Search

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.

Supplementary Figures

Supplementary Tables

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

SARS-CoV-2 infection leads to a highly variable clinical evolution, ranging from asymptomatic to severe disease with acute respiratory distress syndrome, requiring intensive care units (ICU) admission. The optimal management of hospitalized patients has become a worldwide concern and identification of immune biomarkers predictive of the clinical outcome for hospitalized patients remains a major challenge. Immunophenotyping and transcriptomic analysis of hospitalized COVID-19 patients at admission allow identifying the two categories of patients. Inflammation, high neutrophil activation, dysfunctional monocytic response and a strongly impaired adaptive immune response was observed in patients who will experience the more severe form of the disease. This observation was validated in an independent cohort of patients. Using in silico analysis on drug signature database, we identify differential therapeutics that specifically correspond to each group of patients. From this signature, we propose a score—the SARS-Score—composed of easily quantifiable biomarkers, to classify hospitalized patients upon arrival to adapt treatment according to their immune profile.

3517 Background: The WW strategy for patients with rectal cancer who achieved a clinical complete response (cCR) after neoadjuvant therapy (nT) allows to avoid major resection and the associated morbidity and mortality. Standardized criteria to select and monitor WW patients, including biomarkers predicting recurrence after nT, are lacking. The prognostic impact of the immune infiltrate in colorectal cancers is now demonstrated and has been implemented into clinics through the Immunoscore, the first standardized digital-pathology-based assay, recommended by academic institutions. We evidenced that an Immunoscore adapted to biopsies (ISB) performed at diagnosis, predicts the response to nT and the risk of recurrence after nT. Its clinical utility was suggested in a test cohort of WW patients (El Sissy et al., Clin Cancer Res 2020). The aim of this study was to confirm the ability of the ISB to predict clinical outcomes, improve patients’ eligibility for the WW strategy, and optimize a follow-up schedule. Methods: A total of 304 WW patients from 10 centers across 7 countries were included. Tumor biopsies before treatment were immunostained for CD3+ and CD8+ T-cells and converted to ISB using the pre-defined cut-off. The primary endpoint was time-to-recurrence (TTR). Secondary endpoint was disease-free-survival (DFS). As immune response originates in draining lymph nodes, signs of immune activation were carried out in lymph nodes of additional patients managed by radical surgery with complete pathological response (pCR; n = 12) or non-pCR (n = 12) by 3' RNA-Seq and immunofluorescence technologies. Results: High-ISB patients presented with the lowest risk of recurrence after WW. 5-year recurrence-free rates were 97% (92%-100%), 61% (49%-76%), and 56% (44%-73%) with ISB High, Intermediate, and Low, respectively (HR [Low-vs-High] = 14.3, 95% CI 1.8-100). In patients with cCR after nT (n = 209), High-ISB showed a significant association with prolonged TTR and DFS (Logrank P = 0.005 and P = 0.006, respectively). When ISB was evaluated as a continuous variable, the risk of recurrence was increasing along with decreasing ISB (Wald tests, all P < 0.005). In multivariate analyses, ISB was independent of age, sex, location, and cTNM stage and was the single parameter correlated with TTR (HR [ISB High-vs-Low] = 0.08, 95% CI 0.01-0.6; P = 0.015) and DFS (P = 0.013). Unlike for patients with cCR, no difference according to ISB was observed for those with incomplete response (n = 41) or treated with brachytherapy (n = 34). Finally, intranodal signs of T-cell and B-cell activation were only evidenced in patients with pCR. Conclusions: ISB provides a reliable biomarker to predict clinical outcomes, improve eligibility, and optimize patients’ follow-up. Intranodal T-cell and B-cell activation further supports the immune benefit of both organ and lymph node preservation.

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.

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.

