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Purpose: To determine whether the tumor immune infiltrate, as recently evaluated with the Immunoscore methodology, could be a useful prognostic marker in patients with rectal cancers.Experimental design: The influence of the immune infiltrate on patient's outcome was investigated in patients with or without preoperative chemoradiation therapy (pCRT). The density of total (CD3+) and cytotoxic (CD8+) T lymphocytes was evaluated by immunohistochemistry and quantified by a dedicated image analysis software in surgical specimens of patients with rectal cancer (n = 111) who did not receive pCRT and in tumor biopsies performed before pCRT from additional 55 patients. The results were correlated with tumor recurrence, patient's survival, and response to pCRT.Results: The densities of CD3+ and CD8+ lymphocytes and the associated Immunoscore (from I0 to I4) were significantly correlated with differences in disease-free and overall survival (HR, 1.81 and 1.72, respectively; all P < 0.005). Cox multivariate analysis supports the advantage of the Immunoscore compared with the tumor–node–metastasis (TNM) staging in predicting recurrence and survival (all P < 0.001). Lymph node ratio added information in a prognostic model (all P < 0.05). In addition, high infiltration of CD3+ and CD8+ lymphocytes in tumor biopsies was associated with downstaging of the tumor after pCRT (CD3+ cells; Fisher exact test P = 0.01).Conclusions: The Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of response to pCRT. Clin Cancer Res; 20(7); 1891–9. ©2014 AACR.

PDF file - 83KB, Kaplan Meier curves for the duration of OS according A, to T cell (CD3+) density evaluated in combined tumor regions (CT and IM). B, to T cell (CD8+) density evaluated in combined tumor regions (CT and IM).

Supplementary Table 1: characteristics of the HEGP cohort of rectal cancer patients Supplementary Table 2: Characteristics of the St Spiridon Hospital cohort of patients Supplementary Table 3: Immune infiltration of CD3+ and CD8+ cells in tumor regions and clinical outcome. Cohort of 111 patients eligible to primary surgery. Supplementary Table 4: Patients at risk at each interval in the Kaplan Meier survival curves for the duration of DFS and OS according to the Immunoscore (CD3-CD8). Supplementary Table 5: Repartition of the "surgery cohort" patient's according to Stage and Immunoscore

Purpose:No biomarker to personalize treatment in locally advanced rectal cancer (LARC) is currently available. We assessed in LARC whether a diagnostic biopsy-adapted immunoscore (ISB) could predict response to neoadjuvant treatment (nT) and better define patients eligible to an organ preservation strategy (“Watch-and-Wait”).Experimental Design:Biopsies from two independent cohorts (n1 = 131, n2 = 118) of patients with LARC treated with nT followed by radical surgery were immunostained for CD3+ and CD8+ T cells and quantified by digital pathology to determine ISB. The expression of immune-related genes post-nT was investigated (n = 64 patients). Results were correlated with response to nT and disease-free survival (DFS). The ISB prognostic performance was further assessed in a multicentric cohort (n = 73 patients) treated by Watch-and-Wait.Results:ISB positively correlated with the degree of histologic response (P < 0.001) and gene expression levels for Th1 orientation and cytotoxic immune response, post-nT (P = 0.006). ISB high identified patients at lower risk of relapse or death compared with ISB low [HR, 0.21; 95% confidence interval (CI), 0.06–0.78; P = 0.009]. Prognostic performance of ISB for DFS was confirmed in a validation cohort. ISB was an independent parameter, more informative than pre- (P < 0.001) and post-nT (P < 0.05) imaging to predict DFS. ISB combined with imaging post-nT discriminated very good responders that could benefit from organ preservation strategy. In the “Watch-and-Wait” cohort (n = 73), no relapse was observed in patients with ISB high (23.3%).Conclusions:ISB predicts response to nT and survival in patients with LARC treated by surgery. Its usefulness in the selection of patients eligible for a Watch-and-Wait strategy is strongly suggested.

