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Purpose: Gallbladder carcinoma (GBC) is an uncommon and aggressive disease, which remains poorly defined at a molecular level. Here, we aimed to characterize the molecular landscape of GBC and identify markers with potential prognostic and therapeutic implications. Experimental Design: GBC samples were analyzed using the MSK-IMPACT (Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets) platform (targeted NGS assay that analyzes 505 cancer-associated genes). Variants with therapeutic implications were identified using OncoKB database. The associations between recurrent genetic alterations and clinicopathologic characteristics (Fisher exact tests) or overall survival (univariate Cox regression) were evaluated. P values were adjusted for multiple testing. Results: Overall, 244 samples (57% primary tumors and 43% metastases) from 233 patients were studied (85% adenocarcinomas, 10% carcinomas with squamous differentiation, and 5% neuroendocrine carcinomas). The most common oncogenic molecular alterations appeared in the cell cycle (TP53 63% and CDKN2A 21%) and RTK_RAS pathways (ERBB2 15% and KRAS 11%). No recurrent structural variants were identified. There were no differences in the molecular landscape of primary and metastasis samples. Variants in SMAD4 and STK11 independently associated with reduced survival in patients with metastatic disease. Alterations considered clinically actionable in GBC or other solid tumor types (e.g., NTRK1 fusions or oncogenic variants in ERBB2, PIK3CA, or BRCA1/2) were identified in 35% of patients; 18% of patients with metastatic disease were treated off-label or enrolled in a clinical trial based on molecular findings. Conclusions: GBC is a genetically diverse malignancy. This large-scale genomic analysis revealed alterations with potential prognostic and therapeutic implications and provides guidance for the development of targeted therapies.

Introduction: Gallbladder carcinomas (GBC) are rare and aggressive neoplasms. Previous studies in small GBC cohorts have suggested potential molecular alterations associated with poor prognosis, however, a comprehensive understanding of the recurrent genetic events in this cancer type is very limited. In this study, we aim to characterize in detail the recurrent molecular alterations in GBC and their association with pathologic and clinical characteristics. Material and Methods: We studied the prevalence of somatic mutations and copy number alterations (CNA) in n=244 GBC samples (54% primary, 56% metastatic), collected from 2014 to 2021, and sequenced with the targeted NGS panel MSK-IMPACT. We assessed the correlation of the recurrent genomic variants with several pathologic (e.g., T stage, N stage, grade, histologic subtypes) and clinical characteristics (e.g., clinical stage) using Fisher's exact test. We also used Cox Proportional-Hazards modeling to assess the correlation between genetic variants and patient overall survival (OS). Results: The most common histologic subtypes in this GCB cohort included adenocarcinomas NOS (83%), carcinomas with squamous differentiation (9%), high-grade carcinomas (4%), and carcinomas with neuroendocrine differentiation (3%). Most patients were diagnosed with stage IV (65%) and stage III (11%) disease at the time of biopsy/resection, and the mean OS survival was 29.4 months. The most commonly mutated genes were TP53 (59%), SMAD4 (21%), ARID1A (19%), PIK3CA (10%), KRAS (7%), and ERBB2 (7%). Potentially recurrent oncogenic CNAs included deep deletions in CDKN2A (14%) and CDKN2B (14%), and amplifications in MDM2 (12%), ERBB2 (10%), CCNE1 (9%), MYC (7%), and KRAS (7%). RB1, PBRM1, and CTNNB1 variants were more common in cases with neuroendocrine differentiation, whereas alterations in IKZF1 and AGO2 were enriched in cases with squamous differentiation. The most significant event associated with shorted OS was chromosome 12q13-15 amplification i.e., CDK4 p=0.03 HR=2 [95% CI 1-3.6] and MDM2 p=0.05 HR=1.6 [1-2.5], which associated with a median OS of 20 months (vs. 34 months in the wild-type). Genomic variants associated with longer OS included ERBB2 (p=0.006 HR=0.2 [0.06-0.6]), KMT2C (p=0.03 HR=0.2 [0.1-0.9]) and KMT2D (p=0.01 HR=0.2 [0.04-0.7]) mutations, and CDK12 amplification (p=0.04 HR=0.4 [0.2-0.96]). Although ERBB2 amplification was not associated with prognosis, co-amplification of CDK12 and ERBB2 (chromosome 17q12) was frequently observed (Pearson correlation r=0.8). Conclusions: This large-scale genomic analysis reveals recurrent genomic events potentially associated with prognosis in GBC, including single nucleotide variants in ERBB2, KMT2C, and KMT2D, in addition to CNAs in chromosome 12q13-15 and 17q12 regions. This effort will continue to include a detailed analysis of recurrent structural variants, loss of heterozygosity loci, and analysis of the microbiota in this GBC cohort. Citation Format: Nicolas A. Giraldo, Abhinita Mohanty, Chad Vanderbilt, Rose Brannon, Ryma Benayed, Efsevia Vakiani, Ghassan Abou-Alfa, James Harding, Imane El Dika, Nikolaus Schultz, Bob Li, Michael F. Berger, Marc Ladanyi, Eileen O'Reilly, William Jarnagin, Olca Basturk, Maria Arcila. Molecular characterization of gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 82.

