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PDF file - 56K, P values corresponding to different cutoff (minimum P value, first, second and third quartile) in CRC and RCC lung metastases. {section sign}P values were corrected by the formula proposed by Altman et al.

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).

PDF file - 83K, Baseline characteristics of 52 patients with RCC lung metastasis. * determined by Heng et al.

PDF file - 90K, Baseline characteristics of 140 patients with CRC lung metastasis. The stage was determined by pathological examination at the time of diagnosis.

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: If immune cells are involved in tumor surveillance and have a prognostic impact in most primary tumors, little is known about their significance in metastases. Because patients' survival is heterogeneous, even at metastatic stages, we hypothesized that immune cells may be involved in the control of metastases. We therefore characterized the tumor immune microenvironment and its prognostic value in colorectal and renal cell carcinoma (RCC) metastases, and compared it to primary tumors.Experimental Design: We analyzed by immunohistochemistry (n = 192) and qPCR (n = 32) the immune environments of colorectal carcinoma and RCC lung metastases.Results: Metastases from colorectal carcinoma and RCC have different immune infiltrates. Higher densities of DC-LAMP+ mature dendritic cells (P < 0.0001) and lower densities of NKp46+ NK cells (P < 0.0001) were observed in colorectal carcinoma as compared to RCC metastases, whereas densities of T cells were similar. High densities of CD8+ and DC-LAMP+ cells correlated with longer overall survival (OS) in colorectal carcinoma (P = 0.008) and shorter OS in RCC (P < 0.0001). High NK-cell densities were associated with improved survival in RCC (P = 0.002) but not in colorectal carcinoma. Densities of immune cells correlated significantly from primary to relapsing metastases for the same patient. A TH1 orientation was found in colorectal carcinoma metastases, whereas a heterogeneous immune gene expression was found in RCC metastases.Conclusions: Our results show a major prognostic value of the immune pattern (CD8+/DC-LAMP+ cell densities) in colorectal carcinoma and RCC, reproducible from primary to metastatic tumors, although with opposite clinical impacts, and highlight the role of the tumor cell in shaping its immune environment. Clin Cancer Res; 19(15); 4079–91. ©2013 AACR.

PDF file - 101K, Influence of pre-operative patient treatments on the distribution of CD8+, DC-LAMP+ and NKp46+ immune cells in CRC (a, b and c) and RCC lung metastases (d, e and f). 63/140 patients with CRC lung metastases have been treated with neo-adjuvant chemotherapy and 9/52 patients with RCC lung metastases have been treated with IL2/IFN. Whiskers length represents 10-90 percentile. ns, not significant (Mann-Whitney test).

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

PDF file - 88K, Gene expression in lung metastases from CRC and RCC. Expression of genes related to (a) immune cell populations, (b) Th1/Th2 orientations, (c) inflammation and angiogenesis, (d) immunosuppression, (e) cytotoxicity, (f) chemokines/chemokine receptors in lung metastases from CRC (grey bars) and RCC (white bars). Expression levels of genes were determined using threshold cycle (Ct) values normalized to actin B ACTB (ΔCt). Whiskers length represents 10-90 percentile. ns, not significant; *P

PDF file - 107K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of CRC lung metastases. Statistical comparison was performed by the log-rank test.

PDF file - 148K, Kaplan-Meier curves for the duration of OS according to the presence of CD8+ T cells in the center of the tumor (CT) (a), in the invasive margin (IM) (b) and in combined regions (CT+IM) (c) of RCC lung metastases. Representation of the Kaplan-Meier curves for the duration of OS according to the presence of NKp46+ cells in the CT (d), in the IM (e) and in combined region (CT+IM) (f) of RCC lung metastases. Statistical comparison was performed by the log-rank test.

PDF file - 81K

Supplementary Tables

Correlation between CD3+ TIL densities calculated from whole slides and from TMA samples.

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]

Correlation between frameshift mutations and CD8+ TIL density in the second series.

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

Frequency of frameshift mutations within target genes and proportion of tumors harboring these mutations in all malignant cells.

Association between clinical characteristics and number of frameshift mutations.

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 Figure Legend

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

Supplementary Figure 3. In vitro cytotoxic activity of peripheral TLs from donors harboring or not mutations in TGFBR2, TAF1B and ASTE1 genes.

