive or false-negative uptake in some clinical settings. She therefore described several probes (e.g., [18F] FLT, [18F] FAZA, [11C] acetate, [11C] PK11195, [18F] C-7, [18F] F-FAC, [18F] interleukin) with higher sensitivity, which have been developed by different research groups to improve the specificity of detection of the different metabolic pathways in cancer. Lymphocyte activation gene-3 (LAG-3) is a CD4-related cell surface molecule expressed by activated CD4+, CD8+ T and NK cells. This molecule has opposite functions: On one side, it negatively regulates T cells proliferation, on the other side, it induces APC activation trough MHC class II. Chiara Camisaschi (University of Milan Bicocca, Milan, Italy) explored the role of pDC in human melanoma. This population of dendritic cells, deputed to the INF-alpha release, may exert both activating and suppressive activities. Evidences were provided suggesting that, in the tumor microenvironment, pDC displayed an alternative activating pathway consistent with a partial tumor-induced dysfunction, possibly leading to the development and maintenance of immunosuppression. Viviana Vallacchi (Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy) presented a pilot study of gene expression and miRNA profiling to identify signatures in exoMDSC, a cell population in vitro generated from the co-culture of normal monocytes with melanoma exosomes. Results showed that exo-MDSC exclusively express 66 genes (42 upregulated and 24 down-regulated) and 11 miRNA (7 up-regulated and 4 down-regulated) compared to normal monocytes. Several genes up-regulated in MDSC are involved in immune response processes, including CCL7, CCL13 and IFNrelated genes. Up-regulated miRNA included hsa-miR-150, known to regulate different immune cells. She reported that several pro-tumorigenic and immunosuppressive cytokines/ chemokines [vascular endothelial growth factor (VEGF)A, CCL2, IL8 and TGF-beta1], also present in melanoma exosomes, are released by exo-MDSC, suggesting that they may have a role in the skewing of monocytes to MDSC.
Session 4: Immunomodulation in the course of CTLA‑4 blockade Monitoring patients’ immune system is crucial for the immune-guiding and to identify correlates of cancer
Cancer Immunol Immunother (2013) 62:1851–1858
treatment efficacy. This session focused on the immunemonitoring platform established within the NIBIT to investigate changes in the phenotypic profile of PBMC and in the anti-tumor-specific humoral and/or cellular functional immune response(s) induced in cancer patients during the CTLA-4 blockade therapy. In particular, Luana Calabrò (University Hospital of Siena, Siena, Italy), Licia Rivoltini (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy), Pier Franc‑ esco Ferrucci (European Institute of Oncology, Milan, Italy), Cristina Maccalli (San Raffaele Scientific Institute, Milan, Italy) and Elisabetta Fratta (Centro di Riferimento Oncologico, Aviano, Italy) showed preliminary data using different strategies in immunological monitoring within the translational studies associated with the clinical trial NIBIT-M1, utilizing the anti-CTLA-4 mAb ipilimumab in association with fotemustine in metastatic melanoma patients with or without brain metastasis and to the clinical trial involving patients with advanced melanoma treated with ipilimumab within a compassionate use program at the Division of Medical Oncology and Immunotherapy Department of Oncology University Hospital of Siena.
Session 5: Immune‑engineering for cancer therapy Stefano Ugel (University of Verona, Verona, Italy) demonstrated that the spleen has a fundamental role in tumorinduced tolerance in mice. In fact, splenectomy restores T cells activity and leads to a significant increase in EG-7 tumor-bearing mice survival. He also showed that the elimination of splenic CD11b+ Gr-1int myeloid cells by different chemotherapeutic agents enhances the antitumor efficacy of ACT in mice. He demonstrated that the CCL2-mediated accumulation of immunosuppressive CD11b+ Gr-1intLy6ChiCCR2+ cells, in response to CCL2, in the marginal zone of the spleen during tumor growth, leads to direct cross-presentation of tumor antigens to memory CD8+ T lymphocytes in a tolerogenic manner. Finally, he reported that CCL2 serum levels are predictive of the clinical response in patients responding to cancer vaccination. Andrea Brendolan (San Raffaele Scientific Institute, Milan, Italy) explored the molecular and cellular mechanisms at the basis of secondary lymphoid organ development, in terms of stromal cell differentiation and lymphocytes compartmentalization. A study conducted in mice demonstrated that the different spleen stromal cell subsets, including follicular dendritic cells, arise from embryonic mesenchymal precursors of the Nkx2.5+/Isl1+ lineage. This stromal cell lineage was shown to regenerate the splenic stromal microenvironment and to support the formation of artificial lymphoidlike structures (lymphoid
1855
neogenesis), containing functional T and B cell areas. Furthermore, Nicolas van Baren (Ludwig Institute for Cancer Research, Brussels, Belgium) focused on the lymphoid neogenesis in melanoma. He reported the presence of ectopic lymphoid structures in 15–20 % of cutaneous metastases, but never in primary tumors, of melanoma patients. These structures, defined as lymphoid follicles, associate clusters of B lymphocytes, with high endothelial venules and with clusters of DCs and T cells. The molecular characterization of the immunoglobulin (Ig) gene repertoire expressed in lymphoid follicles showed evidence of clonal amplification and diversity of B lymphocytes, as well as isotype switching, hallmarks of immunoglobulin affinity maturation, indicating that adaptive immune responses can occur within the melanoma environment. Anna Mondino (San Raffaele Scientific Institute, Milan, Italy) highlighted the therapeutic synergy of autologous T cells redirected by T cell receptor (TCR) gene transfer toward a tumor-associated model antigen (i.e., large T antigen, Tag) and a ubiquitously expressed minor H antigen (i.e., Y-encoded Uty). In vivo experiments in prostate cancer bearing TRAMP mice showed that TCRredirected CD8+ IFN-gamma+ T cells invade the prostate after tumor-specific vaccination (with Tag-pulsed DC) and instruct therapeutic CD3+ lymphocyte infiltration. TCRredirected autologous cells are also able to provoke tumor debulking of established adenocarcinomas, in the absence of graft versus host disease. Angelo Corti (San Raffaele Scientific Institute, Milan, Italy) described a new head-to-tail-cyclized hexapeptide containing the isoAsp-Gly-Arg (isoDGR) sequence, chemical conjugated to human serum albumin (HSA). IsoDGRHAS selectively binds αvβ3, an integrin up-regulated in the tumor vasculature, recognizes in vitro tumor endothelial cells and homes in vivo to tumor vessels, inhibiting tumor growth in mice. Moreover, conjugate IsoDGR-HAS peptide with gold nanoparticles to deliver TNF-alpha and other cytokines to tumor vessels gave promising results. Active targeting via isoDGR enhances the interaction of nanoparticles with tumor vessels and the therapeutic activity of this new nanosystem. Massimo Guidoboni (IRST-IRCCS, Meldola, Italy) presented data on gene expression analysis at whole transcriptome level of serial melanoma biopsies before and after vaccination with DC loaded with autologous tumor lysate. Ingenuity pathway analysis showed that several genes are up-regulated in post-vaccine lesions and belong to an “immunological constant of rejection” signature. The concomitant up-regulation of DICER1 and DNA (cytosine5)-methyltransferase 1, genes involved in cell motility and epigenetic reprogramming, respectively, suggests a possible switch of melanoma cells toward a “stemlike” phenotype, triggered by vaccine-induced immune response.
