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Consensus Molecular Subgroup-level statistics on each MCP-counter abundance estimates

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.

Table S1. Demographic and clinical characteristics of the analyzed patients are depicted (Mean {plus minus} SD)

Figure S4. Inverse relation between TLS and immune checkpoints in primary ccRCC : photomicrographs of immunohistochemistry staining of PD-L1(red)/hematoxylin and TLS structures as revealed by CD3(Blue)DC-Lamp(Red), CD21(Blue)/CD20(Red), and of CD8(red)/hematoxylin in PD-L1+and/orPD-L2+ (A) and PD-L1-/PD-L2- (B) primary ccRCC

Table S1. Demographic and clinical characteristics of the analyzed patients; Table S2. Antibodies and conditions used for the IHC studies; Table S3. List of immune-related genes analyzed by Low Density Array; Figure S1. Gating and data analysis strategy; Figure S2. CD4+ and CD8+ T-cell differentiation in ccRCC TIL, and autologous PBL and RIL; Figure S3. Gap statistics according to the possible number of clusters of TIL phenotype. Optimal cut-off according to firstSEmax method (R package: cluster) is displayed (dotted line); Figure S4. PCA analysis including RIL and TIL phenotype. TIL clusters are displayed; Figure S5. Tumor size according to TIL clusters; Figure S6. Percentages of CD4+TIL expressing AM and InR according to tumor clusters. C1, Cluster1; C2, Cluster2; C3, Cluster3; Figure S7. Percentages of CD8+TIL expressing AM and InR according to tumor clusters. C1, Cluster1; C2, Cluster2; C3, Cluster3; Figure S8. Clonality Index and frequency of top 15 clonotypes in CD8+PD-1+ TIL according to tumor clusters. C1, Cluster1; C2, Cluster2; C3, Cluster3; Figure S9. Corrected P values for the differential gene expression between TIL clusters; Figure S10. Correlation matrix including TLS-related genes and immune cells densities in Immune-activated and Immune-regulated tumors; Figure S11. Percentages of CD4+PBL expressing differentiation markers, AM and InR in healthy controls (HC) and ccRCC-bearing patients; Figure S12. Percentages of CD8+PBL expressing differentiation markers, AM and InR in HC and ccRCC-bearing patients; Figure S13. Percentages of CD4+PBL expressing differentiation markers, AM and InR according to PBL Clusters; Figure S14. Percentages of CD8+PBL expressing differentiation markers, AM and InR according to PBL Clusters.

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.

Antibodies used for immunohistochemical analyses

Immune, and other stromal samples analyzed

Legends for supplementary figure and trables

Supplementary Figure 1 : Design and use of the Microenvironment Cell Populations (MCP)-counter algorithm a) Heatmap representation of the expression of selected transcriptomic markers underlying MCP-counter scores in transcriptomic profiles corresponding to microenvironment cell populations. These scores have been quantitatively validated for their ability to predict the abundance of the corresponding cell population from the gene expression profile of a cellularly heterogeneous sample. b) MCP-counter is implemented as an R package, which takes as input transcriptomic profiles of cellularly heterogeneous tissues, such as tumors. It first maps the previously-identified transcriptomic markers to the assayed gene-expression features (as illustrated here for the 'discovery' CIT CRC cohort, and summarize them to output a score per cell population per sample. These scores can then be used to compare samples against each-others and to determine whether a group of samples is more highly infiltrated by a given cell type than another group of samples.

Colorectal cancer samples constituting the 3 cohorts analyzed, along with their molecular subgroups

Levels of expression of genes related to inflammation, angiogenesis and immunomodulation in CRC cohorts

Samples from the Cancer Cell Line Encyclopedia

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.

International audience; In colorectal cancer (CRC), T-cell checkpoint blockade is only effective in patients diagnosed with mismatch repair-deficient (MMR-d) cancers. However, defects in Human Leukocyte Antigen (HLA) class I expression were reported to occur in most MMR-d CRCs, which would preclude antigen presentation in these tumours, considered essential for the clinical activity of this immunotherapeutic modality. We revisited this paradox by characterising HLA class I expression in two independent cohorts of CRC. We determined that loss of HLA class I expression occurred in the majority (73-78%) of MMR-d cases. This phenotype was rare in CRC liver metastases, irrespective of MMR status, whereas weak, inducible expression of HLA class I molecules was frequent in liver lesions. We propose that HLA class I is an important determinant of metastatic homing in CRCs. This observation is paramount to understand CRC carcinogenesis and for the application of immunotherapies in the metastatic setting.

