Seth M. Pollack

NY-ESO-1 is a ubiquitous immunotherapeutic target antigen for patients with myxoid/round cell liposarcoma

Myxoid/round cell liposarcoma (MRCL) is the second most common liposarcoma subtype, accounting for >33% of liposarcomas and approximately 10% of all soft tissue sarcomas. Although MRCL is a chemosensitive subtype, patients with metastatic disease have a poor outcome. NY‐ESO‐1 is a cancer‐testis antigen (also known as cancer germ cell antigen) that has been successfully targeted in vaccine trials and in adoptive T‐cell therapy trials for the treatment of several solid tumors.

Pancreatic ductal adenocarcinoma contains an effector and regulatory immune cell infiltrate that is altered by multimodal neoadjuvant treatment

Objective The immune response to pancreatic ductal adenocarcinoma (PDA) may play a role in defining its uniquely aggressive biology; therefore, we sought to clearly define the adaptive immune infiltrate in PDA. Design We used immunohistochemistry and flow cytometry to characterize the immune infiltrate in human PDA and compared our findings to the patients’ peripheral blood. Results In contrast to the myeloid cell predominant infiltrate seen in murine models, T cells comprised the majority of the hematopoietic cell component of the tumor stroma in human PDA. Most intratumoral CD8+ T cells exhibited an antigen-experienced effector memory cell phenotype and were capable of producing IFN-γ. CD4+ regulatory T cells (Treg) and IL-17 producing T helper cells were significantly more prevalent in tumor than in blood. Consistent with the association with reduced survival in previous studies, we observed higher frequencies of both myeloid cells and Treg in poorly differentiated tumors. The majority of intratumoral T cells expressed the co-inhibitory receptor programmed death-1 (PD-1), suggesting one potential mechanism through which PDA may evade antitumor immunity. Successful multimodal neoadjuvant therapy altered the immunoregulatory balance and was associated with reduced infiltration of both myeloid cells and Treg. Conclusion Our data show that human PDA contains a complex mixture of inflammatory and regulatory immune cells, and that neoadjuvant therapy attenuates the infiltration of intratumoral cells associated with immunosuppression and worsened survival.

Assessment of the Hematopoietic Cell Transplantation Comorbidity Index in Non-Hodgkin Lymphoma Patients Receiving Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation

The hematopoietic cell transplantation comorbidity index (HCT-CI), a weighted index of 17 pretransplantation comorbidities, has been validated in nonmyeloablative and myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) studies, but it has not been specifically tested in patients with non-Hodgkin lymphoma (NHL) receiving reduced-intensity conditioning (RIC). We performed a retrospective analysis to assess the impact of the HCT-CI on outcomes of NHL patients treated with HSCT relative to treatment-related mortality (TRM), disease-related mortality (DRM), with a specific emphasis on overall survival (OS). Individual pretransplantation and disease-related factors also were analyzed with HCT-CI relative to their impact on OS. All patients were uniformly treated with an identical pretransplantation induction regimen and an identical RIC regimen (cyclophosphamide [Cy]/fludarabine [Flu]), and received T cell-replete allografts from HLA-matched siblings. The analysis included 63 NHL patients with a median HCT-CI score of 2 (range, 0 to 11). The HCT-CI (0 to 2 comorbidities vs 3+ comorbidities) demonstrated a potential association with TRM, but not with DRM, at 100 days (4.5% vs 26.3%) and at 1 year (13.6% vs 36.8%) posttransplantation. The factor most strongly associated with OS was response to pretransplantation chemotherapy (P= .0001), based on a composite measure. In a Cox model, pretransplantation chemotherapy response remained the most important factor (P< .0001) relative to OS, and there was a trend (P= .056) toward HCT-CI adding predictive value for OS. Although HCT-CI may be useful for predicting TRM, our data further underscore the importance of response to chemotherapy before transplantation as a predictor of overall transplantation outcome in NHL patients being considered for RIC allogeneic HSCT.

NYESO-1/LAGE-1s and prame are targets for antigen specific t cells in chondrosarcoma following treatment with 5-Aza-2-deoxycitabine

Background Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA) family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157–165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTAs have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC) in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression. Methods We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTAs with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment. Results A minority (36%) of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment. Conclusion These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic chondrosarcoma patients where no proven systemic therapies exist.

