Introduction

Immune checkpoint inhibitors (ICI) represent one of the major therapeutic breakthroughs in the history of medical oncology. In this context, metastatic colorectal cancer (mCRC) represent an intriguing entity, with a minority (4 to 5%) of tumors being highly sensitive to these compounds (i.e. CRC harboring microsatellite instability (MSI) and/or mismatch-repair deficiency (dMMR) [START_REF] Le | PD-1 Blockade in Tumors with Mismatch-Repair Deficiency[END_REF]) but a vast majority are cold tumors refractory to immunotherapeutic strategies [START_REF] Patnaik | Phase I Study of Pembrolizumab (MK-3475; Anti-PD-1 Monoclonal Antibody) in Patients with Advanced Solid Tumors[END_REF][START_REF] Topalian | Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer[END_REF][START_REF] Brahmer | Safety and Activity of Anti-PD-L1 Antibody in Patients with Advanced Cancer[END_REF]. It has been known for years that MSI/dMMR tumors are highly infiltrated by immune cells [START_REF] Jass | Classification of colorectal cancer based on correlation of clinical, morphological and molecular features[END_REF]. These tumors are characterized by a high tumor mutational burden (TMB) with highly immunogenic neoantigens arising from frameshift mutations [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]. Moreover, they are associated with an upregulation of checkpoint inhibitors that exhaust intratumoral cytotoxic T lymphocytes and consequently protect MSI cancer cells from their hostile immune microenvironment [START_REF] Marisa | The Balance Between Cytotoxic T-cell Lymphocytes and Immune Checkpoint Expression in the Prognosis of Colon Tumors[END_REF][START_REF] Llosa | The Vigorous Immune Microenvironment of Microsatellite Instable Colon Cancer Is Balanced by Multiple Counter-Inhibitory Checkpoints[END_REF].

Following the durable complete response of one patient with an MSI/dMMR mCRC treated with anti-PD1 ICI in a phase I study that included various types of unselected solid tumors, research and development of ICI for CRC focused on MSI/dMMR mCRC [START_REF] Brahmer | Phase I Study of Single-Agent Anti-Programmed Death-1 (MDX-1106) in Refractory Solid Tumors: Safety, Clinical Activity, Pharmacodynamics, and Immunologic Correlates[END_REF]. The impressive results of several phase II studies demonstrated that immune checkpoint inhibition is a breakthrough therapeutic strategy for dMMR/MSI mCRC [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]. Pembrolizumab or nivolumab alone or in combination with ipilimumab have been therefore approved by the Food and Drug Administration (FDA) for the treatment of chemoresistant MSI/dMMR mCRC patients. More, the FDA granted accelerated approval to pembrolizumab for advanced MSI/dMMR tumors progressing following prior treatment, in a tissue/site-agnostic fashion [START_REF] Le | Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade[END_REF]. The efficacy of ICI in MSI/dMMR mCRC is now well-established, and trials are currently ongoing to assess ICI efficacy in first line, adjuvant or even neoadjuvant setting.

However, questions remain concerning the role of ICI for the treatment of microsatellite stable (MSS) and mismatch repair-proficient (pMMR) CRC. The combination of ICI with other anticancer drugs is currently being evaluated in MSS/pMMR mCRC. However, the disappointing results of the phase III IMblaze 370 trial (atezolizumab with or without cobimetinib versus regorafenib) raises concerns regarding the testing of therapeutic ICI strategies without a guiding biomarker in these "cold" tumors [START_REF] Eng | Atezolizumab with or without cobimetinib versus regorafenib in previously treated metastatic colorectal cancer (IMblaze370): a multicentre, open-label, phase 3, randomised, controlled trial[END_REF]. It has been shown that TMB could be a biomarker for ICI efficacy in various histologies and some MSS/pMMR CRCs show a high TMB, notably due to mutations within genes coding for DNA polymerase [START_REF] Samstein | Tumor mutational load predicts survival after immunotherapy across multiple cancer types[END_REF][START_REF] Muzny | Comprehensive molecular characterization of human colon and rectal cancer[END_REF]. Furthermore, the consensus molecular subtype (CMS) classification, which allows for better comprehension of CRC molecular heterogeneity, might be a tool worth considering for the development of new immunotherapeutic strategies in MSS/pMMR CRC [START_REF] Guinney | The consensus molecular subtypes of colorectal cancer[END_REF].

In this review, we highlight how MSI/dMMR has emerged as a powerful biomarker for ICI efficacy in mCRC. Then we will focus on the currently explored ways to expand the use of ICI in CRC beyond MSI/dMMR and also in adjuvant and neoadjuvant settings.

Immune checkpoint blockade in colorectal cancer: from disappointment to microsatellite instability

The evaluation of immune checkpoint inhibitors (ICI) in CRC has been initially disappointing. The results of phase I basket trials including molecularly unselected mCRC patients did not show evidence of clinical activity. Notably, no objective responses were observed amongst the 3, 19 and 18 mCRC patients treated with pembrolizumab, nivolumab (anti PD-1 monoclonal antibodies), or BMS-936559 (anti-PD-L1 monoclonal antibody), respectively [START_REF] Patnaik | Phase I Study of Pembrolizumab (MK-3475; Anti-PD-1 Monoclonal Antibody) in Patients with Advanced Solid Tumors[END_REF][START_REF] Topalian | Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer[END_REF][START_REF] Brahmer | Safety and Activity of Anti-PD-L1 Antibody in Patients with Advanced Cancer[END_REF].

Clinical activity of ICI in patients with CRC MSI/dMMR

The breakthrough of immunotherapy in CRC came from one out of the 14 mCRC patients included in the basket phase I trial CA209-001 (NCT00441337), who experienced a durable complete response with nivolumab. This patient's tumor presented an MSI phenotype [START_REF] Brahmer | Phase I Study of Single-Agent Anti-Programmed Death-1 (MDX-1106) in Refractory Solid Tumors: Safety, Clinical Activity, Pharmacodynamics, and Immunologic Correlates[END_REF].

The proof-of-concept phase II study (MK-3475-016, NCT01876511) was designed to assess the efficacy of pembrolizumab in three cohorts of chemoresistant metastatic cancer patients: MSI/dMMR mCRC, MSI/dMMR non-CRC and MSS/pMMR CRC [START_REF] Le | PD-1 Blockade in Tumors with Mismatch-Repair Deficiency[END_REF]. Preliminary results published in the New England Journal of Medicine in 2015 showed a 40%, 57% and 0% objective response rates (ORR), respectively, in the three pembrolizumab-treated cohorts (N = 11; N = 9; N = 21). This was the first step to recognize MSI/dMMR as a major predictive tissue-agnostic biomarker for the efficacy of ICI [START_REF] Le | Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade[END_REF]. The updated data after 28 MSI/dMMR mCRC patients treated with pembrolizumab confirmed these encouraging results, with 11% of complete responses, 46% of partial responses and only 4% of progressive diseases [START_REF] Le | Programmed death-1 blockade in mismatch repair deficient colorectal cancer[END_REF].

Results of immune checkpoint blockade strategy in MSI/dMMR mCRC patients are summarized in Table 1. In the Keynote-164 study, pembrolizumab was evaluated in two distinct cohorts: (i) cohort A, after 2 or more prior lines of therapy including fluoropyrimidine, oxaliplatin and irinotecan, and; (ii) cohort B, after 1 or more prior line of therapy. In these 2 cohorts, pembrolizumab was associated with disease control rate (DCR) of 51% and 44%, and 1-year progression-free survival (PFS) rate of 34% and 41%, respectively [START_REF] Le | KEYNOTE-164: Pembrolizumab for patients with advanced microsatellite instability high (MSI-H) colorectal cancer[END_REF][START_REF] Diaz | Efficacy of pembrolizumab in phase 2 KEYNOTE-164 and KEYNOTE-158 studies of microsatellite instability high cancers[END_REF][START_REF] Le | Phase II Open-Label Study of Pembrolizumab in Treatment-Refractory, Microsatellite Instability-High/Mismatch Repair-Deficient Metastatic Colorectal Cancer: KEYNOTE-164[END_REF].