Prostate cancer (PCa) patients with lymph node invasion at radical prostatectomy are at higher risk of tumor recurrence and receive immediate androgen deprivation therapy (ADT). While approximately 30% of these patients do not experience recurrence, others experience disease recurrence despite ADT, and currently no biomarkers can accurately identify them. We analyzed tumors from 51 patients with node-positive prostate cancer using immunohistochemistry to investigate whether expression of the immune checkpoint ligand PD-L1 by tumor cells or the density of CD8+ or CD20+ cells are associated with clinical progression. Patients with at least 1% PD-L1+ tumor cells had shorter metastasis-free survival than those with PD-L1- tumors (p =0.008, log-rank test). Univariate Cox regression showed that patients with PD-L1+ tumors had almost four times the risk of experiencing distant metastases than those with PD-L1- tumors (hazard ratio 3.90). In addition, we found that PD-L1 expression was significantly associated with CD8+ T-cell density, but not with CD20+ B-cell density. While these results need to be confirmed in larger studies, they show that PD-L1 and CD8 may be used as biomarkers for node-positive patients at high risk of progression. The study also provides a rationale for selecting patients with node-positive PCa who might benefit the most from adjuvant immunotherapies. Patient summary: None of the available biomarkers can identify node-positive prostate cancer that will recur after surgery. We found that expression of PD-L1 by tumor cells and a high density of CD8+ T cells in tumor are associated with a higher risk of clinical progression in men with node-positive prostate cancer. Biological markers predicting tumor recurrence in node-positive prostate cancer (PCa) are currently lacking. By investigating formalin-fixed paraffin-embedded tissue sections from 51 patients with node-positive PCa, we found that patients with tumors expressing PD-L1 and/or with a higher frequency of intratumor CD8+ T cells were at the highest risk of developing PCa metastasis after surgery. These results may open new frontiers for the use of adjuvant immunotherapy in this subset of node-positive patients.

Tumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of the cells. There is a high heterogeneity of the staining within tumors but also between patients. Semi-quantification of the staining in large cohorts of patients allows to investigate the prognostic impact of the local complement production and activation. Here we explain the staining process for C1q, C4, and C3 in human paraffin-embedded tumor sections by immunofluorescence and immunohistochemistry.

Introduction Les patients atteints de neurofibromatose 1 (NF1) developpent des neurofibromes plexiformes (NF) a risque de transformation en tumeur maligne des gaines nerveuses (TMGN). Les NF dysplasiques (dNF) sont un stade intermediaire entre NF et TMGN, contenant un infiltrat inflammatoire de cellules mononuclees. Nous supposons une reponse immune au stade « dysplasique », dont le controle par la tumeur permet l’evolution en TMGN. L’objectif de l’etude est de caracteriser les cellules des infiltrats des dNF et l’expression de cibles potentielles d’immunotherapie. Materiel et methodes Les echantillons (materiel fixe en formol et inclus en paraffine) incluent des tumeurs de patients NF1 suivis au CHU Henri Mondor (n = 73) :69 NF, 24 dNF, 14 TMGN, ainsi que 8 tumeurs sporadiques : 6 dNF, 2 TMGN. L’evaluation quantitative des lymphocytes T et macrophages a ete faite par immunohistochimie (IHC, CD3 et CD163) sur l’ensemble de la cohorte avec quantification automatique (logiciel QuPath). Une analyse transcriptomique (PanCancer IO 360 Panel, Nanostring) a ete effectuee sur un sous-groupe (n = 42) de la cohorte NF1 et les donnees analysees avec le logiciel NSolver. L’etude phenotypique a repose sur des marquages multiples en immunofluorescence (IF): CD3/CD8/Tim-3, CD3/CD163/Tim-3, CD163/Gal9/Tim-3, et par IHC pour la granulysine. Resultats Les infiltrats des dNF sont faits de lymphocytes TCD3+ et de macrophages CD163+, plus abondants dans les dNF NF1 comme sporadiques (p Discussion PD1 et Tim-3, exprimes respectivement par les lymphocytes TCD8+ et macrophages CD163+ infiltrant les dNF, pourraient constituer des cibles d’immunotherapie et le couple Tim-3/Gal9 pourrait maintenir une fonction immunosuppressive dans les macrophages par un mecanisme autocrine/paracrine. Si les traitements anti-PD1 sont connus en oncologie, les anti-Tim-3 sont en developpement et en cours d’essai combines avec les anti-PD1 dans certains cancers. Cette combinaison permettrait, dans le cas des dNF, de cibler les deux types cellulaires principaux des infiltrats. Des etudes fonctionnelles sont requises pour valider le role de ces acteurs moleculaires dans la transformation maligne, et l’interet potentiel de l’immunotherapie dans les dNF.