International audience; The highly complex and heterogenous ecosystem of a tumour not only contains malignant cells, but also interacting cells from the host such as endothelial cells, stromal fibroblasts, and a variety of immune cells that control tumour growth and invasion. It is well established that anti-tumour immunity is a critical hurdle that must be overcome for tumours to initiate, grow and spread and that anti-tumour immunity can be modulated using current immunotherapies to achieve meaningful anti-tumour clinical responses. Pioneering studies in melanoma, ovarian and colorectal cancer have demonstrated that certain features of the tumour immune microenvironment (TME)-in particular, the degree of tumour infiltration by cytotoxic T cells-can predict a patient's clinical outcome. More recently, studies in renal cell cancer have highlighted the importance of assessing the phenotype of the infiltrating T cells to predict early relapse. Furthermore, intricate interactions with non-immune cellular players such as endothelial cells and fibroblasts modulate the clinical impact of immune cells in the TME. Here, we review the critical components of the TME in solid tumours and how they shape the immune cell contexture, and we summarise numerous studies evaluating its clinical significance from a prognostic and theranostic perspective.

IL-1 family cytokines play a dual role in the gut, with different family members contributing either protective or pathogenic effects. IL-36γ is an IL-1 family cytokine involved in polarizing Type-1 immune responses. However, its function in the gut, including in colorectal cancer pathogenesis, is not well appreciated. In a murine model of colon carcinoma, IL-36γ controls tertiary lymphoid structure formation and promotes a Type-1 immune response concurrently with a decrease in expression of immune checkpoint molecules in the tumor microenvironment. Here, we demonstrate that IL-36γ plays a similar role in driving a pro-inflammatory phenotype in human colorectal cancer. We analyzed a cohort of 33 primary colorectal carcinoma tumors using imaging, flow cytometry, and transcriptomics to determine the pattern and role of IL-36γ expression in this disease. In the colorectal tumor microenvironment, we observed IL-36γ to be predominantly expressed by M1 macrophages and cells of the vasculature, including smooth muscle cells and high endothelial venules. This pattern of IL-36γ expression is associated with a CD4(+) central memory T cell infiltrate and an increased density of B cells in tertiary lymphoid structures, as well as with markers of fibrosis. Conversely, expression of the antagonist to IL-36 signaling, IL-1F5, was associated with intratumoral expression of checkpoint molecules, including PD-1, PD-L1, and CTLA4, which can suppress the immune response. These data support a role for IL-36γ in the physiologic immune response to colorectal cancer by sustaining inflammation within the tumor microenvironment. PRECIS: This study reveals the association of intratumoral IL-36γ with established markers of cancer prognosis, indicating the translational relevance of investigating the IL-36 signaling pathway as a therapeutic target in colorectal cancer.

Background We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. Methods Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. Results Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm2, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. Conclusions While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, Tregs, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade. Electronic supplementary material The online version of this article (10.1186/s40425-018-0404-0) contains supplementary material, which is available to authorized users.

Tumors are highly heterogeneous structures where malignant cells interact with a large variety of cell populations, including a clinically-relevant immune component. We review and compare the most recent methods designed to analyze and quantify the composition of immune and stromal microenvironment of tumors and discuss their use in identification of patients for high risk of progression. If the impact of the various immune components on patient's relapse share common rules in most malignancies, clear cell renal cell tumors behave differently with regards to immunity. We focus on this specific pathology to show how the tumor interacts with the host's immune system and how this intricate relationship shapes the clinical outcome.