Nivolumab has been also evaluated in the context of MSI/dMMR mCRC. In the CheckMate-142 phase II trial, nivolumab alone (nivolumab 3 mg/kg every 2 weeks) and its combination with ipilimumab (nivolumab 3 mg/kg every 3 weeks + ipilimumab 1 mg/kg every 3weeks for 4 cycles then nivolumab 3 mg/kg every 2 weeks) until disease progression exhibited a 34% and 58% ORR, respectively, with 36% and 12% RECIST1.1 progressive disease at first evaluation, respectively. The 2-year PFS and OS (overall survival) rates were 60% and 74% with the combination of nivolumab and ipilimumab [START_REF] Overman | Nivolumab (NIVO) + low-dose ipilimumab (IPI) in previously treated patients (pts) with microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) metastatic colorectal cancer (mCRC): Long-term follow-up[END_REF][START_REF] Overman | Nivolumab in patients with DNA mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) metastatic colorectal cancer (mCRC): Long-term survival according to prior line of treatment from CheckMate-142[END_REF]. In both cohorts, patients were resistant or intolerant to at least 1 prior line of chemotherapy before inclusion: 54% and 40% of patients, respectively, received 3 lines of systemic treatment or more before ICI. In a third cohort of the CheckMate-142 trial, 45 patients with no prior treatment for MSI/dMMR mCRC were treated with nivolumab 3 mg/kg every 2 weeks plus low-dose ipilimumab every 6 weeks until disease progression. ORR was 77% and only 1 progressive disease was observed; 1-year PFS and OS rates were 77% and 83%, respectively [START_REF] Lenz | LBA18_PRDurable clinical benefit with nivolumab (NIVO) plus low-dose ipilimumab (IPI) as first-line therapy in microsatellite instability-high/mismatch repair deficient (MSI-H/dMMR) metastatic colorectal cancer (mCRC)[END_REF].

Durvalumab, an anti-PD-L1 monoclonal antibody, has also been tested in the context of MSI/dMMR mCRC. Amongst 36 MSI/dMMR mCRC patients treated with durvalumab 10 mg/kg IV every 2 weeks (NCT01693562 and NCT02227667.), ORR was 22%, DCR for more than 24 weeks was 47% and the 2-year OS rate 54% [START_REF] Segal | Safety and clinical activity of durvalumab monotherapy in patients with microsatellite instability-high (MSI-H) tumors[END_REF]. Interestingly, patients were treated for a maximum of 12 months whereas in other trials ICI treatment was maintained for 2 years or until disease progression.

ICI compared to conventional chemotherapy

Based on the positive results from these clinical trials [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][START_REF] Le | Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade[END_REF], pembrolizumab as well as nivolumab alone or in combination with ipilimumab have been granted accelerated approval by the FDA for chemoresistant MSI/dMMR mCRC patients. However, the European Medicine Agency has not yet approved ICI for these subsets of patients.

Several randomized trials are currently evaluating the efficacy of ICI as compared to standardof-care chemotherapy ± targeted therapy in first or second-line metastatic setting (Table 2).

Notably, the KEYNOTE-177 and COMMIT phase III studies might lead to the approval of pembrolizumab and atezolizumab, respectively, in frontline MSI/dMMR mCRC if they meet their primary endpoint. The ongoing randomized phase II trial PRODIGE 54 -SAMCO( NCT03186326) evaluates the efficacy of avelumab in second-line versus standard of care treatment (chemotherapy ± targeted therapy). In the randomized phase III CA209-8HW study (NCT04008030), patients are randomized to receive nivolumab alone, nivolumab plus ipilimumab or investigator's choice chemotherapy ± targeted therapy. The main objective is to compare the clinical benefit, as measured by PFS, ORR and OS, achieved by nivolumab in combination with ipilimumab or by nivolumab monotherapy in patients with MSI/dMMR mCRC.

Importantly, 2 phase III trials are currently assessing the efficacy of ICI in adjuvant setting.

The Alliance A021502 trial (NCT02912559) is evaluating mFOLFOX6 ± atezolizumab in patients with stage III colon cancer. In the experimental arm, patients receive 6 months of adjuvant mFOLFOX6 concurrently with atezolizumab, followed by 6 additional months of atezolizumab monotherapy. The POLEM trial (EudraCT 2017000370_10) is testing the benefit of adding ICIs after the completion of capecitabine-based adjuvant chemotherapy (3 months of capecitabine plus oxaliplatin or 6 months of capecitabine monotherapy) in MSI/dMMR or POLE-mutated stage III CRC. At the end of adjuvant chemotherapy, patients who are randomized to the investigational arm will receive additional 24 weeks of treatment with avelumab. The primary endpoint is disease-free survival in both trials.

Biomarkers within the MSI/dMMR population

Potential biomarkers of ICI efficacy amongst MSI/dMMR population are summarized in table 3.

Despite high rates of response and durable clinical benefit with ICI, 10 to 50% of MSI/dMMR mCRC tumors exhibit primary resistance to immunotherapy. However, a significant amount of these refractory tumors are mistakenly diagnosed as MSI/dMMR. In a cohort study of 38 mCRC patients included in ICI clinical trials based on a positive MSI and/or dMMR status as determined by local laboratories, misdiagnosis of MSI or dMMR status was responsible for 3 of 5 cases of primary resistance to ICI [START_REF] Cohen | Association of Primary Resistance to Immune Checkpoint Inhibitors in Metastatic Colorectal Cancer With Misdiagnosis of Microsatellite Instability or Mismatch Repair Deficiency Status[END_REF]. Thus, the first concern when observing immediate tumor progression under ICI is the possibility of a misdiagnosed MSI phenotype that requires confirmatory analysis by immunohistochemistry or molecular assay, the alternate hypothesis being tumor pseudo-progression [START_REF] Cohen | Association of Primary Resistance to Immune Checkpoint Inhibitors in Metastatic Colorectal Cancer With Misdiagnosis of Microsatellite Instability or Mismatch Repair Deficiency Status[END_REF][START_REF] Svrcek | MSI/MMRdeficient tumor diagnosis: Which standard for screening and for diagnosis? Diagnostic modalities for the colon and other sites: Differences between tumors[END_REF].

Preliminary results on potential biomarkers for the efficacy of ICI amongst MSI/dMMR mCRC did not find significant predictive impact neither in RAS/RAF mutational status, tumor PD-L1 expression, the inherited (i.e. Lynch syndrome) nor sporadic origin of MMR deficiency nor the tumor mutational load [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][START_REF] O'neil | Safety and antitumor activity of the anti-PD-1 antibody pembrolizumab in patients with advanced colorectal carcinoma[END_REF]. Concerning the mechanism underlying MMR deficiency, it is worthy to note that the results remain questionable since the definition of Lynch syndrome-related cancers and sporadic cases is partly equivoque. In the analysis of ICI clinical trials, MMR deficiency origin was determined by investigators based on past medical history collected from clinical records without a systematic molecular approach that might skew the results [START_REF] Le | PD-1 Blockade in Tumors with Mismatch-Repair Deficiency[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][START_REF] Cohen | Clinical and molecular characterisation of hereditary and sporadic metastatic colorectal cancers harbouring microsatellite instability/DNA mismatch repair deficiency[END_REF]. Moreover, it has been recently proven that Lynch-like tumors (MSI tumors without MLH1 promotor hypermethylation, BRAF mutation, or germline MMR gene mutation) arise from biallelic somatic MMR gene inactivation in approximately 50% of patients [START_REF] Salvador | Comprehensive Paired Tumor/Germline Testing for Lynch Syndrome: Bringing Resolution to the Diagnostic Process[END_REF]. Therefore, further studies are warranted to confirm these findings.

