Dr David Malka
email: david.malka@gustaveroussy.fr

Astrid Lièvre

Thierry André
email: thierry.andre@aphp.fr

Julien Taïeb

Michel Ducreux

Frédéric Bibeau

Immune scores in colorectal cancer: Where are we?

Keywords: Colorectal cancer, prognosis, immunity, immune score

There is growing evidence that the immune system may prevent the occurrence, growth and metastatic diffusion of colorectal cancer (CRC). The role played by the adaptive immune response at the tumor site is critical in the balance between tumor invasion and defense against cancer. Recent data have shown that the evaluation of this immune response may help to define the prognosis and possibly the treatment of localized CRC as well as metastatic CRC (mCRC). Tumor infiltrates with T cells (CD3+), cytotoxic T cells (CD8+), and memory T cells (CD45RO+) are the immune parameters most consistently and strongly associated with good clinical outcome in CRC. Several scoring systems have been developed, including the Immunoscore®, based on the immunohistochemical determination with a digital image analysis system of the density of CD3+ and CD8+ lymphocytes in the center and the invasive margin of the tumor. This review will focus on the different immunoscoring systems developed in CRC, their performance, their limitations and their potential for improving patients' care in the future.

Introduction

Colorectal cancer (CRC) is the fourth most common cancer in the world and the second leading cause of death [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]. Survival is high for patients diagnosed with early-stage disease [START_REF] Taieb | Refining adjuvant therapy for non-metastatic colon cancer, new standards and perspectives[END_REF]. However, approximately half of CRC cases are diagnosed with or will develop metastases, the main cause of death in CRC, with a 5-year overall survival (OS) of approximately 10% [START_REF] Li | The main contributor to the upswing of survival in locally advanced colorectal cancer: an analysis of the SEER database[END_REF]. There is thus a real need for therapeutic improvements in order to reduce the risk of recurrence after surgery or to prolong survival in patients with metastatic colorectal cancer (mCRC).

There is growing evidence that the immune system may play a role in preventing the occurrence, growth and metastatic diffusion of CRC. Understanding the role and mechanisms of immune response in CRC is a major issue and may help to better use immunotherapeutic agents in this disease and to develop new therapeutic approaches.

The crucial role of the immune system in controlling tumor development and the concept of immune surveillance were first demonstrated by the more frequent occurrence of nonpathogen-related tumors in immune-compromised patients such as transplant recipients and patients with AIDS. Registry and meta-analysis of solid organ transplant recipients have shown an increased risk of solid tumors including CRC, with a standardized incidence ratio of 1.2 to 1.8 [START_REF] Engels | Spectrum of cancer risk among US solid organ transplant recipients[END_REF][START_REF] Collett | Comparison of the incidence of malignancy in recipients of different types of organ: a UK Registry audit[END_REF]. Whether patients with AIDS are at higher risk of developing CRC is however more debatable [START_REF] Grulich | Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis[END_REF].

In CRC, innate immune cell infiltration has been reported to be of good prognosis for Natural Killer (NK) cells, Natural killer T (NKT) and γδ T lymphocytes. At the opposite, macrophage infiltrates are generally associated with a poor prognosis in human carcinomas [START_REF] Coca | The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma[END_REF][START_REF] Tachibana | Increased intratumor Valpha24-positive natural killer T cells: a prognostic factor for primary colorectal carcinomas[END_REF][START_REF] Corvaisier | V gamma 9V delta 2 T cell response to colon carcinoma cells[END_REF][START_REF] Edin | Phenotypic skewing of macrophages in vitro by secreted factors from colorectal cancer cells[END_REF]. Specific antitumor response is generated by adaptive immune system and in particular by αβ T lymphocytes. Briefly, after tumor cell lysis by innate immune cells such as NK cells, NKT cells and macrophages, the antigen-presenting cells, mainly dendritic cells, can capture, process and present tumor antigens to CD4 T lymphocytes through major histocompatibility class (MHC) II or to CD8 T lymphocytes through MHC I after a maturation process (Figure 1). Activation of T cells requires three signals: 1) recognition of antigenic peptide presented by the antigen-presenting cell; 2) activation of co-stimulatory molecules (CD80/CD28, CD40/CD40L); 3) recruitment of cytokines (interleukin (IL)-1, IL-2, IL-6, IL-12, interferon (IFN)γ). Activated CD8+ T cells can then recognize and lyse tumor cells (Figure 1). Activated CD4+ T cells can modulate the antitumor immune response. They can differentiate into different cell subgroups: T cell helper 1 (Th1) response allows secretion of cytokines promoting antitumor response such as IL-2 or IFNγ, whereas T cell helper 2 (Th2) response is more associated to tumor growth. A third type of immune response, named T cell helper 17 (Th17), implies the release of large amounts of IL17 but its role in the immune response against cancer is controversial. Finally, the regulatory CD4+ T cells (Treg) can inhibit the antitumor immune response and represent an escape mechanism for the cancerous cells (Figure 1) [START_REF] Pernot | Colorectal cancer and immunity: what we know and perspectives[END_REF].

The role of cytotoxic CD8+ T cells has been widely studied in CRC. Tumor infiltrating lymphocytes (TILs) are major actors of antitumor immune response (Figure 2). The prognostic role of immune response has been first analyzed in a large cohort of resected stage I to IV CRC more than 10 years ago [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF][START_REF] Pages | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF]. It has been originally shown that the absence of pathological signs of early metastatic invasion (venous emboli, lymphatic invasion, perineural invasion: VELIPI) was associated with increased infiltrates of immune cells and increased levels of messenger RNA (mRNA) for products of Th1 effector T cells [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF].

In the same cohort, the density of TILs assessed by CD3 immunostaining predicted OS better than usual histopathological prognostic factors (i.e. UICC-TNM classification) [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF].