13
1856
Alessia Covre (University Hospital of Siena, Siena, Italy) talked about the immunomodulatory activity of in vivo administration of SGI-110, a second generation DNA hypomethylating agent, in acute myeloid leukemia (AML) and myelodysplastic syndromes patients enrolled in a phase I–II trials. The presented data indicate that SGI110 was able to reduce the constitutive methylation levels in promoter regions of investigated cancer–testis antigen (CTA) (i.e., NY-ESO-1 and MAGE-A1) of treated patients in a dose-dependent manner. Moreover, a transient induction/up-regulation of NY-ESO-1, MAGE-A1 and MAGEA3 expression was observed in PBMC of several patients treated with the highest doses of SGI-110.
Session 6: Translational in cancer immunotherapy Cancer immunotherapy is a fast evolving field in the management of cancer patients, and it will likely continue to provide novel and effective therapeutic options in the future. This session was focused on the rapid translation of different immunotherapeutic agents and strategies in the clinical setting. The isoform variant 6 of the adhesion receptor CD44 (CD44v6) is widely expressed in AML and multiple myeloma (MM) and is crucially involved in BM homing and cancer stem cell behavior. In phase I/II trials, targeting CD44v6 with mAb conjugated to chemotherapeutic agents showed substantial efficacy, but also toxicity due to background expression of CD44v6 on the skin. Attilio Bondanza (San Raffaele Scientific Institute, Milan, Italy) demonstrated that thanks to the CAR technology it is now possible to efficiently redirect T cells against virtually any antigen for which a mAb is available. Moreover, recent data on HSCT clearly indicate that suicide gene modification is an efficient tool for controlling the potential off-tumor toxicities of T cells. He developed, in a mouse model, a strategy for the safe targeting of CD44v6 through the coexpression of a suicide gene in CAR-modified T cells for the treatment of AML and MM, showing that CD44v6 CAR-redirected suicidal T cells have the potential to eradicate AML and MM. Hopefully, this strategy will be applied soon after autologous HSCT for sterilizing residual tumor. Ennio Carbone (University of Catanzaro, Catanzaro, Italy) discussed on a new strategy for cancer therapy with NK cells that have been historically classified as potent cytotoxic effectors recognizing allogeneic hematopoietic malignancy and solid tumors derived cell lines. More recently, NK cell-based immunotherapies have been proposed to treat hematopoietic malignancies, but are believed to be less effective against solid tumors. However, carbone provided evidences that (1) NK cells recognize melanoma at various stages of the disease, (2) NK cells subsets are
13
Cancer Immunol Immunother (2013) 62:1851–1858
present and active in the ensuing immune response in melanoma infiltrated lymph nodes, (3) KIR+ CCR7+ CD69+ CD57+ CD56dim NK cell subset expands during melanoma lymph node progression and correlates with the prognosis and (4) freshly purified allogeneic NK cells can recognize and kill CRC derived CIC, while the non-CIC counterpart of the tumors (differentiated tumor cells) either autologous or allogeneic is less susceptible to NK cells. Luigi Aurisicchio (MSD, Rome, Italy) reported results of 2 multicenter phase I trials involving cancer patients with solid tumor of different histotypes at stages II–IV of the disease. In the first study, patients were vaccinated with V930 alone, a DNA electroporation vaccine containing equal amounts of plasmids expressing the extracellular and transmembrane domains of human HER2, and a plasmid expressing carcinoembryonic antigen (CEA) fused to the B subunit of Escherichia coli heat labile toxin (Study 1), while in the second study, patients were treated with V930 followed by V932, a dicistronic adenovirus subtype-6 viral vector vaccine coding for the same antigens. Either V930 vaccination with electroporation alone or in combination with V932 was well tolerated without any serious adverse events (AEs). No measurable cell-mediated immune response to CEA or HER2 was detected in patients by ELISPOT; however, a significant increase in both cellmediated immunity and antibody titer against the bacterial heat labile toxin was observed upon vaccination. An increasing number of evidence clearly indicates that different molecular pathways associated with the cancer pathogenesis do correspond to different subsets of cancer patients. Identification of such different patients’ subsets should be introduced in clinical trials, in order to better assess the classification of all predictive and prognostic factors associated with the disease as well as more accurately address patients to the most effective therapeutic intervention according to their biological and molecular status. Giuseppe Palmieri (National Research Council, Sassari, Italy) discussed about BRAF mutations that can be observed in conjunction with PI3K alterations. He highlighted that none of them may coexist with a mutation in a gene of the RAS family; since BRAF and PI3K kinases act downstream RAS protein, occurrence of RAS mutations activating both MAPK and PI3K-AKT pathways makes unnecessary the further activation of BRAF and PI3K (upstream effectors of the MAPK and PI3K-AKT pathways, respectively). In addition, oncogenic BRAF mutations are able to more intensively activate ERK protein, main last effector downstream the MAPK pathway, when inactivation of the mechanisms controlling senescence and apoptosis concomitantly occurs. Anna Maria Di Giacomo (University Hospital of Siena, Siena, Italy) reported on the trial sponsored by the NIBIT Foundation, the NIBIT-M2 trial entitled: “A randomized,
Cancer Immunol Immunother (2013) 62:1851–1858
phase III study of Fotemustine versus the Combination of Fotemustine and Ipilimumab in Patients with Metastatic Melanoma with brain metastasis.” Recent evidences of activity in patients with brain disease, regardless of prior radiotherapy, from the multicentric, phase II, NIBIT-M1 trial, support a potential role for ipilimumab plus fotemustine in metastatic melanoma with brain disease. The NIBITM2 study was designed to assess the efficacy of the combination of ipilimumab and fotemustine/versus/fotemustine in terms of overall survival (OS) in patients with previously untreated metastatic melanoma with brain metastasis. She referred that the enrollment of a total of 146 subjects (73/ arm) will start soon, will involve ten Italian institutions and will be likely completed in 18 months. Paola Queirolo (Istituto Nazionale per la Ricerca sul Cancro IST, Genoa, Italy) discussed on data regarding efficacy and safety of ipilimumab treatment emerging from an Italian cohort of patients with pretreated advanced melanoma within The European Expanded Access Program (EAP) context. In total, 848 Italian patients participated in the EAP from June 2010 to April 2012 across 53 centers. With a median follow-up of 3 months, the disease control rate among 468 evaluable patients was 31.4 %, including 7 patients with a complete response, 51 with a partial response and 89 with stable disease. As of April 2012, median progression-free survival and OS were 3.1 and 6.2 months (range 0.1–21.4), respectively, with 1-year survival rate of 34 %. In total, 50.1 % patients reported an AEs of any grade, most of which were drug-related (36.2 %). Grade 3/4 AEs were reported by 18.5 % patients and considered drug-related in 8.5 %. Eleven patients discontinued treatment due to toxicity. AEs were generally reversible with treatment as per protocol-specific guidelines. The EAP provided an opportunity to confirm the efficacy and safety of ipilimumab 3 mg/kg in a setting similar to daily clinical practice. The encouraging clinical activity of anti-CTLA-4 antibodies in melanoma treatment has served as a model to exploit the therapeutic potential of CTLA-4 blockade in a variety of human malignancies. Luana Calabrò (University Hospital of Siena, Siena, Italy) made an overview on phase II clinical trials that have been conducted or are currently ongoing in lung cancer and mesothelioma. Results in advanced non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients showed that treatment with ipilimumab combined with chemotherapy in sequential phase of treatment was generally well tolerated and induced a significant improvement in immune-related progression-free survival compared to patients treated with chemotherapy alone. Results from a phase II (MESOTTREM-2008) study with tremelimumab in advanced mesothelioma patients who failed a first line of chemotherapy regimen showed that tremelimumab treatment is active in this setting of patients and can induce durable stabilization
