International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study.

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<0·0001). In the training set, patients with a high Immunoscore had the lowest risk of recurrence at 5 years (14 [8%] patients with a high Immunoscore vs 65 (19%) patients with an intermediate Immunoscore vs 51 (32%) patients with a low Immunoscore; hazard ratio [HR] for high vs low Immunoscore 0·20, 95% CI 0·10-0·38; p<0·0001). The findings were confirmed in the two validation sets (n=1981). In the stratified Cox multivariable analysis, the Immunoscore association with time to recurrence was independent of patient age, sex, T stage, N stage, microsatellite instability, and existing prognostic factors (p<0·0001). Of 1434 patients with stage II cancer, the difference in risk of recurrence at 5 years was significant (HR for high vs low Immunoscore 0·33, 95% CI 0·21-0·52; p<0·0001), including in Cox multivariable analysis (p<0·0001). Immunoscore had the highest relative contribution to the risk of all clinical parameters, including the American Joint Committee on Cancer and Union for International Cancer Control TNM classification system. INTERPRETATION: The Immunoscore provides a reliable estimate of the risk of recurrence in patients with colon cancer. These results support the implementation of the consensus Immunoscore as a new component of a TNM-Immune classification of cancer. FUNDING: French National Institute of Health and Medical Research, the LabEx Immuno-oncology, the Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants and the Society for Immunotherapy of Cancer.

The Immunoscore in the Clinical Practice of Patients with Colon and Rectal Cancers.

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.

Rational bases for the use of the Immunoscore in routine clinical settings as a prognostic and predictive biomarker in cancer patients.

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.

[Focus on the Immunoscore and its potential clinical implications]. / Mise au point sur l'Immunoscore et ses potentielles implications cliniques.

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.

International Validation of the Immunoscore Biopsy in Patients With Rectal Cancer Managed by a Watch-and-Wait Strategy.

PURPOSE: No biomarker capable of improving selection and monitoring of patients with rectal cancer managed by watch-and-wait (W&W) strategy is currently available. Prognostic performance of the Immunoscore biopsy (ISB) was recently suggested in a preliminary study. METHODS: This international validation study included 249 patients with clinical complete response (cCR) managed by W&W strategy. Intratumoral CD3+ and CD8+ T cells were quantified on pretreatment rectal biopsies by digital pathology and converted to ISB. The primary end point was time to recurrence (TTR; the time from the end of neoadjuvant treatment to the date of local regrowth or distant metastasis). Associations between ISB and outcomes were analyzed by stratified Cox regression adjusted for confounders. Immune status of tumor-draining lymph nodes (n = 161) of 17 additional patients treated by neoadjuvant chemoradiotherapy and surgery was investigated by 3'RNA-Seq and immunofluorescence. RESULTS: Recurrence-free rates at 5 years were 91.3% (82.4%-100.0%), 62.5% (53.2%-73.3%), and 53.1% (42.4%-66.5%) with ISB High, ISB Intermediate, and ISB Low, respectively (hazard ratio [HR; Low v High], 6.51; 95% CI, 1.99 to 21.28; log-rank P = .0004). ISB was also significantly associated with disease-free survival (log-rank P = .0002), and predicted both local regrowth and distant metastasis. In multivariate analysis, ISB was independent of patient age, sex, tumor location, cT stage (T, primary tumor; c, clinical), cN stage (N, regional lymph node; c, clinical), and was the strongest predictor for TTR (HR [ISB High v Low], 6.93; 95% CI, 2.08 to 23.15; P = .0017). The addition of ISB to a clinical-based model significantly improved the prediction of recurrence. Finally, B-cell proliferation and memory in draining lymph nodes was evidenced in the draining lymph nodes of patients with cCR. CONCLUSION: The ISB is validated as a biomarker to predict both local regrowth and distant metastasis, with a gradual scaling of the risk of pejorative outcome.

Multi-Institutional Evaluation of Pathologists' Assessment Compared to Immunoscore.

BACKGROUND: The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS: An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS: Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS: The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes.

Multicenter International Study of the Consensus Immunoscore for the Prediction of Relapse and Survival in Early-Stage Colon Cancer.