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A role for the immune system in controlling the progression of solid tumors has been established in several mouse models. However, the effect of immune responses and tumor escape on patient prognosis in the context of human cancer is poorly understood. Here, we investigate the cellular and molecular parameters that could describe in situ immune responses in human colorectal cancer according to clinical parameters of metastatic lymph node or distant organ invasion (META− or META+ patients). Primary tumor samples of colorectal carcinoma were analyzed by integrating large-scale phenotypic (flow cytometry, 39 patients) and gene expression (real time reverse transcription-PCR, 103 patients) data sets related to immune and protumoral processes. In META− colorectal cancer primary tumors with high densities of T cells, we observed significant positive correlations between markers of innate immune cells [tumor-associated macrophages, dendritic cells, natural killer (NK) cells, and NKT cells] and markers of early-activated T cells. Significant correlations were also observed between markers of cytotoxic and effector memory T-cell subpopulations. These correlation profiles were absent in tumors with low T-cell infiltrates and were altered in META+ tumors with high T-cell infiltrates. We show that the coexpression of genes mediating cytotoxicity (GNLY) and Th1 adaptive immune responses (IRF1) accurately predicted patient survival independently of the metastatic status. High intratumoral mRNA expression of the proangiogenic mediator vascular endothelial growth factor was associated with significantly reduced survival rates in patients expressing high mRNA levels of GNLY. Investigation of the colorectal cancer primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response. [Cancer Res 2009;69(6):2685–93]

Supplementary Methods, Figures 4-6 from Coordination of Intratumoral Immune Reaction and Human Colorectal Cancer Recurrence

Supplementary Table 1 from Coordination of Intratumoral Immune Reaction and Human Colorectal Cancer Recurrence

Supplementary Table 3 from Coordination of Intratumoral Immune Reaction and Human Colorectal Cancer Recurrence

The tumor microenvironment includes a complex network of immune T-cell subpopulations. In this study, we systematically analyzed the balance between cytotoxic T cells and different subsets of helper T cells in human colorectal cancers and we correlated their impact on disease-free survival. A panel of immune related genes were analyzed in 125 frozen colorectal tumor specimens. Infiltrating cytotoxic T cells, Treg, Th1, and Th17 cells were also quantified in the center and the invasive margin of the tumors. By hierarchical clustering of a correlation matrix we identified functional clusters of genes associated with Th17 (RORC, IL17A), Th2 (IL4, IL5, IL13), Th1 (Tbet, IRF1, IL12Rb2, STAT4), and cytotoxicity (GNLY, GZMB, PRF1). Patients with high expression of the Th17 cluster had a poor prognosis, whereas patients with high expression of the Th1 cluster had prolonged disease-free survival. In contrast, none of the Th2 clusters were predictive of prognosis. Combined analysis of cytotoxic/Th1 and Th17 clusters improved the ability to discriminate relapse. In situ analysis of the density of IL17+ cells and CD8+ cells in tumor tissues confirmed the results. Our findings argue that functional Th1 and Th17 clusters yield opposite effects on patient survival in colorectal cancer, and they provide complementary information that may improve prognosis. Cancer Res; 71(4); 1263–71. ©2011 AACR.

Supplementary Table 2 from Coordination of Intratumoral Immune Reaction and Human Colorectal Cancer Recurrence

Supplementary Figures 1-5, Tables 1-5 from Clinical Impact of Different Classes of Infiltrating T Cytotoxic and Helper Cells (Th1, Th2, Treg, Th17) in Patients with Colorectal Cancer

Because of the function and anatomical environment of the rectum, therapeutic strategies for local advanced rectal cancer (LARC) must deal with two challenging stressors that are a high-risk of local and distal recurrences and a high-risk of poor quality of life (QoL). Over the last three decades, advances in screening tests, therapies, and combined-modality treatment options and strategies have improved the prognosis of patients with LARC. However, owing to the heterogeneous nature of LARC and genetic status, the patient may not respond to a specific therapy and may be at increased risk of side-effects without the life-prolonging benefit. Indeed, each therapy can cause its own side-effects, which may worsen by a combination of treatments resulting in long-term poor QoL. In LARC, QoL has become even more essential with the increasing incidence of rectal cancer in young individuals. Herein, we analyzed the value of the Immunoscore-Biopsy (performed on tumor biopsy at diagnosis) in predicting outcomes, alone or in association with clinical and imaging data, for each therapy used in LARC.

