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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.

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.

Background: The biological diagnostics of antiphospholipid syndrome (APS) takes into account the persistent positivity for anticardiolipin and/or anti-β2GP1 antibodies and/or presence of lupus anticoagulant (LA). However, some non-conventional antiphospholipid antibodies have emerged that could help in the diagnosis of APS. Objectives: To study the potential usefulness of non-conventional antiphospholipid antibodies in clinical practice. Methods: Eighty-seven patients, aged from 15 to 92 years were included and classified in following groups: 41 patients positive for the conventional antibodies with clinical criterion of APS (31 with primary APS and 10 secondary), 17 seronegative APS (SNAPS) patients (i.e., persistent negativity for the conventional antibodies with a strong clinical suspicion of APS), 11 asymptomatic antiphospholipid antibodies carriers (i.e., persistent positivity for the conventional antibodies without clinical evidence of APS), and 18 patients presenting with a first thrombotic or obstetrical event. IgG and IgM were detected to the following antigens: phosphatidylserine/prothrombin (PS/PT) by ELISA, and phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl-glycerol, phosphatidyl-inositol, phosphatidylserine, annexin V, prothrombin by immunodot. Anti-β2GP1 IgA, and anti-β2GP1 domain 1 IgG were detected by chemiluminescence. Results: Positivity for the non-conventional antibodies was correlated with APS severity; patients with catastrophic APS (CAPS) being positive for 10.7 (Median, Range: 5-14) non-conventional antibodies. 9/17 seronegative patients were positive for at least one of non-conventional antibodies. A study of non-supervised hierarchical clustering of all markers revealed that anti-PS/PT antibodies showed high correlation with the presence of LA. All patients with APS triple positivity (highest risk profile) exhibited also persistent positivity for anti-PS/PT antibodies. Conclusions: Our data obtained from a prospective cohort constituted mainly by patients with primary APS, suggest that non-conventional APS antibodies may be useful for patients classified as SNAPS. They demonstrate the potential value of aPS/PT antibodies as a strong marker of APS. We propose that anti-PS/PT antibodies could be a surrogate APS biological marker of LA to classify in high-risk profile patients treated by direct oral anticoagulants (DOACs), in whom LA detection cannot be achieved.

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

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.

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.

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.

Purpose The prognosis of patients with colorectal cancer has sometimes proved uncertain; thus, the prognostic significance of immune criteria was compared with that of the tumor extension criteria using the American Joint Committee on Cancer/International Union Against Cancer–TNM (AJCC/UICC-TNM) staging system. Patients and Methods We studied the intratumoral immune infiltrates in the center of the tumor and in the invasive margin of 599 specimens of stage I to IV colorectal cancers from two independent cohorts. We analyzed these findings in relation to the degree of tumor extension and to the frequency of recurrence. Results Growth of the primary tumor and metastatic spread were associated with decreased intratumoral immune T-cell densities. Sixty percent of patients with high densities of CD8+ cytotoxic T-lymphocyte infiltrate presented with stage Tis/T1 tumor, whereas no patients with low densities presented with such early-stage tumor. In patients who did not relapse, the density of CD8 infiltrates was inversely correlated with T stage. In contrast, in patients whose tumor recurred, the number of CD8 cells was low regardless of the T stage of the tumor. Univariate analysis showed that the immune score was significantly associated with differences in disease-free, disease-specific, and overall survival (hazard ratio [HR], 0.64, 0.60, and 0.70, respectively; P < .005). Time-dependent receiver operating characteristic curve analysis illustrated the predictive accuracy of the immune parameters (c-index = 65.3%, time-dependent c-index [Cτ] = 66.5%). A final stepwise model for Cox multivariate analysis supports the advantage of the immune score (HR, 0.64; P < .001; Cτ = 67.9%) compared with histopathologic features in predicting recurrence as well as survival. Conclusion Assessment of CD8+ cytotoxic T lymphocytes in combined tumor regions provides an indicator of tumor recurrence beyond that predicted by AJCC/UICC-TNM staging.

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.

Purpose Many patients who present with early-stage colorectal cancer (International Union Against Cancer TNM stages I and II) are nevertheless at high risk of relapse. We hypothesized that intratumoral immune reaction could influence their prognosis. Patients and Methods The intratumoral immune reaction was investigated in 29 tumors by large-scale real-time polymerase chain reaction. Cytotoxic (CD8) and memory (CD45RO) T cells were quantified by immunohistochemical analyses of tissue microarrays from the center (CT) and the invasive margin (IM) of the 602 tumors from two independent cohorts. The results were correlated with tumor recurrence and patient survival. Results Patients with a strong infiltration of CD45RO+ cells in the tumor exhibited an increased expression of T-helper 1 and cytotoxicity-related genes. Densities of CD45RO+ and CD8+ cells in tumor regions (CT/IM) classified the patients into four distinct prognostic groups based on the presence of high density of each marker in each tumor region. The four groups were associated with dramatic differences in disease-free, disease-specific, and overall survival (all P < .0001). Five years after diagnosis, only 4.8% (95% CI, 0.6% to 8.8%) of patients with high densities of CD8+ plus CD45RO+ cells had tumor recurrence, and 86.2% (CI, 79.4% to 93.6%) survived. In contrast, the tumor recurred in 75% (95% CI, 17% to 92.5%) of patients with low densities of these cells, and only 27.5% (95% CI, 10.5% to 72%) survived (all P < .0001). Multivariate analyses showed that the immune criteria had independent effects on the rates of complete remission and survival. Conclusion The combined analysis of CD8+ plus CD45RO+ cells in specific tumor regions could provide a useful criterion for the prediction of tumor recurrence and survival in patients with early-stage colorectal cancer.

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.

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.