Characterizing the tumor immune microenvironment enables the identification of new prognostic and predictive biomarkers, the development of novel therapeutic targets and strategies, and the possibility to guide first-line treatment algorithms. Although the driving elements within the tumor microenvironment of individual primary organ sites differ, many of the salient features remain the same. The presence of a robust antitumor milieu characterized by an abundance of CD8+ cytotoxic T-cells, Th1 helper cells, and associated cytokines often indicates a degree of tumor containment by the immune system and can even lead to tumor elimination. Some of these features have been combined into an ‘Immunoscore’, which has been shown to complement the prognostic ability of the current TNM staging for early stage colorectal carcinomas. Features of the immune microenvironment are also potential therapeutic targets, and immune checkpoint inhibitors targeting the PD-1/ PD-L1 axis are especially promising. FDA-approved indications for anti-PD-1/PD-L1 are rapidly expanding across numerous tumor types and, in certain cases, are accompanied by companion or complimentary PD-L1 immunohistochemical diagnostics. Pathologists have direct visual access to tumor tissue and in-depth knowledge of the histological variations between and within tumor types and thus are poised to drive forward our understanding of the tumor microenvironment. This review summarizes the key components of the tumor microenvironment, presents an overview of and the challenges with PD-L1 antibodies and assays, and addresses newer candidate biomarkers, such as CD8+ cell density and mutational load. Characteristics of the local immune contexture and current pathology-related practices for specific tumor types are also addressed. In the future, characterization of the host antitumor immune response using multiplexed and multimodality biomarkers may help predict which patients will respond to immune-based therapies.

Astronomy accelerates tumor imaging Immunohistochemical stains for individual markers revolutionized diagnostic pathology decades ago but cannot capture enough information to accurately predict response to immunotherapy. Newer multiplex immunofluorescent technologies provide the potential to visualize the expression patterns of many functionally relevant molecules but present numerous challenges in accurate image analysis and data handling, particularly over large tumor areas. Drawing from the field of astronomy, in which petabytes of imaging data are routinely analyzed across a wide spectral range, Berry et al. developed a platform for multispectral imaging of whole-tumor sections with high-fidelity single-cell resolution. The resultant AstroPath platform was used to develop a multiplex immunofluorescent assay highly predictive of responses and outcomes for melanoma patients receiving immunotherapy. Science , aba2609, this issue p. eaba2609

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. Complement is a key factor in tissue inflammation, favoring cancer progression through the production of complement component 5a (C5a). However, the activation pathways that generate C5a in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the components that are part of the classical complement pathway. To understand how the complement cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (n = 106, 154, and 43), ccRCC cell lines, and tumor models in complement-deficient mice were used. High densities of cells producing classical complement pathway components C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The in situ orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and complement activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical complement pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

Background: Multidimensional, spatially resolved analyses of immune and tumor cells within the TME of patients treated with checkpoint inhibitors will provide clinically translatable mechanistic insights and potentiate biomarker discovery. To achieve this goal, information from pathology specimens needs to be captured at a single cell level with high fidelity and in meaningfully sized cohorts. To date, efforts have been limited by inadequate tissue sampling and previously unrecognized errors in staining, imaging and data analysis. Here we describe the ‘AstroPath' platform, where strategies from the field of astronomy were adapted to study pathology specimens and generate large high quality mIF data. Methods: Potential error was identified and addressed at each stage of 6-plex (PD-1, PD-L1, FoxP3, CD163, CD8, tumor marker) mIF assay development. Whole slides from formalin-fixed paraffin embedded tissue specimens were stained with the optimized assay and imaged using a multispectral microscope (Vectra 3.0) with 20% overlap of high power fields (HPFs). The overlaps were used to quantify and correct optical lens distortion, HPF alignment, and illumination variation. Errors from cell segmentation algorithms, batch-to-batch staining variation, and HPF sampling were also addressed. The resultant mIF data were organized and analyzed using a large, relational database. Results: The optimized mIF assay captured equivalent signal compared to gold standard chromogenic immunohistochemistry and 2x more signal for PD-1, PD-L1 and FoxP3 compared to the manufacturer's recommended protocol. Errors and corrections for imaging included: pixel alignment error reduced from ~10 to Conclusion: Here we present an end-to-end pathology workflow with rigorous quality control for creating quantitative, spatially resolved mIF datasets using lessons derived from the field of astronomy. Such approaches will vastly improve standardization and scalability of mIF technologies, enabling cross-site comparisons and eventual clinical translation as biomarker discovery platforms or standard diagnostic tests. Citation Format: Sneha Berry, Nicolas Giraldo, Benjamin Green, Elizabeth Engle, Haiying Xu, Aleksandra Ogurtsova, Daphne Wang, Julie E. Stein, Peter Nguyen, Suzanne Topalian, Angelo DeMarzo, Drew M. Pardoll, Robert A. Anders, Tricia R. Cottrell, Alexander S. Szalay, Janis M. Taube. The ‘AstroPath' platform for spatially resolved, single cell analysis of the tumor microenvironment (TME) using multispectral immunofluorescence (mIF) [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 6584.