Simple Summary Beyond the TNM-staging system, biomarkers are needed to guide cancer treatments. We provide an overview of the Immunoscore, a standardized immune assay based on quantification by digital pathology of CD3+ and CD8+ cytotoxic T cells in tumor tissues. We discuss the usefulness of the Immunoscore (IS) and biopsies-adapted IS (ISB) biomarkers for prediction of clinical outcome and treatment response in colonic and rectal cancers. Abstract Four decades were needed to progress from the first demonstration of the independent prognostic value of lymphocytes infiltration in rectal cancers to the first recommendation from the international guidelines for the use of a standardized immune assay, namely the “Immunoscore” (IS), to accurately prognosticate colon cancers beyond the TNM-system. The standardization process included not only the IS conceptualization, development, fine-tuning, and validation by a large international consortium, but also a demonstration of the robustness and reproducibility across the world and testing of international norms and their effects on the IS. This is the first step of a major change of paradigm that now perceives cancer as the result of contradicting driving forces, i.e., the tumor expansion and the immune response, interacting dynamically and influencing the prognosis and the response to therapies. This prompted us to evaluate and evidence the capacity of the tumor immune status, as reflected by the IS, to accurately predict chemotherapy responses in an international, randomized cohort study of colon cancer. Moreover, we developed a derived IS performed on initial diagnostic biopsies (ISB) to assess response levels to neoadjuvant therapies. In rectal cancer, ISB was positively correlated with the degree of histologic response to neoadjuvant chemoradiotherapy and identified - alone and even more accurately if combined with clinical data- patients eligible for a noninvasive strategy. Based on these results, we are currently setting up an international cohort for confirmation. The potential role of IS with immunotherapies must be anticipated.

Reports on the prognostic role of programmed death-ligand 1 (PD-L1) expression in rectal cancer are controversial. We investigated expression patterns and changes of PD-L1 in rectal cancer patients undergoing neoadjuvant chemoradiotherapy (CRT). Seventy-two patients diagnosed with rectal cancer and/or treated with fluorouracil-based neoadjuvant CRT at the Department of Internal Medicine III of the Paracelsus Medical University Salzburg (Austria) between January 2003 and October 2012 were included. PD-L1 scoring was performed according to the tumor proportion score (TPS), combined positive score (CPS), and immune cell score (IC). PD-L1 TPS prior to neoadjuvant CRT had a statistically significant impact on survival (median: &le, 1%: 95.4 months (95% CI: 51.8&mdash, not reached) vs. &gt, 1%: not reached, p = 0.03, log-rank). Patients with a PD-L1 TPS &le, 1% prior to and after CRT showed an inferior survival compared to all other patients (median: 56.7 months (95% CI: 51.4&mdash, not reached) vs. not reached, p = 0.005, log-rank). In multivariate analysis, PD-L1 TPS prior to neoadjuvant CRT (&gt, 1% vs. &le, 1%, hazard ratio: 0.29 (95% CI: 0.11&ndash, 0.76), p = 0.01) remained independently associated with survival. In conclusion, low PD-L1 TPS was associated with inferior survival in rectal cancer patients undergoing neoadjuvant CRT. A prospective validation of the prognostic value of PD-L1 expression in rectal cancer patients within a clinical trial is necessitated.

BackgroundNew and fully validated tests need to be brought into clinical practice to improve the estimation of recurrence risk in patients with colon cancer. The aim of this study was to assess the analytical performances of the Immunoscore (IS) and show its contribution to prognosis prediction.MethodsImmunohistochemical staining of CD3+ and CD8+ T cells on adjacent sections of colon cancer tissues were quantified in the core of the tumor and its invasive margin with dedicated IS modules integrated into digital pathology software. Staining intensity across samples collected between 1989 and 2016 (n=595) was measured. The accuracy of the IS workflow was established by comparing optical and automatic counts. Analytical precision of the IS was evaluated within individual tumor block on distant sections and between eligible blocks. The IS interlaboratory reproducibility (n=100) and overall assay precision were assessed (n=3). Contribution of the IS to prediction of recurrence based on clinical and molecular parameters was determined (n=538).ResultsOptical and automatic counts for CD3+ or CD8+ were strongly correlated (r=0.94, pConclusionIS is a robust and validated clinical assay leveraging immune scoring to predict recurrence risk of patient with localized colon cancer. The strong and independent prognostic value of IS should pave the way for it use in clinical practice.

