Biomarkers of Response and Resistance to Immunotherapy in Microsatellite Stable Colorectal Cancer: Toward a New Personalized Medicine

e15122 Background: The microsatellite instability-high (MSI-H) phenotype confers good prognosis and greater response to immunotherapy in colorectal cancer(CRC). The mutational landscape of MSI-H CRC is unclear. This study was designed to illustrate the difference mutation profile between the MSI-H and microsatellite stable (MSS) CRC. Methods: Tumor tissue and matched blood samples from 40 patients with colorectal cancer were collected. Microsatellite instability (MSI) status were detected by PCR-amplified for five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). Mutation profiles were sequenced by a cancer gene-targeted NGS panel. Results: The tumor mutation burden(TMB) of the MSI-H CRC patients was significantly higher than those MSS CRC patients. Compared with the MSS CRC, MSI-H CRC involved more genes and pathways. Furthermore, we found the copy number variation (CNV) was different between the two groups. The copy number instability (CNI) score of MSI-H CRC patients was significantly lower than those MSS CRC patients. MSI-H CRC patients showed a higher frequency of TP53 gene CNV gain compared with MSS CRC (41% (7/17) in MSI-H CRC versus 13% (3/23) in MSS CRC). Conclusions: The mutational landscape are different between the MSI-H and MSS colorectal cancer. Compared with MSS colorectal cancer, the MSI-H colorectal cancer patients have higher tumor mutation burden(TMB) and lower copy number instability (CNI) score. Keywords: colorectal cancer, microsatellite instability, tumor mutation burden, copy number instability. Abbreviations CRC, colorectal cancer; TMB, tumor mutation burden; MSI-H, The microsatellite instability-high; MSS, microsatellite stable; CNV, copy number variation.

Lifestyle factors and risk of sporadic colorectal cancer by microsatellite instability status: a systematic review and meta-analyses

Background:Colorectal cancer (CRC) is one of the most common cancers worldwide with high number of mortality every year. Microsatellite instability (MSI) is a considerable feature of CRC which affects prognosis and treatment. High level of MSI or MSI-high (MSI-H) colorectal cancer has better prognosis and immunotherapy response, while microsatellite stable (MSS) CRC has better response to 5-fluorouracil (5-FU)-based chemotherapy. More studies are needed, specifically on MSS CRC which has worse prognosis, to further reveal biological differences and similarities between MSS and MSI colorectal cancer, which may equip us with the knowledge to develop more promising therapeutic approaches to target both types or be more effective for each type. Methods:We aimed to find affected biological processes and their regulators in both type, MSS and MSI-H, of CRC; as well as reveal specific ones in each type. We applied meta- and network analysis on freely available transcriptome data in MSS and MSI-H colorectal cancer from gene expression omnibus (GEO) database to detect common differentially expressed genes (DEGs) and critical biological processes and predict their most significant regulators. Results:Our results demonstrate considerable up and downregulation in cell cycle and lipid catabolism processes, respectively; and introduced MYC and FOXM1 as two central and up-stream regulators of DEGs in both type of CRC. Chemokine-mediated processes displayed up-regulation in MSI-H type, while metastasis-related processes showed more activation in MSS CRC. Additionally, DACH1 and TP53 were detected as two important transcription factors that differentially expressed just in MSS and MSI-H, respectively. Conclusion:Our results can explain why MSI and MSS CRC display different immunotherapy response, prognosis, and metastasis feature. Moreover, our predicted up-stream regulators in the regulatory networks may be promising therapeutic targets.

Colorectal cancer (CRC) is the second most common cancer in both women and men. Microsatellite instability-high (MSI-H) CRC is a molecular subgroup and has distinct clinical and pathologic features from microsatellite stable (MSS) CRC. Studies have suggested an association between hereditary antigens in ABO blood group system and the risk of developing various cancers but the relationship between blood groups and MSI-H CRC has not been investigated. This study aimed to investigate this relationship and its possible effect on clinicopathological features in patients with CRC. This is a retrospective cross-sectional single-center study including pathology-confirmed CRC patients. Demographic and clinicopathological features, blood groups, and microsatellite status were examined among two groups. Microsatellite instability was examined by immunohistochemistry (IHC) in pathology specimen. A total of 144 patients, 72 patients with MSI-H CRC and 72 patients with MSS CRC, were included in the study. Among all patients, median age was 61.7 ± 12.9 (range 27-89) and 57.6% were male. MSI-H and MSS groups were similar in terms of age, gender distribution, and comorbidities. Patients with MSI-H CRC had significantly common O-blood group than control group (44.4% vs 18.1%, p: 0.001). In multivariate analysis, O-blood group was 4.2 times more common in the MSI-H patient group (95% CI: 1.514-11.819, p: 0.006). Also patients with MSI-H CRC were found to have significantly more right-sided, high-grade tumors and early-stage disease. MSI-H CRC is an important subgroup in colon cancer with different molecular and clinicopathological features. It was observed that O-blood group was 4.2 times more common in MSI-H CRC. We believe that clarifying the relationship between microsatellite instability and O-blood group and its possible genetic and epigenetic mechanismsin larger studies will enable us to better understand tumor behavior and prognosis, also affect our treatment choices of these patient groups.