Colorectal cancers with microsatellite instability (MSI) represent 15% of all colorectal cancers, including Lynch syndrome as the most frequent hereditary form of this disease. Notably, MSI colorectal cancers have a higher density of tumor-infiltrating lymphocytes (TIL) than other colorectal cancers. This feature is thought to reflect the accumulation of frameshift mutations in sequences that are repeated within gene coding regions, thereby leading to the synthesis of neoantigens recognized by CD8+ T cells. However, there has yet to be a clear link established between CD8+ TIL density and frameshift mutations in colorectal cancer. In this study, we examined this link in 103 MSI colorectal cancers from two independent cohorts where frameshift mutations in 19 genes were analyzed and CD3+, CD8+, and FOXP3+ TIL densities were quantitated. We found that CD8+ TIL density correlated positively with the total number of frameshift mutations. TIL densities increased when frameshift mutations were present within the ASTE1, HNF1A, or TCF7L2 genes, increasing even further when at least one of these frameshift mutations was present in all tumor cells. Through in vitro assays using engineered antigen-presenting cells, we were able to stimulate peripheral cytotoxic T cells obtained from colorectal cancer patients with peptides derived from frameshift mutations found in their tumors. Taken together, our results highlight the importance of a CD8+ T cell immune response against MSI colorectal cancer–specific neoantigens, establishing a preclinical rationale to target them as a personalized cellular immunotherapy strategy, an especially appealing goal for patients with Lynch syndrome. Cancer Res; 75(17); 3446–55. ©2015 AACR.

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

PURPOSE The recommended duration of adjuvant fluoropyrimidine and oxaliplatin chemotherapy for patients with stage III colon cancer is based on tumor classification into clinically low-risk (T1-3 N1) and high-risk (T4 or N2) groups. We determined whether Immunoscore can enhance prognostication within these risk groups. MATERIALS AND METHODS Patients with stage III colon carcinomas (N = 600) were randomly selected from the infusional fluorouracil, leucovorin, and oxaliplatin arm of adjuvant trial NCCTG N0147 (Alliance for Clinical Trials in Oncology). Tumors were evaluated for Immunoscore that quantifies CD3+ and CD8+ T-cell densities in the tumor center and invasive margin by digital image analysis. Disease-free survival (DFS) by Immunoscore was analyzed using a multivariable Cox regression model in each risk group with adjustment for covariates including KRAS, BRAF V600E, and mismatch repair status. RESULTS Of 559 cancers with Immunoscore data, 299 (53.5%) were classified as clinically low-risk (T1-3 N1) and 260 (46.5%) as clinically high-risk (T4 and/or N2). Among patients with low-risk tumors, those with Immunoscore-Low versus Immunoscore-High tumors had significantly worse 5-year DFS rates (77.5% v 91.8%; hazard ratio, 1.70; 95% CI, 1.03 to 2.79; P = .037). Among patients with high-risk tumors, those with Immunoscore-Low versus Immunoscore-High tumors also had significantly worse DFS (55.3% v 70.3%; hazard ratio, 1.65; 95% CI, 1.11 to 2.47; P = .013). Tumors that were low-risk/Immunoscore-Low had similar outcomes as did tumors that were high-risk/Immunoscore-High ( P = .174). Prognostication was significantly improved in multivariable models where Immunoscore was added to clinical risk parameters and limited biomarkers (likelihood ratio test P = .0003). CONCLUSION Immunoscore can refine patient prognosis beyond clinical risk group classification, suggesting its potential utility for adjuvant decision making.

Background: Tumor-infiltrating lymphocytes are associated with a better prognosis in early triple-negative breast cancer (TNBC). These cells can be enumerated in situ by the “Immunoscore Clinical Research” (ISCR). The original Immunoscore® is a prognostic tool that categorizes the densities of CD3+ and CD8+ cells in both the invasive margin (IM) and center of the tumor (CT) in localized colon cancer, yielding a five-tiered classification (0–4). We evaluated the prognostic potential of ISCR and pathological complete response (pCR) following neoadjuvant chemotherapy (NACT). Methods: The cohort included 53 TNBC, 32 luminal BC, and 18 HER2-positive BC patients undergoing NACT. Pre-treatment tumor biopsies were immune-stained for CD3+ and CD8+ T-cell markers. Quantitative analysis of these cells in different tumor locations was performed using computer-assisted image analysis. Results: The pCR rate was 44%. Univariate analysis showed that primary tumor size, estrogen-receptor negative, progesterone-receptor negative, luminal vs. HER2-positive vs. TNBC, high Ki-67, high densities (cells/mm2) of CD3 CT, CD8+ CT, CD3+ IM, and CD8+ IM cells were associated with a high pCR. ISCR was associated with pCR following NACT. A multivariate model consisting of ISCR and the significant variables from the univariate analysis showed a significant trend for ISCR; however, the low sample size did not provide enough power for the model to be included in this study. Conclusions: These results revealed a significant prognostic role for the spatial distributions of the CD3+, and CD8+ lymphocytes, as well as the ISCR in relation to pCR following NACT.