Tumors contain a complement rich microenvironment in which all cell types (e.g., tumor cells and stromal cells) are able to produce different proteins. We developed immunohistochemistry (IHC) assays allowing to identify on paraffin embedded tumor sections, not only the complement producing cells but also the complement activation fragments which result from activation of complement cascade within the tumor. The local production of complement can be detected by cytoplasmic staining, whereas the activation fragments are localized at the surface of the cells. There is a high heterogeneity of the staining within tumors but also between patients. Semi-quantification of the staining in large cohorts of patients allows to investigate the prognostic impact of the local complement production and activation. Here we explain the staining process for C1q, C4, and C3 in human paraffin-embedded tumor sections by immunofluorescence and immunohistochemistry.

e15041 Background: The treatment of HER2+ breast cancer (BC) has improved substantially with the use of targeted therapies, and patients achieving pathological complete response (pCR) following neoadjuvant therapy have significantly improved disease free survival. However, there is currently no way to determine which patients are most likely to respond to neoadjuvant HER2-targeting treatment. Growing evidence indicates tumour infiltrating lymphocytes influence HER2+ BC outcome. We hypothesize that a deeper investigation of the immune landscape of HER2+ patient tumours may improve patient stratification and identify an immune profile associated with pCR. Methods: 3plex immunohistochemistry panels identifying immune cell subpopulations and tertiary lymphoid structures were applied to HER2+ BC tumors (pre-treatment N=19, post-treatment N=9) of patients of the neoadjuvant TCHL clinical trial (NCT01485926), who received HER2-targeting therapy. Quantitative analysis of CD3+, CD20+, DC-Lamp+, CD4+, and CD8+ immune cells on multi-labelled whole-slide images of TCHL tumor sections was performed using artificial intelligence image analysis models. Patient-specific densities of each cell type in tumor regions were determined to facilitate assessment of pre-treatment differences in target immune subpopulations in complete, partial, and non-responders. Results: Statistical analysis demonstrated a trend for higher densities of each immune subpopulation in pre-treatment biopsies of pCR vs. non-pCR patients. Patient categorisation based on median threshold demonstrated patients achieving pCR are more frequently high for each immune cell type assessed, while non-pCR patients are more frequently in the low category. Investigation of association of pre-treatment immune infiltrate with clinicopathological features demonstrated trends for higher levels of T-cell infiltration in later stage and hormone receptor negative patients. Conclusions: Multiplex IHC is an optimal technique for the concurrent assessment of multiple target markers in patient tissue, which can be quantitatively analyzed in a high throughput manner using AI models. The findings of this preliminary study support recent evidence regarding the role of tumor infiltrating immune cells in HER2+ BC outcome and warrant further investigation in a larger cohort.