Background: The prognostic value of Immunoscore was evaluated in Stage II/III colon cancer (CC) patients, but it remains unclear in Stage I/II, and in early-stage subgroups at risk. An international Society for Immunotherapy of Cancer (SITC) study evaluated the pre-defined consensus Immunoscore in tumors from 1885 AJCC/UICC-TNM Stage I/II CC patients from Canada/USA (Cohort 1) and Europe/Asia (Cohort 2). METHODS: Digital-pathology is used to quantify the densities of CD3+ and CD8+ T-lymphocyte in the center of tumor (CT) and the invasive margin (IM). The time to recurrence (TTR) was the primary endpoint. Secondary endpoints were disease-free survival (DFS), overall survival (OS), prognosis in Stage I, Stage II, Stage II-high-risk, and microsatellite-stable (MSS) patients. RESULTS: High-Immunoscore presented with the lowest risk of recurrence in both cohorts. In Stage I/II, recurrence-free rates at 5 years were 78.4% (95%-CI, 74.4−82.6), 88.1% (95%-CI, 85.7−90.4), 93.4% (95%-CI, 91.1−95.8) in low, intermediate and high Immunoscore, respectively (HR (Hi vs. Lo) = 0.27 (95%-CI, 0.18−0.41); p < 0.0001). In Cox multivariable analysis, the association of Immunoscore to outcome was independent (TTR: HR (Hi vs. Lo) = 0.29, (95%-CI, 0.17−0.50); p < 0.0001) of the patient's gender, T-stage, sidedness, and microsatellite instability-status (MSI). A significant association of Immunoscore with survival was found for Stage II, high-risk Stage II, T4N0 and MSS patients. The Immunoscore also showed significant association with TTR in Stage-I (HR (Hi vs. Lo) = 0.07 (95%-CI, 0.01−0.61); P = 0.016). The Immunoscore had the strongest (69.5%) contribution χ2 for influencing survival. Patients with a high Immunoscore had prolonged TTR in T4N0 tumors even for patients not receiving chemotherapy, and the Immunoscore remained the only significant parameter in multivariable analysis. CONCLUSION: In early CC, low Immunoscore reliably identifies patients at risk of relapse for whom a more intensive surveillance program or adjuvant treatment should be considered.

Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation.

BACKGROUND: Cases of the spontaneous regression of multiple pulmonary metastases, after radiofrequency ablation (RFA), of a single lung metastasis, have been documented to be mediated by the immune system. The interaction of immune checkpoints, e.g., PD-1/PD-L1 and CTLA-4/CD80, may explain this phenomenon. The purpose of this study is to identify and quantify immune mechanisms triggered by RFA of pulmonary metastases originating from colorectal cancer. METHODS: We used two-site time-resolved Förster resonance energy transfer as determined by frequency-domain FLIM (iFRET) for the quantification of receptor-ligand interactions. iFRET provides a method by which immune checkpoint interaction states can be quantified in a spatiotemporal manner. The same patient sections were used for assessment of ligand-receptor interaction and intratumoral T-cell labeling. CONCLUSION: The checkpoint interaction states quantified by iFRET did not correlate with ligand expression. We show that immune checkpoint ligand expression as a predictive biomarker may be unsuitable as it does not confirm checkpoint interactions. In pre-RFA-treated metastases, there was a significant and negative correlation between PD-1/PD-L1 interaction state and intratumoral CD3+ and CD8+ density. The negative correlation of CD8+ and interactive states of PD-1/PD-L1 can be used to assess the state of immune suppression in RFA-treated patients.

The "Immunoscore" in rectal cancer: could we search quality beyond quantity of life?

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

Therapeutic Implications of the Immunoscore in Patients with Colorectal Cancer.

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

Prognostic assessment of resected colorectal liver metastases integrating pathological features, RAS mutation and Immunoscore.

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 < 0.0000; and 2-year TTR rate I 0: 25.7% (95% CI: 16.3-40.5) versus I 3-4: 60% (95% CI: 47.2-76.3), HR = 2.87 (95% CI: 1.73-4.75), p = 0.0000). Immunoscore was also prognostic for OS (HR [I 3-4 versus I 0] = 4.25, 95% CI: 1.95-9.23; p = 0.0001). Immunoscore (HR [I 3-4 versus I 0] = 0.27, 95% CI: 0.12-0.58; p = 0.0009) and RAS mutation (HR [mutated versus WT] = 1.66, 95% CI: 1.06-2.58; p = 0.0265) were significant for OS. In conclusion, PS including relevant clinicopathological parameters and Immunoscore permit stratification of stage IV colorectal cancer patient prognosis in terms of TTR and identify patients with higher risk of recurrence. Immunoscore remains the major prognostic factor for OS.

Chemoradiation triggers antitumor Th1 and tissue resident memory-polarized immune responses to improve immune checkpoint inhibitors therapy.