Surgical resection of colorectal liver metastases combined with systemic treatment aims to maximize patient survival. However, recurrence rates are very high postsurgery. In order to assess patient prognosis after metastasis resection, we evaluated the main patho‐molecular and immune parameters of all surgical specimens. Two hundred twenty‐one patients who underwent, after different preoperative treatment, curative resection of 582 metastases were analyzed. Clinicopathological parameters, RAS tumor mutation, and the consensus Immunoscore (I) were assessed for all patients. Overall survival (OS) and time to relapse (TTR) were estimated using the Kaplan–Meier method and compared by log‐rank tests. Cox proportional hazard models were used for uni‐ and multivariate analysis. Immunoscore and clinicopathological parameters (number of metastases, surgical margin, histopathological growth pattern, and steatohepatitis) were associated with relapse in multivariate analysis. Overall, pathological score (PS) that combines relevant clinicopathological factors for relapse, and I, were prognostic for TTR (2‐year TTR rate PS 0–1: 49.8.% (95% CI: 42.2–58.8) versus PS 2–4: 20.9% (95% CI: 13.4–32.8), hazard ratio (HR) = 2.54 (95% CI: 1.82–3.53), p

International audience; Colorectal cancers (CRCs) with microsatellite instability (MSI) are due to a defect in the DNA mismatch repair (MMR) system resulting in an accumulation of frame-shift mutations. They are characterized by a tumor microenvironment richer in cytotoxic CD8 T-cells (CTLs) and a better prognosis compared to microsatellite stable (MSS) CRCs. The mechanisms by which defective MMR system may influence tumor-infiltrating immune cells and their impact on patient survival were still unclear. Thus, we performed a comprehensive analysis of MSI colorectal tumors. We found that the numbers of frame-shift mutations potentially resulting in neo-epitopes were positively correlated to the density of tumor infiltrating CD8 T-cells but were lower than expected at random. We also evidenced that MSI patients could naturally harbor CTLs targeting frame-shift mutation-derived antigens. This favors the hypothesis of an active immunosurveillance in MSI colorectal tumors leading to the genetic evidence of an immunoediting. To evaluate the link between MSI tumor immune contexture and prognosis, we took advantage of a standardized assay that we developed to quantify tumor-infiltrating T-cells, the Immunoscore. Multivariate analyses revealed an advantage of Immunoscore over MSI in predicting recurrence and survival. Our data suggests that the prognostic value of MSI could be attributed to major underlying differences of infiltrating immune cells. Immunotherapeutic treatments, that are more efficient in patients with a preexisting anti-tumor immunity, were approved in MSI patients following successful clinical trials. We suggest that the Immunoscore could be used not only for colorectal tumor prognosis but also for predicting responses to immunotherapies.

(1) Background&mdash, The five-year overall survival (OS) of muscle-invasive bladder cancer (MIBC) with neoadjuvant chemotherapy and cystectomy is around 50%. There is no validated biomarker to guide the treatment decision. We investigated whether the Immunoscore (IS) could predict the pathologic response to neoadjuvant chemotherapy and survival outcomes. (2) Methods&mdash, This retrospective study evaluated the IS in 117 patients treated using neoadjuvant chemotherapy for localized MIBC from six centers (France and Greece). Pre-treatment tumor samples were immunostained for CD3+ and CD8+ T cells and quantified to determine the IS. The results were associated with the response to neoadjuvant chemotherapy, time to recurrence (TTR), and OS. (3) Results&mdash, Low (IS-0), intermediate (IS-1&ndash, 2), and high (IS-3&ndash, 4) ISs were observed in 36.5, 43.7, and 19.8% of the cohort, respectively. IS was positively associated with a pathologic complete response (pCR, p-value = 0.0096). A high IS was found in 35.7% of patients with a pCR, whereas it was found in 11.3% of patients without a pCR. A low IS was observed in 48.4% of patients with no pCR and in 21.4% of patients with a pCR. Low-, intermediate-, and high-IS patients had five-year recurrence-free rates of 37.2%, 36.5%, and 72.6%, respectively. In the multivariable analysis, a high IS was associated with a prolonged TTR (high vs. low: p = 0.0134) and OS (high vs. low: p = 0.011). (4) Conclusions&mdash, This study showed the significant prognostic and predictive roles of IS regarding localized MIBC.