Concurrent inactivating mutations and expression losses of RGS2, HNF1A, and CAPN12 candidate tumor suppressor genes in colon cancers.

Colorectal Cancer Risks in Relatives of Young-Onset Cases: Is Risk the Same Across All First-Degree Relatives?

In this study, we investigated the clinical relevance of CD274 (PD-L1) protein expression by tumor cells and tumor-infiltrating immune cells in colorectal cancer (CRC). To this end, 186 microsatellite instability-high (MSI-H) and 153 microsatellite stable (MSS) CRCs were subjected to immunohistochemistry (IHC) analysis for the expression of CD274 and mismatch repair proteins. CD274 expression was evaluated in tumor cells at the center (TC) and periphery (TP), and immune cells at the center (IC) and periphery (IP) of CRC. IHC slides stained for CD3 and CD8 were scanned using an Aperio ScanScope for precise calculation of tumor-infiltrating T cell density. Additionally, samples were screened for the B-Raf (BRAF)-V600E mutation using a Cobas 4800 System and IHC. In total, CD274TC, CD274TP, CD274IC, and CD274IP were observed in 43 (23.1%), 47 (25.3%), 107 (57.5%), and 102 (54.8%) of the MSI-H CRCs examined, and in three (2.0%), four (2.6%), 47 (30.7%), and 56 (36.6%) of the 153 MSS CRCs tested. Meanwhile, intratumoral heterogeneity of CD274 expression in tumor cells and immune cells was detected in 24 (12.9%) and 47 (25.3%) MSI-H CRCs, respectively. Notably, in both MSI-H and MSS CRC, CD274IC and CD274IP were independently associated with improved prognosis (P<0.05), while BRAF mutation was associated with CD274TP, poor differentiation, sporadic type, and hMLH1(-)/hMSH2(+)/hMSH6(+)/PMS2(-) in MSI-H CRC (P<0.006). In conclusion, CD274 expression in tumor-infiltrating immune cells was an independent factor for improved prognosis in CRC patients. A deeper understanding of CD274 status may yield improved responses to future CRC immunotherapies.

575 Background: Microsatellite instability (MSI) testing in colorectal cancer (CRC) provides prognosis, predicts chemotherapy response, and guides diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. MSI can be sporadic or hereditary, arising from somatic or germline mutations in DNA mismatch repair (MMR) genes respectively. The clinical implications of these distinct mechanisms are uncertain. Methods: Patients who underwent MSI testing for CRC between 2000 and 2011 were identified. MSI-high (MSH) CRCs were defined by: pathogenic mutation in MMR genes; &gt;30% of markers with allelic shift in PCR-based MSI testing; or loss of expression in at least 1 MMR protein on immunohistochemistry. The subset with MLH1 gene promoter methylation, BRAF mutation, or EPCAM mutation was considered sporadic. Clinicopathologic features and disease-free survival (DFS) were examined in reference to microsatellite stable (MSS) CRCs. Results: MSH CRC’s, 92 germline and 49 sporadic, were compared with 105 MSS CRCs. Compared to MSS CRCs, both germline and sporadic MSH CRCs more commonly arose in the proximal colon (63% and 92%, vs. 30%; p&lt;.001), exhibited mucinous/signet ring histology (40% and 47%, vs. 16%; p&lt;.001) and lymphocytic infiltrate/Crohn’s like reaction (15% and 49%, vs. 8%; p&lt;.001). Further comparison between germline vs. sporadic MSH CRCs revealed significant differences in median age (44 yrs. vs. 66; p&lt;.001), proximal colon tumor location (63% vs. 91%; p&lt;.001), AJCC stage (33% vs. 51% Stage III or IV; p&lt;.025) and presence of lymphocytic/Crohn’s like reaction (49% vs. 15%, p&lt;.001). Moreover, sporadic MSH CRCs more often had poor prognostic features including poor differentiation (51% vs. 28% in germline, p&lt;.025) and lymphovascular invasion (57% vs. 34% in germline, p&lt;.007). No difference was observed in stage-stratified DFS between germline vs sporadic MSH CRCs. Conclusions: Patients with sporadic MSH CRCs exhibit distinct clinicopathologic features compared to those with germline MSH tumors. Despite poor prognostic features, no apparent survival difference was observed. Further characterization of these distinct groups is warranted to explain the discordance between risk factors and outcomes.