BACKGROUND: Multiple synergistic combination approaches with cancer drugs are developed to overcome primary resistance to immunotherapy; however, the mechanistic rationale to combine chemoradiotherapy (CRT) with immune checkpoint inhibitors remains elusive. METHODS: This study described the immunological landscape of tumor microenvironment (TME) exposed to CRT. Tumor samples from patients with rectal cancer (n=43) treated with neoadjuvant CRT or radiotherapy were analyzed by nanostring and immunohistochemistry. Studies in mice were performed using three syngeneic tumors (TC1, CT26 and MC38). Tumor-bearing mice were treated either with platinum-based CRT, radiotherapy or chemotherapy. Anti-CTLA-4 and/or anti-Programmed Cell Death Receptor-1 (PD-1) therapy was used in combination with CRT. The therapy-exposed TME was screened by RNA sequencing and flow cytometry and tumor-infiltrating T lymphocyte functionality was evaluated by interferon (IFN)-γ ELIspot and intracellular cytokine staining. RESULTS: Front-to-front comparison analysis revealed the synergistic effect of CRT to establish a highly inflamed and Th1-polarized immune signature in the TME of patients and mice. In both settings, CRT-exposed TMEs were highly enriched in newly-infiltrated tumor-specific CD8+ T cells as well as tissue resident memory CD103+CD8+ T cells. In mice, CD8 T cells were involved in the antitumor response mediated by CRT and were primed by CRT-activated CD103+ dendritic cells. In the three tumor models, we showed that concurrent combination of CRT with a dual CTLA-4 and PD-1 blockade was required to achieve an optimal antitumor effect and to establish a broad and long-lasting protective antitumor T cell immunity. CONCLUSIONS: Our results highlight the ability of CRT to stimulate strong antitumor T-cell-mediated immunity and tissue resident memory T activation in TME, to foster immune checkpoint inhibitors action. These findings have implications in clinic for the design clinical trials combining chemoradiation with immunotherapy.

Differential association between inflammatory cytokines and multiorgan dysfunction in COVID-19 patients with obesity.

To investigate the mechanisms underlying the SARS-CoV-2 infection severity observed in patients with obesity, we performed a prospective study of 51 patients evaluating the impact of multiple immune parameters during 2 weeks after admission, on vital organs' functions according to body mass index (BMI) categories. High-dimensional flow cytometric characterization of immune cell subsets was performed at admission, 30 systemic cytokines/chemokines levels were sequentially measured, thirteen endothelial markers were determined at admission and at the zenith of the cytokines. Computed tomography scans on admission were quantified for lung damage and hepatic steatosis (n = 23). Abnormal BMI (> 25) observed in 72.6% of patients, was associated with a higher rate of intensive care unit hospitalization (p = 0.044). SARS-CoV-2 RNAaemia, peripheral immune cell subsets and cytokines/chemokines were similar among BMI groups. A significant association between inflammatory cytokines and liver, renal, and endothelial dysfunctions was observed only in patients with obesity (BMI > 30). In contrast, early signs of lung damage (ground-glass opacity) correlated with Th1/M1/inflammatory cytokines only in normal weight patients. Later lesions of pulmonary consolidation correlated with BMI but were independent of cytokine levels. Our study reveals distinct physiopathological mechanisms associated with SARS-CoV-2 infection in patients with obesity that may have important clinical implications.

Usefulness and robustness of Immunoscore for personalized management of cancer patients.

This review details the analytical performance characteristics of the consensus Immunoscore, measuring the immune response to cancer, improving the estimation of risk of recurrence, and predicting response to treatment for patients with colon cancer. The analytical validation of Immunoscore has been documented. Immunoscore is a robust, reproducible, quantitative, and standardized immune assay, with a high prognostic performance, independent of all of the prognostic markers currently used in clinical practice. Immunoscore evaluation within the tumor microenvironment is clinically relevant, and Immunoscore was recently introduced into ESMO Clinical Practice Guidelines for colon cancer and into the WHO classification of the Digestive System Tumors. This paves the way for the use of Immunoscore in clinical practice in colorectal tumors and likely soon in many other solid tumors.

Immunoscore assay for the immune classification of solid tumors: Technical aspects, improvements and clinical perspectives.

