Over-expression of B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some cancer patients and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genomescale T-cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating myeloid cells, and its expression is mutually exclusive to B7-H1, partially due to its induction by M-CSF and downregulation by IFN-γ. We demonstrate that Siglec-15 suppresses antigen-specific T-cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 amplifies anti-tumor immunity in the TME and inhibits tumor Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Purpose: To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non-small cell lung cancer (NSCLC). Experimental Design: Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment. Results: PD-1, LAG-3, and TIM-3 were detected in tumorinfiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progressionfree survival. Conclusions: PD-1, LAG-3, and TIM-3 have distinct tissue/ cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.

Purpose Determine the localized expression pattern and clinical significance of VISTA/PD-1H in human NSCLC. Experimental Design Using multiplex quantitative immunofluorescence (QIF), we performed localized measurements of VISTA, PD-1 and PD-L1 protein in 758 stage I-IV NSCLCs from 3 independent cohorts represented in tissue microarray format. The targets were selectively measured in cytokeratin+ tumor epithelial cells, CD3+ T-cells, CD4+ T-helper cells, CD8+ cytotoxic T-cells, CD20+ B-lymphocytes and CD68+ tumor-associated macrophages. We determined the association between the targets, clinico-pathological/molecular variables and survival. Genomic analyses of lung cancer cases from TCGA was also performed. Results VISTA protein was detected in 99% of NSCLCs with a predominant membranous/cytoplasmic staining pattern. Expression in tumor and stromal cells was seen in 21% and 98% of cases, respectively. The levels of VISTA were positively associated with PD-L1, PD-1, CD8+ T-cells and CD68+ macrophages. VISTA expression was higher in T-lymphocytes than in macrophages; and in cytotoxic T-cells than in T-helper cells. Elevated VISTA was associated with absence of EGFR mutations and lower mutational burden in lung adenocarcinomas. Presence of VISTA in tumor compartment predicted longer 5-year survival. Conclusions VISTA is frequently expressed in human NSCLC and shows association with increased TILs, PD-1 axis markers, specific genomic alterations and outcome. These results support the immuno-modulatory role of VISTA in human NSCLC and suggests its potential as therapeutic target.

Importance There are at least four immunohistochemistry assays for PD-L1 at various stages of interaction with the FDA as companion or complementary diagnostic tests for benefit from PD-1 axis therapies. The performance of each assay for selection of patients that respond to therapy has been published, but no data has been published that compares the assays to one another or to direct measurements of PD-L1. Objective To determine whether the antibody reagents are interchangeable, we quantitatively compared expression of PD-L1 protein using six monoclonal antibodies (SP142, E1L3N, 9A11, SP263, 22c3 and 28-8). Design To test for protein measurement, rather than clinical utility, we created a PD-L1 index tissue microarray including cell line and tissue controls, in addition to 30 NSCLC cases with full dynamic range of PD-L1 expression. We then validated our results on a commercially available, genetically defined PD-L1 engineered cell line array with a range of controlled protein expressing cell lines. Protein levels were measured by both quantitative immunofluorescence and quantitative chromogenic assessment. Results Concordance between 4 antibodies showed regression (R2 values) between 0.42-0.91 for tumor tissue cores and 0.83-0.97 for cells line cores by QIF in the PD-L1 index tissue microarray. All six antibodies showed high levels of concordance (R2 ranging from 0.76 to 0.99) when using chromogenic staining in isogenic cell lines. Conclusions and Relevance Since the antibodies are highly concordant, these results suggest that assays based on the use of these antibodies could yield concordant results. They further suggest that previously described differences in PD-L1 expression in tissue is independent of the antibody utilized and likely due to tumor heterogeneity, assay/platform-specific variables or other factors.

Purpose: Protein expression in formalin-fixed, paraffinembedded tissue is routinely measured by IHC or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital spatial profiling (DSP) allows spatially informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma. Experimental Design: The NanoString GeoMx DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in non-small cell lung cancer (NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 patients with immunotherapy-treated melanoma on a tissue microarray using a 44-plex immune marker panel measured in three compartments (macro-phage, leukocyte, and melanocyte) generating 132 quantitative variables. Results: The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared with QIF for stromal CD3, CD4, CD8, CD20, and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression-free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68-positive cells (macrophages) and not in tumor cells was a predictive marker for PFS, OS, and response. Conclusions: DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity, we found a series of expression patterns associated with outcome, including that the expression of PD-L1 in macrophages is associated with response.