Based on these results, different assessments of TILs have been elaborated and developed during the past decade [START_REF] Pages | In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer[END_REF][START_REF] Mlecnik | Biomolecular network reconstruction identifies Tcell homing factors associated with survival in colorectal cancer[END_REF][START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF][START_REF] Allard | Linear quantification of lymphoid infiltration of the tumor margin: a reproducible method, developed with colorectal cancer tissues, for assessing a highly variable prognostic factor[END_REF]. However, despite impressive results on disease prognostication, tumoral and peritumoral infiltration by immune cells is not yet used in clinical practice. This review will focus on the different immunoscoring systems developed in CRC, their performance, their limitations and their potential for improving patients' care in the future.

Methods

Search strategy

A MEDLINE systematic literature search was conducted for English language studies published between January 2002 and May 2019. Conference abstracts published between January 2016 and June 2019 were searched for the following congresses: the American Association for Cancer Research, the American Society of Clinical Oncology (ASCO), the ASCO Gastrointestinal Cancers Symposium, the European Society for Medical Oncology (ESMO), and the ESMO World Congress on Gastrointestinal Cancer. A search strategy was developed for use in MEDLINE consisting of medical subject headings (MeSH) combined with text words for CRC and those associated with immune score, immune system, immune infiltrated cells, tumor infiltrating lymphocytes, CD3 and immunotherapy. This strategy was adapted for use in other databases. Additional relevant publications identified by the authors were also included.

Eligibility criteria

Eligible studies evaluated immune infiltrates in patients with non-metastatic and metastatic CRC, and included endpoints related to oncological outcomes such as response rate and survival. Concomitant patients' clinical-pathological features and disease molecular assessments (mismatch repair status: Microsatellite Stable (MSS) or Microsatellite Instable (MSI); BRAF and RAS mutations) available for multivariable testing were also analyzed.

Prospective and retrospective studies with a minimum of 30 patients published between January 2002 and May 2019 (manuscripts), or between January 2016 and June 2019 (congress abstracts) were included. We also included post-hoc assessments of randomized controlled trials assessing immune cells infiltrates in CRC patients and additional studies felt to be of interest by the authors. Preclinical studies were excluded along with narrative reviews, editorials, opinions, letters, non-English language publications and congress abstracts for which insufficient methodological details were reported to allow critical appraisal of study quality and/or the endpoints of interest.

Data synthesis

Methodological aspects of immune scores

Studies evaluating the clinical impact of immune infiltrates in CRC have been based on different morphological techniques. Most of the studies rely on immunohistochemical (IHC) techniques based on monoclonal antibodies against T lymphocytes (CD3), cytotoxic T lymphocytes (CD8) and memory T lymphocytes (CD45R0), usable on formalin-fixed paraffinembedded tissue. Different evaluation methods have been used: the only use of CD3, the joint use of CD3+/CD45RO+ or CD3+/CD8+ or CD8+/CD45RO+, a microarray tissue approach (consisting of duplicated or triplicated tissue cores of 0.6 to 1 mm in diameter from tumor samples) [START_REF] Pages | In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer[END_REF][START_REF] Mlecnik | Biomolecular network reconstruction identifies Tcell homing factors associated with survival in colorectal cancer[END_REF] or a whole tumor tissue section approach [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF]; quantification on the invasion front and/or in the tumor or a so-called linear quantification [START_REF] Allard | Linear quantification of lymphoid infiltration of the tumor margin: a reproducible method, developed with colorectal cancer tissues, for assessing a highly variable prognostic factor[END_REF]. In most cases, quantification was performed by image analysis and only some studies used a semiquantification approach [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF][START_REF] Pages | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF][START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF][START_REF] Allard | Linear quantification of lymphoid infiltration of the tumor margin: a reproducible method, developed with colorectal cancer tissues, for assessing a highly variable prognostic factor[END_REF][START_REF] Mlecnik | Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability[END_REF][START_REF] Anitei | Prognostic and predictive values of the immunoscore in patients with rectal cancer[END_REF][START_REF] Kwak | Immunoscore encompassing CD3+ and CD8+ T cell densities in distant metastasis is a robust prognostic marker for advanced colorectal cancer[END_REF][START_REF] Emile | Prospective validation of a lymphocyte infiltration prognostic test in stage III colon cancer patients treated with adjuvant FOLFOX[END_REF][START_REF] Yoon | Intertumoral Heterogeneity of CD3(+) and CD8(+) T-Cell Densities in the Microenvironment of DNA Mismatch-Repair-Deficient Colon Cancers: Implications for Prognosis[END_REF][START_REF] Mlecnik | Comprehensive Intrametastatic Immune Quantification and Major Impact of Immunoscore on Survival[END_REF][START_REF] Tanis | Prognostic impact of immune response in resectable colorectal liver metastases treated by surgery alone or surgery with perioperative FOLFOX in the randomised EORTC study 40983[END_REF][START_REF] Park | Tumour invasiveness, the local and systemic environment and the basis of staging systems in colorectal cancer[END_REF]. The methodological aspects of the immune infiltrate assessment are summarized in table 1.

A standardization of the evaluation of lymphocyte immune infiltrates, called Immunoscore®, has been proposed, based on an international multicenter study [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF]. Immunoscore® is based on the determination of the intratumoral density of lymphocyte populations (CD3+/CD45RO+ or CD3+/CD8+ or CD8+/CD45RO+) in the core of tumor (CT) and in the tumor's invasive margin (IM) (figure 3). This is achieved by an IHC technique on tissue sections coupled with an image analysis system. For each case, CD3+ and CD8+ cell densities in colon tumor's core and invasive margin regions were compared. The mean of four percentiles (two markers, two regions) was calculated and converted into a score called Immunoscore® (figure 3). In a three-category Immunoscore® analysis, a 0-25% density was scored as low, a density between 25% and 70% was scored as intermediate, and a density between 70% and 100% density was scored as high. In a two-category Immunoscore® analysis, a 0-25% density was scored as low, and a density between 25% and 100% was scored as intermediate-high. The lack of reproducibility of the Immunoscore® outside expert hands could be the main weakness of the method. Its reproducibility was thus tested in a study involving 13 centers around the world [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF]. The Immunoscore® was highly reproducible among centers and investigators (r=0.97 for the evaluation of the Immunoscore® at the tumor center; r=0.97 for its evaluation at the invasive margin; p<0.0001).