For a long time, cancer therapy has had as its sole objective the direct elimination of tumor cells. In case of nonmetastatic disease, this is accomplished by surgery, which removes the primary tumor. Radiotherapy and conventional chemotherapies also aimed at targeting tumor cells preferentially. The high capacity of tumor cells to divide as compared to the normal cells makes them more sensitive to agents that physically, in the case of radiotherapy, or chemically, in the case of chemotherapy, attack DNA and lead to cell death. Chemotherapies targeting mutations in tumor cells such as BRAF have been developed as well. However, these approaches also destroy the nonmalignant cells and have local and/or systemic consequences. To increase specificity toward the tumor cells, cytotoxic agents have been coupled to antibodies that bind to the tumor cells in order to allow their specific targeting to the tumor and not to the normal cells. However, the entry of such constructs into tumors still remains a major issue.

International audience; Tumors are highly heterogeneous tissues where malignant cells are surrounded by and interact with a complex tumor microenvironment (TME), notably composed of a wide variety of immune cells, as well as vessels and fibroblasts. As the dialectical influence between tumor cells and their TME is known to be clinically crucial, we need tools that allow us to study the cellular composition of the microenvironment. In this focused research review, we report MCP-counter, a methodology based on transcriptomic markers that assesses the proportion of several immune and stromal cell populations in the TME from transcriptomic data, and we highlight how it can provide a way to decipher the complex mechanisms at play in tumors. In several malignancies, MCP-counter scores have been used to show various prognostic impacts of the TME, which we also show to be linked with the mutational burden of tumors. We also compared established molecular classifications of colorectal cancer and clear-cell renal cell carcinoma with the output of MCP-counter, and show that molecular subgroups have different TME profiles, and that these profiles are consistent within a given subgroup. Finally, we provide insights as to how knowing the TME composition may shape patient care in the near future.

Purpose: The efficacy of PD-1 checkpoint blockade as adjuvant therapy in localized clear cell renal cell carcinoma (ccRCC) is currently unknown. The identification of tumor microenvironment (TME) prognostic biomarkers in this setting may help define which patients could benefit from checkpoint blockade and uncover new therapeutic targets. Experimental Design: We performed multiparametric flow cytometric immunophenotypic analysis of T cells isolated from tumor tissue [tumor-infiltrating lymphocytes (TIL)], adjacent non-malignant renal tissue [renal-infiltrating lymphocytes (RIL)], and peripheral blood lymphocytes (PBL), in a cohort of patients (n = 40) with localized ccRCC. Immunophenotypic data were integrated with prognostic and histopathologic variables, T-cell receptor (TCR) repertoire analysis of sorted CD8+PD-1+ TILs, tumor mRNA expression, and digital quantitative immunohistochemistry. Results: On the basis of TIL phenotypic characterization, we identified three dominant immune profiles in localized ccRCC: (i) immune-regulated, characterized by polyclonal/poorly cytotoxic CD8+PD-1+Tim-3+Lag-3+ TILs and CD4+ICOS+ cells with a Treg phenotype (CD25+CD127−Foxp3+/Helios+GITR+), that developed in inflamed tumors with prominent infiltrations by dysfunctional dendritic cells and high PD-L1 expression; (ii) immune-activated, enriched in oligoclonal/cytotoxic CD8+PD-1+Tim-3+ TILs, that represented 22% of the tumors; and (iii) immune-silent, enriched in TILs exhibiting RIL-like phenotype, that represented 56% of patients in the cohort. Only immune-regulated tumors displayed aggressive histologic features, high risk of disease progression in the year following nephrectomy, and a CD8+PD-1+Tim-3+ and CD4+ICOS+ PBL phenotypic signature. Conclusions: In localized ccRCC, the infiltration with CD8+PD-1+Tim-3+Lag-3+ exhausted TILs and ICOS+ Treg identifies the patients with deleterious prognosis who could benefit from adjuvant therapy with TME-modulating agents and checkpoint blockade. This work also provides PBL phenotypic markers that could allow their identification. Clin Cancer Res; 23(15); 4416–28. ©2017 AACR.