Purpose: No biomarker to personalize treatment in locally advanced rectal cancer (LARC) is currently available. We assessed in LARC whether a diagnostic biopsy-adapted immunoscore (ISB) could predict response to neoadjuvant treatment (nT) and better define patients eligible to an organ preservation strategy (“Watch-and-Wait”). Experimental Design: Biopsies from two independent cohorts (n1 = 131, n2 = 118) of patients with LARC treated with nT followed by radical surgery were immunostained for CD3+ and CD8+ T cells and quantified by digital pathology to determine ISB. The expression of immune-related genes post-nT was investigated (n = 64 patients). Results were correlated with response to nT and disease-free survival (DFS). The ISB prognostic performance was further assessed in a multicentric cohort (n = 73 patients) treated by Watch-and-Wait. Results: ISB positively correlated with the degree of histologic response (P < 0.001) and gene expression levels for Th1 orientation and cytotoxic immune response, post-nT (P = 0.006). ISB high identified patients at lower risk of relapse or death compared with ISB low [HR, 0.21; 95% confidence interval (CI), 0.06–0.78; P = 0.009]. Prognostic performance of ISB for DFS was confirmed in a validation cohort. ISB was an independent parameter, more informative than pre- (P < 0.001) and post-nT (P < 0.05) imaging to predict DFS. ISB combined with imaging post-nT discriminated very good responders that could benefit from organ preservation strategy. In the “Watch-and-Wait” cohort (n = 73), no relapse was observed in patients with ISB high (23.3%). Conclusions: ISB predicts response to nT and survival in patients with LARC treated by surgery. Its usefulness in the selection of patients eligible for a Watch-and-Wait strategy is strongly suggested.

International audience; Background: The Immunoscore (IS), which prognostically classifies stage I–III colon cancer (CC) patients, was evaluated in the International Duration Evaluation of Adjuvant Therapy (IDEA) France cohort study investigating 3 versus 6 months of oxaliplatin-based adjuvant chemotherapy in stage III CC patients.Patients and methods: Densities of CD3+ and CD8+ T cells in the tumor and invasive margin were determined by immunohistochemistry, quantified by digital pathology, and converted to IS. Mismatch repair status was determined by immunohistochemistry or by pentaplex PCR. Prediction of disease-free survival (DFS) by IS was analyzed by a multivariable Cox regression model in each study arm. Harrell's C-statistics were used to investigate the IS performance.Results: Samples of 1322 patients were available. IS Low, Intermediate (Int), and High were observed in 43.6%, 47.0%, and 9.4% of patients, respectively. IS Low identified patients at higher risk of relapse or death compared with Int + High [hazard ratio (HR) = 1.54; 95% confidence interval (CI) 1.24–1.93, P = 0.0001]. The 3-year DFS was 66.80% (95% CI 62.23–70.94) for IS Low and 77.14% (95% CI 73.50–80.35) for IS Int + High. In multivariable analysis, IS remained significantly independently associated with DFS (P = 0.003) when adjusted for sex, histological grade, T/N stage, and microsatellite instability. For mFOLFOX6-treated patients (91.6% of the cohort), a statistical significant interaction was observed for the predictive value of IS for treatment duration (3 versus 6 months) in terms of DFS (P = 0.057). IS Int + High significantly predicted benefit of 6 months of treatment (HR = 0.53; 95% CI 0.37–0.75; P = 0.0004), including clinically low- and high-risk stage III CC (all P < 0.001). Conversely, patients with IS Low (46.4%) did not significantly benefit from the 6-month mFOLFOX6 versus the 3-month mFOLFOX6.Conclusions: The prognostic value of IS for DFS was confirmed in patients with stage III CC treated with oxaliplatin-based chemotherapy. Its predictive value for DFS benefit of longer duration of mFOLFOX6 adjuvant treatment was found in IS Int + High. These results will be validated in an external independent cohort. ClinicalTrials.gov registration: NCT03422601; EudraCT Number: 2009-010384-16.