Several potential mechanisms of resistance are under evaluation, such as notably deleterious mutations of JAK (found in approximately 10 to 20% of MSI/dMMR tumors), loss of major histocompatibility complex (MHC) molecules or B2M (beta-2-microglobulin) loss-of-function mutations (20-30% of MSI CRC) [START_REF] Kopetz | Exploratory analysis of Janus kinase 1 (JAK1) loss-of-function (LoF) mutations in patients with DNA mismatch repair-defıcient/microsat-ellite instability-high (dMMR/MSI-H) metastatic colorectal cancer (mCRC) treated with nivolumab plus ipilimumab in CheckMate-142[END_REF][START_REF] Sveen | Multilevel genomics of colorectal cancers with microsatellite instability-clinical impact of JAK1 mutations and consensus molecular subtype 1[END_REF][START_REF] Zaretsky | Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma[END_REF][START_REF] Shin | Primary Resistance to PD-1 Blockade Mediated by JAK1/2 Mutations[END_REF]. The latter was not found to be associated with ICI resistance in a cohort of 13 B2M-mutated MSI/dMMR mCRC with ORR of 85% (11/13).

Remarkably, the authors showed that MHC class I expression is variable in B2M-mutated MSI CRC [START_REF] Middha | Majority of B2M-Mutant and -Deficient Colorectal Carcinomas Achieve Clinical Benefit From Immune Checkpoint Inhibitor Therapy and Are Microsatellite Instability-High[END_REF] but not associated with response to ICI. To note, there is currently no available data on the impact of gut microbiota in MSI/dMMR cancer patients treated with ICI [START_REF] Routy | Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors[END_REF][START_REF] Gopalakrishnan | Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients[END_REF]. Histopathological characteristics such as the amount of extracellular mucin and PD-L1 expression at the invasive front might be associated with resistance to ICI [START_REF] Llosa | Immunopathologic Stratification of Colorectal Cancer for Checkpoint Blockade Immunotherapy[END_REF].

The impact of tumor mutational load remains controversial within the MSI/dMMR population. Important recently published works detected a positive correlation between tumor mutational load and the efficacy of ICI amongst MSI/dMMR population [START_REF] Mandal | Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response[END_REF][START_REF] Schrock | Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSI-high metastatic colorectal cancer[END_REF]. However, the sample sizes remain small, with potential tumors misdiagnosed as MSI/dMMR amongst ICI-resistant cases [START_REF] Cohen | Association of Primary Resistance to Immune Checkpoint Inhibitors in Metastatic Colorectal Cancer With Misdiagnosis of Microsatellite Instability or Mismatch Repair Deficiency Status[END_REF]. Moreover, the threshold of tumor mutational load to be considered as predictive biomarker may be different from other tumor types. Larger translational studies are therefore warranted to establish the role of TMB in MSI/dMMR tumors. Finally, intra-and inter-tumoral heterogeneity might negatively influence ICI efficacy on sporadic MSI/dMMR tumors especially in cases of mixed dMMR/pMMR tumors [START_REF] Tachon | Heterogeneity of mismatch repair defect in colorectal cancer and its implications in clinical practice[END_REF][START_REF] Kim | Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer[END_REF].

The quality of the neoantigens seems capital as a potential new biomarker, as demonstrated in MSI/dMMR tumors treated by ICI [START_REF] Le | Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade[END_REF]. Even in the context of high TMB and predicted neoantigens, very few effective immune responses directed against tumor associated neoantigens are detected, suggesting that immune response to ICI may rely only on a few highly immunogenic antigens. These antigens are derived mostly from insertion/deletion mutations (InDels), a hallmark of dMMR tumors [START_REF] Mandal | Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response[END_REF][START_REF] Campbell | Comprehensive Analysis of Hypermutation in Human Cancer[END_REF]. Importantly, this phenomenon may be difficult to overcome in MSS/pMMR tumors where InDels are rarely detected . Criteria in Solid Tumors criteria (RECIST)) followed by measurable tumor regression, sometimes months to years after therapy initiation. The frequency of pseudo-progression in MSI/dMMR population is currently unknown but it may be common in this highly sensitive population. Amongst patients from the CheckMate-142 trial with progression of disease as best response (n=14) and who continued treatment beyond progression (N = 11), those with a reduction (N = 3) or stabilization of target lesions (N = 3) were more likely to survive more than 12 months [START_REF] Overman | Nivolumab (NIVO) + low-dose ipilimumab (IPI) in previously treated patients (pts) with microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) metastatic colorectal cancer (mCRC): Long-term follow-up[END_REF]. Distinguishing pseudo-progression from true disease progression is tricky, and irRECIST (Immune-related RECIST) or iRECIST (immune RECIST) criteria can be helpful [START_REF] Gerwing | The beginning of the end for conventional RECIST -novel therapies require novel imaging approaches[END_REF]. Clinical parameters such as the improvement of performance status, improvement of symptoms or decrease of serum tumor marker levels may help in the clinician's decision to continue the ICI and wait for later radiologic evaluations. To note, the KEYNOTE-016 trial used irRECIST to assess response, meanwhile others such asCheckMate-142 and KEYNOTE-164 used RECIST v.1.1. This difference in radiologic evaluation criteria might explain the difference in ORR across studies [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][START_REF] Le | Programmed death-1 blockade in mismatch repair deficient colorectal cancer[END_REF][START_REF] Le | KEYNOTE-164: Pembrolizumab for patients with advanced microsatellite instability high (MSI-H) colorectal cancer[END_REF][START_REF] Le | Phase II Open-Label Study of Pembrolizumab in Treatment-Refractory, Microsatellite Instability-High/Mismatch Repair-Deficient Metastatic Colorectal Cancer: KEYNOTE-164[END_REF].

Concerning secondary resections of primary tumor or metastasis after ICI treatment, the data are scarce. Some case reports suggested that pre-operative immunotherapy followed by resection of primary tumor and or metastatic sites is associated with high rates of pathological complete response, despite the presence of residual lesions on radiological exams [START_REF] Sehdev | Pathological complete response with anti-PD-1 therapy in a patient with microsatellite instable high, BRAF mutant metastatic colon cancer: a case report and review of literature[END_REF]. This brings to light some issues in the therapeutic management of ICI-treated patients with MSI/dMMR cancer: (i) what is the best therapeutic strategy for long responder patients: ICI continuation, surgcal removal of residual lesions or ICI discontinuation without surgery?, and ; (ii) Would those patients experiencing disease progression confined to a single site benefit from surgery? These issues are currently emerging since the number of ICI-treated MSI/dMMR mCRC patients is growing exponentially.

Another emerging crucial concern is the optimal duration of treatment for patients who respond to ICI. The strategy varies across trials, with a fixed duration of ICI (usually limited to 1 or 2 years of treatment) or the continuation of treatment until progression or unacceptable toxicity, which is a burden for patients and healthcare systems. The question remains unanswered and clinical trials are needed to find the optimal treatment duration.

Immunotherapy in colorectal cancer beyond MSI/dMMR

Ongoing clinical trials evaluating therapeutic strategies with ICI for MSS/pMMR CRC patients are displayed in table 4.

Hypermutability amongst microsatellite stable colorectal cancer: DNA polymerase mutations

The biological substratum of ICI efficacy on MSI/dMMR tumors relies on immunogenic frameshift mutations and high TMB. Several studies have shown that MSI/dMMR CRC could be detected through TMB evaluation with similar accuracy as polymerase chain reaction or immunohistochemistry [START_REF] Stadler | Reliable Detection of Mismatch Repair Deficiency in Colorectal Cancers Using Mutational Load in Next-Generation Sequencing Panels[END_REF][START_REF] Vanderwalde | Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients[END_REF]. Moreover, there is a high-TMB population that do not exhibit MSI/dMMR but displays an ultramutated phenotype with significantly higher TMB than MSI/dMMR CRC: all these tumors harbor mutations in DNA polymerase genes (Figure 1) [START_REF] Muzny | Comprehensive molecular characterization of human colon and rectal cancer[END_REF][START_REF] Stadler | Reliable Detection of Mismatch Repair Deficiency in Colorectal Cancers Using Mutational Load in Next-Generation Sequencing Panels[END_REF][START_REF] Ahn | The somatic POLE P286R mutation defines a unique subclass of colorectal cancer featuring hypermutation, representing a potential genomic biomarker for immunotherapy[END_REF].