Soft-tissue sarcomas represent a heterogeneous group of cancer, with more than 50 histological subtypes1,2. The clinical presentation of patients with different subtypes is often atypical, and responses to therapies such as immune checkpoint blockade vary widely3,4. To explain this clinical variability, here we study gene expression profiles in 608 tumours across subtypes of soft-tissue sarcoma. We establish an immune-based classification on the basis of the composition of the tumour microenvironment and identify five distinct phenotypes: immune-low (A and B), immune-high (D and E), and highly vascularized (C) groups. In situ analysis of an independent validation cohort shows that class E was characterized by the presence of tertiary lymphoid structures that contain T cells and follicular dendritic cells and are particularly rich in B cells. B cells are the strongest prognostic factor even in the context of high or low CD8+ T cells and cytotoxic contents. The class-E group demonstrated improved survival and a high response rate to PD1 blockade with pembrolizumab in a phase 2 clinical trial. Together, this work confirms the immune subtypes in patients with soft-tissue sarcoma, and unravels the potential of B-cell-rich tertiary lymphoid structures to guide clinical decision-making and treatments, which could have broader applications in other diseases. Immune profiling of the tumour microenvironment of soft-tissue sarcoma identifies a group of patients with high levels of B-cell infiltration and tertiary lymphoid structures that have improved survival and a high response rate to immune checkpoint blockade therapy.

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.

The oncogenic events involved in breast implant-associated anaplastic large cell lymphoma (BI-ALCL) remain elusive. To clarify this point, we have characterized the genomic landscape of 34 BI-ALCLs (15 tumor and 19 in situ subtypes) collected from 54 BI-ALCL patients diagnosed through the French Lymphopath network. Whole-exome sequencing (n = 22, with paired tumor/germline DNA) and/or targeted deep sequencing (n = 24) showed recurrent mutations of epigenetic modifiers in 74% of cases, involving notably KMT2C (26%), KMT2D (9%), CHD2 (15%), and CREBBP (15%). KMT2D and KMT2C mutations correlated with a loss of H3K4 mono- and trimethylation by immunohistochemistry. Twenty cases (59%) showed mutations in ≥1 member of the JAK/STAT pathway, including STAT3 (38%), JAK1 (18%), and STAT5B (3%), and in negative regulators, including SOCS3 (6%), SOCS1 (3%), and PTPN1 (3%). These mutations were more frequent in tumor-type samples than in situ samples (P = .038). All BI-ALCLs expressed pSTAT3, regardless of the mutational status of genes in the JAK/STAT pathway. Mutations in the EOMES gene (12%) involved in lymphocyte development, PI3K-AKT/mTOR (6%), and loss-of-function mutations in TP53 (12%) were also identified. Copy-number aberration (CNA) analysis identified recurrent alterations, including gains on chromosomes 2, 9p, 12p, and 21 and losses on 4q, 8p, 15, 16, and 20. Regions of CNA encompassed genes involved in the JAK/STAT pathway and epigenetic regulators. Our results show that the BI-ALCL genomic landscape is characterized by not only JAK/STAT activating mutations but also loss-of-function alterations of epigenetic modifiers.

Purpose: The impact of tertiary lymphoid structures (TLS) in hepatocellular carcinoma (HCC) progression is being extensively investigated. However, their presence during the early steps of human liver carcinogenesis remains unknown. We thus aimed to determine whether TLS are induced in preneoplastic/early hepatic lesions (EHL), and whether they are associated with a particular immune profile. Experimental Design: A series of 127 EHLs (low/high-grade dysplastic nodules, early HCC, and small and progressed HCC) was included in the study. TLSs were investigated by pathologic reviewing. Densities of immune cells were assessed using IHC. A subset of lesions was microdissected and gene expression profiling was performed with a custom NanoString panel. Results: Compared with surrounding cirrhotic nodules, EHL of all stages displayed increased densities of T cells, B cells, and dendritic cells. Immature TLSs were identified in 24% of EHL. Gene expression profiling identified a subset of EHL with elevated mRNA levels of various cytokines involved in immune cells' recruitment and TLS induction. This subgroup of EHL also showed overexpression of genes related to T- and B-cells' activation and antigen presentation, as well as those related to immunosuppression and immune exhaustion. Conclusions: Local immune activation occurs in the very early steps of liver carcinogenesis; however, it may not be fully efficient and paradoxically favor immune evasion and progression to full-blown HCC. These results have implications for the development of anti-HCC chemopreventive strategies in cirrhotic patients.

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.

