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BackgroundThe expression of SYK in cancer cells has been associated with both tumor promoting and tumor suppressive effects. Despite being proposed as anticancer therapeutic target, the possible role of SYK in modulating local adaptive antitumor immune responses remains uncertain. Using detailed analysis of primary human tumors and in vitro models, we reveal the immunomodulatory effect of SYK protein in human solid cancer.MethodsWe spatially mapped SYK kinase in tumor cells, stromal cells and tumor-infiltrating leukocytes (TILs) in 808 primary non-small cell lung carcinomas (NSCLCs) from two cohorts and in 374 breast carcinomas (BCs) from two independent cohorts. We established the associations of localized SYK with clinicopathologic variables and outcomes. The immunomodulatory role of SYK on tumor cells was assessed using in vitro cytokine stimulation, transcriptomic analysis and selective SYK blockade using a small molecule inhibitor. Functional responses were assessed using cocultures of tumor cells with peripheral blood lymphocytes. T cell responses in baseline and post-treatment biopsies from patients with BC treated with a SYK inhibitor in a phase I clinical trial were also studied.ResultsElevated tumor cell or leukocyte SYK expression was associated with high CD4+ and CD8+ TILs and better outcome in both NSCLC and BC. Tumor cell SYK was associated with oncogenic driver mutations in EGFR or KRAS in lung adenocarcinomas and with triple negative phenotype in BC. In cultured tumor cells, SYK was upregulated by TNFα and required for the TNFα-induced proinflammatory responses and T cell activation. SYK blockade after nivolumab in a phase I clinical trial including three patients with advanced triple negative BC reduced TILs and T cell proliferation. Our work establishes the proinflammatory function of tumor cell SYK in lung and breast cancer. SYK signaling in cultured tumor cells is required for T cell activation and SYK blockade limits adaptive antitumor immune responses and tumor rejection in patients with cancer.ConclusionsTogether, our results establish the immunomodulatory role of SYK expression in human solid tumors. This information could be used to develop novel biomarkers and/or therapeutic strategies.

Despite unique genetic alterations within brain metastases (BrMs) and an immunologically distinct surrounding microenvironment, the composition and functional properties of tumor-infiltrating lymphocytes within BrM remain largely unexplored. In particular, the expression of coinhibitory receptors, such as programmed cell death 1 (PD-1), T cell immunoglobulin mucin receptor 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3), within BrMs is unknown. Using multiplexed quantitative immunofluorescence (QIF), this study evaluates the localized expression of PD-L1, level and functional profile of major T cell subsets, and coinhibitory receptors within lung cancer-associated BrMs and primary lung tumors. Clinicopathologically annotated samples from 95 patients with lung cancer between 2002 and 2015 were represented in a tissue microarray format. Spatially resolved and multiplexed QIF was used to evaluate PD-L1 protein, phenotype markers for major T cell subsets (CD3, CD4, CD8, and FOXP3), cell-localized activation and proliferation markers (granzyme B and Ki67), and coinhibitory receptors (PD-1, LAG-3, and TIM-3). The signal for each marker was measured in marker-selected tissue compartments, and associations between marker levels, tumor location, and major clinicopathological variables were studied. In total, 41 primary lung tumors and 65 BrMs were analyzed, including paired samples from 11 patients. Levels of tumor PD-L1 expression were comparable between BrMs and primary lung tumors. BrMs had significantly lower levels of all T cell subsets relative to primary lung tumors, and T cells in BrMs displayed lower levels of granzyme B than primary lesions. PD-1, TIM-3, and LAG-3 levels in CD3+ T-cells were also significantly lower in BrMs. Marker expression in patients with paired samples from BrMs and primary lung tumors showed comparable results. High CD3+ T-cells, as well as high levels of TIM-3 and LAG-3 in CD3+ T-cells, were associated with longer overall survival in BrMs but not primary lung tumors. Lung cancer-associated BrMs display lower T cell infiltration, markers of cytolytic function, and immune regulatory signals than primary lung tumors. Despite these differences, high TIM-3 and high LAG-3 expressions in CD3+ T-cells were associated with longer survival. These features are accompanied by comparable levels of PD-L1 protein expression compared with primary lung tumors. These results highlight unique aspects of the tumor immune microenvironment within the brain and provide further support for intracranially focused therapies.