PURPOSE The purpose of this study was to evaluate the prognostic value of Immunoscore in patients with stage III colon cancer (CC) and to analyze its association with the effect of chemotherapy on time to recurrence (TTR). METHODS An international study led by the Society for Immunotherapy of Cancer evaluated the predefined consensus Immunoscore in 763 patients with American Joint Committee on Cancer/Union for International Cancer Control TNM stage III CC from cohort 1 (Canada/United States) and cohort 2 (Europe/Asia). CD3+ and cytotoxic CD8+ T lymphocyte densities were quantified in the tumor and invasive margin by digital pathology. The primary end point was TTR. Secondary end points were overall survival (OS), disease-free survival (DFS), prognosis in microsatellite stable (MSS) status, and predictive value of efficacy of chemotherapy. RESULTS Patients with a high Immunoscore presented with the lowest risk of recurrence, in both cohorts. Recurrence-free rates at 3 years were 56.9% (95% CI, 50.3% to 64.4%), 65.9% (95% CI, 60.8% to 71.4%), and 76.4% (95% CI, 69.3% to 84.3%) in patients with low, intermediate, and high immunoscores, respectively (hazard ratio [HR; high v low], 0.48; 95% CI, 0.32 to 0.71; P = .0003). Patients with high Immunoscore showed significant association with prolonged TTR, OS, and DFS (all P < .001). In Cox multivariable analysis stratified by participating center, Immunoscore association with TTR was independent (HR [high v low], 0.41; 95% CI, 0.25 to 0.67; P = .0003) of patient’s sex, T stage, N stage, sidedness, and microsatellite instability status. Significant association of a high Immunoscore with prolonged TTR was also found among MSS patients (HR [high v low], 0.36; 95% CI, 0.21 to 0.62; P = .0003). Immunoscore had the strongest contribution χ2 proportion for influencing survival (TTR and OS). Chemotherapy was significantly associated with survival in the high-Immunoscore group for both low-risk (HR [chemotherapy v no chemotherapy], 0.42; 95% CI, 0.25 to 0.71; P = .0011) and high-risk (HR [chemotherapy v no chemotherapy], 0.5; 95% CI, 0.33 to 0.77; P = .0015) patients, in contrast to the low-Immunoscore group ( P > .12). CONCLUSION This study shows that a high Immunoscore significantly associated with prolonged survival in stage III CC. Our findings suggest that patients with a high Immunoscore will benefit the most from chemotherapy in terms of recurrence risk.

International audience; Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies.

While our knowledge about the roles of microbes and viruses in the ocean has increased tremendously due to recent advances in genomics and metagenomics, research on marine microbial eukaryotes and zooplankton has benefited much less from these new technologies because of their larger genomes, their enormous diversity, and largely unexplored physiologies. Here, we use a metatranscriptomics approach to capture expressed genes in open ocean Tara Oceans stations across four organismal size fractions. The individual sequence reads cluster into 116 million unigenes representing the largest reference collection of eukaryotic transcripts from any single biome. The catalog is used to unveil functions expressed by eukaryotic marine plankton, and to assess their functional biogeography. Almost half of the sequences have no similarity with known proteins, and a great number belong to new gene families with a restricted distribution in the ocean. Overall, the resource provides the foundations for exploring the roles of marine eukaryotes in ocean ecology and biogeochemistry., Marine microbial eukaryotes and zooplankton display enormous diversity and largely unexplored physiologies. Here, the authors use metatranscriptomics to analyze four organismal size fractions from open-ocean stations, providing the largest reference collection of eukaryotic transcripts from any single biome.