Purpose: The tumor microenvironment is formed by many distinct and interacting cell populations, and its composition may predict patients' prognosis and response to therapies. Colorectal cancer is a heterogeneous disease in which immune classifications and four consensus molecular subgroups (CMS) have been described. Our aim was to integrate the composition of the tumor microenvironment with the consensus molecular classification of colorectal cancer. Experimental Design: We retrospectively analyzed the composition and the functional orientation of the immune, fibroblastic, and angiogenic microenvironment of 1,388 colorectal cancer tumors from three independent cohorts using transcriptomics. We validated our findings using immunohistochemistry. Results: We report that colorectal cancer molecular subgroups and microenvironmental signatures are highly correlated. Out of the four molecular subgroups, two highly express immune-specific genes. The good-prognosis microsatellite instable–enriched subgroup (CMS1) is characterized by overexpression of genes specific to cytotoxic lymphocytes. In contrast, the poor-prognosis mesenchymal subgroup (CMS4) expresses markers of lymphocytes and of cells of monocytic origin. The mesenchymal subgroup also displays an angiogenic, inflammatory, and immunosuppressive signature, a coordinated pattern that we also found in breast (n = 254), ovarian (n = 97), lung (n = 80), and kidney (n = 143) cancers. Pathologic examination revealed that the mesenchymal subtype is characterized by a high density of fibroblasts that likely produce the chemokines and cytokines that favor tumor-associated inflammation and support angiogenesis, resulting in a poor prognosis. In contrast, the canonical (CMS2) and metabolic (CMS3) subtypes with intermediate prognosis exhibit low immune and inflammatory signatures. Conclusions: The distinct immune orientations of the colorectal cancer molecular subtypes pave the way for tailored immunotherapies. Clin Cancer Res; 22(16); 4057–66. ©2016 AACR.

Purpose: Selecting patients with metastatic clear-cell renal cell carcinoma (m-ccRCC) who might benefit from treatment with targeted tyrosine kinase inhibitors (TKI) is a challenge. Our aim was to identify molecular markers associated with outcome in patients with m-ccRCC treated with sunitinib. Experimental Design: We performed global transcriptome analyses on 53 primary resected ccRCC tumors from patients who developed metastatic disease and were treated with first-line sunitinib. We also determined chromosome copy-number aberrations, methylation status, and gene mutations in von Hippel–Lindau and PBRM1. Molecular data were analyzed in relation with response rate (RR), progression-free survival (PFS), and overall survival (OS). Validation was performed in 47 additional ccRCC samples treated in first-line metastatic setting with sunitinib. Results: Unsupervised transcriptome analysis identified 4 robust ccRCC subtypes (ccrcc1 to 4) related to previous molecular classifications that were associated with different responses to sunitinib treatment. ccrcc1/ccrcc4 tumors had a lower RR (P = 0.005) and a shorter PFS and OS than ccrcc2/ccrcc3 tumors (P = 0.001 and 0.0003, respectively). These subtypes were the only significant covariate in the multivariate Cox model for PFS and OS (P = 0.017 and 0.006, respectively). ccrcc1/ccrcc4 tumors were characterized by a stem-cell polycomb signature and CpG hypermethylation, whereas ccrcc3 tumors, sensitive to sunitinib, did not exhibit cellular response to hypoxia. Moreover, ccrcc4 tumors exhibited sarcomatoid differentiation with a strong inflammatory, Th1-oriented but suppressive immune microenvironment, with high expression of PDCD1 (PD-1) and its ligands. Conclusions: ccRCC molecular subtypes are predictive of sunitinib response in metastatic patients, and could be used for personalized mRCC treatment with TKIs, demethylating or immunomodulatory drugs. Clin Cancer Res; 21(6); 1329–39. ©2015 AACR.

We recently reported a 56% objective response rate in patients with advanced MCC receiving pembrolizumab (anti-PD-1) as first-line therapy. However, a tumor biomarker predicting clinical response was not defined. The purpose of this study was to determine potential associations of anti-PD-1 response (RECIST 1.1, analysis 8/1/16) and the presence of MCPyV, with the density and distribution of CD8+, PD-1+ and PD-L1+ cell populations in the tumor microenvironment (TME). Pretreatment FFPE tumor specimens were stained for CD8 (n=23) and PD-L1 (n=16, 22C3 assay) with immunohistochemistry (IHC). Immunofluorescence (IF) was used to detect PD-1 (n=16). Intratumoral (IT), peritumoral (PT, 100 um zone) and total (PT+IT) densities of PD-1+ and CD8+ immune cells (IC) were determined with digital image analysis. PD-L1+ cases had >1% tumor cells (TC) expressing PD-L1. Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ cells in the IT, PT, and IT+PT regions when compared to non-responders (Mann-Whitney test, p-value=0.03, 0.06, 0.03, respectively). There was no significant association of response with CD8+ IC densities (IT, PT or total) or TC PD-L1 expression. Similarly, when we quantified the number of PD-1+ IC located within 15 um from a PD-L1+ cell (TC or IC), an association was observed between PD-1 and PD-L1 proximity and clinical response (Mann-Whitney test, p-value =0.04), but not CD8 and PD-L1 proximity. Because viral neoantigens can elicit a strong immune response, we also studied these TME factors for their potential associations with MCPyV. We found the presence of virus was significantly associated with increased densities of PT CD8+ cells (Mann-Whitney test, p-value=0.008) and TC PD-L1 expression (Fisher's test, p-value=0.04), but not PD-1+ IC. A more extensive multiplex IF panel (CD8, PD-1, PD-L1, CD68, FoxP3, NSE) analysis was performed on select pre-treatment specimens using the Vectra multispectral imaging system (Perkin Elmer) to explore this divergence. We found that only ~60% of observed total PD-1 expression was displayed by CD8+ cells. Although preliminary, our results suggest a relationship between PD-1+ cells and response to anti-PD-1 therapy and highlight that lymphocyte subsets other than CD8+ T-cells may contribute to the observed response. Further studies in larger cohorts are needed to validate these results. Citation Format: Nicolas A. Giraldo, Genevieve J. Kaunitz, Tricia R. Cottrell, Sneha Berry, Joel C. Sunshine, Peter Nguyen, Haiying Xu, Aleksandra Orgutsova, Candice D. Church, Natalie J. Miller, Jennifer H. Yearley, Evan J. Lipson, Ludmila Danilova, Paul T. Nghiem, Suzanne L. Topalian, Janis M. Taube. The differential association of PD-1, PD-L1, and CD8+ cells with response to pembrolizumab and presence of Merkel cell polyomavirus (MCPyV) in patients with Merkel cell carcinoma (MCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2017-662