In the TCGA cohort (N = 276), 16% of CRC samples (N=44) exhibited hypermutation (defined as a TMB greater than 12 mutations per 10 6 bases). Among these, all MSS tumors (N=7) harbored exonucleasic proofreading domain POLE-mutations (DNA epsilon polymerase) and presented the highest TMB. Similarly, in a study by Stadler and colleagues, 31 out of 224 CRC samples were hypermutated, of which 3 MSS POLE-mutated tumors presented a TMB 3-fold higher than MSI/dMMR cases [START_REF] Stadler | Reliable Detection of Mismatch Repair Deficiency in Colorectal Cancers Using Mutational Load in Next-Generation Sequencing Panels[END_REF]. In a study by Vanderwalde and colleagues, 93 out of 1395 CRC (6.7%) samples were hypermutated (TMB high if ≥ 17 mutations per megabase (mt/mb)) and 80 (5.7%) were MSI (and only 4 MSI tumors had low TMB) [START_REF] Vanderwalde | Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients[END_REF]. DNA polymerase enzymes are involved in DNA synthesis and repair during S phase of cell cycle. Delta and epsilon polymerases (POLD and POLE) are the main DNA polymerases in humans, with a proofreading exonuclease domain activity allowing error correction during replication. Deleterious mutations in the exonuclease domain of POLD and POLE genes are responsible for a dramatic genetic instability associated with an ultramutated tumor phenotype. Germline mutations of POLD and POLE genes are associated with an autosomic dominant familial cancer syndrome favoring notably endometrial and colorectal cancers occurrence (PPAP: polymerase proofreading-associated polyposis). Somatic POLE gene mutations may also occur [START_REF] Rayner | A panoply of errors: polymerase proofreading domain mutations in cancer[END_REF][START_REF] Bourdais | Polymerase proofreading domain mutations: New opportunities for immunotherapy in hypermutated colorectal cancer beyond MMR deficiency[END_REF]. Interestingly, POLE mutations have been described in MSI/dMMR cancers, but arise distantly to the exonuclease domain and are therefore considered as passenger mutations due to the high genetic instability of MSI/dMMR tumors. This hypothesis is supported by the higher TMB of POLE-mutated MSS CRC compared to POLE-mutated MSI CRC tumors [START_REF] Campbell | Comprehensive Analysis of Hypermutation in Human Cancer[END_REF][START_REF] Domingo | Somatic POLE proofreading domain mutation, immune response, and prognosis in colorectal cancer: a retrospective, pooled biomarker study[END_REF].

Exonucleasic domain POLE-mutated CRCs typically arise from left colon and rectum in younger male patients. Not all POLE mutations seem to be driving hypermutagenesis and the high mutation load phenotype seems restricted to specific hotspots. These mutations are observed in 1% of stage II and III tumors and are associated with favorable prognosis. This population is very rare in the metastatic setting, accounting for less than 1% of cases [START_REF] Ahn | The somatic POLE P286R mutation defines a unique subclass of colorectal cancer featuring hypermutation, representing a potential genomic biomarker for immunotherapy[END_REF][START_REF] Guerra | POLE somatic mutations in advanced colorectal cancer[END_REF],

and its prognosis and response to conventional treatments are still poorly understood. Considering ICI in this population, the literature is scarce but nevertheless promising with case reports suggesting major clinical activity for POLE-mutated CRC patients [START_REF] Gong | Response to PD-1 Blockade in Microsatellite Stable Metastatic Colorectal Cancer Harboring a POLE Mutation[END_REF][START_REF] Santin | Regression of Chemotherapy-Resistant Polymerase ε (POLE) Ultra-Mutated and MSH6 Hyper-Mutated Endometrial Tumors with Nivolumab[END_REF][START_REF] Bhangoo | Tumor Mutational Burden Guides Therapy in a Treatment Refractory POLE-Mutant Uterine Carcinosarcoma[END_REF].

Several ongoing clinical trials are dedicated to this population (NCT03827044, NCT02715284, NCT03012581).

Tumor Mutational Burden and efficacy of ICI in CRC

TMB is an exonic genomic measure of non-synonymous mutations in tumor cells. Using algorithms comparing DNA sequences from control tissues to tumor samples, TMB helps to predict the number of somatic mutations within tumors. Therefore, it is an indirect measure of tumoral predicted neoantigens [START_REF] Schumacher | Neoantigens in cancer immunotherapy[END_REF]. The usefulness of TMB is based on the concept that when it is high, the likelihood of observing an effector T cell response directed against a specific tumor antigen is increased. This concept seems to be verified in MSI/dMMR tumors, as both are hypermutated and rich in effector immune cells. A recent study evaluated the predictive value of TMB in MSI/dMMR tumors treated by anti PD-1 [START_REF] Kim | Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer[END_REF]. Authors showed that patients with high TMB (> 37 mt/Mb) tumors displayed an ORR of 100% (N = 13) and improved PFS compared with patients with low TMB tumors (N = 9) [START_REF] Schrock | Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSI-high metastatic colorectal cancer[END_REF]. While these results are promising, confirmation in prospective larger cohorts is needed before being translated to clinical practice because, as previously mentioned, the impact of the mutation load varies from one study to another. MSS/pMMR CRC tumors may also display a high TMB but with a low frequency, including exonucleasic domain POLE-mutated tumors and other CRCs in which the mechanism of hypermutation remains unclear. A recent 2:1 randomized phase II clinical trial, CCTG CO.26, assessed the combination of tremelimumab, an anti-CTLA-4 and durvalumab, versus Best Supportive Care (BSC) in 180 MSS chemoresistant mCRC patients [START_REF] Chen | .26: Updated analysis and impact of plasma-detected microsatellite stability (MSS) and tumor mutation burden (TMB) in a phase II trial of durvalumab (D) plus tremelimumab (T) and best supportive care (BSC) versus BSC alone in patients (pts) with refractory metastatic colorectal carcinoma (rmCRC)[END_REF]. The trial reached its primary endpoint, with a median OS of 6.6 months for the ICI combination versus 4.1 months for BSC (HR 0.72 P = 0.07 for a preplanned threshold <0.1 for statistical significance) but without any improvement in the median PFS which were 1.8 and 1.9 months, respectively. DCR was 22.7% for the experimental arm versus 6.6% for BSC. TMB was assessed on cell free DNA (cfDNA) using a 500-gene next generation sequencing panel [START_REF] Artyomenko | Microsatellite instability detection by targeted sequencing of cell-free DNA[END_REF], and, excluding 2 MSI/dMMR patients, the mean TMB was 20.4 ± 16.3 mts/Mb (range: 0.96 -114.0). Interestingly, 21% of tumors had a high TMB (defined as ≥28 mt/Mb), which was associated with better OS compared to patients with low or intermediate TMB tumors (5.5 month versus 3 months respectively, HR 0.34, 90% CI 0.18-0.63). Nevertheless, the pre-specified threshold of 20 mt/Mb was not predictive of benefit either for DCR, PFS or OS.

Genotyping of circulating tumor DNA (ctDNA) allows for a non-invasive quantification and characterization of the tumoral molecular profile and can indicate tumor heterogeneity and changes in TMB in real time. High blood TMB (bTMB) has been recently associated with an increase of response and survival in other tumor types treated with immunotherapy becoming a promising biomarker for ICI [START_REF] Wang | Evaluation of POLE and POLD1 Mutations as Biomarkers for Immunotherapy Outcomes Across Multiple Cancer Types[END_REF][START_REF] Gandara | Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab[END_REF][START_REF] Khagi | Hypermutated Circulating Tumor DNA: Correlation with Response to Checkpoint Inhibitor-Based Immunotherapy[END_REF]. However, bTMB shows higher number of mutations per megabase compared to classic TMB assessed in tissue resulting in variable concordance data between tissue and liquid samples. Moreover, in CRC it remains unclear why a benefit is observed in OS but not in other outcomes. Further prospective studies are needed for implementation of bTMB into clinical practice.

The theoretical presence of tumoral neoantigens may not be able to independently predict their immunogenicity. A high number of neoantigens and/or high quality neoantigens are necessary to induce a durable antitumor immune response. A recent study underlined that non-hypermutated MSS CRC have a very low predicted number of neoantigens (45 ± 22) compared to MSI (635 ± 308), or hypermutated MSS CRC (1651 ± 1475) [START_REF] Angelova | Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy[END_REF]. Notably, the number of predicted neoantigens decreases from early to advanced stage in the context of an active immunoediting, resulting in the elimination of the immunoreactive subclones and selection of immune privileged CRC subclones [START_REF] Angelova | Evolution of Metastases in Space and Time under Immune Selection[END_REF]. Specific translational immunological studies should be performed at latter stage to better understand the immune escape mechanisms in mCRC.