The past two decades witnessed the appreciation of the importance of specific tumor-infiltrating immune cells in influencing tumor evolution. The discovery that a favorable immune contexture is linked to a prolonged patients' survival, and more specifically that intratumoral cytotoxic T lymphocytes hold powerful prognostic value, provided the foundations for the development of the Immunoscore. Immunoscore is a digital pathology, IHC-based immune assay measuring the densities of CD3+ and CD8+ T cells at different tumor locations, linking them with patients' clinical outcome. The present chapter provides a detailed overview of the assay development and procedure, from the bench to the data analysis, and of the internationally approved and validated guidelines to perform a consensus Immunoscore for colon cancer patients. Assay strengths and limitations are also discussed, as well as the clinical implications of its introduction in routine practice.

Adaptive and Innate Immune Cells in Fetal Human Cytomegalovirus-Infected Brains.

BACKGROUND: The understanding of the pathogenesis of cytomegalovirus (CMV)-induced fetal brain lesions is limited. We aimed to quantify adaptive and innate immune cells and CMV-infected cells in fetal brains with various degrees of brain damage. METHODS: In total, 26 archived embedded fetal brains were studied, of which 21 were CMV-infected and classified in severely affected (n = 13) and moderately affected (n = 8), and 5 were uninfected controls. The respective magnitude of infected cells, immune cells (CD8+, B cells, plasma cells, NK cells, and macrophages), and expression of immune checkpoint receptors (PD-1/PD-L1 and LAG-3) were measured by immunochemistry and quantified by quantitative imaging analysis. RESULTS: Quantities of CD8+, plasma cells, NK cells, macrophages, and HCMV+ cells and expression of PD-1/PD-L1 and LAG-3 were significantly higher in severely affected than in moderately affected brains (all p values < 0.05). A strong link between higher number of stained cells for HCMV/CD8 and PD-1 and severity of brain lesions was found by component analysis. CONCLUSIONS: The higher expression of CD8, PD-1, and LAG-3 in severely affected brains could reflect immune exhaustion of cerebral T cells. These exhausted T cells could be ineffective in controlling viral multiplication itself, leading to more severe brain lesions. The study of the functionality of brain leucocytes ex vivo is needed to confirm this hypothesis.

Analytical validation of the Immunoscore and its associated prognostic value in patients with colon cancer.

BACKGROUND: New 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. METHODS: Immunohistochemical 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). RESULTS: Optical and automatic counts for CD3+ or CD8+ were strongly correlated (r=0.94, p<0.001 and r=0.92, p<0.001, respectively). CD3 and CD8 staining intensities were not altered by the age of the tumor block over a period of 30 years. Neither the position of tested tissue sections within a tumor block nor the selection of the tissue blocks affected the IS. Reproducibility of the IS was not affected by multiple variables (eg, antibody lots, DAB revelation kits, immunohistochemistry automates and operators). Interassay repeatability of the IS was 100% and interlaboratory reproducibility between two testing centers was 93%. Finally, in a case series of patients with stage II-III colon cancer, the relative proportion of variance for time to recurrence was greatest for the IS (53% of prognostic variability) in a model that included IS, T-stage, microsatellite instability status and total number of lymph nodes. CONCLUSION: IS 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.

A Diagnostic Biopsy-Adapted Immunoscore Predicts Response to Neoadjuvant Treatment and Selects Patients with Rectal Cancer Eligible for a Watch-and-Wait Strategy.

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 (n 1 = 131, n 2 = 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.

Multicenter International Society for Immunotherapy of Cancer Study of the Consensus Immunoscore for the Prediction of Survival and Response to Chemotherapy in Stage III Colon Cancer.

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.

Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer.

The prognosis of colon cancer (CC) is dictated by tumor-infiltrating lymphocytes, including follicular helper T (TFH) cells and the efficacy of chemotherapy-induced immune responses. It remains unclear whether gut microbes contribute to the elicitation of TFH cell-driven responses. Here, we show that the ileal microbiota dictates tolerogenic versus immunogenic cell death of ileal intestinal epithelial cells (IECs) and the accumulation of TFH cells in patients with CC and mice. Suppression of IEC apoptosis led to compromised chemotherapy-induced immunosurveillance against CC in mice. Protective immune responses against CC were associated with residence of Bacteroides fragilis and Erysipelotrichaceae in the ileum. In the presence of these commensals, apoptotic ileal IECs elicited PD-1+ TFH cells in an interleukin-1R1- and interleukin-12-dependent manner. The ileal microbiome governed the efficacy of chemotherapy and PD-1 blockade in CC independently of microsatellite instability. These findings demonstrate that immunogenic ileal apoptosis contributes to the prognosis of chemotherapy-treated CC.