1857
of disease, warranting further investigation. The AEs observed were consistent with tremelimumab safety profile in other indications, in addition treatment associates with major changes in T cell subpopulations. Michele Maio (University Hospital of Siena, Siena, Italy) discussed about different immunotherapeutic agents that have demonstrated to induce survival benefit in cancer patients. In particular, the mAb BMS-936559 anti-programmed cell death-1 (PD-1) and mAb MEDI4736 antiprogrammed cell death 1 ligand 1 (PD-L1) acting on the PD-1/PD-L1 blockade pathway showed promising results of clinical activity of in subjects with solid tumors. Overall, lessons from the development of ipilimumab contributed to a new clinical paradigm for cancer immunotherapy. Andrea Volonté (San Raffaele Foundation Scientific Institute, Milan, Italy) focused on the characterization of the immunosuppressive properties of CIC isolated from glioblastoma multiforme or CRC, showing that CSCs display an immune-suppression activity of anti-tumor T cell responses and demonstrating that the indoleamine 2,3-dioxygenase (IDO)-mediated tryptophan catabolism pathway is responsible of this phenomenon. Interestingly, IDO-mediated activity was inhibited by treatment of these cells with the specific inhibitor 1-methyl tryptophan (1-MT) as suggested by the capacity of 1-MT to restore the proliferation of autologous PBL. Volontè also demonstrated that CRC CICs overexpress IL-4 compared to their FBS cultured nonCIC (FBS tumor cells) counterpart and that the neutralization of IL-4 can rescue the efficiency of anti-tumor immune responses. Elena Jachetti (San Raffaele Foundation Scientific Institute, Milan, Italy) provided experimental evidence that cancer stem cells (CSC) lines established from the prostate of TRAMP mice expressed prostate cancer-associated antigens, MHC I and MHC II molecules and ligands for NK cells. Indeed, CSC was targets of NK and cytotoxic T lymphocytes both in vitro and in vivo. Vaccination with DC pulsed with irradiated CSC induced a tumor-specific immune response that was stronger than the one induced by DC pulsed with differentiated tumor cells, delayed tumor growth in mice challenged with prostate CSC and caused tumor regression in TRAMP mice. Thus, CSC is targets of both innate and adaptive immune responses and might be exploited for the design of novel immunotherapeutic approaches against cancer. Moreover, the identification and targeting of putative CSC within solid tumors are relevant and still unmet issues as CSC are considered responsible for chemo-resistance and disease relapses. Dario Sangiolo (Institute for Cancer Research and Treatment Candiolo, Turin, Italy) proposed a new gene transfer strategy to visualize putative CSC and to investigate their potential killing by immunotherapy with autologous cytokine-induced killer (CIK) cells. The results demonstrated that CIK cells are able to efficiently kill
13
1858
autologous CSC of metastatic melanomas and sarcomas. Overall, the data presented suggest CIK cells as promising candidates for effective immunotherapy in currently incurable clinical settings. Human HSP105 is a high molecular weight chaperone constitutively expressed at low levels within the cytoplasm that can also be induced in the nucleus by various forms of stress. It is overexpressed in several solid tumors, including melanoma, breast, thyroid and gastroenteric cancers as well as in B cell non-Hodgkin lymphoma (B-NHLs). Roberta Zappasodi (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) presented data on the functional role of HSP105 in B-NHLs by stably silencing its expression in the Namalwa aggressive lymphoma cell line. In particular, results showed that (1) aggressive B-NHL cell lines survive HSP105 knockdown, (2) HSP105-silencing significantly delays Namalwa cell growth by increasing cell doubling time, (3) in vitro cell growth delay of siHSP105 versus MOCK cultures is confirmed in vivo, (4) BCL-6 and c-Myc are down-regulated in function of HSP105 knockdown levels in siHSP105 Namalwa clones and (5) HSP105 coimmunoprecipitates with Bcl-6 and c-Myc and is expressed at higher levels in primary Bcl-6+ or c-Myc+ B-NHLs than in other aggressive lymphoma that do not overexpress these oncoproteins.
Conclusion As summarized by Ruggero Ridolfi (IRST-IRCCS, Meldola, Italy), the NIBIT 2012 Annual Meeting ended after 2 days of top-notch presentations reporting new data and enhancing our knowledge about the complex relationship between cancer and the host immune system. Many of these involved basic research on cell cultures and animal models, investigating the still open question of cancerrelated immune suppression and unveiling fine details of the mechanisms involved. Some of these have also been evaluated in humans, with a special focus on the effects of cancer vaccines and immunotherapeutic drugs.
13
Cancer Immunol Immunother (2013) 62:1851–1858
The event was closed by a round-table discussion with representatives of other Italian scientific societies and of the Istituto Superiore di Sanità, thus reinforcing the intrinsic vocation of NIBIT for open scientific cooperation. The panel pointed out that, despite the recent efforts of regulatory authorities to improve collaboration with clinical researchers, the rules regulating both the production of advanced therapies and the introduction of new drugs into clinical practice can delay decisions and, therefore, potentially damage both researchers and patients. Cellular therapies have actually been recognized as drugs: Therefore, no variation is allowed, unless specifically validated and authorized after a cumbersome and expensive procedure. The negative impact on spontaneous research on this promising field can be dramatic, eventually leading to a loss of therapeutic opportunities for the patients. The latter is true especially in case for some new drugs, which require long bureaucracy before being commercially available. This question has also been raised by the Italian Association of Medical Oncology and other medical societies. There is also an urgent need to communicate the recent advances in immunology and cancer immunotherapy to medical oncologists. Effective education should therefore be pursued. To achieve this important aim an active collaboration with the Italian Society of Cancerology, committed especially to basic research, and other scientific societies is warranted. Actually, recent data demonstrate the central role played by the immune system, since it can also affect the response to conventional treatments such as chemo- or radiotherapy. For these reasons, NIBIT will continue to search for important national and international scientific cooperation in the next few years. Acknowledgments This work was supported in part by grants from: Associazione Italiana per la Ricerca sul Cancro (Hugues Jean Marie Nicolay fellow) and Istituto Toscano Tumori. A special thanks to the NIBIT members and meeting speakers for their contribution to the manuscript and the conference. Conflict of interest The authors declare that they have no conflict of interest.