Contains fulltext : 193579.pdf (Publisher’s version ) (Closed access) BACKGROUND: The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS: An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrell's C-statistics was used to assess model performance. FINDINGS: Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0.97 for colon tumour; r=0.97 for invasive margin; p

Objectives: The aim of the study was to determine the molecular mechanisms underlying the quasi-equilibrium between HIV and its host in the model of functional cure represented by elite controllers who spontaneously maintain exceptionally low levels of HIV reservoirs. Design: Whole-genome transcriptional study and quantification of the cell-associated HIV DNA and HIV RNA levels of the four major resting CD4 þ T-cell subsets in HIV-1infected elite controllers, viremic long-term nonprogressors (vir-LTNPs), and uninfected individuals. Methods: We compared the whole-genome transcriptional profiles (ArrayExpress accession number E-MTAB-1480) of the four major resting CD4 þ T-cell subsets [naive (TN), central-memory (TCM), transitional-memory (TTM), and effector-memory (TEM)] from 14 HIV-1-infected individuals including seven elite controllers (E-LTNPs) and seven vir-LTNPs, and from seven uninfected individuals. The HIV-1 cellular DNA and mRNA levels were quantified in parallel in each sorted subset. Results: Host genetranscriptomes followed subset differentiation and viremia exceptin E-LTNPs wherein TCM, the main CD4 þ cell compartment, showed the highest activity with three specific signatures involving overexpression of T-cell receptor and costimulation signaling pathways, overexpression of the PRDM-1/Blimp-1 transcriptional repressor, and downmodulation of type-I IFN-related genes. Among subsets, the PRDM1/Blimp-1 upregulation was associated with lower levels of both cellular HIV-DNA and HIV mRNA levels. Conclusion: This unique Blimp-1 transcriptional repressor signature and the contrast between host and virus transcriptional activities in TCM from elite controllers suggest Blimp-1 might be involved in controlling the HIV reservoirs in the key TCM subset. 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2014, 28:1567‐1577

SummaryThe complex interactions between tumors and their microenvironment remain to be elucidated. Combining large-scale approaches, we examined the spatio-temporal dynamics of 28 different immune cell types (immunome) infiltrating tumors. We found that the immune infiltrate composition changed at each tumor stage and that particular cells had a major impact on survival. Densities of T follicular helper (Tfh) cells and innate cells increased, whereas most T cell densities decreased along with tumor progression. The number of B cells, which are key players in the core immune network and are associated with prolonged survival, increased at a late stage and showed a dual effect on recurrence and tumor progression. The immune control relevance was demonstrated in three endoscopic orthotopic colon-cancer mouse models. Genomic instability of the chemokine CXCL13 was a mechanism associated with Tfh and B cell infiltration. CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis correlating with survival. This integrative study reveals the immune landscape in human colorectal cancer and the major hallmarks of the microenvironment associated with tumor progression and recurrence.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) tumor, nodes, metastasis (TNM) classification system based on tumor features is used for prognosis estimation and treatment recommendations in most cancers. However, the clinical outcome can vary significantly among patients within the same tumor stage and TNM classification does not predict response to therapy. Therefore, many efforts have been focused on the identification of new markers. Multiple tumor cell-based approaches have been proposed but very few have been translated into the clinic. The recent demonstration of the essential role of the immune system in tumor progression has allowed great advances in the understanding of this complex disease and in the design of novel therapies. The analysis of the immune infiltrate by imaging techniques in large patient cohorts highlighted the prognostic impact of the in situ immune cell infiltrate in tumors. Moreover, the characterization of the immune infiltrates (e.g. type, density, distribution within the tumor, phenotype, activation status) in patients treated with checkpoint-blockade strategies could provide information to predict the disease outcome. In colorectal cancer, we have developed a prognostic score ('Immunoscore') that takes into account the distribution of the density of both CD3(+) lymphocytes and CD8(+) cytotoxic T cells in the tumor core and the invasive margin that could outperform TNM staging. Currently, an international retrospective study is under way to validate the Immunoscore prognostic performance in patients with colon cancer. The use of Immunoscore in clinical practice could improve the patients' prognostic assessment and therapeutic management.

In the fine balance between tumor invasion and our defensive systems, the role played by the adaptive immune response at the tumor site is critical. Beyond the fact that all the immune components of the innate and adaptive response can be observed to varying degrees in the tumor microenvironment, it appears that a high density of T cytotoxic and memory lymphocytes, in a context of Th1 immune orientation in the tumor and its invasion front, provides a prognostic marker of paramount importance for colorectal cancer and more generally all solid tumors. The understanding of the role of immunity in cancer, tailored during one century of intensive research, has led to a complete paradigm shift.based on a sharp dissection In order to show the major impact of this conceptual revolution, we herein retrace through the example of colorectal cancer, how an effective immune test, namely the "Immunoscore", has been developed. We also provide up to date data demonstrating the capacity of the Immunoscore to prognosticate with a better accuracy than the TME classification clinical outcomes and to guide therapeutic strategies.