Correlation with clinical-pathological characteristics

A few studies have assessed the association between tumor T-cell infiltration and clinicalpathological parameters. In a series of 152 cases of resected CRC, no correlation was found between tumor CD8+ densities and age or tumor location [START_REF] Prall | Prognostic role of CD8+ tumor-infiltrating lymphocytes in stage III colorectal cancer with and without microsatellite instability[END_REF]. In an ancillary study of the adjuvant PETACC8 phase III trial, the density of lymphocyte infiltration determined in 1220 stage III colon cancers with an automated counting of CD3 lymphocytes within the tumor margin was also not correlated with age, gender, WHO performance status, bowel obstruction or perforation and tumor location or grade [START_REF] Emile | Prospective validation of a lymphocyte infiltration prognostic test in stage III colon cancer patients treated with adjuvant FOLFOX[END_REF]. Galon et al. showed that CRC cases with a high density of infiltrating memory (CD45RO+) and effector (CD3+CD8+) T cells were less likely associated with positive regional lymph nodes, vascular emboli, lymphatic or perineural invasion ("VELIPI" status), which are all considered as early steps of the metastatic process [START_REF] Pages | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF]. In two independent cohorts of patients, the same team reported a correlation between high densities of CD8+ cytotoxic T-lymphocyte infiltrates and early-stage Tis/T1 disease [START_REF] Mlecnik | Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction[END_REF]. While it was also significantly correlated with pT stage, the CD3-based lymphocyte infiltration developed by Emile et al. was not associated with pN stage or with vascular invasion or lymphatic infiltration [START_REF] Emile | Prospective validation of a lymphocyte infiltration prognostic test in stage III colon cancer patients treated with adjuvant FOLFOX[END_REF], with the limits of a pure stage III population.

Prognostic impact

In human cancers, the presence of TILs generally indicates a favorable prognosis. In CRC, Pages et al. have found in a first cohort of 958 resected stage I to IV CRC patients an association between T-cell immune response within the tumor, the absence of early metastatic invasion (VELIPI-negative status) and increased survival [START_REF] Pages | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF]. Using the same cohort of patients, they showed that the type, density and location of immune cells within the tumor may influence the clinical outcome of CRC patients. The adaptive immune reaction composed of T cells (CD3+) with cytotoxic (CD8+) and memory (CD45RO+) phenotypes in the CT and IM was a highly significant parameter to predict time to recurrence and OS. These initial results were subsequently validated in two additional patient populations [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF] (table 2). After standardization of their method, the same team showed the usefulness of an Immunoscore® based on the combined evaluation of memory and cytotoxicity markers for the prediction of tumor recurrence and survival, specifically in early-stage (UICC/TNM stage I and II) CRC [START_REF] Pages | In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer[END_REF]. Then, Mlecnik et al. investigated the relationship between the extent of immune cell density (CD45RO+ and CD8+ T cells) in the CT and IM of the tumors, the degree of tumor extension (pT and pN stages) and the frequency of tumor recurrence in two independent cohorts of 415 and 184 specimens of stage I to IV CRC [START_REF] Mlecnik | Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction[END_REF]. In the subgroup of stage I to III CRC patients, they showed a better prognosis (i.e. better DFS and OS) associated with a strong density of both CD45RO+ and CD8+ T cells in CT and IM. In multivariate analysis of DFS, including pT and pN stage, age, sex, total number of lymph nodes, tumor grade, mucinous type, occlusion and bowel perforation, only the Immunoscore® and bowel perforation remained significant prognostic markers (table 2). These data suggest that the Immunoscore® may accurately predict recurrence in addition to the TNM classification. Interestingly, in patients who did not relapse, the density of CD8 infiltrates was inversely correlated with T stage whereas in patients who relapsed the number of CD8 cells was low regardless of the T stage of the tumor, suggesting an inverse correlation between in situ response and tumor progression.

Two other large studies, including 843 and 768 patients respectively, have shown that tumor immune infiltrate patterns are significant prognostic biomarkers, even after adjustment for stage, lymph node involvement and well-established prognostic tumor molecular markers including MSI status or BRAF mutation [START_REF] Nosho | Tumour-infiltrating T-cell subsets, molecular changes in colorectal cancer, and prognosis: cohort study and literature review[END_REF][START_REF] Ogino | Lymphocytic reaction to colorectal cancer is associated with longer survival, independent of lymph node count, microsatellite instability, and CpG island methylator phenotype[END_REF]. Two large CRC population-based studies have suggested that, in addition to TILs, high peritumoraL diffuse lymphoid inflammation (lymphoid cell infiltrates within stromal tissue along the invasive front) and Crohn's diseaselike lymphoid reaction (lymphoid aggregates corresponding to tertiary lymphoid structures), which are frequent features of MSI tumors, are both significantly associated with reduced risks of CRC-specific survival and OS independently of traditional prognostic markers including the MSI status [START_REF] Rozek | Tumor-Infiltrating Lymphocytes, Crohn's-Like Lymphoid Reaction, and Survival From Colorectal Cancer[END_REF][START_REF] Hynes | Back to the future: routine morphological assessment of the tumour microenvironment is prognostic in stage II/III colon cancer in a large population-based study[END_REF] (table 2). In 1220 stage III colon cancer patients treated with adjuvant FOLFOX included in the PETACC8 phase III study [START_REF] Emile | Prospective validation of a lymphocyte infiltration prognostic test in stage III colon cancer patients treated with adjuvant FOLFOX[END_REF], lymphocyte infiltration was analyzed using a linear quantification of CD3 density of the tumor margin previously demonstrated as an independent prognostic factor in a study of 119 stage II-III colon cancers [START_REF] Allard | Linear quantification of lymphoid infiltration of the tumor margin: a reproducible method, developed with colorectal cancer tissues, for assessing a highly variable prognostic factor[END_REF]. Patients with high lymphocyte infiltration had a lower 2-year time-to-recurrence (14% vs 21% p=0.02) and longer 3-year DFS (81% vs 72%; HR = 0.69; p=0.01) as and 5-year OS (89% vs 80%, HR=0.58; p=0.0048). Lymphocyte infiltration remained with tumor stage, grade, RAS and BRAF status an independent prognostic factor in multivariate analysis (HR=0.6; 95%CI: 0.4-0.98; p=0.04) [START_REF] Emile | Prospective validation of a lymphocyte infiltration prognostic test in stage III colon cancer patients treated with adjuvant FOLFOX[END_REF] (table 2).