Meeting Report
Tenth annual meeting of the Italian Network for Tumor Biotherapy (NIBIT), SIENA, Italy, November 5–7, 2012 Michele Maio · Hugues Jean Marie Nicolay · Paolo Antonio Ascierto · Filippo Belardelli · Roberto Camerini · Mario Paolo Colombo · Paola Queirolo · Ruggero Ridolfi · Vincenzo Russo · Giulia Parisi · Ornella Cutaia · Ester Fonsatti · Giorgio Parmiani
Received: 17 May 2013 / Accepted: 24 October 2013 / Published online: 5 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Keywords Cancer · Immunology · Immunotherapy · Networks · NIBIT Abbreviations 1-MT 1-Methyl tryptophan AML Acute myeloid leukemia ACT Adoptive cell therapy AEs Adverse events Haplo-HSCT Haploidentical hematopoietic stem cell transplantation
This study was conducted on behalf of Italian Network for Tumor Biotherapy (NIBIT), Siena, Italy. M. Maio (*) · H. J. M. Nicolay · G. Parisi · O. Cutaia · E. Fonsatti Division of Medical Oncology and Immunotherapy, Department of Oncology, University Hospital of Siena, Viale Mario Bracci n. 16, 53100 Siena, Italy e-mail: [email protected] P. A. Ascierto Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, Istituto Nazionale Tumori Fondazione “G. Pascale”, Naples, Italy F. Belardelli Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy R. Camerini Clinical Research, Sigma Tau S.p.A., Pomezia, Italy
APC Antigen-presenting cells APM Antigen processing and presentation machinery B-NHLs B cell non-Hodgkin lymphoma BLI Bioluminescence imaging BM Bone marrow CIC Cancer initiating cells CSC Cancer stem cells CTA Cancer–testis antigen CEA Carcinoembryonic antigen CCL Chemokine (C–C motif) ligand CAR Chimeric antigen receptors CLL Chronic lymphocytic leukemia CRC Colorectal cancer cells CREB cAMP response element-binding protein P. Queirolo Oncologia Medica A, IRCCS Ospedale San Martino-Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy R. Ridolfi Immunotherapy and Somatic Cell Therapy Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Forlì, Italy V. Russo Cancer Gene Therapy Unit, Division of Molecular Oncology, San Raffaele Foundation Center, Milan, Italy G. Parmiani Unit of Immunobiotherapy of Melanoma and Solid Tumors, Division of Molecular Oncology, San Raffaele Foundation Centre, Milan, Italy
M. P. Colombo Molecular Immunology Unit, Department of Experimental Oncology, Fondazione Istituto Ricovero e Cura a Carattere Scientifico, Istituto Nazionale dei Tumori, Milan, Italy
13
1852
CIK Cytokine-induced killer CTLA-4 Cytotoxic T-lymphocyte antigen 4 DC Dendritic cells T-bodies Engineered T cells EAP Expanded access program GVHD Graft versus host disease VH CDR3 Heavy complementarity-determining region 3 HLA Human leukocyte antigen hPSMA Human prostate-specific membrane antigen HAS Human serum albumin Ig Immunoglobulin IDO Indoleamine 2,3-dioxygenase isoDGR IsoAsp-Gly-Arg KIR Killer cell immunoglobulin-like receptor LAG-3 Lymphocyte activation gene-3 LN Lymph node MHC Major histocompatibility complex MC Mast cells MMP-9 Metalloproteinase-9 mAb Monoclonal antibody MM Multiple myeloma MDSC Myeloid-derived suppressor cells NK Natural killer NSCLC Non-small-cell lung cancer OS Overall survival PBMC Peripheral blood mononuclear cell PD-1 Programmed cell death-1 PD-L1 Programmed cell death-1 ligand 1 TGF Transforming growth factor TRAMP Transgenic adenocarcinoma of the mouse prostate TAA Tumor-associated antigen TNF-alpha Tumor necrosis factor alpha TCR T cell receptor Treg Regulatory T cells CD44v6 Variant 6 of the adhesion receptor CD44 VEGF Vascular endothelial growth factor
Introduction NIBIT (acronym for the Network Italiano per la Bioterapia dei Tumori-Italian Network for Tumor Biotherapy) is a non-profit association created in 2004 to promote and foster scientific interactions among Italian and international professionals working in the field of cancer immunotherapy. The aim of the NIBIT annual meeting is to share and discuss the more recent preclinical and clinical results among researchers engaged in the field of cancer immunology and biotherapy. The meeting included more than 40 Italian and international leading groups representing academia, biotechnology
13
Cancer Immunol Immunother (2013) 62:1851–1858
and pharmaceutical industry. It covered the following topics related to immunology and immunotherapy of cancer: (1) gene mutations as targets of immunobiotherapy, (2) recent insights from preclinical models of cancer immunotherapy, (3) shaping the tumor microenvironment by inflammation and altered metabolism, (4) immunomodulation in the course of CTLA-4 blockade, (5) immunoengineering for cancer therapy and (6) translation in cancer immunotherapy.
Session 1: Gene mutations as targets of immunobiotherapy Unique tumor-associated antigens (TAAs), encoded by somatically mutated cancer genes, generate strong immunogenic epitopes, and cancer vaccination with mutated TAAs can result in increased clinical benefit. Daniele Mennonna (San Raffaele Scientific Institute, Milan, Italy) focused on the identification of unique colorectal cancer antigens by next generation sequencing of somatically mutated genes. Preliminary results obtained in colorectal cancer cells (CRC) show that the frame shift mutation, occurring in the antigen-presenting cell (APC) genes, generates a neoepitope that is presented by human leukocyte antigen (HLA)–DR molecules, and it is recognized by specific CD4+ T cells. Peptides corresponding to the non-synonymous point mutations in TP53 and SMAD4 genes elicit CD8+ and CD4+ T cells that specifically react against CRC cells. These results support the feasibility of this approach to identify unique TAAs in CRC by massive sequencing and reverse immunology. Cristina Maccalli (San Raffaele Scientific Institute, Milan, Italy) discussed about the immunoglobulin gene repertoire in chronic lymphocytic leukemia (CLL). She reported that this repertoire is remarkably restricted with >30 % of cases carrying quasi-identical (stereotyped) heavy complementarity-determining region 3 (VH CDR3) sequences. She suggested that peptides from stereotyped VH CDR3 sequences can represent candidate antigens to elicit anti-CLL T cell responses, especially in poor prognosis, where novel therapeutic interventions are needed. Although preliminary, the presented results may provide the proof of principle for the design of new immunotherapeutic protocols for CLL patients, including both active vaccination and adoptive cell therapy. Franco Locatelli (Bambino Gesù Children’s Hospital, Rome, Italy) presented data on haploidentical hematopoietic stem cell transplantation (haplo-HSCT) and innate immunity. He showed that in haplo-HSCT, alloreactive natural killer (NK) cells mediate potent anti-leukemia effects, which alloreactivity can be predicted by killer cell immunoglobulin-like receptor (KIR)/KIR-ligand genotypic analysis of donor/recipient cells. Also, the identification, the size
Cancer Immunol Immunother (2013) 62:1851–1858
of the NK alloreactive subset and the presence of activating KIR are important for the optimal donor selection. He finally discussed as failure to reconstitute the invariant NK T cell repertoire after haplo-HSCT is associated with high risk of leukemia relapse. By using proteome-based analyses and/or cDNA microarrays of RAS and/or HER-2/neu transformants candidate genes/proteins, Barbara Seliger (Martin-Luther University, Halle, Germany) showed that the overexpression of biglycan in oncogene-transformed cells leads to a transcriptional up-regulation of antigen processing machinery (APM) components associated with an increased major histocompatibility complex (MHC) class I surface expression. These data suggest that deficient biglycan expression in neoplastic cells may promote MHC class I down-regulation and allow their escape from immune surveillance. Finally, both inhibition of cAMP response element-binding protein (CREB) and biglycan overexpression lead to reduced tumor formation in oncogene-transformed cells. Silvia Carluccio (San Raffaele Scientific Institute, Milan, Italy) showed that tumor cell lines and dendritic cells (DC) were successfully obtained from CRC patients to stimulate T cells isolated from autologous PBMC. To date, ELISPOT was performed for two patients: Statistically significant INF-gamma secretion was detected at third, fourth and fifth stimulation for one patient, while no relevant cytokine secretion was found for the other patient. These data demonstrate the feasibility to generate tumorspecific CTL from peripheral blood to be useful for supporting adoptive cell therapy in patients with CRC. Peptide-based vaccines, although safe in patients, have problems related to HLA restriction and rapid clearance. To overcome these issues, Gilberto Filaci (University of Genoa, Genoa, Italy) set a telomerase-based vaccine, named GX301, constituted by four different peptides (i.e., hTERT540–548, hTERT611–626, hTERT672–686 and hTERT766–780) and two adjuvants (i.e., Montanide ISA51 and Imiquimod). Preliminary results, from a phase I/II clinical trial enrolling stage IV prostate and renal cancer patients resistant to conventional therapies, indicate that this therapeutic strategy is safe and induces efficient immune responses against potentially weak immunogenic antigens such as self-tumor antigens.