Immunoscore® and efficacy of ICI in CRC

The immunoscore® is a measure of the tumor-associated inflammatory stroma, assessing pan-/ cytotoxic lymphocyte densities (CD3 + and CD8 + , respectively) inside the tumor and at the invasion margin [START_REF] Galon | Type, Density, and Location of Immune Cells Within Human Colorectal Tumors Predict Clinical Outcome[END_REF]. The immunoscore® is a validated independent prognostic factor in early stage CRC to predict disease-free survival. Patients with a low immunoscore®, i.e. low infiltration of CD3 + and CD8 + cells, have higher risk of recurrence thereby justifying its use as a stratification factor in adjuvant clinical trials [START_REF] Pagès | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF]. In this publication, when stratified into two immunoscore® categories (0-1, and 2-4), 49 MSI tumors (16%) had a low immunoscore® (0-1) and 255 (84%) had intermediate and high immunoscore® [START_REF] Patnaik | Phase I Study of Pembrolizumab (MK-3475; Anti-PD-1 Monoclonal Antibody) in Patients with Advanced Solid Tumors[END_REF][START_REF] Topalian | Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer[END_REF][START_REF] Brahmer | Safety and Activity of Anti-PD-L1 Antibody in Patients with Advanced Cancer[END_REF]. Patients with intermediate and high immunoscore® had prolonged DFS and OS, irrespective of their microsatellite status (unadjusted HR for high versus low immunoscore® in MSI tumors 0.56, 95%CI 0.34-0.90; P = 0.0150). DFS of patients with immunoscore® high was similar for MSS and MSI tumors (around 80% at 3 years) and DFS of patients with immunoscore® low was similar for MSS and MSI tumor (around 60%).

The usefulness of immunoscore® in advanced disease seems more complex, as it relies on the assessment of the immune contexture of the invasion margin. Indeed, discordances between the primary tumor and the metastases has been observed and only the immunoscore® of metastases remained an independent prognostic factor in multivariate analyses [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 other tumors, the lymphocytic infiltration or intratumoral PD-L1 expression is not always associated with a benefit from immunotherapy, which is better predicted by transcriptional signatures where interferon gamma (IFN-γ) response genes are included [START_REF] Sun | Regulation and Function of the PD-L1 Checkpoint[END_REF]. The immunoscore® correlation with IFN-γ response remains to be established and, if confirmed, would allow the prospective assessment of ICI according to immunoscore® both in dMMR and pMMR tumors.

Molecular alterations and immune escape mechanisms in CRC

The international consensus molecular classification of CRC has been proposed, with 4 subtypes described so far: CMS1 (immune), CMS2 (canonical), CMS3 (metabolic), and CMS4 (mesenchymal) [START_REF] Guinney | The consensus molecular subtypes of colorectal cancer[END_REF]. Interestingly, the CMS are associated with specific immune and stromal profile [START_REF] Becht | Immune and Stromal Classification of Colorectal Cancer Is Associated with Molecular Subtypes and Relevant for Precision Immunotherapy[END_REF][START_REF] Karpinski | Immunological landscape of consensus clusters in colorectal cancer[END_REF]. The CMS was developed using a set of localized tumors and its value in the metastatic setting remains controversial: (i) spatial CMS heterogeneity has been described, as some tumors have a homogeneous CMS and others have a heterogeneous CMS, which complicates the allocation of tumors to a specific group and modulate the prognosis [START_REF] Laurent-Puig | 60PDColon cancer molecular subtype intratumoral heterogeneity and its prognostic impact: An extensive molecular analysis of the PETACC-8[END_REF]; (ii) CMS modifications has been observed within metastatic tumor under active antitumoral treatment such as anti-EGFR agents [START_REF] Woolston | Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer[END_REF]. Consequently, CMS clinical applicability in the metastatic setting remains limited and even reinforced by the complexity of bioinformatical analyses. Nevertheless, CMS helped to identify at least three main immune escape mechanisms in CRC that are related to distinct molecular pathway alterations.

Importantly, there is also an increasing body of evidence showing that the genomic landscape of CRC correlates with specific immune profiles and immune escape mechanisms, which are consistent with the one described in the CMS. Figure 2 proposes an immune classification of CRC based on molecular pathway alterations and the potential treatment strategies to immunomodulate each tumor subtype.

Firstly, tumors with DNA repair impairment, such as MMR deficiency or mutations within the polymerases exonucleasic domain, are prone to accumulate immunogenic mutations. These tumors are immune inflamed and characterized by high infiltration in CD8 cells, a high number of neoantigens, and increased expression of CMH-I and immune checkpoints such as PD-1, PD-L1, CTLA-4, resulting in an immune profile that is highly favorable for ICI activity.

Secondly, CRC with TGF-β pathway dependency presents a strong angiogenesis activation and an immune inflamed profile. The role of TGF-β in colorectal carcinogenesis is complex as it may activate multiple transduction pathways and has a dual anti-and pro-tumoral role from early stage to late stage cancer [START_REF] Jung | Transforming Growth Factor β Superfamily Signaling in Development of Colorectal Cancer[END_REF]. Multiple alterations in genes implicated in TGF-β transduction pathways have been described in CRC, such as in the SMAD family genes, BMPR1A or TGFb receptors itself. The impact of these alterations on immune cell recruitment in human CRC remains to be elucidated, but multiple preclinical models have shown that impairment of these genes results in a chronic inflammation favoring colorectal cancer progression [START_REF] Jung | Transforming Growth Factor β Superfamily Signaling in Development of Colorectal Cancer[END_REF]. Even if immune inflamed, the immune contexture of TGF-β dependent tumors is different from DNA repair impaired tumors and is characterized by an imbalance between the pro-and anti-tumoral immune cells with higher Treg, M0 and M2 macrophages and lower number of CD8 + cells. Enrichment in immunosuppressive and complement factors such as high expression of chemokines attracting myeloid cells is also observed, reinforcing the pro-tumoral immune response. Recent preclinical studies suggest that targeting the TGF-β pathway in CRC favors the recruitment of T cells and enhances the Th1 response leading to an increased sensitivity to ICI [START_REF] Tauriello | TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis[END_REF]. Another seductive target is angiogenesis, as Vascular Endothelial Growth Factor (VEGF) pathway inhibitors have shown encouraging results in other tumor types in combination with ICI [START_REF] Mcdermott | Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma[END_REF][START_REF] Wallin | Atezolizumab in combination with bevacizumab enhances antigen-specific T-cell migration in metastatic renal cell carcinoma[END_REF]. Indeed, in CRC, anti-VEGF agents were shown to increase CD8+ cells infiltration and decrease Treg in resected metastases, giving a rationale for combination strategies with ICI [START_REF] Manzoni | Immunological effects of bevacizumab-based treatment in metastatic colorectal cancer[END_REF].

Finally, tumors with WNT/β-catenin pathway activation derived from APC or CTNNB1 mutations have been shown to be poorly infiltrated by immune cells through the lack of immune cells trafficking [START_REF] Grasso | Genetic Mechanisms of Immune Evasion in Colorectal Cancer[END_REF] and characterized by: (i) few nonsynonymous mutations; (ii) low intratumoral immune infiltrate with a global decrease in lymphocytes, CD8+ cells, Th1, activated NK, and PD-L1+ mono/macrophages, and; (iii) low CMH-1 and B2M expression, suggesting poor antigen presentation. Preclinical models of CRC suggested that inhibition of WNT/β-catenin pathway could increase the immunological "hotness" of these tumors and generate sensitivity to ICI [START_REF] Xiao | DKK2 imparts tumor immunity evasion through β-catenin-independent suppression of cytotoxic immune-cell activation[END_REF].

Compared to CMS assessment, immuno-genomic profiling could be more easily transposable in clinical practice as it relies on DNA sequencing and/or pathological assessment. This immuno-genomic profiling should probably be repeated overtime during the course of the metastatic disease and after exposure to treatments due to constant immunoediting, which actively modulates the immuno-genomic phenotype [START_REF] Angelova | Evolution of Metastases in Space and Time under Immune Selection[END_REF].