The role of the immune response at the tumor site is now recognized as crucial in the clinical course of patients with cancer. The importance of the immune cell type, their functional orientation, their density and location within the tumor's regions (tumor/invasion margin) has recently been shown and were grouped together under the term "immune contexture". A strong infiltration by cytotoxic and memory T cells in a Th1-polarized tumor microenvironment appears to have a major prognosis impact. A test called Immunoscore taking into account these various parameters has been suggested to measure in a simple, reproducible and robust manner the intra- and peritumoral immune response. The prognostic value of Immunoscore has recently been validated in colon cancers by a large international retrospective study under the aegis of the Society for Immunotherapy of Cancer (SITC). The Immunoscore could have several potential clinical applications such as prognostic as well as theranostic.

Immunotherapies targeting co-inhibitory receptors recently open a new promising approach of cancer treatment. Indeed, an objective clinical response was observed after treatment by anti-CTLA-4 and anti-PD-1 in many indications but the treatment still failed in 70 to 80 % of cases treated. Given the adverse effects and the high cost of these therapies, there is a need for the development of biomarkers. This review focus on potential predictive biomarkers. In peripheral blood, high level of Il-2 soluble receptor at baseline and absence of ICOS+ CD4-T lymphocytes induction may be associated with the absence of clinical response for melanoma patients treated by ipilimumab (anti-CTLA-4). PD-L1 - PD-1 ligand- expression on cancer lung adenocarcinoma and melanoma is associated with an improved clinical response to anti-PD-1/PD-L1. Nevertheless, a standardization of the biological assays is needed before a clinical translation. CD8-T cell tumor infiltration seems to be a prerequisite to an optimal clinical response after anti-PD-1/PD-L1 administration. In situ high mutational load is associated with a CD8-T cell infiltration and a higher rate of anti-PD-1 and anti-CTLA-4 response. If we consider a more holistic approach, the role of the gut microbiota in the response to these treatments is now well established in pre-clinical experiments. The universal marker is not identified so far, but the reliable marker should be in the tumor compartment and combining multiples markers could be suitable to predict response in different contexts.

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.

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

2628 Background: We investigated whether an adaptation to rectal biopsies of the recently validated consensus Immunoscore, could predict the response to neoadjuvant treatment and delineate clinical responders that could benefit from a “Watch and Wait” (W&W) strategy with acceptable outcomes. Methods: Initial biopsies from 273 patients with locally advanced rectal cancer (LARC) treated by neoadjuvant chemoradiotherapy (nCRT) followed by Total Mesorectal Excision (TME), were immunostained for CD3+ and cytotoxic CD8+ T cells and quantified by digital pathology to determine the Immunoscore within pre-treatment Biopsy (ISB). Expression level of 44 immune related genes post-neoadjuvant treatment was investigated by Nanostring technology (n = 64 patients). Results were correlated with response to neoadjuvant treatment, disease free survival (DFS) and time to recurrence (TTR). Prognostic performance of ISB was finally assessed in 73 LARC treated by W&W strategy. Results: ISB Low, Intermediate and High were respectively observed in 23.3, 50.4 and 26.3 % of the cohort. ISB was positively and significantly correlated with the response to nCRT, as evaluated by Dworak classification (P = .0034), ypTNM (P = .0003), down-staging (P = .0014), and neoadjuvant rectal (NAR) score, (P < .0001). ISB status was also positively associated with the degree of local immune activation post-neoadjuvant treatment. ISB High patients were at low risk of relapse, with 5-year DFS rates of 81.1 % (CI, 71.3-92.1 %) as compared to 57.8 % (CI, 45.9-72.9 %) in ISB low patients. In multivariate analysis, ISB was the only significant parameter at presentation associated with DFS (High vs Low: P = .001). Among W&W patients, significant difference was observed for TTR according to ISB status (High vs Low: P = .025). Conclusions: ISB could provide a reliable estimate of the response to nCRT and risk of recurrence in LARC patients' treated by TME or W&W strategy.

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.

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.