In parallel, an independent international panel of expert laboratories has started to work on large cohorts of colon cancers to promote the Immunoscore® (a trade mark test) based on the density of CD3+/CD8+ T cells evaluated by immunohistochemistry in routine clinical practice [START_REF] Galon | Cancer classification using the Immunoscore: a worldwide task force[END_REF] and evaluate its relationship with MSI and the respective prognostic impact of these two related parameters. Mlecnik et al. demonstrated, from the analysis of two cohorts comprising more than 900 CRC patients, that MSI tumors, but also a subgroup of MSS tumors, are characterized by a prominent gene immune expression [START_REF] Mlecnik | Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability[END_REF]. In this study, the Immunoscore®, but not MSI and TNM parameters, was significantly predictive of patient's disease recurrence and survival in multivariate analysis [START_REF] Mlecnik | Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability[END_REF]. These findings indicate that assessment of the Immunoscore® provides a potent indicator of tumor recurrence and could be an important guide, in addition to TNM stage and MSI status, for adjuvant chemotherapy after resection of CRC.

A prospective validation of the Immunoscore® in clinical practice has been recently published by Pages et al. [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF] thanks to an international consortium that analyzed 2681 resected stage I-III colon cancers from 14 centers in 13 countries. This study confirmed the very good intra-and inter-observer reproducibility of the Immunoscore® and its powerful prognostic value, with a 8% recurrence rate at 5 years in patients with high Immunoscore® compared to 19% and 32% in cases of intermediate and low Immunoscore® respectively (HR 0.20, p <0.0001). In multivariate analysis, the prognostic impact of Immunoscore® on DFS was independent of age, gender, T and N stage, but also of MSI. This prognostic value was maintained in the subgroup of 1434 stage II colon cancers (hazard ratio (HR) high versus low 0.20; p<0.0001). Immunoscore® had the highest relative contribution to the risk of recurrence among all clinical-pathological parameters (table 2). These results support the implementation of the Immunoscore® as a new component of the prognostic classification of CRC.

Predictive impact on adjuvant chemotherapy benefit

Whether the immune contexture of the primary tumor predicts the benefit from adjuvant chemotherapy is of high importance for patient clinical management. Some studies have reported an excellent prognosis of CRC patients with a strong immune component receiving 5-fluorouracil/leucovorin as adjuvant treatment [START_REF] Prall | Prognostic role of CD8+ tumor-infiltrating lymphocytes in stage III colorectal cancer with and without microsatellite instability[END_REF]. However, the high cytotoxic immune response may not be predictive of response to adjuvant chemotherapy, but rather reflects the fact that the host-immune response within the tumor protects the patient and prolongs patient survival. In patients with stage III colon cancer treated with oxaliplatin-based chemotherapy, the prognostic value of the Immunoscore® for DFS was confirmed in the 3-vs 6-month adjuvant trial IDEA-France [START_REF] Pages | Validation of the Immunoscore prognostic value in stage III colon cancer patients treated with oxaliplatin in the prospective IDEA France cohort study (PRODIGE-GERCOR)[END_REF]. Patients treated with mFOLFOX6 (90% of the patients in the trial) with high or intermediate Immunoscore® had better DFS in the 6-month arm than in the 3-month arm, whether they have a high-risk (T4N2) or low-risk (T1-3N1) stage III cancer (table 2). By contrast, the duration of adjuvant chemotherapy did not seem to make a difference in patients with low Immunoscore® treated with the same regimen. These data suggest that the duration of adjuvant chemotherapy could be guided by the Immunoscore® (at least for mFOLFOX6). Albeit counterintuitive (patients with a good prognosis needing a longer adjuvant treatment), these data may reflect a higher sensitivity of tumors infiltrated by immune cells to oxaliplatin-based adjuvant treatment or conversely a resistance to treatment in tumors without any immune infiltrates. These data regarding adjuvant mFOLFOX6 need to be confirmed in other IDEA studies, which should also explore whether there is any difference in patients treated with CAPOX (received by only 10% of patients in IDEA-France study), the other standard adjuvant regimen. Besides these results on adjuvant treatment duration, we still lack any clear published data regarding the predictive value of the Immunoscore®. Another important data currently missing is the potential interest of neo-adjuvant or adjuvant immunotherapy in patients with a high Immunoscore®, whose survival is good but could be improved by immunotherapy. Until we have these data, the Immunoscore® is in our opinion unlikely to be incorporated into routine clinical practice.

Immunoscore and deficient mismatch repair and/or microsatellite instability (MSI)

MSI tumors, including CRC, accumulate insertions and deletions in DNA repeat sequences.

About two-thirds of MSI tumors are sporadic, while one-third is inherited (Lynch syndrome).

The high mutational load (i.e. hypermutated phenotype) and frequent frameshift mutations that characterize MSI tumors lead to the production of many neo-antigens recognized by the immune system, which can trigger lymphocyte infiltration in these tumors. However, these immune infiltrates may differ in quantity and quality within MSI patients [START_REF] Llosa | The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints[END_REF][START_REF] Maby | Correlation between Density of CD8+ T-cell Infiltrate in Microsatellite Unstable Colorectal Cancers and Frameshift Mutations: A Rationale for Personalized Immunotherapy[END_REF]. MSI tumors represent approximately 15% to 20% of stage II and 10% of stage III CRC cases, and are associated with better prognosis compared to MSS tumors [START_REF] Zaanan | Role of Deficient DNA Mismatch Repair Status in Patients With Stage III Colon Cancer Treated With FOLFOX Adjuvant Chemotherapy: A Pooled Analysis From 2 Randomized Clinical Trials[END_REF]. By contrast, the prognostic value of MSI status is debated in mCRC, and MSI tumors represent only 5% of mCRC cases [START_REF] Lenz | Impact of Consensus Molecular Subtype on Survival in Patients With Metastatic Colorectal Cancer: Results From CALGB/SWOG 80405 (Alliance)[END_REF][START_REF] Innocenti | Mutational Analysis of Patients With Colorectal Cancer in CALGB/SWOG 80405 Identifies New Roles of Microsatellite Instability and Tumor Mutational Burden for Patient Outcome[END_REF][START_REF] Taieb | Prognosis of microsatellite instability and/or mismatch repair deficiency stage III colon cancer patients after disease recurrence following adjuvant treatment: results of an ACCENT pooled analysis of seven studies[END_REF][START_REF] Heinemann | Somatic DNA mutations, tumor mutational burden (TMB), and MSI Status: Association with efficacy in patients (pts) with metastatic colorectal cancer (mCRC) of FIRE-3 (AIO KRK-0306)[END_REF].