Session 2: Recent insights from preclinical models of cancer immunotherapy Antonio Rosato (University of Padua, Padua, Italy) focused on the importance of tracking T cells in vivo to assess the persistence, expansion and homing properties of injected T cells in ACT protocols. He presented data on the development of a second generation chimeric antigen
1853
receptor (CAR) directed against the human prostate-specific membrane antigen (hPSMA), to engineer human peripheral blood mononuclear cell (PBMC) for immunotherapeutic strategies in prostate cancer. Lentiviral vectors were used as gene transfer method, designed with a bidirectional promoter for the expression of both CAR and firefly luciferase, enabling the in vivo monitoring of engineered T cells (T-bodies) with bioluminescence imaging (BLI). T-bodies expressed high levels of CAR, a memory phenotype and the ability to kill hPSMA-expressing cells. However, data from BLI showed limited in vivo persistence and homing properties of transferred T-bodies in mouse model. In the last decade, several types of vaccines for boosting the body’s immune response to cancer cells are being tested in clinical trials with limited therapeutic efficacy. Matteo Bellone (San Raffaele Scientific Institute, Milan, Italy) reported on different effects of booster vaccinations in prophylactic and therapeutic settings. In vivo data demonstrated that priming with DC-based vaccines and the correct timing of booster injections favor the persistence of antigen-specific memory T cells and correlate with tumor rejection, thus increasing anti-tumor immunity in prophylactic settings. In contrast, prime/boost vaccination proved to be of no advantage or even detrimental in therapeutic settings in B16F1 and transgenic adenocarcinoma of the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse models. Paola Pittoni (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) discussed the role of mast cells (MC) in mouse and human prostate cancer and the possible application of MC-targeted cancer therapy. She showed that MC display different functions according to tumor stage. In fact, MC support the growth of early-stage tumors, providing metalloproteinase-9 (MMP-9), while prostate tumors after epithelial-to-mesenchymal transition produce MMP-9 and become independent from MC. Moreover, results highlighted a new, opposite role of MC in suppressing the occurrence of aggressive c-Kit+ neuroendocrine tumor variants. These findings support the hypothesis that c-Kit tyrosine kinase inhibitors, as imatinib, could block both adenocarcinoma-promoting MC (stroma-targeting) and neuroendocrine tumor variants (tumor-targeting) in prostate cancer. Elena Quaglino (University of Turin, Turin, Italy) reported on the identification of oncoantigens with a critical role in mammary carcinogenesis in ErbB2+ transgenic (BALB-neuT) mice. The results obtained from the analysis of the oncoantigens ErbB2 (expressed on the tumor cell surface) and Amot (expressed by the endothelia of angiogenic tumor tissues) demonstrated that in BALBneuT mice: (1) DNA vaccination targeting ErbB2 persistently inhibits cell proliferation of early mammary lesions, (2) DNA vaccination targeting Amot induces an antibody
13
1854
response that significantly halts the progression of incipient carcinomas and alters tumor vessel structure and permeability. These findings suggest the importance to extend the investigation on cancer initiating cells (CIC), responsible for tumor progression, metastasis, resistance to therapy and tumor recurrence. Marcella Tazzari (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) discussed the immunomodulating potential of targeted therapies in patients with solitary fibrous tumor. The study revealed a reduction in the circulating immunosuppressive cells myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs) by treatment with sunitinib, a multi-targeted receptor tyrosine kinase inhibitor. This peripheral immune suppression was associated with the modulation of the immunological status at tumor site resulting in the acquisition of immunological traits compatible with the setting of an adaptive response.
