ALBERT

Description: Introduction: BCMA targeted CAR T cell therapy has shown promising results in patients with relapsed/refractory multiple myeloma (MM). Herein, we report on the safety and efficacy of MCARH171, a second generation, human derived BCMA targeted autologous 4-1BB containing CAR T cell therapy, including a truncated epidermal growth factor receptor safety system (Smith EL. Mol Ther 2018). Methods: This is a phase I first in human, dose escalation trial of MCARH171. Patients received conditioning chemotherapy with cyclophosphamide (Cy) 3 gm/m2 as a single dose or fludarabine 30 mg/m2 daily and Cy 300 mg/m2 daily for 3 days followed by MCARH171 infusion in 1-2 divided doses. The trial followed a standard 3+3 design with 4 dose levels where patients received the following mean doses per cohort: (1) 72x106, (2) 137x106, (3) 475x106, (4) 818x106 viable CAR+ T cells. The primary objective was to demonstrate safety, and secondary objectives included efficacy and expansion, and persistence of CAR T cells using PCR from the peripheral blood. The last accrued patient received MCARH171 on Dec 6, 2017 and the data cut-off is July 16, 2018. The study is closed to accrual. Results: 11 patients with relapsed and/or refractory MM were treated. Median number of prior lines of therapy was 6 (range: 4-14), and all patients received prior therapy with a proteasome inhibitor, IMiD, anti-CD38 monoclonal antibody, and high dose melphalan/stem cell transplant. Nine (82%) patients had high-risk cytogenetics and 9 (82%) were refractory to their immediate prior line of treatment. One patient was not evaluable for DLTs given the need for early radiation and steroids for impending spinal cord compression by tumor. There are no DLTs reported. Cytokine release syndrome (CRS) grade 1-2 occurred in 4 patients (40%), grade 3 occurred in 2 (20%), and there was no grade 4-5 CRS. Grade 2 encephalopathy occurred in 1 patient (10%) in the setting of high fevers which resolved in less than 24 hours. There was no grade 3 or higher neurotoxicity observed. Tocilizumab was administered to 3 patients; 2 in cohort 2, and 1 in cohort 3. Laboratory values correlating with CRS reaching grade 3 or requiring Tocilizumab (N=4) compared to those with no or milder CRS (N=6) included peak CRP (mean: 28.5 vs 4.6 mg/dL, p

Description: Abstract 4055 Cancer-testis (CT) antigens are a family of proteins normally expressed in immune privileged sites such as testicular germ cells and placenta, but are overexpressed in various malignant tumors (Scanlan et al. Immunological Rev. 2002). CT antigens are therefore useful markers of malignancy as well as potential targets for antigen specific cancer immunotherapy. In multiple myeloma, CT 7, CT10 and MAGE-A are homogenously expressed in up to 75% of cases, and their expression increases with disease stage and cell proliferation (Jungbluth et al. Blood, 2005). In addition, CT-7 and MAGE-A3 play a role in plasma cell proliferation and chemosensitivity (Atanackovic et al. Haematologica 2010). Immunogenicity of CT antigens is evidenced by spontaneous humoral responses against CT antigens in patients with multiple myeloma (Cohen et al. ASH abstract 2008). In addition, anti-CT antigen immune responses of donor derived T and B cells have been reported following allogeneic stem cell transplantation, suggesting CT antigens may serve as a natural target for a graft-versus myeloma effect (Atanackovic et al. Blood 2007). Systemic light-chain (AL) amyloidosis is a plasma cell dyscrasia related to multiple myeloma characterized by small numbers of non-proliferating, clonogenic plasma cells producing pathologic light chains. In this study, we investigated the expression of several CT antigens in patients with AL amyloidosis to identify potential targets for immunotherapy and determine their prognostic significance. Methods: Fifteen cases of AL amyloidosis were studied employing standard IHC techniques on paraffin-embedded archival