Purpose: The immunomodulatory activity of DNA hypomethylating agents (DHAs) suggests they may improve the effectiveness of cancer immunotherapies. The phase Ib NIBIT-M4 trial tested this hypothesis using the next-generation DHA guadecitabine combined with ipilimumab. Patients and Methods: Patients with unresectable stage III/IV melanoma received escalating doses of guadecitabine 30, 45, or 60 mg/m2/day subcutaneously on days 1 to 5 every 3 weeks, and ipilimumab 3 mg/kg intravenously on day 1 every 3 weeks, starting 1 week after guadecitabine, for four cycles. Primary endpoints were safety, tolerability, and MTD of treatment; secondary were immune-related (ir) disease control rate (DCR) and objective response rate (ORR); and exploratory were changes in methylome, transcriptome, and immune contextures in sequential tumor biopsies, and pharmacokinetics. Results: Nineteen patients were treated; 84% had grade 3/4 adverse events, and neither dose-limiting toxicities per protocol nor overlapping toxicities were observed. Ir-DCR and ir-ORR were 42% and 26%, respectively. Median CpG site methylation of tumor samples (n = 8) at week 4 (74.5%) and week 12 (75.5%) was significantly (P < 0.05) lower than at baseline (80.3%), with a median of 2,454 (week 4) and 4,131 (week 12) differentially expressed genes. Among the 136 pathways significantly (P < 0.05; Z score >2 or ←2) modulated by treatment, the most frequently activated were immune-related. Tumor immune contexture analysis (n = 11) demonstrated upregulation of HLA class I on melanoma cells, an increase in CD8+, PD-1+ T cells and in CD20+ B cells in posttreatment tumor cores. Conclusions: Treatment of guadecitabine combined with ipilimumab is safe and tolerable in advanced melanoma and has promising immunomodulatory and antitumor activity.

2549 Background: DNA hypomethylating agents show broad immuno-modulatory activity in neoplastic cells, and may improve the effectiveness of cancer immunotherapies. The phase 1b NIBIT-M4 trial investigated a previously unexplored therapeutic strategy using the next-generation DNA hypomethylating agent guadecitabine sequenced with ipilimumab for the treatment of advanced melanoma. Methods: Patients with unresectable Stage III/IV melanoma received escalating doses of guadecitabine 30, 45 or 60 mg/m2 subcutaneously on Days 1–5 every three weeks, and ipilimumab 3 mg/kg intravenously on Day 1 every three weeks, starting one week after guadecitabine, for four cycles. Primary endpoints were the safety, tolerability and maximum tolerated dose of treatment; secondary endpoints included immune-related disease control and objective response. Genome-wide methylation, RNA sequencing, and immunohistochemistry analyses were performed on tumor samples collected at baseline, W4 and W12. (NCT02608437). Results: 19 patients were treated and evaluable for safety and efficacy. The most common treatment-related adverse events of any grade were myelotoxicity (n = 17; 89%) and immune-related adverse events (n = 12; 63%). Grade 3 or 4 myelotoxicity occurred in 15 (79%) patients. There were no dose limiting toxicities. Rates of immune-related disease control and objective response were 8/19 (42%) and 5/19 (26%), respectively. Exploratory analyses of tumour samples (n = 8) showed that median CpG site methylation at Week 4 (74.5%) and Week 12 (75.5%) was significantly lower (p < 0.05) than at baseline (80.3%), with a median of 2454 (Week 4) and 4131 (Week 12) differentially expressed genes identified compared to baseline; among the 136 pathways significantly modulated by treatment, the most frequently activated were immune-related. Tumour immune contexture analysis (n = 11) demonstrated up-regulation of Human Leukocyte Antigen (HLA) class I molecules on melanoma cells, and an increase in CD8+, PD-1+ T cells and in CD20+ B cells in post-treatment tumour core specimens. Conclusions: Sequential guadecitabine and ipilimumab is safe and tolerable in patients with metastatic melanoma, and has promising immunological and anti-tumour activity. Clinical trial information: NCT02608437.