Session 3: Shaping the tumor microenvironment by inflammation and altered metabolism In her keynote lecture, Licia Rivoltini (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) gave an overview of the role of MDSC in cancer. She showed that at least 2 MDSC subsets can be identified (monocytic and granulocytic), involved in different phases of tumor progression. In particular, MDSC accumulation was reported to be associated with bad prognosis in stage II–III melanoma patients and in kidney cancer patients. She illustrated a hypothesis on genesis of tumor-induced MDSC accumulation in peripheral blood, due to selective tumor factors delivered as embedded in nanovesicular structures (exosomes), selectively homing to the bone marrow (BM) to influence myelopoiesis. Finally, data were presented on the pharmacologic down-modulation of MDSC in cancer patients induced by sunitinib, esomeprazole and BRAF inhibitors, suggesting the use of these therapeutic agents in combination with immune-based strategies to achieve more effective immune responses and disease control. Andrea Anichini (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) provided evidence of presence of CD8+ FOXP3+ T cells, which do not present an “early effector” profile (CD127−, KLRG1−, HLADR+, CD38+, T-bet+, perforin+) within neoplastic tissues from primary and metastatic lesions derived from melanoma patients. This subset of CD8+ FOXP3+ T cells is neither exhausted nor senescent and do not exhibit a regulatory [cytotoxic T-lymphocyte antigen 4 (CTLA-4), chemokine (C–C motif) ligand (CCL)-4, IL-10, transforming growth factor (TGF)-beta-1 phenotype]. He suggested that blockade of CTLA-4 by the monoclonal antibody (mAb) ipilimumab promotes CD3+ T cell infiltration, the presence of CD1a+ DC and a lower frequency of FOXP3+ lymphocytes, mimicking the early
13
Cancer Immunol Immunother (2013) 62:1851–1858
stages of T cell-mediated responses at the tumor site of responding lesions. Rosa Maria Moresco (University of Milan Bicocca, Milan, Italy) discussed on the development of specific tracers for positron emission tomography-based molecular imaging of metabolism and inflammation in cancer. She underlined that [18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) tracer, largely used for patients staging and followup, may result in false-positive or false-negative uptake in some clinical settings. She therefore described several probes (e.g., [18F] FLT, [18F] FAZA, [11C] acetate, [11C] PK11195, [18F] C-7, [18F] F-FAC, [18F] interleukin) with higher sensitivity, which have been developed by different research groups to improve the specificity of detection of the different metabolic pathways in cancer. Lymphocyte activation gene-3 (LAG-3) is a CD4-related cell surface molecule expressed by activated CD4+, CD8+ T and NK cells. This molecule has opposite functions: On one side, it negatively regulates T cells proliferation, on the other side, it induces APC activation trough MHC class II. Chiara Camisaschi (University of Milan Bicocca, Milan, Italy) explored the role of pDC in human melanoma. This population of dendritic cells, deputed to the INF-alpha release, may exert both activating and suppressive activities. Evidences were provided suggesting that, in the tumor microenvironment, pDC displayed an alternative activating pathway consistent with a partial tumor-induced dysfunction, possibly leading to the development and maintenance of immunosuppression. Viviana Vallacchi (Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy) presented a pilot study of gene expression and miRNA profiling to identify signatures in exoMDSC, a cell population in vitro generated from the co-culture of normal monocytes with melanoma exosomes. Results showed that exo-MDSC exclusively express 66 genes (42 upregulated and 24 down-regulated) and 11 miRNA (7 up-regulated and 4 down-regulated) compared to normal monocytes. Several genes up-regulated in MDSC are involved in immune response processes, including CCL7, CCL13 and IFNrelated genes. Up-regulated miRNA included hsa-miR-150, known to regulate different immune cells. She reported that several pro-tumorigenic and immunosuppressive cytokines/ chemokines [vascular endothelial growth factor (VEGF)A, CCL2, IL8 and TGF-beta1], also present in melanoma exosomes, are released by exo-MDSC, suggesting that they may have a role in the skewing of monocytes to MDSC.
Session 4: Immunomodulation in the course of CTLA‑4 blockade Monitoring patients’ immune system is crucial for the immune-guiding and to identify correlates of cancer
Cancer Immunol Immunother (2013) 62:1851–1858
treatment efficacy. This session focused on the immunemonitoring platform established within the NIBIT to investigate changes in the phenotypic profile of PBMC and in the anti-tumor-specific humoral and/or cellular functional immune response(s) induced in cancer patients during the CTLA-4 blockade therapy. In particular, Luana Calabrò (University Hospital of Siena, Siena, Italy), Licia Rivoltini (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy), Pier Franc‑ esco Ferrucci (European Institute of Oncology, Milan, Italy), Cristina Maccalli (San Raffaele Scientific Institute, Milan, Italy) and Elisabetta Fratta (Centro di Riferimento Oncologico, Aviano, Italy) showed preliminary data using different strategies in immunological monitoring within the translational studies associated with the clinical trial NIBIT-M1, utilizing the anti-CTLA-4 mAb ipilimumab in association with fotemustine in metastatic melanoma patients with or without brain metastasis and to the clinical trial involving patients with advanced melanoma treated with ipilimumab within a compassionate use program at the Division of Medical Oncology and Immunotherapy Department of Oncology University Hospital of Siena.
Session 5: Immune‑engineering for cancer therapy Stefano Ugel (University of Verona, Verona, Italy) demonstrated that the spleen has a fundamental role in tumorinduced tolerance in mice. In fact, splenectomy restores T cells activity and leads to a significant increase in EG-7 tumor-bearing mice survival. He also showed that the elimination of splenic CD11b+ Gr-1int myeloid cells by different chemotherapeutic agents enhances the antitumor efficacy of ACT in mice. He demonstrated that the CCL2-mediated accumulation of immunosuppressive CD11b+ Gr-1intLy6ChiCCR2+ cells, in response to CCL2, in the marginal zone of the spleen during tumor growth, leads to direct cross-presentation of tumor antigens to memory CD8+ T lymphocytes in a tolerogenic manner. Finally, he reported that CCL2 serum levels are predictive of the clinical response in patients responding to cancer vaccination. Andrea Brendolan (San Raffaele Scientific Institute, Milan, Italy) explored the molecular and cellular mechanisms at the basis of secondary lymphoid organ development, in terms of stromal cell differentiation and lymphocytes compartmentalization. A study conducted in mice demonstrated that the different spleen stromal cell subsets, including follicular dendritic cells, arise from embryonic mesenchymal precursors of the Nkx2.5+/Isl1+ lineage. This stromal cell lineage was shown to regenerate the splenic stromal microenvironment and to support the formation of artificial lymphoidlike structures (lymphoid
1855
neogenesis), containing functional T and B cell areas. Furthermore, Nicolas van Baren (Ludwig Institute for Cancer Research, Brussels, Belgium) focused on the lymphoid neogenesis in melanoma. He reported the presence of ectopic lymphoid structures in 15–20 % of cutaneous metastases, but never in primary tumors, of melanoma patients. These structures, defined as lymphoid follicles, associate clusters of B lymphocytes, with high endothelial venules and with clusters of DCs and T cells. The molecular characterization of the immunoglobulin (Ig) gene repertoire expressed in lymphoid follicles showed evidence of clonal amplification and diversity of B lymphocytes, as well as isotype switching, hallmarks of immunoglobulin affinity maturation, indicating that adaptive immune responses can occur within the melanoma environment. Anna Mondino (San Raffaele Scientific Institute, Milan, Italy) highlighted the therapeutic synergy of autologous T cells redirected by T cell receptor (TCR) gene transfer toward a tumor-associated model antigen (i.e., large T antigen, Tag) and a ubiquitously expressed minor H antigen (i.e., Y-encoded Uty). In vivo experiments in prostate cancer bearing TRAMP mice showed that TCRredirected CD8+ IFN-gamma+ T cells invade the prostate after tumor-specific vaccination (with Tag-pulsed DC) and instruct therapeutic CD3+ lymphocyte infiltration. TCRredirected autologous cells are also able to provoke tumor debulking of established adenocarcinomas, in the absence of graft versus host disease. Angelo Corti (San Raffaele Scientific Institute, Milan, Italy) described a new head-to-tail-cyclized hexapeptide containing the isoAsp-Gly-Arg (isoDGR) sequence, chemical conjugated to human serum albumin (HSA). IsoDGRHAS selectively binds αvβ3, an integrin up-regulated in the tumor vasculature, recognizes in vitro tumor endothelial cells and homes in vivo to tumor vessels, inhibiting tumor growth in mice. Moreover, conjugate IsoDGR-HAS peptide with gold nanoparticles to deliver TNF-alpha and other cytokines to tumor vessels gave promising results. Active targeting via isoDGR enhances the interaction of nanoparticles with tumor vessels and the therapeutic activity of this new nanosystem. Massimo Guidoboni (IRST-IRCCS, Meldola, Italy) presented data on gene expression analysis at whole transcriptome level of serial melanoma biopsies before and after vaccination with DC loaded with autologous tumor lysate. Ingenuity pathway analysis showed that several genes are up-regulated in post-vaccine lesions and belong to an “immunological constant of rejection” signature. The concomitant up-regulation of DICER1 and DNA (cytosine5)-methyltransferase 1, genes involved in cell motility and epigenetic reprogramming, respectively, suggests a possible switch of melanoma cells toward a “stemlike” phenotype, triggered by vaccine-induced immune response.