tissues. Presence of plasma cells was verified by CD138 immunostain. The following monoclonal antibodies (to the following CT Antigens) were used: mAb MA454 (MAGE-A1), 6C1 (several MAGE-A antigens), 57B (MAGE-A4), E978 (NY-ESO-1), CT7-33(CT7), CT10#5 (CT10), #26 (GAGE). Immunopositivity was graded based on the amount of IHC-positive plasma cells. Results: All 15 patients had a confirmed diagnosis of AL amyloidosis with an average plasma cell burden of 12.7% of the cells in the marrow. Eighty-seven percent (13/15) had lambda disease and 13% (2/15) kappa disease. Organ involvement included kidney (n=7)), heart (n=8), peripheral nervous system (n=2), and GI/liver (n=2). Five patients (33%) had multi-organ involvement. All patients were treated uniformly with risk-adapted melphalan as their initial therapy. CT7 was present in 9/15 (60%) while CT10 was demonstrated in only 1/15 AL amyloid cases. Plasma cells did not stain with any other anti-CT mAb. There were no significant differences with regard to organ involvement, response to treatment or prognosis and CT antigen positivity in this small sample set. Discussion: This is the first study identifying CT7 as the prevalent CT antigen in plasma cells of patients with AL amyloidosis. The almost exclusive presence of CT7 in AL amyloidosis may have clinical significance. Further studies are planned on additional samples to confirm the prevalence of CT7 expression in AL amyloidosis, determine its immunogenicity and further investigate prognostic implications. Additional studies are needed to determine the biology of CT antigens in AL amyloidosis and their value as a potential target for immunotherapy. Disclosures: Comenzo: Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Elan Pharmaceuticals: Consultancy; Genzyme: Research Funding; Celgene: Research Funding; Ortho: Research Funding.

Description: The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis family of tumor-associated antigens and are found in a broad range of solid and hematologic malignancies. We previously showed that the type I MAGE proteins CT7 (MAGE-C1) and MAGE-A3 were commonly detected in primary myeloma by both RT-PCR and immunohistochemistry (IHC). Higher levels of MAGE protein expression had a positive correlation with abnormally elevated proliferation as measured by the Plasma Cell Proliferation Index (PCPI, percentage of Ki-67+ cells in the CD138+ myeloma cell compartment). These findings suggest that MAGE may play a role in abnormal cell cycle regulation in myeloma. We explored this hypothesis by examining type I MAGE gene expression and proliferation by IHC in 46 newly-diagnosed, untreated and 35 relapsed myeloma patients, based on the clinical observation that relapsed patients exhibit lower response rates to therapy and shorter time to progression, indicative of more aggressive disease. PCPI was significantly higher in relapsed patients (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p

Description: Background: Primary testicular diffuse large B cell lymphoma (DLBCL) is an uncommon malignancy portending a poor prognosis with increased risk of central nervous system disease. Phenotypically, most primary testicular lymphomas have a non-germinal center B-cell like (non-GCB) origin. To identify the genetic characteristics of testicular DLBCL, we evaluated DNA copy number and mutational profiling using SNP array and a next generation targeted sequencing platform. Methods: Twelve cases of testicular DLBCL with patient consent for tissue specimens and sufficient tumor tissue were retrospectively identified. Cell of origin was determined by Hans immunohistochemistry (IHC) model. We performed a custom, targeted deep-sequencing assay of 585 cancer genes (HemePACT) on matched tumor and normal pairs. Barcoded pools were sequenced on Illumina HiSeq 2500 to 500-1000x coverage per sample Sequencing was compared to a matched normal tissue control (N=10) if available or alternatively a pooled normal tissue control. We