Although distant metastases account for most of the deaths in cancer patients, fundamental questions regarding mechanisms that promote or inhibit metastasis remain unanswered. We show the impact of mutations, genomic instability, lymphatic and blood vascularization, and the immune contexture of the tumor microenvironment on synchronous metastases in large cohorts of colorectal cancer patients. We observed large genetic heterogeneity among primary tumors, but no major differences in chromosomal instability or key cancer-associated mutations. Similar patterns of cancer-related gene expression levels were observed between patients. No cancer-associated genes or pathways were associated with M stage. Instead, mutations of FBXW7 were associated with the absence of metastasis and correlated with increased expression of T cell proliferation and antigen presentation functions. Analyzing the tumor microenvironment, we observed two hallmarks of the metastatic process: decreased presence of lymphatic vessels and reduced immune cytotoxicity. These events could be the initiating factors driving both synchronous and metachronous metastases. Our data demonstrate the protective impact of the Immunoscore, a cytotoxic immune signature, and increased marginal lymphatic vessels, against the generation of distant metastases, regardless of genomic instability.

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.

The role of tumor-infiltrating immune cells in the early metastatic invasion of colorectal cancer is unknown.We studied pathological signs of early metastatic invasion (venous emboli and lymphatic and perineural invasion) in 959 specimens of resected colorectal cancer. The local immune response within the tumor was studied by flow cytometry (39 tumors), low-density-array real-time polymerase-chain-reaction assay (75 tumors), and tissue microarrays (415 tumors).Univariate analysis showed significant differences in disease-free and overall survival according to the presence or absence of histologic signs of early metastatic invasion (P0.001). Multivariate Cox analysis showed that an early conventional pathological tumor-node-metastasis stage (P0.001) and the absence of early metastatic invasion (P=0.04) were independently associated with increased survival. As compared with tumors with signs of early metastatic invasion, tumors without such signs had increased infiltrates of immune cells and increased levels of messenger RNA (mRNA) for products of type 1 helper effector T cells (CD8, T-BET [T-box transcription factor 21], interferon regulatory factor 1, interferon-gamma, granulysin, and granzyme B) but not increased levels of inflammatory mediators or immunosuppressive molecules. The two types of tumors had significant differences in the levels of expression of 65 combinations of T-cell markers, and hierarchical clustering showed that markers of T-cell migration, activation, and differentiation were increased in tumors without signs of early metastatic invasion. The latter type of tumors also had increased numbers of CD8+ T cells, ranging from early memory (CD45RO+CCR7-CD28+CD27+) to effector memory (CD45RO+CCR7-CD28-CD27-) T cells. The presence of high levels of infiltrating memory CD45RO+ cells, evaluated immunohistochemically, correlated with the absence of signs of early metastatic invasion, a less advanced pathological stage, and increased survival.Signs of an immune response within colorectal cancers are associated with the absence of pathological evidence of early metastatic invasion and with prolonged survival.

3599 Background: Pre-operative CRT followed by total mesorectal excision (TME) is nowadays the standard of care for patient with LARC (cT3-T4N0 or cTxN+). Currently, pathologic complete response occurs in +/- 15% after CRT. Colorectal cancer T-cell infiltration is a strong prognostic factor for survival after primary tumor resection. Our aim was to determine whether T-cell infiltration in PTB could be predictive of tumor response and relapse after CRT + TME. Methods: Between 1999 and 2012, patients with LARC who underwent CRT + TME and with available clinical follow-up and PTB (with sufficient tumor cells density) were identified at the Cliniques universitaires St-Luc. The density of CD3 (T cells) and CD8 (cytotoxic) was quantified on immunostained PTB slides and analyzed with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Cumulative disease-free survival (DFS) was performed using the Kaplan-Meier estimator and compared by log-rank tests. Cox regression we used for uni- and multi-variate analysis. P value of less than 0.05 was considered statistically significant. Results: 154 patients (sex ratio M/F 1.8; mean age 65 years-old; upper (20%), mid (29%) and low rectum (51%), synchronous metastases (11%)) were analyzed. High CD3 and CD8 PTB densities were significantly associated with a higher pathological response (Dworak 3-4) and lower ypTNM stage after CRT +TME (p < 0,05). Higher CD3 and CD8 PTB densities were associated with higher patient DFS (CD3: HR = 2,30 (CI95%:1,15-4,59) p = 0,02; CD8: HR = 1,95 (CI95%: 1,01-3,75) p = 0,04). These results were confirmed in uni and multivariate analysis. CD3 and CD8 PTB densities added to pathological response (ypTNM/Dworak) but also clinical response (ycTNM) after CRT + TME increases significantly the accuracy prediction of tumor relapse. Conclusions: Pretherapeutic T-cell infiltration of LARC is predictive of tumor response and relapse after CRT +TME. This biomarker could be helpful for patient treatment decision. It must be validated in larger patient cohorts.