Purpose: To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non–small cell lung cancer (NSCLC). Experimental Design: Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell–specific defects, and clinicopathologic/survival associations of β2 microglobulin (β2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II β chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts. Results: Cancer cell–specific downregulation of HLA markers was identified in 30.4% of cases. β2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of β2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in Conclusions: Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival.

Introduction. VISTA/PD-1H is an Ig domain-containing type I transmembrane protein able to suppress T-cell activation and is being evaluated as a candidate anti-cancer immunotherapy target. VISTA can bind P-selectin glycoprotein ligand-1 (PSGL1) and V-set and immunoglobulin domain containing 3 (VSIG3) suggesting these interactions could mediate VISTA's immunomodulatory effect. We studied the tissue distribution of VISTA, PSGL1 and VSIG3 in human non-small cell lung cancer (NSCLC). Methods We used multiplexed quantitative immunofluorescence (QIF) to simultaneously measure DAPI, cytokeratin (CK), VISTA, PSGL1 and VSIG3 in 48 paired tumor/normal lung samples and in 850 stage I-IV NSCLCs from four independent cohorts represented in tissue microarray format. The first 2 NSCLC cohorts (Cohorts #1, n=382 and #2, n=282) included cases treated with standard of care non-immunotherapy. Cohort #3 included 137 lung adenocarcinomas with analysis of mutant oncogenic drivers; and Cohort #4 included 49 NSCLC cases treated with PD-1 axis blockers. The targets were selectively measured in CK+ tumor cells and CK-negative stromal cells. We also determined the spatial association between VISTA and its binding partners by fluorescence signal co-localization. The association between target levels, clinicopathologic/molecular variables, tumor infiltrating lymphocytes (TILs), PD-L1 expression and survival was established. Results PSGL1 was located predominantly in stromal/immune cells and VSIG3 was detected in both tumor and stromal compartments. Both targets were expressed at higher levels in NSCLC than in non-tumor lung tissue and showed a positive association with VISTA in cancer tissues. Using the visual detection threshold PSGL1 and VSIG3 expression were detected in >90% of cases from cohorts #1 and #2 and showed positive association with CD3+ TILs and PD-L1 levels. PSGL1 was higher in lung adenocarcinomas harboring EGFR mutations than in tumors with KRAS variants or cases lacking mutations in both oncogenes. Elevated VISTA/PSGL1 co-localization was significantly associated with longer 5-year overall survival in cases not treated with immunostimulatory therapy (Cohorts #1 and #2). However, an opposite association was seen in cases treated with PD-1 axis blockers, where elevated VISTA/PSGL1 co-expression was associated with shorter survival. Conclusions PSGL1 and VSIG3 are frequently expressed in human NSCLC. Expression of PSGL1 is associated with increased tumor immune infiltration and activating EGFR mutations. High baseline VISTA/PSGL1 co-expression is associated with adverse outcome after PD-1 axis blockers. The latter suggest VISTA/PSGL1 as a dominant immune evasion pathway independent from PD-1/PD-L1 axis in a subset of human NSCLC. Validation of these findings is ongoing. Citation Format: Alicia Ding, Franz Villarroel-Espindola, Adam Ducler, Brian S. Henick, Shruti Desai, Nicole Gianino, Jon Zugazagoitia, David L. Rimm, Alain Robert, Francisco Cruzalegui, Pierre Ferré, Roy Herbst, Miguel Sanmamed, Lieping Chen, Kurt A. Schalper. VISTA/PSGL1 axis as a dominant immunomodulatory pathway in human non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5525.