Mlecnik et al. demonstrated that the Immunoscore® was superior to MSI in predicting disease recurrence and survival in patients with stage I-III CRC [START_REF] Mlecnik | Biomolecular network reconstruction identifies Tcell homing factors associated with survival in colorectal cancer[END_REF]. In this study, among 105 patients with MSI tumor, patients with high Immunoscore® (n=71) had prolonged diseasespecific survival and OS as compared to patients with low Immunoscore® (n=34). The DFS of low Immunoscore® patients was similar in MSS and MSI tumors as well as the DFS of high Immunoscore® patients (approximately 60% and 80% at 3 years, respectively) [START_REF] Mlecnik | Biomolecular network reconstruction identifies Tcell homing factors associated with survival in colorectal cancer[END_REF]. These findings suggest that assessment of Immunoscore® may help to decide adjuvant therapy in patients with early-stage, MSS but also MSI CRC. It is also possible that only MSI patients with high Immunoscore® might benefit from checkpoint inhibitors in future clinical trials. Of note, 32% of MSI patients had a low Immunoscore® in this study.

In the IDEA-France study, among the 93 patients with MSI tumors evaluable for Immunoscore®, 20 (21.5%) had low Immunoscore®, 56 (60.2%) had intermediate or high Immunoscore® and 17 (18.3%) had inconclusive results [START_REF] Pages | Validation of the Immunoscore prognostic value in stage III colon cancer patients treated with oxaliplatin in the prospective IDEA France cohort study (PRODIGE-GERCOR)[END_REF]. Among the 304 patients with MSI tumors in the international validation study of the Immunoscore®, 49 patients (16%) had low Immunoscore® and 255 (84%) had intermediate and high Immunoscore® [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF].

Patients with intermediate and high Immunoscore® had prolonged DFS and OS, irrespective of their microsatellite status (unadjusted hazard ratio for high versus low Immunoscore® in MSI patients: 0.56, 95%CI 0.34-0.90; p=0.015). In multivariable analysis, the Immunoscore® predicted time to recurrence independently from T and N stage, but also MSI [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF].

Yoon et al. examined CD3+ and CD8+ T cells in stage III colon cancer from patients included

in the adjuvant study N0147 (FOLFOX6 ± cetuximab). MSI tumors, despite a higher CD3+ and CD8+ lymphocytic infiltration in the tumor micro-environment, had greater inter-tumor patient heterogeneity in terms of density of CD3+ and CD8+ T cells than MSS tumors [START_REF] Yoon | Intertumoral Heterogeneity of CD3(+) and CD8(+) T-Cell Densities in the Microenvironment of DNA Mismatch-Repair-Deficient Colon Cancers: Implications for Prognosis[END_REF].

Patients whose tumor had a low T cell-density had a shorter OS compared with patients harboring tumors with a high T-cell density. Around 30% of MSI tumors exhibited T-cell densities as low as in MSS tumors. CD3+ at the IM provided robust stratification for prognosis and identified both MSI and MSS tumors with low CD3+ IM infiltrates and shorter OS. Another study demonstrated higher heterogeneities in MSI than in MSS CRC [START_REF] Taieb | Prognosis of microsatellite instability and/or mismatch repair deficiency stage III colon cancer patients after disease recurrence following adjuvant treatment: results of an ACCENT pooled analysis of seven studies[END_REF].

Altogether these publications stress the variability of immune infiltrate density within MSI tumors and identify 16-32% of MSI CRC with low lymphocyte infiltrates.

Differences in mutation burden and variability in neoantigen profiles and unstable microsatellites may explain the heterogeneity of immune infiltrates among MSI tumors. Lynch-associated MSI tumors present with more somatic mutations and neoantigens compared with sporadic MSI tumors, which may result in stronger immune reactions and different outcomes. Immune checkpoint expression may cancel the prognostic relevance of tumor-infiltrating T cells in highly immunogenic colon tumors and predicts a poor outcome in MSI CRC patients [START_REF] Marisa | The Balance Between Cytotoxic T-cell Lymphocytes and Immune Checkpoint Expression in the Prognosis of Colon Tumors[END_REF]. At early stage, MSI leads to the synthesis of aberrant and potentially immunogenic neoantigens. A consequence is that MSI tumors are heavily infiltrated with active cytotoxic T cells and that immune response is efficacious to avoid tumor growth and metastatic dissemination. This phenomenon explains the good prognosis of MSI stage I to III colon cancer. However, for some tumors, overexpression of immune checkpoints could allow immune escape, tumor growth and development of distant metastases. Once disease is metastatic, MSI has been shown to be a major predictive biomarker for the efficacy of immune checkpoint inhibitors among mCRC patients, as well as other metastatic MSI tumors. Clinical responses and long survival were observed across all biomarker groups assessed (PD-L1 tumor expression, BRAF or KRAS mutations, or a clinical history of Lynch syndrome) [START_REF] Le | PD-1 Blockade in Tumors with Mismatch-Repair Deficiency[END_REF][START_REF] Overman | Durable Clinical Benefit With Nivolumab Plus Ipilimumab in DNA Mismatch Repair-Deficient/Microsatellite Instability-High Metastatic Colorectal Cancer[END_REF][START_REF] Overman | Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study[END_REF] but no data are available yet regarding immune scores in patients with MSI mCRC treated with immune checkpoint inhibitors and the predictive value of immune scores in MSS mCRC, though trials are on-going.