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.

471 Background: To date the anatomic extent of tumor (TNM classifications) has been by far the most important factors to predict the prognosis of cancer patients. However, the impact of immune responses and tumor escape on patient prognosis in human cancer is poorly understood. Methods: We analyzed large retrospective cohorts of colorectal cancer patients. Results: We showed that tumors from human colorectal cancer with a high density of infiltrating memory and effector memory T-cells (T-EM) are less likely to disseminate to lymphovascular and perineural structures and to regional lymph-nodes (New Engl J Med, 2005). We showed that the combination of immune parameters associating the nature, the density, the functional orientation and the location of immune cells within the tumor was essential to accurately define the impact of the local host immune reaction on patients prognosis (Science, 2006). We proposed to define these immune criteria as “immune contexture.” Analysis of patients with early-stage colorectal cancer confirmed the major role of cyotoxic effector T cells in predicting the prognosis of the patients (J Clin Oncol, 2009). Investigation of the primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response. We described four major immune coordination profiles within primary tumors depending on the balance between tumor escape and immune coordination. Recent advances analyzing mechanisms responsible for lymphocytic infiltration will be discussed. Conclusions: The density and the immune-cell location within the tumor have a prognostic value that is superior of those of the TNM classifications. Tumor invasion is statistically dependent on the host-immune reaction. No significant financial relationships to disclose.

To date the anatomic extent of tumor (TNM classifications) has been by far the most important factors to predict the prognosis of colorectal cancer patients. However, the impact of immune responses and tumor escape on patient prognosis in human cancer is poorly understood. We showed that tumors from human colorectal cancer with a high density of infiltrating memory and effector memory T-cells (TEM) are less likely to disseminate to lymphovascular and perineural structures and to regional lymph-nodes. We showed that the combination of immune parameters associating the nature, the density, the functional orientation and the location of immune cells within the tumor was essential to accurately define the impact of the local host immune reaction on patients prognosis. We proposed to define these immune criteria as "immune contexture". Investigation of the CRC primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response. We described four major immune coordination profiles within CRC primary tumors depending on the balance between tumor escape and immune coordination. In conclusion, the density and the immune-cell location within tumor regions had a prognostic value that was superior of those of the TNM classifications. Tumor invasion was dependent on the host immune reaction.

Implications immunologiques potentielles du curage ganglionnaire : Exemple du cancer colorectal F. Pages, A. Berger, F. Zinzindohoue, A. Kirilovsky, J. Galon, W.-H. Fridman La chirurgie du cancer colorectal comporte un curage ganglionnaire qui complete la resection tumorale et permet une evaluation pronostique basee sur l’analyse histologique de l’extension tumorale. Les investigations de recherche ont montre la presence d’une reaction immunitaire au site tumoral qui atteste de la persistance d’un dialogue entre la tumeur et nos systemes de defense. Le ganglion constitue une structure immunitaire de premiere importance pouvant initier une reponse de l’hote vis-a-vis de sa tumeur, ou a l’inverse participer a un etat local d’immunosuppression. Cette revue detaille les donnees actuellement disponibles sur le statut immunitaire intra-tumoral et ganglionnaire des cancers colorectaux, et tente d’evaluer les implications immunologiques potentielles du curage ganglionnaire.