Setting aside the issue of the CMS classification, a recent early phase I trial assessing the combination of nivolumab and regorafenib (anti-VEGF Receptor inhibitor) in MSS mCRC showed encouraging results with an ORR of 33% and a median PFS of 6.3 months warranting further development [START_REF] Fukuoka | Regorafenib plus nivolumab in patients with advanced gastric (GC) or colorectal cancer (CRC): An openlabel, dose-finding, and dose-expansion phase 1b trial (REGONIVO, EPOC1603)[END_REF], suggesting that angiogenesis inhibitors and ICI are synergistic in a subset of CRC, possibly the TGFβ/angiogenesis subgroup. Nevertheless, the use of other kinase inhibitors, such as MEK inhibitors, in combination with anti PD-L1 failed to show an improvement compared to regorafenib monotherapy in MSS mCRC tumors, despite showing promising results in phase Ib trials [START_REF] Eng | Atezolizumab with or without cobimetinib versus regorafenib in previously treated metastatic colorectal cancer (IMblaze370): a multicentre, open-label, phase 3, randomised, controlled trial[END_REF].

Conclusion and perspectives

ICI has been a major oncologic breakthrough in dMMR/MSI CRC but showed very limited results in low-TMB pMMR/MSS patients, with disappointing results in several clinical trials.

Despite dramatic responses to pembrolizumab or nivolumab in patients with dMMR/MSI tumors, nearly half of the patients would finally progress, and no biomarker is currently validated to correctly predict the resistance or the benefit derived from ICI, precluding further clinical development. Potential biomarkers are controversial because they are assessed retrospectively or in post hoc translational studies of small cohorts. Two examples are (i) B2M mutations thought to be predictive of resistance in early studies and not confirmed in latter studies, and (ii) the discrepancies between mutation load and TMB correlations with clinical outcome, potentially explained by technical limitations and non-consensual bioinformatical analyses. Combination of anti-PD-1/PD-L1 and anti CTLA-4 agents seems to increase the benefit with an acceptable safety profile in selected patients with MSI/dMMR mCRC.

Currently, there are still many unresolved questions concerning the clinical management of patients with ICI-treated MSI/dMMR mCRC such as duration of therapy, surgical removal of post-immunotherapy residual lesions, the best predictive factors of efficacy, or the choice of the best treatment scheme approach. POLE/D exonucleasic domain mutations are an important alternative biomarker to select best candidates for ICI in MSS/pMMR mCRC. POLE-mutated tumors seem rare and confirming ICI activity in this subset may be challenging prospectively as the pathogenicity of these mutations remains unclear outside of the described hotspots. TMB is a seductive biomarker to offer ICI to patients with MSI/pMMR CRC with high mutation load. First results seem to indicate a limited benefit on OS but not on other outcomes. However, TMB assessment remains expensive, without a clear threshold and technical/bioinformatical pitfalls, and its predictive value remains to be established both for MSS/pMMR and MSI/dMMR tumors. The Immunoscore® and IFN-γ response signature are also emerging biomarkers but preclinical/translational studies are still necessary to confirm their value in CRC, especially in pMMR patients before designing prospective clinical trials. Finally, immuno-genomic classification may be useful, especially to separate MSS/pMMR CRC in clinical trials in accordance with immune escape mechanisms. Incorporating immune-genomic profiling in future studies assessing immunomodulation/targeted therapeutic combinations would be of added value to identify subsets of patients benefiting from these treatments and move to individualized immunotherapeutic strategies in CRC.

CD-ON-MEDI4736

2. 4 .

 4 Controversies in the clinical management of MSI/dMMR mCRC patients treated with ICI Pseudo-progression is a noted phenomenon of ICI therapy. Pseudo-progression is an initial radiographic tumor enlargement (consistent with progression per Response Evaluation

FOLFIRIFigure 1 :Figure 2 :Figure 1 :

 121 FIGURE CAPTIONS

Table 2 : Randomized trials with immune checkpoint inhibitors for MSI colorectal cancer 1

 2

	-
	1108 [24]
	Durvalumab	≥ 1	36 -	22	38	54	29
	NCT02227667 [24]
	Durvalumab	≥ 1	11 -	27	36		30

CR: complete response; PR: partial response; SD: stable disease; NE: not evaluable; PFS: progression-free survival; OS: overall survival 1

ACKNOWLEGMENT

We would like to thank Michelle Lamendola-Essel for editing assistance and English editing.

Funding:

No external funding was used in the preparation of this manuscript.

Conflict of interest:

Benoît Rousseau served in a consulting/advisory role for Bayer, Roche, Novartis, Gilead, Servier and has received travel, accommodations, and expenses from Bayer, Servier, Astellas.

Joana Vidal has received honoraria for advisory role, speaker or travel grants from Amgen, Hoffman La-Roche, Merck-Serono, Sanofi and Sysmex Inostics. Romain Cohen has received honoraria from Amgen, Sanofi and Servier, and travel fees from Sanofi. Thierry André reported receiving honoraria from Roche, Genentech, Ventana, Sanofi, Bristol-Myers Squibb, MSD Oncology, Servier, Pierre Fabre; consulting or advisory role for Roche, Amgen, Bristol-Myers Squibb, HalioDX; speakers' bureau for Bristol-Myers Squibb and Servier; travel, accommodations, and expenses from Roche, Genentech, Amgen, Bristol-Myers Squibb.

Raphaël Colle and Luis A Jr Diaz declare that they have no conflicts of interest that might be relevant to the contents of this manuscript.

COMPLIANCE WITH ETHICAL STANDARDS


L Méès

Denis Lebrun

Didier Fréchou

Romuald Boucheron

Interferometric Laser imaging technique applied to nuclei size measurements in cavitation tunnel

Interferometric Laser Imaging Technique (ILIT) is applied to nuclei size measurements in cavitation tunnel. The use of a large collection angle optics is needed to allow nuclei size measurement down to 10 µm at 1 m distance. The negative effect of spherical aberration in such a situation is exhibited. A lens assembly, free from spherical aberrations and specifically designed for this application is presented. Measurements in a cavitation tunnel shows nuclei size distributions in the range 10-200µm. In addition to bubble sizes, ILIT can provide information on bubble concentration. To do so, a measurement volume is defined from statistical information extracted from the ILIT results themselves.

INTRODUCTION

The operational requirements for naval and research vessels has seen an increasing demand for quieter ships. Hence, the management of a ship's hydrodynamic signature requires better and more reliable estimates of propeller noise. One of the major sources of radiated noise is the occurrence of cavitation on the ship propeller. This makes the cavitation inception prediction a critical issue to assess for propeller model scale testing in hydrodynamic tunnels.

The water quality or nuclei content control is essential in a hydrodynamic tunnel dedicated to cavitation studies. Several studies [START_REF] Gindroz | Influence of the nuclei on the cavitation inception for different types of cavitation on ship propellers[END_REF][START_REF] Billet | Report of the Cavitation Committee[END_REF] have confirmed the merit of the nuclei control for cavitation tests. Most of the cavitation tunnels control the nuclei content indirectly by controlling the dissolved air content of the water. The water quality usually varies with time and tests sequences. The French large cavitation tunnel is one of the rare facilities in which the nuclei content is controlled through micro-bubbles injection [START_REF] Lecoffre | Le Grand Tunnel Hydrodynamique (GTH)[END_REF][START_REF] Fréchou | Marine Propulsor noise investigation in the hydroacoustic water tunnel GTH[END_REF] independently of the dissolved air content control. Other tunnels like AMC in Australia, LEGI in Grenoble France and pump or turbine loops use the same technique. This method guarantees the repeatability of the test conditions over time.

The nuclei content measurement is required to ensure that the critical pressure of the water is close to the vapor pressure and to know the density of active nuclei. This implies the measurement of bubbles sizes in the typical range 20µm -100µm. The Interferometric Laser Imaging Technique (ILIT) has been largely developed for droplets sizing, mainly because there are more industrial applications with droplets than with micro-bubbles. The technique is then often called ILIDS for Interferometric Laser imaging Droplet Sizing. But the ILIT principle remains valid for any transparent particles with quasispherical shape (or circular section).