The Complex Portal (www.ebi.ac.uk/complexportal) is a manually curated, encyclopaedic database of macromolecular complexes with known function from a range of model organisms. It summarizes complex composition, topology and function along with links to a large range of domain-specific resources (i.e. wwPDB, EMDB and Reactome). Since the last update in 2019, we have produced a first draft complexome for Escherichia coli, maintained and updated that of Saccharomyces cerevisiae, added over 40 coronavirus complexes and increased the human complexome to over 1100 complexes that include approximately 200 complexes that act as targets for viral proteins or are part of the immune system. The display of protein features in ComplexViewer has been improved and the participant table is now colour-coordinated with the nodes in ComplexViewer. Community collaboration has expanded, for example by contributing to an analysis of putative transcription cofactors and providing data accessible to semantic web tools through Wikidata which is now populated with manually curated Complex Portal content through a new bot. Our data license is now CC0 to encourage data reuse. Users are encouraged to get in touch, provide us with feedback and send curation requests through the ‘Support’ link.

3596 Background: Surgical resection of CRLM aims to maximize patient survival. However, recurrence rates remain high post-surgery. We previously reported the prognostic relevance of tumor regression grading (TRG) and HGP of resected CRLM. Several studies reported the association of tumoral heterogeneity with anti-cancer drug resistance and prognosis. This study aims to explore tumoral heterogeneity for TRG and HGP in patients resected for CRLM and its prognostic implication. Methods: Tumor homogeneity for PR and HGP was evaluated in 2 independent cohorts. Cohort 1 included 57 patients (159 CRLMs) resected after chemotherapy/bevacizumab (prospective BEV-ONCO trial). Cohort 2 included 221 patients (582 CRLMs) operated after preoperative treatment or not. TRG (1 to 5 according complete to no response), HGP (desmoplastic, pushing, replacement or mixed) were evaluated for each CRLM. Max-TRG (higher TRG among all the CRLM) was used to define PR. Homogenous TRG (TRG-h) and HGP (HGP-h) was defined when all CRLMs had the same TRG or HGP pattern. HGP homogeneous desmoplastic (HGP-hd) was defined when all CRLM had a desmoplastic HGP. TRG-h, HGP-h and HGP-hd were combined into a homogeneity score (H-score: 0 to 3, 1 point given for each parameter and summed-up). Overall survival (OS for both cohorts), progression-free survival (PFS for cohort 1) and time to relapse (TTR for cohort 2) were estimated using the Kaplan–Meier method and compared by log-rank tests. Cox proportional hazard models were used for univariate and multivariate analyses. Results: Patient and disease characteristics were comparable in both cohorts excepted for preoperative treatment. In cohort 1, TRG-h and HGP-h were significantly associated with a longer PFS (HR = 0.21; 95CI:0.10-0.43, p < 0.001; HR = 0.27; 95CI = 0.14-0.54, p < 0.001) and better OS (HR = 0.23; 95CI = 0.07-0.70, p = 0.010; HR = 0.32; 95CI = 0.10-0.93, p = 0.037). Interestingly, the same significant results were observed in cohort 2 for TTR (TRG-h: HR = 0.60; 95CI = 0.43-0.85, p = 0.004; HGP-h: HR = 0.68; 95CI = 0.49-0.94, p = 0.017) and OS (TRG-h: HR = 0.51;95CI = 0.33-0.80, p = 0.003; HGP-h: HR = 0.63; 95CI = 0.41-0.97, p = 0.034). HGP-h reported a significant association with TRG-h, a Max-TRG < = 3, the absence of HGP replacement and mixed, a desmoplastic pattern, and the absence of sinusoidal obstruction syndrome in both cohorts. H-score was significantly associated with TTR (score 1-2: HR = 0.57; 95CI = 0.38-0.85, p = 0.004; score 3: HR = 0.4; 95CI = 0.24-0.64, p < 0.001) and OS (score 3: HR = 0.31; 95CI = 0.15-0.64, p < 0.001) in univariate analysis and with OS (HR = 0.74; 95CI = 0.59-0.94, p = 0.011) in multivariate analysis (cohort 2). Conclusions: TRG-h and HGP-h are strongly associated with patient’s survival. H-score could be an easy morphological and prognostic score to assess. Validation studies are needed.