Soft tissue sarcoma (STS) are rare mesenchymal-originated tumors with more than 50 different histologies identified. Not every histology in STS responds to immunotherapy and immunologic predictive markers are lacking. The purpose of this study is to establish an immune classification of STS by analysis of the transcriptome. This was performed by using a deconvolution method that allowed us to quantify 8 immune populations and endothelial cells. As a secondary objective, we searched for a surrogate biomarker that could be assessable in the clinic. We analyzed transcriptomic data of four publicly available datasets, accounting for 608 complex genomic STS, including leiomyosarcoma (LMS, 35.4%), dedifferentiated liposarcoma (DDLPS, 33.9%) and undifferentiated pleomorphic sarcoma (UPS, 30.8%). By using the MCP-Counter deconvolution method, we characterized the tumor microenvironment (TME) of these tumors and established a robust immune classification that is consistent through various cohorts. We classified the patients into 5 Sarcoma Immune Classes (SIC), labeled as A1, A2, B, C1 and C2. The A1 and A2 groups are associated with very low to low immune infiltrates. Conversely, SIC C1 and C2 tumors are characterized by strong to very strong expression of signatures associated to all immune cells. SIC B tumors are characterized by a high expression of endothelial cell signature, an intermediate presence of neutrophils, and a rather low infiltration by other immune cell types. Regarding functional orientation of the TME, gene signatures associated with immune cells chemotaxis activation and survival, expression of major histocompatibility complex class I, and regulatory T-cells are highly expressed in SIC C1 and C2, modestly expressed in B and A2, and very lowly expressed in A1. Interestingly, immune checkpoint genes exhibited strong expression differences between SICs. SIC C2 had a strong expression of PD-1, PD-L2, CTLA-4 and TIM-3 genes. We also found that the lymphoid structure-associated B cell chemoattractant chemokine CXCL13 is remarkably highly expressed in C2 class. CXCL13 is associated with the presence of tertiary lymphoid structures (TLS). Although all histologies are distributed in each SIC group, LMS are more commonly found in the immune low SIC A1 and A2 groups, and we also extended our analysis to other histologies such as synovial sarcoma or gastrointestinal stromal tumors. Our classification is associated with clinical outcome, and SIC group C (C1/C2) has the longest overall survival, as compared to SIC A group (A1/A2) (p = 0.015). We then validated SIC classification using STS FFPE samples (n=32). SIC classification by RNA expression was correlated with quantitative immunohistochemistry (IHC) of CD3 (T-cells), DC-Lamp (activated dendritic cells), CD20 (B cells), CD8 (CD8+ T-cells), and CD34 (endothelial cells). Densities of CD3 (p=0.0033), CD8 (p=0.004) and CD20 (p=0.00043) were significantly higher in SIC C tumors. Tumor SIC B groups have a higher, albeit nonsignificant (p=0.6) expression of CD34+ endothelial cells. In the validation cohort, SIC group C is associated with a better prognosis (p=0.053). We further investigated whether TLS were a marker of the immune-high SIC C group. Not surprisingly, TLS-positive tumors were found only in SIC C2 (100%, 9/9) and C1 (50%, 3/6) groups of the validation cohort. We expanded the validation cohort (n = 93) to analyze the correlation between TLS and CD3+/CD8+/CD20+ tumor-infiltrating lymphocytes. We found that the presences of each type of TILs all are significantly associated with the presence of TLS (CD3, CD8, CD20, all p values < 0.0001). This validation finding suggests that TLS may be a good surrogate marker for identification of SIC C groups and could be validated in the future to correlate with the efficacy of immunotherapy in STS. Citation Format: Wei-Wu Tom Chen, Florent Petitprez, Cheng-Ming Sun, Laetitia Lacroix, Aurélien de Reyniès, Antoine Italiano, Maud Toulmonde, Carlo Lucchesi, Yec'han Laizet, Catherine Sautès-Fridman, Wolf Herve Fridman. Immune-based classification of soft-tissue sarcoma is associated with clinical outcome and unveils tertiary lymphoid structures as surrogate biomarker for the clinic [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR03.

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.