ILIT PRINCIPLE

The ILIT technique uses a laser sheet that illuminates the particles in the flow and a camera that records images of particles passing through the Laser sheet. This technique has been first demonstrated for isolated droplets by Ragucci [START_REF] Ragucci | Drop sizing by Laser light Scaterring Exploiting Intensity Angular oscillation in Mie Regime[END_REF] but it has been applied to a droplet spray, showing its real potential, by Glover et al [START_REF] Glover | Interferometric Laser imaging for droplet sizing: a method for droplet-size measurement in sparse spray systems[END_REF]. The scattered diagram (intensity versus scattering angle) of a transparent spherical particle shows oscillations. These oscillations are due to interferences between diffracted, reflected light and refracted light. In case of droplets and in the forward scattering region between 20° and 70° (standard ILIDS configuration [START_REF] Glover | Interferometric Laser imaging for droplet sizing: a method for droplet-size measurement in sparse spray systems[END_REF]), these oscillations are mainly produced by interference between reflection and first order refraction. The fringe's angular frequency depends on the droplet size (interferometric sizing principle). In the case of bubbles, at a scattered angle around 90° between the laser propagation direction and the observation direction, fringes are mainly produced by interferences between refection and second order refraction (see fig. 1). In any case, the fringe pattern produced around a given angle can be calculated by using Lorenz-Mie Theory (LMT) to establish a relation between the fringe frequency and the particle size [START_REF] Pu | Development of interferometric methods for particle fields characterisation[END_REF]. on the ccd array where each particle image take a circular shape (the shape of the lens), containing a fringes pattern, depending on the bubble size. The location of the circle is related to the bubble location in the laser sheet, ensuring a separation of different bubble images. Figure 3 gives an example of a droplet spray ILIT image. where the coefficient κ can be determined theoretically, by geometrical optics (with some assumptions) or by LMT (assuming a perfect spherical shape).

BUBBLE SIZE LIMITATIONS AND SPHERICAL ABERRATIONS

The lower limit of ILIT for bubble size measurement is fixed by the lower number of fringes measurable in bubbles images. Assuming that N=2 fringes is a reasonable limit, from Eq. ( 1), it can be seen that a large collection angle α is needed to measure small bubbles. Considering the large distance at which the collection optics is set in our application (about 1 m), large lenses diameters are needed. Figure 5 shows the minimum measurable diameter as a function of the collection angle α. To reach the objective of bubble size measurement lower than 20 µm, a collection angle of about 8° is then needed.

Considering a distance of 1m (equivalent in air) between the bubble flow (laser sheet) and the lens, a lens with effective aperture approaching 200 mm is required, that is to say 10 times the standard lenses aperture. A large spherical lens (diameter A=200 mm and focal lens f=450 mm) is first consider. Figure 6 shows a schematic view of optical rays, emitted from a single point located in the object plane and refocused by the lens in the image plane to form a point image. The upper part of the figure represents the ideal case of a perfectly stigmatic lens. All rays intersect in a single image point. The lower part of the figure represents rays behavior through a "real" spherical lens. In this case, rays coming from a single point no more intersect in a single image point. The paraxial rays (close to the lens center) intersect in the image plane but edge rays intersect at closer distances. This stigmatism default is well known as spherical aberrations. As a consequence, in the image plane, the point image is no more a single point but a point with some dimensions, surrounded by a halo of light. The effect of spherical aberration on out of focus images (ILIT) is illustrated in figure 6. Out of focus images of a glass fiber are represented for different out of focus plane locations. For very large out of focus (out of focus plane close to the lens), the fiber image shows 8 fringes, only 7 on the second out of focus plane, and so on. When moving the recording plane toward the image plane, the boundary fringes progressively overlap, leading to a dramatic under estimation of the fringes number. Unfortunately, a large out of focus degree it is not reasonable. In case of droplets or bubbles, the out of focus images are full circles and not a line as for the glass fiber. With the larger out of focus degree considered in figure 6 (plane 1), the line length (circle diameter for spherical particles) equal half the full image size, leading to an important (and quasi systematic) overlapping between bubble images, prohibiting the extraction of individual sizes from fringes frequency. One can deal with some amount of overlapping (depending on the image processing) but in a reasonable limit.

In conclusion, spherical aberrations cannot be eliminated without reducing the lens aperture (increasing the lower measurable size) or without using a very large out of focus, prohibiting simultaneous sizing of several bubbles.

ABERRATION FREE SPECIFIC LENS

Our aim is to perform small nuclei size measurement at large distance by ILIT. The problem to solve is to ensure a large collection angle at large distance while avoiding the negative effects of spherical aberrations. Spherical aberration can be reduced or eliminated by using lenses with non-spherical surfaces. A specific lens assembly has been designed and manufactured to eliminate spherical aberration and to keep a large effective collection angle. One of the lenses has an aspherical profile in order to eliminate spherical aberrations. The optical arrangement consists of two plano-convex lenses in order to reduce the other geometrical aberrations, responsible for distortions of images at the edges of the sensor (departing from the optical axis). The lens assembly has been designed to work at a fixed optical distance (equivalent in air) of 1 m, allowing measurement through 1 m in water and a glass window of about 100 mm. The effective aperture of the optics has been experimentally checked to be 190 mm, as expected. The corresponding minimum bubble diameter (for two fringes) is 12.68 µm. The effect of manufacturing default on the lenses surfaces have been observed on the out of focus images but frequency measurement is not strongly affected. Note that chromatic aberrations have not been considered because the Laser illumination is monochromatic. The design has been done for a given wavelength (λ=532 nm).

EXPERIMENTAL SET-UP AND CALIBRATION

Figure 7 shows the experimental set-up. If we assume the theoretical value of coefficient κ w and refractive index value of water n w , the only needed calibration consists in evaluating the effective collection angle α. Generally, ILIT (or ILIDS) measurement are performed using a standard commercial lens. A numerical aperture of the lens is usually specified and the collection angle could be theoretically deduced from it and from the focal length and the working distance. However, the numerical aperture is usually defined for use at infinity (object distance >>focal length) and most of the lens are not completely free from spherical aberration. Returning to figure 3, showing an example of out of focus images recorded at 500 mm with a 50 mm standard lens, with a positive out of focus (camera moves toward the lens), the effect of spherical aberration is visible as the bright circle bordering the circular images. In practice, the effective collection angle is different from the theoretical one and varies with the degree of defocusing. A full calibration procedure is usually recommended for ILIT. But in case of bubble size measurements, this calibration procedure is complicated.

In our case, we first moved backward the ensemble camera/lens from the tunnel, in order to image a calibration target in air. The distance between the target and the lens is fixed at 1 m and the distance between the CCD sensor and the lens is adjusted to focus on the target. A magnification ratio is deduced. The measured magnification ratio equals 0.465. The magnification ratio predicted from the lens assembly properties (optical design) equals 0.464. Next, the camera is moved forward to focus on the laser sheet (on illuminated bubbles). This procedure ensures that the optical distance between the laser sheet and the lens is 1 m with a precision easily better that 1%. Next, the ccd sensor is moved toward the lens to set the out of focus position (d x =3 mm in our case). To determine the collection angle α, the effective aperture of the lens assembly must be precisely estimated. The lens diameter is 200 mm but its manufacture and design guaranty only an effective diameter of 190 mm. A 190 mm aperture (part of the support) is then placed in front of the first lens.

The key point was to check the effective aperture of the lens. To do so, apertures (mask) of different sizes (80 and 100 mm) have been placed (in air) in front of the lens. Out of focus images of bubbles illuminated by a very thin laser sheet have been recorded, with the masks and without mask. For a given out of focus, the image circle diameter is theoretically proportional to the aperture diameter.

Then the ratios between image circle diameters (in pixel) have been compared to the ratio between physical apertures diameters. This simple procedure would have evidenced if the effective lens aperture was smaller than expected because of spherical aberrations. The lens assembly aperture has been checked to be equal to 190 mm ± 1 mm and the collection angle is then α=10.85° ± 0.1°.