3595 Background: Immune checkpoint inhibitors demonstrated poor efficacy in MSS mCRC. Cetuximab (anti-EGFR inhibitor) could initiate, independently from RAS mutation, an immunogenic tumor cell death and mediate antitumor immune response. In this trial, we aim to explore the clinical efficacy and safety of avelumab (anti-PDL1) combined with cetuximab and irinotecan for treatment refractory MSS mCRC and to understand its mechanisms of action through associated translational research. Methods: AVETUXIRI trial (NCT03608046) enrols MSS, chemorefractory (fluoropyrimidine, oxaliplatin, irinotecan and anti-EGFR treatment if RAS wt tumor) mCRC patients in 2 cohorts (A: RAS-wt, n = 10 – B: RAS-mut, n = 13). Primary endpoints are safety and tumor response rate ((i)RECIST1.1). Secondary endpoints include disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). According to a Simon 2-stage design, 23 patients have been included in the first stage of the trial. Multiplex immunofluorescence and RNA sequencing were realized on metastasis biopsies performed before, during and after the treatment. Densities of CD3+ (T cells) and CD8+ (cytotoxic) were quantified and analyzed with to generate an immunoscore (IS). RNA-seq data was used to perform differential expression analysis (DESeq2), gene set enrichment analysis (GSEA), deconvolution analysis (ConsensusTME) and gene ontology analysis (GO). Results:: No unexpected safety signals were observed. 3/10 tumor responses were observed in cohort A, 0/13 in cohort B. DCR was 60.0% and 61.5% in cohort A and B, respectively. 6-months PFS and 12-months OS rates were respectively 40.0% and 50.0% (cohort A) and 38.5% and 46.2% (cohort B). Independently of RAS mutation, patients with a high IS (metastasis biopsy, baseline) had significantly higher tumor shrinkage (OR = 18.67 p = 0.019), median PFS (6.9 vs 3.4 months; HR = 0.16, p = 0.002) and median OS (13.7 vs 7.9 months, HR = 0.26, p = 0.009). Similarly, tumor shrinkage and survival outcome (PFS > 6 months, OS > 12 months) were associated with upregulation of an adaptive immune response signature (including Th1, chemokine, adhesion molecules, immune checkpoints and T-cell activation genes, p. adj = 0.009) and the GSEA hallmark of epithelial to mesenchymal transition (p. adj = 0.045). Few modifications of IS and gene expression profiles were observed on the different metastasis biopsies performed overtime in the included patients. Conclusions: AVETUXIRI met its preliminary primary efficacy endpoint for RAS-wt mCRC pts, justifying its current continuation. Encouraging survival data observed in RAS-mut cohort allow the opening of a new cohort (PFS as primary endpoint). IS and adaptive immune response signature evaluated on metastases biopsies were associated with treatment efficacy and survival. Clinical trial information: NCT03608046.

Background The American Joint Committee on Cancer staging and other prognostic tools fail to account for stage-independent variability in outcome. We developed a prognostic classifier adding Immunoscore to clinicopathological and molecular features in patients with stage III colon cancer. Methods Patient (n = 559) data from the FOLFOX arm of adjuvant trial NCCTG N0147 were used to construct Cox models for predicting disease-free survival (DFS). Variables included age, sex, T stage, positive lymph nodes (+LNs), N stage, performance status, histologic grade, sidedness, KRAS/BRAF, mismatch repair, and Immunoscore (CD3+, CD8+ T-cell densities). After determining optimal functional form (continuous or categorical) and within Cox models, backward selection was performed to analyze all variables as candidate predictors. All statistical tests were two-sided. Results Poorer DFS was found for tumors that were T4 vs T3 (hazard ratio [HR] = 1.76, 95% confidence interval [CI] = 1.19 to 2.60; P = .004), right- vs left-sided (HR = 1.52, 95% CI = 1.14 to 2.04; P = .005), BRAF V600E (HR = 1.74, 95% CI = 1.26 to 2.40; P Conclusions The Immunoscore can enhance the accuracy of survival prediction among patients with stage III colon cancer.