The tumor microenvironment includes a complex network of immune T-cell subpopulations. In this study, we systematically analyzed the balance between cytotoxic T cells and different subsets of helper T cells in human colorectal cancers and we correlated their impact on disease-free survival. A panel of immune related genes were analyzed in 125 frozen colorectal tumor specimens. Infiltrating cytotoxic T cells, Treg, Th1, and Th17 cells were also quantified in the center and the invasive margin of the tumors. By hierarchical clustering of a correlation matrix we identified functional clusters of genes associated with Th17 (RORC, IL17A), Th2 (IL4, IL5, IL13), Th1 (Tbet, IRF1, IL12Rb2, STAT4), and cytotoxicity (GNLY, GZMB, PRF1). Patients with high expression of the Th17 cluster had a poor prognosis, whereas patients with high expression of the Th1 cluster had prolonged disease-free survival. In contrast, none of the Th2 clusters were predictive of prognosis. Combined analysis of cytotoxic/Th1 and Th17 clusters improved the ability to discriminate relapse. In situ analysis of the density of IL17+ cells and CD8+ cells in tumor tissues confirmed the results. Our findings argue that functional Th1 and Th17 clusters yield opposite effects on patient survival in colorectal cancer, and they provide complementary information that may improve prognosis. Cancer Res; 71(4); 1263–71. ©2011 AACR.

International audience; In large regions of the open ocean, iron is a limiting resource for phytoplankton. The reduction of iron quota and the recycling of internal iron pools are among the diverse strategies that phytoplankton have evolved to allow them to grow under chronically low ambient iron levels. Phytoplankton species also have evolved strategies to cope with sporadic iron supply such as long-term storage of iron in ferritin. In the picophytoplanktonic species Ostreococcus we report evidence from observations both in the field and in laboratory cultures that ferritin and the main iron-binding proteins involved in photosynthesis and nitrate assimilation pathways show opposite diurnal expression patterns, with ferritin being maximally expressed during the night. Biochemical and physiological experiments using a ferritin knock-out line subsequently revealed that this protein plays a central role in the diel regulation of iron uptake and recycling and that this regulation of iron homeostasis is essential for cell survival under iron limitation.

The role of the adaptive immune response in controlling the growth and recurrence of human tumors has been controversial. We characterized the tumor-infiltrating immune cells in large cohorts of human colorectal cancers by gene expression profiling and in situ immunohistochemical staining. Collectively, the immunological data (the type, density, and location of immune cells within the tumor samples) were found to be a better predictor of patient survival than the histopathological methods currently used to stage colorectal cancer. The results were validated in two additional patient populations. These data support the hypothesis that the adaptive immune response influences the behavior of human tumors. In situ analysis of tumor-infiltrating immune cells may therefore be a valuable prognostic tool in the treatment of colorectal cancer and possibly other malignancies.

Purpose: To determine whether the tumor immune infiltrate, as recently evaluated with the Immunoscore methodology, could be a useful prognostic marker in patients with rectal cancers. Experimental design: The influence of the immune infiltrate on patient's outcome was investigated in patients with or without preoperative chemoradiation therapy (pCRT). The density of total (CD3+) and cytotoxic (CD8+) T lymphocytes was evaluated by immunohistochemistry and quantified by a dedicated image analysis software in surgical specimens of patients with rectal cancer (n = 111) who did not receive pCRT and in tumor biopsies performed before pCRT from additional 55 patients. The results were correlated with tumor recurrence, patient's survival, and response to pCRT. Results: The densities of CD3+ and CD8+ lymphocytes and the associated Immunoscore (from I0 to I4) were significantly correlated with differences in disease-free and overall survival (HR, 1.81 and 1.72, respectively; all P < 0.005). Cox multivariate analysis supports the advantage of the Immunoscore compared with the tumor–node–metastasis (TNM) staging in predicting recurrence and survival (all P < 0.001). Lymph node ratio added information in a prognostic model (all P < 0.05). In addition, high infiltration of CD3+ and CD8+ lymphocytes in tumor biopsies was associated with downstaging of the tumor after pCRT (CD3+ cells; Fisher exact test P = 0.01). Conclusions: The Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of response to pCRT. Clin Cancer Res; 20(7); 1891–9. ©2014 AACR.