Metastatic disease

Tumor-infiltrating lymphocyte counts and T-cell subsets (CD4+, CD8+, Tregs, FOXp3+) in liver metastases have been reported to be independent prognostic indicators of survival after CRC liver resection [START_REF] Halama | Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy[END_REF][START_REF] Katz | Regulatory T cell infiltration predicts outcome following resection of colorectal cancer liver metastases[END_REF][START_REF] Nakagawa | Low infiltration of peritumoral regulatory T cells predicts worse outcome following resection of colorectal liver metastases[END_REF][START_REF] Maker | Genetic evidence that intratumoral T-cell proliferation and activation are associated with recurrence and survival in patients with resected colorectal liver metastases[END_REF]. T-cell counts and subset ratios in liver metastases were shown to predict recurrence and survival [START_REF] Katz | Regulatory T cell infiltration predicts outcome following resection of colorectal cancer liver metastases[END_REF][START_REF] Nakagawa | Low infiltration of peritumoral regulatory T cells predicts worse outcome following resection of colorectal liver metastases[END_REF][START_REF] Katz | T cell infiltrate predicts long-term survival following resection of colorectal cancer liver metastases[END_REF][START_REF] Brunner | Prognosis according to histochemical analysis of liver metastases removed at liver resection[END_REF][START_REF] Brudvik | Regulatory T-cell-mediated inhibition of antitumor immune responses is associated with clinical outcome in patients with liver metastasis from colorectal cancer[END_REF]. In a comparison of patients who survived ≤ 2 and ≥ 10 years following resection of CRC liver metastases, high CD8+ T cell counts and low CD4+ T-cell counts were associated with 10-year survival rate following surgery [START_REF] Nakagawa | Low infiltration of peritumoral regulatory T cells predicts worse outcome following resection of colorectal liver metastases[END_REF]. Another study showed that low CD45RO infiltration at the IM and fibrotic capsule formation independently predicted prolonged survival. The number of peritumoral Tregs was shown to predict cancer-specific survival and DFS in mCRC patients undergoing liver resection. A scoring system using TIL densities based on CD3+, CD8+, granzyme B+ and FOXp3+ immune cells at the IM of liver metastases predicted the response to chemotherapy with a sensitivity of 79% and specificity of 100% [START_REF] Halama | Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy[END_REF].

The evolution patterns during metastatic progression depend on the immune contexture at the metastatic site [START_REF] Angelova | Evolution of Metastases in Space and Time under Immune Selection[END_REF]. Van den Eynde et al. quantified immune cell types on whole slides of 603 resected, synchronous or metachronous metastases and 97 primary tumors in a retrospective series of 222 mCRC patients [START_REF] Van Den Eynde | The Link between the Multiverse of Immune Microenvironments in Metastases and the Survival of Colorectal Cancer Patients[END_REF]. They showed the intra-and inter-tumor lesions and inter-patient heterogeneity of immune T-cell (CD3, CD8, CD45RO, FOXP3) and Bcell infiltrates for both primary tumors and metastases. As a consequence, the Immunoscore® was generally heterogeneous between metastases from the same patient, whereas interestingly, comparisons of Immunoscore® within lung versus liver metastases, and within synchronous versus metachronous metastases showed no difference. Mutational profiling showed heterogeneity between metastases and primary tumors, in concordance with the idea that tumors grow by clonal expansion and accumulating genetically diverse subpopulations. A high Immunoscore® was associated with a significantly lower number of metastases per patient. In particular, the patients with the fewest metastases had the highest immune intra-metastatic density in its least-infiltrated metastasis. CD3 and CD8 densities were higher in metastases compared with the primary tumors of the same patients showing no general loss of immunity within metastases. This could also reflect the development of an immunosuppressive microenvironment within the primary tumors over time, and this is supported by the higher FOXP3 densities observed within the CT of the primary tumors, particularly in anti-EGFR-treated patients. However, metastases from patients with highly infiltrated primary tumors also had higher densities of immune cells suggesting a more active systemic tumor-specific immune response.

The liver and brain are metastatic sites considered "immunoprivileged" because of their relatively low basic immune infiltration and resistance to immune response [START_REF] Halama | Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy[END_REF][START_REF] Katz | Regulatory T cell infiltration predicts outcome following resection of colorectal cancer liver metastases[END_REF]. This phenomenon may explain why "cold liver metastases" with poor T-cell infiltrates were more numerous and associated with a more unfavorable prognosis the work of Van den Eynde et al [START_REF] Overman | Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study[END_REF]. Immune densities, Immunoscore® and T-and B-cell score (TB score) (CD8 and CD20) were investigated in relation to patient outcome. In both training and validation cohorts, high immune infiltrate, Immunoscore® and TB score were significantly associated with prolonged DFS and OS, even in patients with large metastases. The least-infiltrated metastasis showed the strongest association with patient outcome (HR > 2, p < 0.05) compared with the mean metastatic infiltrate or a randomly selected metastasis, and no association with the patient survival was observed for the most-infiltrated metastasis. In another publication derived from the same patient population, patients with a high Immunoscore® in their least-infiltrated metastasis had a statistically significantly lower risk of relapse (DFS rate 27.9% vs 12.3%; HR 0.45 [95% CI 0.28-0.70], P = 0.02) and a prolonged OS (OS rate 64.6% vs 32.5%; HR 0.32 [95% CI 0.15-0.66], P = 0.001) compared with patients with a low Immunoscore® [START_REF] Mlecnik | Comprehensive Intrametastatic Immune Quantification and Major Impact of Immunoscore on Survival[END_REF]. Similar results were obtained for the TB score. Among patients not responding to preoperative systemic therapy according to their histological tumor regression grade, those with strong immune infiltrates had better DFS (HR 0.28, 95% CI 0.15-0.52, P = 0.001) and better OS (HR 0.25, 95% CI 0.1-0.62, P = 0.001) than those with low immune infiltrates. In a multivariable analysis of relevant clinical and immune parameters including TB score or Immunoscore®, node stage, preoperative treatment type, metastasis resection margin status, tumor regression grade and RAS status, only the TB score (HR 0.20, 95% CI 0.08-0.48, P < 0.001) or the Immunoscore® (HR 0.24, 95% CI 0.10-0.58, P = 0.001) remained statistically significantly associated with OS. The TB score was also statistically significantly associated with DFS (HR 0.42, 95% CI 0.25-0.71, P = 0,001), as was the number of metastases per patient (≤3 vs >3; HR 2.28, 95% CI 1.38-3.77, P = 0,001).