Purpose: Clear cell renal cell carcinoma (ccRCC) has shown durable responses to checkpoint blockade therapies. However, important gaps persist in the understanding of its immune microenvironment. This study aims to investigate the expression and prognostic significance of immune checkpoints in primary and metastatic ccRCC, in relation with mature dendritic cells (DC) and T-cell densities. Experimental Design: We investigated the infiltration and the localization of CD8+ T cells and mature DC, and the expression of immune checkpoints (PD-1, LAG-3, PD-L1, and PD-L2) in relation with prognosis, in 135 primary ccRCC tumors and 51 ccRCC lung metastases. RNA expression data for 496 primary ccRCC samples were used as confirmatory cohort. Results: We identify two groups of tumors with extensive CD8+ T-cell infiltrates. One group, characterized by high expression of immune checkpoints in the absence of fully functional mature DC, is associated with increased risk of disease progression. The second group, characterized by low expression of immune checkpoints and localization of mature DC in peritumoral immune aggregates (tertiary lymphoid structures), is associated with good prognosis. Conclusions: The expression of the immune checkpoints and the localization of DC in the tumor microenvironment modulate the clinical impact of CD8+ T cells in ccRCC. Clin Cancer Res; 21(13); 3031–40. ©2015 AACR.

A high density of intratumoral effector memory CD8+/Th1 T cells is associated with favorable prognosis in most cancers and may be induced or increased by immunotherapy. Efficient adaptive immune reactions are shaped in tumor adjacent tertiary lymphoid structures, which exhibit all characteristics of immunity generating lymphoid formations in reactive lymph nodes. Malignant tumor cells impact favorably or deleteriously their immune microenvironment if they bear genetic mutations that result in neo-antigens or by producing chemokines and cytokines that recruit lymphocytes and myeloid cells or increase inflammation and neo-angiogenesis. This intricate network of interactions results in control or escape of tumors, and its understanding will help define goals to monitor efficiency of immunotherapies.

Tumors grow within a complex microenvironment composed of immune cells, fibroblasts, endothelial cells, and other non-malignant cells. The study of the composition of tumor microenvironments has led to classifications with prognostic and theranostic values, as well as to treatments modulating its composition and its functional orientation. Concurrently, molecular classifications of tumors have proposed taxonomies that define groups of patients with different prognosis and which predict responses to treatments. The density, location, and functional orientation of tumor-infiltrating lymphocytes form the immune contexture, the composition of which is positively correlated in most cases with patient survival. Colorectal cancer represents a paradigm for tumor immunology, as it is the human cancer in which it was exemplified that an adaptive immune response can control tumor growth and metastasis. A high infiltration of Th1/cytotoxic T cells is associated with longer disease or progression free and/or overall survival both in primary and metastatic sites. There are, howewer, exceptions to this rule, such as renal cell carcinoma where high CD8 T cell infiltration correlates with shorter survival. High infiltration by myeloid cells and fibroblats is generally associated with poor prognosis in cancer. We developed and validated a method, called MCP-Counter, which simultaneously quantifies the proportions of 10 different cellular populations in human tissues and applied it to human cancers. The method enabled a microenvironment-based classification of cancers that we correlated with known molecular classifications in colorectal and clear cell renal cell cancers. We confirmed our data by quantifying tumor infiltrating cells by immunohistochemistry. In colorectal cancer, the molecular and immune classifications confirmed that not only microsatellite-instable (MSI) tumors, but also a subgroup of microsatellite-stable (MSS) tumors, are characterized by a favorable immune contexture with high Th1/cytotoxic infiltration. Other subtypes exhibited poor immune infiltation or, in the worst prognostic case, high T cell infiltration in the context of a major inflammatory, angiogenic, and desmoplastic reaction. A group of experts met under the auspices of Fight Colorectal Cancer and the Cancer Research Institute. This group has recently elaborated therapeutic strategies for the different subtypes of colorectal cancer. In clear cell renal cell cancer, we identified a poor prognosis subgroup with high infiltration of CD8 T cells which express checkpoint inhibitors in the presence of PDL-1 and/or PDL-2 expressing tumor cells. In addition, using multiparametric immunophenotyping of tumor-infiltrating T cells, we characterized the lymphocyte populations that correlate with poor prognosis. Our analyses form the basis of a unification of molecular and immune classifications of human cancers, challenge our current views of the relationship between the composition of the tumor microenvironment and patient's prognosis, and suggest immunotherapeutic approaches that could benefit subgroups of patients in different cancers. Citation Format: Etienne Becht, Nicolas A. Giraldo, Aurélien De Reynies, Pierre Laurent-Puig, Benoit Beuselinck, Jessica Zucman-Rossi, Catherine Sautès-Fridman, Wolf H. Fridman. Tumor microenvironments: Prognostic and theranostic impacts [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA10.