excluded all mutations either present at a high variant allele frequency in the matching germline samples, present in two databases of inherited variants (DBSNP and 1000 genomes) or present in one databases of inherited variants and absent from COSMIC. We evaluated copy number and allelic imbalance with an Affymetrix OncoScan SNP-array. IHC was performed for select genes. Results were compared to a panel of non-testicular DLBCL previously described (N=78). Results: The median age of the patients was 55.1 years (range 21.9-77.9). Patients had clinical stage IE (50%) and IV (50%) disease. All samples were sequenced from pre-treatment biopsies. Eleven of 12 patients were initially treated with R-CHOP chemotherapy, intrathecal methotrexate and radiation. Treatment history for one patient was unknown. We identified 124 mutations in 12 cases of testicular DLBCL. The most common mutation was MYD88 occurring in 10/12 patients (83%) with 6 mutations in non-GCB and 2 mutations in GCB (Fig 1A). The MYD88 L265P allele was most frequent and occurred in 9/12 patients (75%). The median MYD88 L265P variant allele frequency was 0.36 (range 0.07-0.51) with normal copy number status at that loci. In contrast, MYD88 mutations were less frequent in DLBCL without testicular involvement, 12/37 (32%) non-GCB and 3/41 (7%) GCB DLBCL, p

Description: Multiple myeloma is a malignancy of plasma cells. Vaccine immunotherapy is among the novel therapeutic strategies under investigation for this disease. To identify myeloma-associated antigens as potential targets for vaccine immunotherapy, we surveyed a comprehensive panel of bone marrow specimens from patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma for expression of cancer-testis (CT) antigens. Immunohistochemistry (IHC) demonstrated that 82% of stage-III myeloma specimens expressed the CT antigen CT7 (also known as melanoma antigen C1 [MAGE-C1]) and 70% expressed MAGE-A3/6. Messenger RNA for CT7 and MAGE-A family members was detected in 87% and 100% of stage-III samples, respectively. CT7 protein expression increased with advanced stage of disease. Higher levels of CT7 and MAGE-A3/6 proteins also correlated with elevated plasma-cell proliferation. These results show that CT7 and MAGE-A3/6 are promising myeloma-associated antigens for application in vaccine immunotherapy. Furthermore, the common expression and correlation with proliferation suggest a possible pathogenic role for these proteins in myeloma.

Description: Immunotherapies using cancer-testis (CT) antigens as targets represent a potentially useful treatment in patients with multiple myeloma (MM) who commonly show recurrent disease following chemotherapy. We analyzed the expression of 11 CT antigens in bone marrow samples from patients with MM (n = 55) and healthy donors (n = 32) using reverse transcriptase–polymerase chain reaction (RT-PCR). CT antigens were frequently expressed in MM with 56% (MAGEC2), 55% (MAGEA3), 35% (SSX1), 20% (SSX4, SSX5), 16% (SSX2), 15% (BAGE), 7% (NY-ESO-1), and 6% (ADAM2, LIPI) expressing the given antigen. Importantly, CT antigens were not expressed in healthy bone marrow. Analyzing patients with MM (n = 66) for antibody responses against MAGEA3, SSX2, and NY-ESO-1, we found strong antibody responses against CT antigens preferentially in patients who had received allogeneic stem cell transplantation (alloSCT). Antibody responses against NY-ESO-1 correlated with NY-ESO-1–specific CD4+ and CD8+ T-cell responses against peptide NY-ESO-151-62 and CD4+ responses against NY-ESO-1121-140 in 1 of these patients. These allogeneic immune responses were not detectable in pretransplantation samples and in the patients' stem cell donors, indicating that CT antigens might indeed represent natural targets for graft-versus-myeloma effects. Immune responses induced by alloSCT could be boosted by active CT antigen–specific immunotherapy, which might help to achieve long-lasting remissions in patients with MM.