13
1856
Alessia Covre (University Hospital of Siena, Siena, Italy) talked about the immunomodulatory activity of in vivo administration of SGI-110, a second generation DNA hypomethylating agent, in acute myeloid leukemia (AML) and myelodysplastic syndromes patients enrolled in a phase I–II trials. The presented data indicate that SGI110 was able to reduce the constitutive methylation levels in promoter regions of investigated cancer–testis antigen (CTA) (i.e., NY-ESO-1 and MAGE-A1) of treated patients in a dose-dependent manner. Moreover, a transient induction/up-regulation of NY-ESO-1, MAGE-A1 and MAGEA3 expression was observed in PBMC of several patients treated with the highest doses of SGI-110.
Session 6: Translational in cancer immunotherapy Cancer immunotherapy is a fast evolving field in the management of cancer patients, and it will likely continue to provide novel and effective therapeutic options in the future. This session was focused on the rapid translation of different immunotherapeutic agents and strategies in the clinical setting. The isoform variant 6 of the adhesion receptor CD44 (CD44v6) is widely expressed in AML and multiple myeloma (MM) and is crucially involved in BM homing and cancer stem cell behavior. In phase I/II trials, targeting CD44v6 with mAb conjugated to chemotherapeutic agents showed substantial efficacy, but also toxicity due to background expression of CD44v6 on the skin. Attilio Bondanza (San Raffaele Scientific Institute, Milan, Italy) demonstrated that thanks to the CAR technology it is now possible to efficiently redirect T cells against virtually any antigen for which a mAb is available. Moreover, recent data on HSCT clearly indicate that suicide gene modification is an efficient tool for controlling the potential off-tumor toxicities of T cells. He developed, in a mouse model, a strategy for the safe targeting of CD44v6 through the coexpression of a suicide gene in CAR-modified T cells for the treatment of AML and MM, showing that CD44v6 CAR-redirected suicidal T cells have the potential to eradicate AML and MM. Hopefully, this strategy will be applied soon after autologous HSCT for sterilizing residual tumor. Ennio Carbone (University of Catanzaro, Catanzaro, Italy) discussed on a new strategy for cancer therapy with NK cells that have been historically classified as potent cytotoxic effectors recognizing allogeneic hematopoietic malignancy and solid tumors derived cell lines. More recently, NK cell-based immunotherapies have been proposed to treat hematopoietic malignancies, but are believed to be less effective against solid tumors. However, carbone provided evidences that (1) NK cells recognize melanoma at various stages of the disease, (2) NK cells subsets are
13
Cancer Immunol Immunother (2013) 62:1851–1858
present and active in the ensuing immune response in melanoma infiltrated lymph nodes, (3) KIR+ CCR7+ CD69+ CD57+ CD56dim NK cell subset expands during melanoma lymph node progression and correlates with the prognosis and (4) freshly purified allogeneic NK cells can recognize and kill CRC derived CIC, while the non-CIC counterpart of the tumors (differentiated tumor cells) either autologous or allogeneic is less susceptible to NK cells. Luigi Aurisicchio (MSD, Rome, Italy) reported results of 2 multicenter phase I trials involving cancer patients with solid tumor of different histotypes at stages II–IV of the disease. In the first study, patients were vaccinated with V930 alone, a DNA electroporation vaccine containing equal amounts of plasmids expressing the extracellular and transmembrane domains of human HER2, and a plasmid expressing carcinoembryonic antigen (CEA) fused to the B subunit of Escherichia coli heat labile toxin (Study 1), while in the second study, patients were treated with V930 followed by V932, a dicistronic adenovirus subtype-6 viral vector vaccine coding for the same antigens. Either V930 vaccination with electroporation alone or in combination with V932 was well tolerated without any serious adverse events (AEs). No measurable cell-mediated immune response to CEA or HER2 was detected in patients by ELISPOT; however, a significant increase in both cellmediated immunity and antibody titer against the bacterial heat labile toxin was observed upon vaccination. An increasing number of evidence clearly indicates that different molecular pathways associated with the cancer pathogenesis do correspond to different subsets of cancer patients. Identification of such different patients’ subsets should be introduced in clinical trials, in order to better assess the classification of all predictive and prognostic factors associated with the disease as well as more accurately address patients to the most effective therapeutic intervention according to their biological and molecular status. Giuseppe Palmieri (National Research Council, Sassari, Italy) discussed about BRAF mutations that can be observed in conjunction with PI3K alterations. He highlighted that none of them may coexist with a mutation in a gene of the RAS family; since BRAF and PI3K kinases act downstream RAS protein, occurrence of RAS mutations activating both MAPK and PI3K-AKT pathways makes unnecessary the further activation of BRAF and PI3K (upstream effectors of the MAPK and PI3K-AKT pathways, respectively). In addition, oncogenic BRAF mutations are able to more intensively activate ERK protein, main last effector downstream the MAPK pathway, when inactivation of the mechanisms controlling senescence and apoptosis concomitantly occurs. Anna Maria Di Giacomo (University Hospital of Siena, Siena, Italy) reported on the trial sponsored by the NIBIT Foundation, the NIBIT-M2 trial entitled: “A randomized,
Cancer Immunol Immunother (2013) 62:1851–1858
phase III study of Fotemustine versus the Combination of Fotemustine and Ipilimumab in Patients with Metastatic Melanoma with brain metastasis.” Recent evidences of activity in patients with brain disease, regardless of prior radiotherapy, from the multicentric, phase II, NIBIT-M1 trial, support a potential role for ipilimumab plus fotemustine in metastatic melanoma with brain disease. The NIBITM2 study was designed to assess the efficacy of the combination of ipilimumab and fotemustine/versus/fotemustine in terms of overall survival (OS) in patients with previously untreated metastatic melanoma with brain metastasis. She referred that the enrollment of a total of 146 subjects (73/ arm) will start soon, will involve ten Italian institutions and will be likely completed in 18 months. Paola Queirolo (Istituto Nazionale per la Ricerca sul Cancro IST, Genoa, Italy) discussed on data regarding efficacy and safety of ipilimumab treatment emerging from an Italian cohort of patients with pretreated advanced melanoma within The European Expanded Access Program (EAP) context. In total, 848 Italian patients participated in the EAP from June 2010 to April 2012 across 53 centers. With a median follow-up of 3 months, the disease control rate among 468 evaluable patients was 31.4 %, including 7 patients with a complete response, 51 with a partial response and 89 with stable disease. As of April 2012, median progression-free survival and OS were 3.1 and 6.2 months (range 0.1–21.4), respectively, with 1-year survival rate of 34 %. In total, 50.1 % patients reported an AEs of any grade, most of which were drug-related (36.2 %). Grade 3/4 AEs were reported by 18.5 % patients and considered drug-related in 8.5 %. Eleven patients discontinued treatment due to toxicity. AEs were generally reversible with treatment as per protocol-specific guidelines. The EAP provided an opportunity to confirm the efficacy and safety of ipilimumab 3 mg/kg in a setting similar to daily clinical practice. The encouraging clinical activity of anti-CTLA-4 antibodies in melanoma treatment has served as a model to exploit the therapeutic potential of CTLA-4 blockade in a variety of human malignancies. Luana Calabrò (University Hospital of Siena, Siena, Italy) made an overview on phase II clinical trials that have been conducted or are currently ongoing in lung cancer and mesothelioma. Results in advanced non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients showed that treatment with ipilimumab combined with chemotherapy in sequential phase of treatment was generally well tolerated and induced a significant improvement in immune-related progression-free survival compared to patients treated with chemotherapy alone. Results from a phase II (MESOTTREM-2008) study with tremelimumab in advanced mesothelioma patients who failed a first line of chemotherapy regimen showed that tremelimumab treatment is active in this setting of patients and can induce durable stabilization