Epigenetic modulation to enable antigen-specific T-cell therapy of colorectal cancer

Development of specific immunotherapy for colorectal cancer (CRC) will require identification of antigens selectively or exclusively expressed on CRC cells and strategies to induce and enhance immune responses against these antigenic targets. Cancer-testis (C-T) antigens are proving to be excellent targets for immunotherapy of solid tumors such as melanoma, but their clinical utility for treatment of CRC has to date been limited by their infrequent expression in CRC cells. Here we report that the hypomethylating agent 5-aza-2′-deoxycytidine (DAC) induces expression of NY-ESO-1 and other C-T genes in CRC cells both in vitro and in vivo in a dose-dependent manner but has negligible effects on the expression of C-T genes in normal nontransformed cells such as fibroblasts. The induction by DAC of NY-ESO-1 expression in CRC cells persists over 100 days after DAC exposure and is associated with increased levels of NY-ESO-1 protein. CRC cells exposed to DAC at concentrations that can be readily achieved in vivo are rendered susceptible to major histocompatibility complex-restricted recognition by CD8+ NY-ESO-1-specific T cells. We also demonstrate that retroviral transduction of polyclonal peripheral blood T cells from a metastatic CRC patient with the T-cell receptor &agr;-chain and &bgr;-chain genes encoding a human leukocyte antigen-A2-restricted, NY-ESO-1157-165-specific T-cell receptor can be used to generate both CD8+ and CD4+ NY-ESO-1157-165-specific T cells that selectively recognize DAC-treated CRC but not nontransformed cells. Collectively, these results suggest that the combination of epigenetic modulation and adoptive transfer of genetically engineered T lymphocytes may enable specific immunotherapy for CRC.

Immune-Based Therapies for Sarcoma

Immunotherapy has shown promise in a number of tumor types, but its exact role in sarcoma remains to be defined. Advanced bone and soft tissue sarcomas are challenging diseases to treat with an unmet need for effective systemic therapy. Previous reports have suggested that immune-based treatments may be effective in sarcoma, but such approaches have not yet become part of standard clinical practice. A number of sarcoma subtypes express targets known as cancer testis antigens and hence may be excellent targets for immunotherapy. This paper will focus on the recent advances and understanding of cancer testis antigens in sarcoma and also clinical data of immunotherapeutic approaches in these diseases.

Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8 + T cells

BackgroundAdoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL).MethodsCTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties.ResultsFollowing in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8+, tetramer+ T cells with a memory phenotype that recognized endogenous NY-ESO-1.ConclusionThis study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy.

Molecular profiling of soft tissue sarcomas using next-generation sequencing: a pilot study toward precision therapeutics

Next-generation sequencing (NGS) can provide in-depth detection of numerous gene alterations. To date, there are very few reports describing the use of this technique in soft tissue sarcomas. Herein, we aim to test the utility of NGS in identifying targetable mutations in these tumors. NGS was performed using a clinically validated multiplexed gene sequencing panel interrogating the full coding sequence of 194 cancer-related genes. A custom bioinformatics pipeline was developed to detect all classes of mutations directly from the NGS data, including single-nucleotide variants, small insertions and deletions, copy number variation, and complex structural variations. Twenty-five soft tissue sarcomas were analyzed; 18 of these patients had metastatic disease and 7 primary locally advanced tumors. Targetable mutations for which clinical trials are available were identified in 60% of the cases. MAP2K4, AURKA, AURKB, and c-MYC amplification were recurrent events in leiomyosarcomas. Frequent non-targetable variants included copy losses of the TP53 (24%), PTEN (16%), and CDKN2A (20%). Additional frameshift mutations, deletion mutations, and single-nucleotide variants involving numerous genes, including RB1, NOTCH1, PIK3CA, PDGFRB, EPHA5, KDM6A, NF1, and FLT4 genes, were also identified. NGS is useful in identifying targetable mutations in soft tissue sarcomas that can serve as a rationale for inclusion of patients with advanced disease in ongoing clinical trials and allow for better risk stratification.

T-cell infiltration and clonality correlate with programmed cell death protein 1 and programmed death-ligand 1 expression in patients with soft tissue sarcomas

Patients with metastatic sarcomas have poor outcomes and although the disease may be amenable to immunotherapies, information regarding the immunologic profiles of soft tissue sarcoma (STS) subtypes is limited.

The evolution of systemic therapy in sarcoma

Approximately 50% of patients with localized soft tissue sarcomas will develop recurrent disease after complete surgical resection, requiring alternative means of treatment. Conventional chemotherapy comprising of doxorubicin and ifosfamide has shown benefit in advanced disease, however, there remains a clear need for more effective, less toxic, therapies for the treatment of this heterogeneous group of mesenchymal malignancies. Recently, greater emphasis has been placed on the underlying biology of individual sarcoma subtypes, with the development and evaluation of novel therapies both in common and in rare subtypes. In addition, there is a greater specificity in the selection of chemotherapy agents based on activity in individual histological subtypes. Despite these advances the management of sarcoma, and in particular of rare subtypes, remains a major challenge. Some histological subtypes are resistant to conventional chemotherapy and patients with these diseases should be offered participation in early phase clinical trials of novel drugs.