Markers included in the OS final model were also tested for DFS, and similar results were obtained for both TB score and Immunoscore®.

Quantification of TILs in a small region of a randomly selected metastasis has previously been reported to be associated with survival. Several reports underlined the possible limitation of tumor sampling using single biopsies for patient treatment decision. Van den Eynde et al. showed that computer simulation of a single biopsy correctly identified lowinfiltrated metastases in more than 90% of the cases, but that due to the heterogeneity of the metastatic disease, overall intra-metastatic immune infiltrate might be better estimated with multiple biopsies or sampling of larger tumor areas [START_REF] Van Den Eynde | The Link between the Multiverse of Immune Microenvironments in Metastases and the Survival of Colorectal Cancer Patients[END_REF].

Van den Eynde et al. also explored differences in the immune infiltration of lesions according to the type of neoadjuvant treatment received in a subset of 48 patients [START_REF] Van Den Eynde | The Link between the Multiverse of Immune Microenvironments in Metastases and the Survival of Colorectal Cancer Patients[END_REF]. Patients treated with chemotherapy and anti-EGFR had greater infiltration of T lymphocytes (CD8, CD45RO, FOXP3) into the CT of metastases and higher Immunoscore® than patients who received chemotherapy and anti-VEGF. Strikingly, the strongest increase of immune densities in the treated patient samples was observed in the highest infiltrated metastasis, while the mean infiltration of metastases showed only a slight increase after anti-EGFR treatment, and the least-infiltrated metastasis showed no significant changes. These results suggest that the treatment could be more efficient in increasing the intra-metastatic immune reaction in metastases with pre-existing immune infiltrate. They also observed upregulation of B cell pathways (immune cell densities and gene expression) in primary tumors after chemotherapy plus anti-VEGF treatment.

In a retrospective study of resected CRC liver metastases in 249 patients, a high immune score was associated with fewer liver metastases (P = 0.020), smaller lesion sizes (P = 0.028) and lower Fong's clinical risk score [START_REF] Fong | Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer: analysis of 1001 consecutive cases[END_REF](CRS) (P = 0.033). Patients with high immune score had longer RFS (21.4 vs 8.7 months, P < 0.001; 3-year RFS rate, 42.4 vs. 17.0%; P < 0.001) and OS (not reached vs 28.7 months, P < 0.001; 5-year OS rate, 59.7 vs. 25.9%, P < 0.001) than those with low immune score [START_REF] Wang | The Immunoscore system predicts prognosis after liver metastasectomy in colorectal cancer liver metastases[END_REF]. The prognostic value of the Immunoscore for OS persisted after stratification on CRS.

In another series of 196 mCRC patients, the densities of CD3+, CD4+, CD8+, FOXP3+, CD68+ and CD163+ immune cells were determined in both primary tumor and metastases. High values of primary tumor Immune score, metastatic immune score (immune score determined in a distant metastasis added to primary tumor immune score) and macrophage immune score (CD163+ density in the CT and IM of primary tumor added to primary tumor immune score; with high density of tumor-associated macrophages recorded as score 0, contrarily to CD3+ and CD8+ TILs) were significantly associated with a better prognosis [START_REF] Kwak | Immunoscore encompassing CD3+ and CD8+ T cell densities in distant metastasis is a robust prognostic marker for advanced colorectal cancer[END_REF].

In multivariate analysis, only the metastatic immune score retained an independent value (low vs high, HR 1.86 (95% CI 1.14-3.02), p=0.012).

Conclusion

Different scoring systems evaluating lymphocyte infiltrates in colorectal cancer (primary tumor and/or metastases) have been elaborated and developed. The Immunoscore® has been the most widely studied and is achieved by an IHC technique on tissue section coupled with an image analysis system. Its prognostic performance has been shown in colon and rectal cancer, primary and metastatic lesions, MSI and MSS tumors and confirmed each time in multivariate analyses. This Immunoscore® may also become in the future a useful tool in the prediction of efficacy of immunotherapy in patients with MSI or MSS CRC. The role of Immunoscore® in the management of CRC patients in the adjuvant setting, including postresection of metastases, needs more data, ideally through biomarker-driven prospective clinical trials, to better define its impact on the need, type and duration of adjuvant therapies. The antitumor microenvironment encompasses CD8+ cytotoxic lymphocytes and type 1 helper (Th1) T cells in the tumor core and the invasive margin. In the stroma, tertiary lymphoid structures characterized by an association of B cells, CD8+ cytotoxic lymphocytes and dendritic cells mimicking a lymph node can also be observed. This contexture mainly reflects an adaptive immune response. Besides these cell populations, pro-tumorigenic cells can be encountered such as regulatory T cells (Tregs). Macrophages have an anti-or protumor role, depending on their phenotype, M1 or M2 respectively.

Figure 3 The Immunoscore® design in colorectal cancer

The Immunoscore® is a standardized approach to characterizing T-cell infiltration of surgical pathology tumor specimen, notably colorectal cancer. Left panels: Digital representation of tissue areas. The border between the advancing tumor edge and normal tissue is annotated on a colorectal carcinoma specimen. A 500-μm distance on either side of this border is designated the 'invasive margin' (IM, outlined in yellow). The adjacent healthy tissue is excluded (outlined in green and blue). The remainder of the tumor is designated as the 'tumor core' (TC, outlined in red). Upper right panels: Immunohistochemistry for CD3 and CD8 used to quantify cell densities for each of these immune cell subsets in both the IM and TC. Lower right panels: The density of each region is labeled 'high' or 'low' density for each marker. The mean percentile of the four immune parameters is calculated, resulting in a -Data confirmed regardless of MMR status: MSI and MSS with a low IS associated with a higher risk of relapse, shorter DSS and OS compared to high IS

Figure 1 .