The ccd sensor array is a 1600×1200 pixels with square pixels of 7.4µm. The lens assembly focal length is about 327 mm, the magnification ratio is 0.465 and the field of view 25.4 mm×19 mm. The out of focus distance is fixed at 3 mm toward the lens. The laser sheet is located 1 m (equivalent in air) from the lens. The collection angle α is 10.85°, corresponding to a minimum measurable diameter of about 12.7 µm (2 fringes). There is also an upper limit for bubble size. It depends not only on the collection angle but also on pixel size and out of focus degree. The greater measurable size corresponds to a number of fringes of about half the image circle diameter. In our configuration, the image circle diameter equals 166 pixels and the corresponding bubble diameter should be greater than 500 µm. However, bigger bubbles have a very bright image and the limited dynamic range of the camera, associated with the necessity of a high laser power to make visible the smaller bubbles, leads to an effective upper limit of about 200 µm.

IMAGE PROCESSING

Figure 8 shows an ILIT image recorded in the tunnel. Two particle images are clearly visible in this image: a bubble image showing horizontal fringes as predicted by the ILIT model and a particle image showing a more irregular pattern. This second image corresponds to a solid particle. ILIT is generally applied to droplets sprays and to droplet size of several hundreds of microns. It is then unusual to face solid particle mixed with the objects of interest. In the present situation, we investigated very small bubbles (down to 10 µm) and solid particles in the same size range are then visible. Fortunately, the horizontal fringes analyzed in ILIT are the consequence of transparency and spherical shape of bubbles. Solid particles produce complex interference pattern, due to their irregular shapes. It is then possible to separate solid particle from bubbles with ILIT by analyzing the fringes pattern of particle images.

The key steps of the image processing we adopted are the following:

1. A background image is subtracted and the image dimension is extended to a 2048×2048 image in order to allow the use of Fast Fourier Transform algorithms.

2. The resulting image is normalized and filtered (see figure 9-a) in order to smooth the fringes, to facilitate the detection of the circular images (step 4).

3. A spatial derivative of the filtered image is computed to extract the boundaries of particle images (figure 9-b).

Circles are located by computing a convolution product between the boundary image and a

ring whose diameter is defined by the optical configuration parameter (166 pixels in the present case). This step is an iterative process. The first detected particle images are removed from the boundary image which is normalized again and a new convolution product is computed to detect less contrasted particle images. Such an interactive process is needed to detect simultaneously the pale images of the smallest bubbles and the bright images of the biggest. In figure 9-c, the particle images already detected has been subtracted from the boundary image. A third particle image is now clearly visible. it will be detected at the third iteration.

5. The fringes patterns are then analyzed by means of 2D Fourier transforms. Bubble images are validated, on the basis of fringes frequency angle and contrast criterions. In figure 9-d, images 1 and 3 correspond to bubbles whose diameters equal 62.2 µm and 11.2 µm respectively (9.75 and 1.76 fringes). Image 2 corresponds to a solid particle and is not validated. Note that the image circle size actually varies by a few pixels from one bubble to another. This variation will be discussed later in the paper but the real size of the image circle is measured before the fringes frequency analysis, to accurately determine the fringes number and bubble diameter.

Finally, note that about 1 hour is needed to process 100 images on a laptop.

RESULTS AND DISCUSSION

The French large cavitation tunnel has 2 test sections. Measurements presented below have been done in the large test section (2 m x 1,35 m). The ILIT set-up is the one illustrated in figure 7. 2000 images have been recorded for 4 different pressure conditions in the tunnel (207, 567, 927 and 1287 mbar). Figure 10 shows the size distribution obtained by ILIT for these 4 conditions. No bubble smaller than 10 µm are considered, because the corresponding number of fringes would be less than 1.57.

For such a small number of fringes it is not possible to distinguish solid particle images from bubble images. Results are organized in terms of particle size ranges 5 µm wide. Note that the number of validated bubbles with respect to the total number of particle images detected (solid particles + bubbles) is in the order of 5%.

Until now, nuclei content of the cavitation tunnel was measured only by means of the Centerbody Susceptibility Meter (CSM) [START_REF] Pham | A new design of the Cavitation Susceptibility Meter[END_REF]. The CSM provides bubble cumulative distributions which are the only reference we have to compare our ILIT results. Moreover the CSM provides cumulative distribution in concentration units (number of nuclei per cm 3 ). To express our results in terms of concentration, the measurement volume must be estimated.

CONCENTRATION MEASUREMENT

It is well known that techniques based on laser sheet illumination are not suitable for concentration measurements. In such techniques, the measurement volume must be estimated from the field of view and the laser sheet width. But a laser sheet generally shows a Gaussian profile and the exact limit of the measurement volume is very hard to define. Moreover, the intensity of light scattered by particles strongly depends on the particle size. As a consequence, the measurement volume should vary with particle size (increasing with particle diameter). Figure 11 shows the laser sheet profile as it has been measured after the measurement campaign. A rough evaluation of the measurement volume can be deduced from the profile width and from the field of view but we have to assume a threshold value under which the bubble image will be not bright enough to be detected. In other words we have to chose an arbitrary value for this threshold. Some variations with respect to the 166 theoretical values are visible in images and the real size of any image can be extracted. But these variations are not only due to the z coordinate of the bubble. Indeed, these circle diameter variations are also due, for a part, to the non spherical geometrical aberrations. Bubbles located at the edges of the imaged field have weakly distorted images. The image is no more bounded by a perfect circle but by an oval shape. An average circle diameter can be however defined and a correction to the measured diameter is applied as a function of the bubble position. After applying this correction, average circle diameter and standard deviation have been computed for each particle size range. Figure 13

Figure 1 :

 1 Figure 1: scattering diagram (scattered intensity versus scattering angle) of a 100 µm diameter air bubble immersed in water (relative refractive index of 0.75), computed by LMT. Around 90°, fringes are mainly produced by interferences between reflection and second order refraction. ILIT combines the advantages of interferometric techniques for the size measurement precision, and imaging techniques for object localization and the ability to isolate different objects from each other.

Figure 2 ,

 2 Figure 2, gives a schematic view of ILIT experiments, for a scattering angle θ=90°, a collecting lens of diameter A (collection angle α), and the sensor array (CCD of the camera) placed in an out of focus plane distant by d x from the image plane. The fringes generated by bubbles are projected by the lens

Figure 2 :

 2 Figure 2: ILIT configuration. The size of the circle delimiting the particle image only depends on the out of focus degree (distance d x in figure 2), and the number of fringe in the circular shape depends only on the particle size. The image circle contains the same number of fringes than the lens surface. The lens diameter A and the circle diameter a verify

Figure 3 :

 3 Figure 3: Out of focus images of a droplet spray recorded with a standard 50 mm lens.

Figure 4 :

 4 Figure 4: Collection angle change through the tank wall.

Figure 5 :

 5 Figure 5: minimum measurable diameter (corresponding to 2 fringes) as a function of the collection angle α.

Figure 6 :

 6 Figure 6: Effect of spherical aberrations on out of focus images.

Figure 7 :

 7 Figure 7: Experimental set-up in cavitation tunnel.

Figure 8 :

 8 Figure 8: ILIT recorded image.

Figure 9 :

 9 Figure 9: key steps of the image processing.

Figure 10 :

 10 Figure 10: Bubble size distribution in the cavitation tunnel measured by ILIT.

Figure 11 : 2 where 1 .Figure 12 :

 112112 Figure 11: Measured Laser sheet profile.

2 ,

 2 displays the standard deviation of the image circle diameter computed for each bubble size range (20 µm wide), on the results presented figure 10 (all the 4 cases together). A quasi linear evolution of the standard deviation can be extracted from figure13. Deviation from this tendency for greater bubble is due to the small number of bubble constituting the sample in these size ranges. The standard deviation σ(d B ) in image circle diameter can be easily associated to a measurement volume, function of the bubble diameter d B , the field of view area.

Figure 13 : 3 ,

 133 Figure 13: Standard deviation of the image circle diameter as a function of the bubble diameter.

Figure 14 :

 14 Figure 14: Cumulative distribution of bubble in the cavitation tunnel test section.

Figure 15 :

 15 Figure 15: Cumulative nuclei distribution obtained by CSM measurements in similar conditions.