Introduction: The anti-CTLA-4 monoclonal antibody (mAb) ipilimumab (Ipi) has firstly shown the therapeutic potential of targeting immune checkpoints in metastatic melanoma (MM) patients (pts), though with limited objective clinical responses, ranging from 10 to 13% (Hodi, NEJM 2010; Ascierto, TLO 2017). Targeting of CTLA-4 is of interest due to its role in the priming phase of the immune response; thus, anti-CTLA-4 mAb are being tested in different combinations to improve their clinical efficacy also by overcoming tumor resistance to treatment. We have thoroughly demonstrated that epigenetic remodelling of cancer cells with DNA hypomethylating agents (DHA) improves tumor immunogenicity and immune recognition (Maio M, CCR 2015). These findings identified DHA as highly intriguing “partner agents” to improve the therapeutic index of checkpoint blockers, including CTLA-4 mAb. This hypothesis is being tested in the ongoing NIBIT-M4 study (NCT02608437). Methods: The NIBIT-M4 is a phase 1b, dose-escalation study sequencing treatment-naïve/pre-treated, unresectable Stage III/IV MM pts, with the next generation DHA guadecitabine followed by Ipi. The dose escalation follows a 3+3 design: guadecitabine (at 30, 45 or 60 mg/m2) is administered s.c. on Week (W) 0, 3, 6, 9, days 1-5 and Ipi i.v. at 3 mg/kg on W1, 4, 7 and 10 q21d. Tumor assessment per immune-related response criteria is performed at screening (W0), W12, 18, 24 and q12 weeks. Patients-wise genome-wide methylation, RNA sequencing, whole-exome sequencing, and immunohistochemistry (IHC) analyses were performed on tumor samples collected at W0, 4 and 12. Canonical pathways and upstream regulators were investigated with IPA. Preliminary results: Fourteen out of the 19 planned pts have been enrolled so far, with no dose limiting toxicities. Among the 11 evaluable pts, the objective response rate is 27%. A reduction in tumor CpG sites methylation, from W0 (median 81.18%) to W4 (median 76.54%) and W12 (median 76.89%), was identified in the initial 8 pts enrolled. Transcriptomic analyses identified 3,897 ± 3,577 and 5,414 ± 3,569 differentially expressed genes (DEG) (q 1.0) at W4 and W12 compared to W0, respectively. Among the 137 pathways that were significantly modulated in all tumor samples at either W4 and/or W12, the most frequently activated ones were immune related (i.e., Th1 and Th2, iCOS-iCOSL and CD28 Signaling in T Helper Cells, and Interferon (IFN) Signaling). In addition, the top predicted upstream regulators were genes involved in the interferon IFN pathway (i.e., IFNG, STAT1, IRF7, IFNA2, IRF1). Though limited by the current sample size investigated, a greater number of significantly (p Conclusions: Epigenetic tumor immune-remodeling seems to be a promising strategy to improve the efficacy of CTLA-4 blockade, to be further pursued also with other immunomodulating mAb. *AMDG and AC equally contributed. Citation Format: Anna M. Di Giacomo, Alessia Covre, Francesca Finotello, Dietmar Rieder, Luca Sigalotti, Carolina Fazio, Ornella Cutaia, Christoph Bock, Florent Petitprez, Laetitia Lacroix, James N. Lowder, Wolf H. Fridman, Catherine Sautès-Fridman, Zlatko Trajanoski, Michele Maio. Epigenetic tumor remodelling to improve the efficacy of immune checkpoint blockade: the NIBIT-M4 clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT059.