BACKGROUND & AIMS: Colorectal cancer is a complex disease involving immune defense mechanisms within the tumor. Herein, we used data integration and biomolecular network reconstruction to generate hypotheses about the mechanisms underlying immune responses in colorectal cancer that are relevant to tumor recurrence. METHODS: Mechanistic hypotheses were formulated on the basis of data from 108 patients and tested using different assays (gene expression, phenome mapping, tissue-microarrays, T-cell receptor [TCR] repertoire). RESULTS: This integrative approach revealed that chemoattraction and adhesion play important roles in determining the density of intratumoral immune cells. The presence of specific chemokines (CX3CL1, CXCL10, CXCL9) and adhesion molecules (ICAM1, VCAM1, MADCAM1) correlated with different subsets of immune cells and with high densities of T-cell subpopulations within specific tumor regions. High expression of these molecules correlated with prolonged disease-free survival. Moreover, the expression of certain chemokines associated with particular TCR repertoire and specific TCR use predicted patient survival. CONCLUSIONS: Data integration and biomolecular network reconstruction is a powerful approach to uncover molecular mechanisms. This study shows the utility of this approach for the investigation of malignant tumors and other diseases. In colorectal cancer, the expression of specific chemokines and adhesion molecules were found as being critical for high densities of T-cell subsets within the tumor and associated with particular TCR repertoire. Intratumoral-specific TCR use correlated with the prognosis of the patients.

A role for the immune system in controlling the progression of solid tumors has been established in several mouse models. However, the effect of immune responses and tumor escape on patient prognosis in the context of human cancer is poorly understood. Here, we investigate the cellular and molecular parameters that could describe in situ immune responses in human colorectal cancer according to clinical parameters of metastatic lymph node or distant organ invasion (META− or META+ patients). Primary tumor samples of colorectal carcinoma were analyzed by integrating large-scale phenotypic (flow cytometry, 39 patients) and gene expression (real time reverse transcription-PCR, 103 patients) data sets related to immune and protumoral processes. In META− colorectal cancer primary tumors with high densities of T cells, we observed significant positive correlations between markers of innate immune cells [tumor-associated macrophages, dendritic cells, natural killer (NK) cells, and NKT cells] and markers of early-activated T cells. Significant correlations were also observed between markers of cytotoxic and effector memory T-cell subpopulations. These correlation profiles were absent in tumors with low T-cell infiltrates and were altered in META+ tumors with high T-cell infiltrates. We show that the coexpression of genes mediating cytotoxicity (GNLY) and Th1 adaptive immune responses (IRF1) accurately predicted patient survival independently of the metastatic status. High intratumoral mRNA expression of the proangiogenic mediator vascular endothelial growth factor was associated with significantly reduced survival rates in patients expressing high mRNA levels of GNLY. Investigation of the colorectal cancer primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response. [Cancer Res 2009;69(6):2685–93]

Summary: We have developed ClueGO, an easy to use Cytoscape plug-in that strongly improves biological interpretation of large lists of genes. ClueGO integrates Gene Ontology (GO) terms as well as KEGG/BioCarta pathways and creates a functionally organized GO/pathway term network. It can analyze one or compare two lists of genes and comprehensively visualizes functionally grouped terms. A one-click update option allows ClueGO to automatically download the most recent GO/KEGG release at any time. ClueGO provides an intuitive representation of the analysis results and can be optionally used in conjunction with the GOlorize plug-in. Availability: http://www.ici.upmc.fr/cluego/cluegoDownload.shtml Contact: jerome.galon@crc.jussieu.fr Supplementary information: Supplementary data are available at Bioinformatics online.