This study aimed to decipher the potential mechanisms linking increased CD8+ T cell infiltration with an adverse clinical outcome in ccRCC. Clear cell Renal Cell Carcinoma (ccRCC) appears to be one of the most sensitive tumors to checkpoint blockade therapies. However, contrary to most cancers, some studies suggest that high densities of infiltrating CD8+ T cells correlate with patients' poor prognosis. In this study we determined: 1) the prognosis associated with the expression of immune checkpoints and its coordination with dendritic cell (DC) and CD8+ cell infiltration, and 2) the major phenotypic traits of CD8+ tumor infiltrating lymphocytes (CD8+ TILs). The prognosis associated with CD8+ and dendritic cell (DC) infiltrations, in addition to the expression of immune checkpoints were investigated in a cohort of 135 primary ccRCC by quantitative immunohistochemistry (IHC). We found that the densities of CD8+, PD-1+ and LAG-3+ cells were closely inter-correlated, and independently associated with decreased PFS and OS. In addition, patients whose tumors presented both high densities of PD-1+ infiltrating cells and PD-L1+ and/or L2+ tumor cells (>5% positive cells), displayed the worst clinical outcome [9% 5-year disease-free survival (DFS) vs. 64% in the other group, P These results suggest that in the absence of an immune response orchestrated by TLS-DC, the CD8+ T cells in ccRCC display an inhibited phenotype, associated with more advanced tumor stages and poor clinical outcome. To functionally characterize the CD8+ T cell infiltrates in ccRCC, we investigated the major phenotypic traits of freshly isolated lymphocytes from 21 primary tumors obtained by partial or radical nephrectomy. Non-supervised analyses of the CD8+ TIL phenotype revealed two different types of tumors: The first group or “inhibited group” (8/21) was characterized by the over-expression of inhibitory molecules (PD-1 and TIM-3) and activation molecules (CD69 and CD38), and the expansion of the effector memory cell subpopulation (CCR7-CD45RA-) in the CD8+ TIL; the second group (14/21) was characterized by the expansion of the CD8+EMRA subpopulation (CCR7-CD45RA+) in addition to a reduced expression of activation or inhibitory markers. Preliminary data suggest that the “inhibited group” of tumors is characterized by more advanced tumor stages, higher Fuhrman grades and increased CD8+ cell densities (as determined by IHC), and thus shares major characteristics with tumors associated with patients' worst clinical outcome in the retrospective cohort. The functional orientation and cytotoxic potential of TIL are under investigation in both groups of tumors to further characterize the exhaustion and/or suppressive features of CD8+ T cells associated with poor clinical outcome in primary ccRCC. In summary, we demonstrated that the infiltration with CD8+ TILs in ccRCC is accompanied by the enhanced expression of immune checkpoints in a subgroup of tumors displaying more aggressive features and advanced tumors stages. This immune profile defines a poor prognosis group of patients that should be suitable to receive immune checkpoint inhibitors. Citation Format: Nicolas A. Giraldo, Becht Etienne, Laetitia Lacroix, Yann Vano, Rafael Sanchez-Salas, Pierre Validire, Virginie Verkarre, Arnaud Mejean, Marie-Caroline Dieu-Nosjean, Stephane Oudard, Xavier Cathelineau, Wolf H. Fridman, Catherine Sautès-Fridman. Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary clear cell renal cell cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A085.