Soft tissue sarcomas (STS) form a group of rare cancers which accounts for around 1% of non-pediatric solid tumors. Recent clinical trials with check-point blockade immunotherapies reported a response in up to 15% of patients, but there are no biomarkers predicting response of STS to checkpoint blockade therapies yet. We analyzed transcriptomic data of publicly available cohorts, accounting for more than 600 complex genomic STS, including leiomyosarcoma (LMS, 35.4%), dedifferentiated liposarcoma (DDLPS, 33.9%) and undifferentiated pleomorphic sarcoma (UPS, 30.8%). Using a deconvolution method to estimate the tumor microenvironment composition with accurate scores depicting the immune profile of tumors, we established a robust immune classification that is consistent through different datasets. We divided the patients into 5 Sarcoma Immune Classes, labelled A1, A2, B, C1 and C2, which exhibit different extents and composition of immune infiltrate. Two groups (A1: 23.3% and A2: 27.1% of all tumors) exhibit a very low to low immune infiltrate for all cell types. Another class (B: 14.5% of all tumors) displays moderate immune infiltrate and a strong presence of endothelial cells. Finally, the remaining two groups (C1: 19.4% and C2: 15.6%) are highly infiltrated by all immune cell types. All histologies were found in all five immune classes, although LMS were enriched in classes A1 and A2. The immune-high groups exhibit a significantly prolonged overall survival compared to the other groups. Notably, the immune high group C2 is characterized by an extremely rich immune infiltrate containing both lymphocytes and myeloid cells, and is associated with a striking overexpression of several immune checkpoints genes: PDCD1 (PD-1), CD274 (PD-L1), PDCD1LG2 (PD-L2), LAG3, HAVCR2 (TIM-3), CTLA4. This group also contains tumors with a strong expression of the B cell attractant chemokine CXCL13, often associated with the presence of tertiary lymphoid structures (TLS). We validated the relevance of TLS as a biomarker of this group by quantitative immunohistochemistry (IHC) using stainings for CD3, DC-Lamp, CD8, and CD20 on a 95-patient STS cohort (LMS, 34%; DDLPS, 33%; UPS, 34%). Tumors with visible TLS had a higher density of CD3+, CD8+ and CD20+ cells in the tumor core and the invasive margin, hence showing the possibility to identify the immune-high group by using IHC. The expression of immune checkpoint molecules was also detected by multiplex immunofluorescence for CD3, CD20, PD-1 and Lag3 in these tumors, confirming the presence of checkpoint molecules in the immune-high group. To check whether the immune-high group C2 was associated with enhanced response to checkpoint blockade therapy, we intend to analyze tumors from a recently published phase 2 clinical trial in which patients underwent treatment with anti-PD-1 pembrolizumab. Citation Format: Florent Petitprez, Tom Wei-Wu Chen, Cheng-Ming Sun, Julien Calderaro, Li-Ping Hsiao, Laetitia Lacroix, Ivo Natario, Maud Toulmonde, Carlo Lucchesi, Yec'han Laizet, Antoine Italiano, Aurélien de Reyniès, Catherine Sautès-Fridman, Wolf H. Fridman. A novel transcriptomic-based immune classification of soft tissue sarcoma (STS) and its association with molecular characteristics, clinical outcome and response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4045.

Tertiary lymphoid structures (TLS) form at sites of chronic inflammation, and locally prime the immune system independently of secondary lymphoid organs. The intratumoral presence of TLS has been associated with a positive prognostic outcome for patients with solid tumors, including colorectal carcinoma. Thus, the therapeutic induction of TLS would be hypothesized to confer clinical benefit to patients. Indeed, the therapeutic introduction of the IL-1 family cytokine IL-36 gamma into the tumor microenvironment (TME) of murine MC38 colon carcinoma leads to delayed tumor progression and the development of TLS at 5 days post-treatment. The TLS present with characteristics of secondary lymphoid organs, including PNAd+ high endothelial venules (HEV) surrounded by CD3+ T cells and CD11c+ dendritic cells, but are considered as “non-classical” due to the absence of a defined B cell zone. The formation of TLS is dependent upon the adaptive immune response, as IL-36 gamma-treated tumors grown in Rag2−/− animals fail to present with PNAd+ vasculature within the tumor. Thus, crosstalk between the immune system and cells of the vasculature is key to the efficacy of the IL-36 gamma-induced anti-tumor immune response. In human colorectal carcinoma, IL-36 gamma was also observed to promote the local anti-tumor immune response. Expression of IL-36 gamma by vascular endothelial cells of HEV was associated with an increased density of CD20+ B cells in TLS. Specific IL-36 gamma production by CD68+ macrophages was associated with an increased infiltration of CD4+ central memory T cells into the TME. Therefore, IL-36 gamma is a pro-inflammatory mediator involved in the anti-tumor immune response in both murine and human colorectal cancer.

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: 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.

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), 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.

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.