Description: While the emergence of WT1-specific cytotoxic T lymphocytes (WT1-CTL) has been correlated with better relapse-free survival after allogeneic stem cell transplantation in patients with myeloid leukemias, little is known about the role of these cells in multiple myeloma (MM). We examined the significance of WT1-CTL responses in patients with relapsed MM and high-risk cytogenetics who were undergoing allogeneic T cell–depleted hematopoietic stem cell transplantation (alloTCD-HSCT) followed by donor lymphocyte infusions. Of 24 patients evaluated, all exhibited WT1-CTL responses before allogeneic transplantation. These T-cell frequencies were universally correlated with pretransplantation disease load. Ten patients received low-dose donor lymphocyte infusions beginning 5 months after transplantation. All patients subsequently developed increments of WT1-CTL frequencies that were associated with reduction in specific myeloma markers, in the absence of graft-versus-host disease. Immunohistochemical analyses of WT1 and CD138 in bone marrow specimens demonstrated consistent coexpression within malignant plasma cells. WT1 expression in the bone marrow correlated with disease outcome. Our results suggest an association between the emergence of WT1-CTL and graft-versus-myeloma effect in patients treated for relapsed MM after alloTCD-HSCT and donor lymphocyte infusions, supporting the development of adoptive immunotherapeutic approaches using WT1-CTL in the treatment of MM (registered at http://clinicaltrials.gov, ID: NCT01131169).

Description: Abstract 785 The type I Melanoma Antigen GEne (MAGE) MAGE-A3 is commonly present in primary multiple myeloma cells and its expression is correlated with advanced disease and proliferation. MAGE-A3 belongs to the Cancer-Testis antigen (CTAg) family of tumor-associated proteins, which are present in many cancers, but their normal expression is limited to developing germ cells and placental trophoblast. This unique expression pattern fuels speculation on a role for CTAg in oncogenesis; however, very little is known about their function. In gene expression analyses of primary myeloma cells, CTAg were associated with proliferative gene signatures and poor clinical outcome, suggesting they contribute to the pathogenesis or progression of this disease through effects on survival and/or proliferation of myeloma cells. To investigate this, we examined the impact of MAGE-A on disease progression, proliferation, and apoptosis in primary myeloma specimens and human myeloma cell lines (HMCL). MAGE-A3 protein expression was examined by immunohistochemistry in a new, independent set of myeloma bone marrow specimens from two critical clinical milestones, newly diagnosed, untreated patients and patients who relapsed after chemotherapy. MAGE-A3 was detected in a higher percentage of tumor specimens from relapsed patients (77%) compared to those from newly diagnosed patients (36%, p=0.0003). The percentage of proliferating myeloma cells, as measured by staining for the proliferation marker Ki-67, was significantly higher in relapsed specimens (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p=0.0002), demonstrating a correlation between MAGE-A3, progression of disease and proliferation. The mechanisms for MAGE-A3 activity were investigated by silencing this gene in primary myeloma cells and HMCL by shRNA interference. Targeted lentiviral shRNA transduction efficiently knocked down MAGE-A3 mRNA and protein in MM.1r (p53+/+) and ARP-1 (p53−/−) HMCL and in primary myeloma cells by 48 hours, and this effect was maintained up to 96 hours. Silencing of MAGE-A did not affect cell cycling, as this intervention did not affect the phosphorylation of the Retinoblastoma gene product (Rb) that is required for progression through the G1 cell cycle checkpoints and entry into S phase. In contrast, MAGE-A was required for survival of proliferating myeloma cells. Silencing of MAGE-A led to a precipitous loss of viable cells within 48–72 hrs compared to controls. This was due to activation of intrinsic apoptosis, as demonstrated by increased annexin V staining, loss of mitochondrial membrane polarization, and cleavage/activation of caspase-9. These effects of MAGE-A knock-down were completely reversed by the pan-caspase inhibitor Quinoline-Val-Asp-CH2-OPh. Apoptosis after MAGE-A silencing appeared to be mediated by at least two distinct mechanisms; p53-dependent activation of pro-apoptotic Bax and Bak expression and reduced expression of the Inhibitor of Apoptosis Protein survivin through both p53-dependent and independent mechanisms. These results demonstrate that MAGE-A plays a role in the survival of proliferating multiple myeloma cells through the regulation of two critical apoptotic mechanisms. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1993 Wilm's tumor protein-1 (WT1) is over-expressed in a number of solid and hematologic malignancies including multiple myeloma (MM). The emergence of WT1-specific T cells has been shown to correlate with better relapse-free survival after allogeneic stem cell transplantation in patients (pts) with hematologic malignancies, such as leukemia. In MM, the expression of WT1 in the bone marrow has been shown to correlate with numerous negative prognostic factors, including disease stage and M protein ratio. Taken together, these findings suggest that immunotherapeutic augmentation of WT1-specific immune responses, such as adoptive transfer of WT1-specific T cells, may be capable of eradicating minimal residual disease and preventing relapse in MM. Thus, we examined the significance of WT1-specific cellular immune responses in pts with relapsed MM and high-risk cytogenetics who are undergoing allogeneic T cell-depleted hematopoietic stem cell transplantation (TCD HSCT). In this study, pts were eligible to receive low doses of donor lymphocyte infusions (DLI, 5×105-1×106 CD3+/kg) no earlier than 5 months post TCD HSCT. WT1-specific T-cell frequencies were measured in freshly isolated peripheral blood and bone marrow specimens. Frequencies were detected by staining for intracellular IFN-γ production in response to WT1 peptides, and/or by tetramer analysis, where available. Of 17 pts evaluated, all pts exhibited low frequencies of WT1-specific T-cell responses pre TCD HSCT. Ten of these pts received DLI post TCD HSCT. All 10 pts developed WT1-specific T cell responses post DLI. These increments in WT1-specific T-cell frequencies were associated with reduction in circulating myeloma proteins in all pts. Long-term evaluation demonstrated fluctuations in persisting WT1-specific T-cell frequencies following DLI. In one representative patient, a peak of 3.5% (72/ml) WT1-specific CD8+ T cells were detected in the peripheral blood by staining with the tetramer HLA-A*0201 RMF. This peak T-cell response occurred post TCD HSCT and DLI, and coincided with disease regression. This patient has remained in complete remission for more than 3 years post transplant, with fluctuating levels of WT1-specific CD8+ T cells ranging from 0.3–1.5% still persisting. Findings from concurrent molecular chimerism studies conducted on isolated T cells post TCD HSCT suggest that the WT1-specific T cells are of donor origin. Immunohistochemical analyses of WT1 and CD138 staining in MM bone marrow specimens demonstrated consistent co-expression within malignant plasma cells. WT1 expression in the bone marrow of all 6 pts tested correlated with the extent of malignant plasma cell infiltration. In contrast, no WT1 expression was observed when disease was low or absent. Taken together, our findings suggest a correlation between the emergence of WT1-specific T cells post DLI, and disease regression in pts being treated for relapsed MM. The present data support the development of adoptive immunotherapeutic approaches utilizing WT1-specific T cells for pts with MM. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Despite improvements with combination chemotherapy and/or radiation, up to one third of patients with classical Hodgkin lymphoma (cHL) relapse following primary therapy. The standard of care for these patients is salvage chemotherapy followed by autologous stem cell transplantation; however, this carries a high risk of treatment-related morbidity and is curative in only about one-half of patients. Histopathologically, HL is characterized by rare tumor-initiating Reed-Sternberg (RS) cells surrounded by a dense but functionally incapacitated inflammatory infiltrate. This immunosuppression is thought to be mediated by multiple mechanisms, including genetic amplification of PD-L1 expression on RS cells, loss of MHC class I and class II expression, and infiltration of regulatory T cells. Whether any components of the immune microenvironment are associated with disease recurrence or long-term outcomes in HL remains unknown. Methods: We identified 76 patients with newly diagnosed and 50 patients with relapsed cHL from 2000 to 2014 who had formalin-fixed, paraffin-embedded tissue available for immunohistochemical (IHC) analysis. IHC staining was performed as per standard protocols against B2M, MHC-I, MHC-II, FoxP3, and PD-L1. Scoring for B2M, MHC-I, and MHC-II was reported in a binary fashion. FoxP3 expression was scored as low (50 cells/HPF). PD-L1 expression was scored both within Reed-Sternberg (RS) cells and within the tumor microenvironment (TME) as low (