In the vast majority of cancer types, as demonstrated and exemplified in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC), a high density of CD8+ T cells associated with high densities of tertiary lymphoid structures (TLS) in the tumor microenvironment correlates with a good prognosis for the patient. However, an opposite association has been reported in primary clear cell renal cell carcinoma (RCC) and several other cancers. Our team studied the immune infiltrates of pulmonary metastases from CRC and RCC. As in the primary tumors, a high density of CD8+ T cells correlated with good prognosis for CRC metastases, while it correlated with a bad prognosis for RCC metastases. These results suggested that the identity of the tumor cells, rather than the organ where they grow, is critical for shaping the immune contexture of a given tumor. Transcriptomic analyses of large cohorts of human CRC and RCC allow to define molecular subgroups associated with distinct risks of tumor progression. We therefore characterized the immune microenvironments of the molecular subgroups of CRC and RCC. In CRC, the MSI-enriched subgroup identified patients with high cytotoxic T cells infiltration and favorable prognosis. In CRC and RCC, we confirmed that patients with low tumoral T and B cell infiltrations presented with high risk of death; In addition, in both cancer types, we identified subgroups of poor-prognosis patients with high tumoral lymphocyte infiltration, in the context of high expression of genes related to inflammation, immunosuppression, and angiogenesis. Integration of molecular and immune tumor phenotypes refines prognostic cancer groups. Moreover, these classifications allow to select immunotherapies appropriate to specific, potentially responding, groups of patients. Citation Format: Etienne Becht, Nicolas A. Giraldo, Aurelien De Reynies, Pierre Laurent-Puig, Benoit Beuselinck, Jessica Zucman-Rossi, Marie Caroline Dieu-Nosjean, Catherine Sautes-Fridman, Wolf H. Fridman. Cancer subtypes and their immune microenvironments. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA20.

In the vast majority of cancer types, as demonstrated and exemplified in colorectal cancer (CRC), a high density of CD8+ T cells in the tumor microenvironment correlates with a good prognosis for the patient. However, an opposite correlation has been reported in primary clear cell renal cell carcinoma (RCC) tumors. Our team studied the immune infiltrates of pulmonary metastases from CRC and RCC. We reported that despite the advanced stage of the disease in these patients, the immune infiltrate of pulmonary metastases remained a prognostic factor. As in the primary tumors, a high density of CD8+ T cells correlated with good prognosis for CRC metastases while it correlated with a bad prognosis for RCC metastases. These results suggest that the identity of the tumor cell is critical in shaping the immune contexture of a given tumor. We therefore investigated the molecular characteristics of the tumor cells which could be responsible for this opposite clinical impact in RCC versus CRC. First, we assessed the prognostic value associated with the expression of genes specific for different immune populations in several cohorts of primary CRC (n=177) and RCC tumors (n=72 and n=479), and confirmed the opposite impact of infiltrating lymphocyte densities on patient survival. In addition, RCC tumors showed a higher expression of genes related to inflammation, immunosuppression and angiogenesis. By comparing transcriptomic data from RCC and CRC cell lines, we observed that RCC tumor cells also expressed a significantly higher amount of these transcripts when compared to CRC tumor cells. Altogether, these results support the hypothesis that RCC malignant cells produce factors which are able to modify and determine the functional orientation of their immune microenvironment. Identifying which factors and pathways are critical for the RCC-related detrimental impact of CD8+ T cells on prognosis will be a major breakthrough, enabling the identification of new immunomodulatory drug targets and guide researchers and clinicians in immunotherapy protocols. Citation Format: Etienne Becht, Nicolas A. Giraldo, Romain Remark, Aurelien de Reynies, Laetitia Lacroix, Diane Damotte, Catherine Sautès-Fridman, Wolf-Herman Fridman. Molecular determinants of colon and renal cancers' immune contextures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1075. doi:10.1158/1538-7445.AM2014-1075

We introduce the Microenvironment Cell Populations-counter (MCP-counter) method, which allows the robust quantification of the absolute abundance of eight immune and two stromal cell populations in heterogeneous tissues from transcriptomic data. We present in vitro mRNA mixture and ex vivo immunohistochemical data that quantitatively support the validity of our method’s estimates. Additionally, we demonstrate that MCP-counter overcomes several limitations or weaknesses of previously proposed computational approaches. MCP-counter is applied to draw a global picture of immune infiltrates across human healthy tissues and non-hematopoietic human tumors and recapitulates microenvironment-based patient stratifications associated with overall survival in lung adenocarcinoma and colorectal and breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1070-5) contains supplementary material, which is available to authorized users.