1857
of disease, warranting further investigation. The AEs observed were consistent with tremelimumab safety profile in other indications, in addition treatment associates with major changes in T cell subpopulations. Michele Maio (University Hospital of Siena, Siena, Italy) discussed about different immunotherapeutic agents that have demonstrated to induce survival benefit in cancer patients. In particular, the mAb BMS-936559 anti-programmed cell death-1 (PD-1) and mAb MEDI4736 antiprogrammed cell death 1 ligand 1 (PD-L1) acting on the PD-1/PD-L1 blockade pathway showed promising results of clinical activity of in subjects with solid tumors. Overall, lessons from the development of ipilimumab contributed to a new clinical paradigm for cancer immunotherapy. Andrea Volonté (San Raffaele Foundation Scientific Institute, Milan, Italy) focused on the characterization of the immunosuppressive properties of CIC isolated from glioblastoma multiforme or CRC, showing that CSCs display an immune-suppression activity of anti-tumor T cell responses and demonstrating that the indoleamine 2,3-dioxygenase (IDO)-mediated tryptophan catabolism pathway is responsible of this phenomenon. Interestingly, IDO-mediated activity was inhibited by treatment of these cells with the specific inhibitor 1-methyl tryptophan (1-MT) as suggested by the capacity of 1-MT to restore the proliferation of autologous PBL. Volontè also demonstrated that CRC CICs overexpress IL-4 compared to their FBS cultured nonCIC (FBS tumor cells) counterpart and that the neutralization of IL-4 can rescue the efficiency of anti-tumor immune responses. Elena Jachetti (San Raffaele Foundation Scientific Institute, Milan, Italy) provided experimental evidence that cancer stem cells (CSC) lines established from the prostate of TRAMP mice expressed prostate cancer-associated antigens, MHC I and MHC II molecules and ligands for NK cells. Indeed, CSC was targets of NK and cytotoxic T lymphocytes both in vitro and in vivo. Vaccination with DC pulsed with irradiated CSC induced a tumor-specific immune response that was stronger than the one induced by DC pulsed with differentiated tumor cells, delayed tumor growth in mice challenged with prostate CSC and caused tumor regression in TRAMP mice. Thus, CSC is targets of both innate and adaptive immune responses and might be exploited for the design of novel immunotherapeutic approaches against cancer. Moreover, the identification and targeting of putative CSC within solid tumors are relevant and still unmet issues as CSC are considered responsible for chemo-resistance and disease relapses. Dario Sangiolo (Institute for Cancer Research and Treatment Candiolo, Turin, Italy) proposed a new gene transfer strategy to visualize putative CSC and to investigate their potential killing by immunotherapy with autologous cytokine-induced killer (CIK) cells. The results demonstrated that CIK cells are able to efficiently kill
13
1858
autologous CSC of metastatic melanomas and sarcomas. Overall, the data presented suggest CIK cells as promising candidates for effective immunotherapy in currently incurable clinical settings. Human HSP105 is a high molecular weight chaperone constitutively expressed at low levels within the cytoplasm that can also be induced in the nucleus by various forms of stress. It is overexpressed in several solid tumors, including melanoma, breast, thyroid and gastroenteric cancers as well as in B cell non-Hodgkin lymphoma (B-NHLs). Roberta Zappasodi (Fondazione IRCCS “Istituto Nazionale dei Tumori,” Milan, Italy) presented data on the functional role of HSP105 in B-NHLs by stably silencing its expression in the Namalwa aggressive lymphoma cell line. In particular, results showed that (1) aggressive B-NHL cell lines survive HSP105 knockdown, (2) HSP105-silencing significantly delays Namalwa cell growth by increasing cell doubling time, (3) in vitro cell growth delay of siHSP105 versus MOCK cultures is confirmed in vivo, (4) BCL-6 and c-Myc are down-regulated in function of HSP105 knockdown levels in siHSP105 Namalwa clones and (5) HSP105 coimmunoprecipitates with Bcl-6 and c-Myc and is expressed at higher levels in primary Bcl-6+ or c-Myc+ B-NHLs than in other aggressive lymphoma that do not overexpress these oncoproteins.
Conclusion As summarized by Ruggero Ridolfi (IRST-IRCCS, Meldola, Italy), the NIBIT 2012 Annual Meeting ended after 2 days of top-notch presentations reporting new data and enhancing our knowledge about the complex relationship between cancer and the host immune system. Many of these involved basic research on cell cultures and animal models, investigating the still open question of cancerrelated immune suppression and unveiling fine details of the mechanisms involved. Some of these have also been evaluated in humans, with a special focus on the effects of cancer vaccines and immunotherapeutic drugs.
13
Cancer Immunol Immunother (2013) 62:1851–1858
The event was closed by a round-table discussion with representatives of other Italian scientific societies and of the Istituto Superiore di Sanità, thus reinforcing the intrinsic vocation of NIBIT for open scientific cooperation. The panel pointed out that, despite the recent efforts of regulatory authorities to improve collaboration with clinical researchers, the rules regulating both the production of advanced therapies and the introduction of new drugs into clinical practice can delay decisions and, therefore, potentially damage both researchers and patients. Cellular therapies have actually been recognized as drugs: Therefore, no variation is allowed, unless specifically validated and authorized after a cumbersome and expensive procedure. The negative impact on spontaneous research on this promising field can be dramatic, eventually leading to a loss of therapeutic opportunities for the patients. The latter is true especially in case for some new drugs, which require long bureaucracy before being commercially available. This question has also been raised by the Italian Association of Medical Oncology and other medical societies. There is also an urgent need to communicate the recent advances in immunology and cancer immunotherapy to medical oncologists. Effective education should therefore be pursued. To achieve this important aim an active collaboration with the Italian Society of Cancerology, committed especially to basic research, and other scientific societies is warranted. Actually, recent data demonstrate the central role played by the immune system, since it can also affect the response to conventional treatments such as chemo- or radiotherapy. For these reasons, NIBIT will continue to search for important national and international scientific cooperation in the next few years. Acknowledgments This work was supported in part by grants from: Associazione Italiana per la Ricerca sul Cancro (Hugues Jean Marie Nicolay fellow) and Istituto Toscano Tumori. A special thanks to the NIBIT members and meeting speakers for their contribution to the manuscript and the conference. Conflict of interest The authors declare that they have no conflict of interest.