 1 Figure 1. Schematic illustration of the anti-tumor response Innate immunity involves cells such as NK cells, NKT cells and macrophages, leading to tumor cell lysis. The released antigens are processed by dendritic cells which are presented to CD4 T lymphocytes through major histocompatibility class (MHC) II or to CD8 T lymphocytes through MHC I. Then activated cytotoxic CD8+ T cells can recognize and lyse tumor cells. This immune response can be counterbalanced by immunosuppressive cells, immunosuppressive molecules and exhaustion molecules (abbreviations: DC: dendritic cells, Mφ: Macrophages, MDSC: myeloid derived suppressive cells NK, Natural Killer; NKT Natural Killer T, Treg: regulatory T cells).

Figure 2 .

 2 Figure 2. The immune microenvironment of colorectal cancer

Figure 3

 3 Figure 3.

---FOXP3-----

  -CD8+ associated with better OS (HR 0.43; p=0.009) and DFS (HR 0.56; p= 0.047) in MVA -CD8+ high/MSI associated with better OS (HR 0.65; p=0.023) in MVA Pages 2005 (13) I-IV (959) (I-III: 750) CD45RO -IS associated with VELIPI-negative tumors in MVA -CD45RO+ associated with better DFS (53.2 vs 20.6 mo, p<0.001) and OS (36.5 vs 11.1 mo, p<0.001) -CD45RO+: independent prognostic factor for OS (p=0.02) in MVA including T, CD3CT/CD3IM, CD8CT/CD8IM, CD45ROCT/CD45ROIM (separated or combined) associated with better DFS and OS overall and in stage I-III -CD3CT/CD3IM: independent prognostic factor for DFS and OS in MVA including T, N and grade Pages 2009 CD8CT/CD8IM, CD45ROCT/CD45ROIM (separated or combined) associated with better DFS and OS -CD8CT/IM, CD45ROCT/IM: independent prognostic factors of DFS and OS in MVA including T TIL associated with better CRC-specific survival in UVA but not in MVA -Lymphocytic reaction associated with better 5-yr CRC-specific OS (p=0.002) and OS (p=0.002) in MVA including N, KRAS/BRAF status, MSI, CIMP and other clinical-pathological factors # MSI independently associated with CD45RO+ Density of CD8+, CD45RO+ and FOXP3+ associated with OS in UVA -CD45RO+ (but not CD3+, CD8+ and FOXP3+) associated with CRC-specific OS (p=0.0032) and OS (p=0.0036) in MVA including KRAS/BRAF status, MSI, CIMP and other clinical-pathological factors £ CD8+ associated with more frequent Tis/T1 stage tumors In stage I-III CRC: -IS (CD3CT/CD3IM, CD8CT/CD8IM, CD45ROCT/CD45ROIM) associated with better DFS and OS (p<0.005) in UVA -IS and bowel perforation: the only prognostic factors in MVA (including T and N, age, sex, total number of lymph nodes, grade, mucinous colloid type, occlusion and bowel perforation) T, N, LN ratio, VE, PNI, CD3+ associated with better DFS in UVA -CD3+ (HR: 6.02; p<0.001) and metastatic LN ratio: independent prognostic factors for DFS in MVA High IS (high CD8CT/IM CD45ROCT/IM infiltration) increased in MSI tumors (p<0.02) but ∼50% of MSS with a high IS -High IS associated with better DSS (HR 6.21; p<0.001), DFS (HR 6.35; p<0.001) and OS (HR 3.96; p<0.001)

Table 1 . Immune scoring systems: methodological aspects.

 1 cancer. CRC, colorectal. CT, center of the tumor. IM, invasive margin. NA, not available. RC, rectal cancer. TMA, tissue microarray. TNI, Tumor normal interface. WTS, whole-slide serial tissue section. * On primary tumor and liver metastases. ** On liver metastases.

	First author	Year	Primary Tumor	Patients Markers	Material	Tumor region	Technique
	[reference]			stage				analyzed
	Pagès [13]	2005	CRC	I-IV	959	CD45RO	TMA (n=415)	NA*	Image analysis
	Galon [12]	2006	CRC	I-IV	603	CD3/CD8/CD45RO	TMA	CT/IM	Image analysis
	Pagès [14]	2009	CRC	I-II	623	CD8/CD45RO	TMA	CT/IM	Image analysis
	Mlecnik [27] 2011	CRC	I-IV	599	CD3/CD8/CD45	TMA	CT/IM	Image analysis
	Allard [17]	2012	CRC	II-III	117	CD3/CD45RO	WTS	IM	Image analysis
	Anitei [18]	2014	RC	I-IV	166	CD3/CD8	TMA/WTS (biopsies) CT/IM	Image analysis
	Tanis [24]	2015	CRC	IV	82	CD3/CD8	WTS*	CT/TNI**	Image analysis
	Kwak [19]	2016	CRC	IV	196	CD3/CD8/CD163	TMA**	CT/IM	Image analysis
	Mlecnik [20] 2016	CRC	I-IV	964	CD3/CD8/CD45	TMA	CT/IM	Image analysis
	Park [25]	2017	CRC	III	331	CD3/CD8	WTS	CT/IM	Semi-quantitative
	Emile [21]	2017	CRC	III	1220	CD3	WTS	CT/TNI**	Image analysis
	Yoon [22]	2018	CC	III	561	CD3/CD8	WTS	CT/IM	Image analysis
	Mlecnik [23] 2018	CRC	IV	153	CD3/CD8; CD8/CD20 WTS*	CT/IM	Image analysis
	Pagès [16]	2018	CC	I-III	2681	CD3/CD8	WTS	CT/IM	Image analysis
	CC, colon

Table 2 . Selected studies of immune scores in the colorectal cancer adjuvant setting.

 2

possible Immunoscore ranging from I0 to I4, or three categories (Low, Intermediate, High) (abbreviations: IM, invasive margin; TC, tumor core).