BackgroundMicrosatellite instability-high (MSI-H) is a form of genomic instability present in 15% of colorectal cancer (CRC) cases. Several differential gene analyses have been conducted on CRC; however, none have specifically explored the differentially expressed genes in MSI-H CRC. Research on the different gene expressions between MSI-H CRC and microsatellite stable (MSS) CRC, and their different patterns of metastasis will provide invaluable insights for diagnosis, prognosis, and treatment.MethodsIn this study, the differential expression of 46,602 genes were analyzed across 613 different tissue samples from The Cancer Genome Atlas (TCGA)-colon adenocarcinoma (COAD) and TCGA-rectum adenocarcinoma (READ) as part of a gene association analysis. R package TCGAbiolinks (version 2.18.0) was used to download the data set, and DESeq2 (version 1.30.1) was used for the differential gene analysis. The resulting genes were then analyzed for shared pathways with R package clusterProfiler (version 3.0.4).ResultsA total of 237 significantly differentially expressed genes (Padj<0.05) were found between MSI-H and MSS CRC. Differentially expressed genes include insulin like growth factor 2 (IGF2) and fibroblast growth factor 3 (FGF3), and the enriched pathways mostly involve hearing, digestive regulation, and neurogenesis.463 differentially expressed genes were found between metastatic and non-metastatic CRC. Notably differentially expressed genes in metastatic CRC include DEAD-box helicase 53 (DDX53) and adiponectin, C1Q and collagen domain containing (ADIPOQ), and enriched pathways include the immune system, cell adhesion, and cell signaling. For MSI-H CRC, a total of 34 genes were significantly differently expressed between metastatic and non-metastatic CRC. These include notum, palmitoleoyl-protein carboxylesterase (NOTUM), serpin family B member 2 (SERPINB2), and several keratin (KRT) genes, and the pathway analysis showed the major enrichment of the hormonal and secretion and regulation pathways. Of the differentially expressed genes in metastatic CRC, 25 were immunity related and include fatty acid binding protein 4 (FABP4), and the pathway analysis showed the enrichment of humoral immunity and lymphocyte regulation.ConclusionsOf the biologically plausible differentially expressed genes, the most notable were NOTUM, KRT6A, KRT14, SERPINB2, and serum amyloid A1 (SAA1). NOTUM, KRT6A, and KRT14 are active in the Wnt pathway. All five are also involved in various inflammation pathways.

Introduction/Objective Microsatellite stable (MSS) and Instable colorectal cancer (CRC) as primary colon cancers differ in their pathological features and biological behavior including response to treatment. In general, MSI-high CRC has a better prognosis and a more favorable response to treatment. To our knowledge, the co-occurrence of the microsatellite stable and unstable CRCs in the same person has not been reported in the literature. Herein, we present the case report of 78 year African American female who presented to us with this condition. Methods A literature search of publication up to March 2020 in PubMed was conducted on the occurrence of microsatellite stable and unstable CRCs in the same individual. The literature search was limited to English language only. The following words were used in the search engine of PubMed: “Microsatellite stable colorectal cancer + unstable colorectal cancer and colorectal cancer + same patient”. No articles were identified. Results A search using the terms “coexistence of MSS and MSI CRC in the same individual” identified 45 articles. No case report or article discussing the presence of coexisting MSS and MSI CRC in the same individual was identified Conclusion Identification of MSI CRC is important, as MSI may serve as a screening tool for detecting Lynch Syndrome, a prognostic marker for patient outcome, and as a predictive marker for response to chemotherapy and to immunotherapy. Whether coexistence of MSI and MSS favorably affects the prognosis of the disease is unknown.

Introduction: Recent data have demonstrated that patients with MSI-H metastatic CRC are more likely to respond to a PD-1 inhibitor as a single agent compared to patients with MSS CRC. Effective immunotherapy approaches for MSS CRC remain a critical unmet need. There is great interest in investigating immune checkpoint inhibition in combination with novel agents, but preclinical studies have been hampered by current models. To gain a better understanding of immune responses and facilitate preclinical evaluation of combination strategies with immunotherapy, we developed a “hematopoietic” humanized mouse model with the intent of leveraging this model for the development of rational combinations in MSS CRC patients. Methods: BRGS (in house breeding) newborn pups were transplanted with CD34+ cells purified from (HLA unmatched) umbilical cord blood at 48 hours. In order to better understand the immune context of these novel animal models, we selected an MSI-high CRC (MDA-C0999-203) and a MSS (CRC172) humanized PDX model. At 16 weeks, MDA-C0999-203 (MSI-H) and CRC172 (MSS) were implanted on both flanks of humanized mice and nonhumanized controls. When the average tumor size reached ~150-300 mm3, both groups of mice were randomized into vehicle and nivolumab (30mg/kg IP twice weekly) stratified by % chimerism (human CD45+ &amp;gt;45%). At the end of the treatment, a portion of the tumor was immediately fixed in formalin for immunohistochemistry and pharmacodynamic analyses with seven-color multispectral imaging using the Perkin Elmer Vectra- 3 instrument, and compared to a surgical specimen from an untreated patient with an MSI-H tumor. Results: In the MSI-H model we observed tumor growth inhibition in the nivolumab-treated, humanized mice with respect to the humanized vehicle-treated control and the nonhumanized nivolumab-treated group (TGII 3.5% vs 76%). In a separate experiment, an MSS CRC humanized PDX model (CRC172) was treated with nivolumab as described above. Although initial control of tumor growth was observed, this was followed by rapid tumor progression (TGII 138.4%). In addition to flow cytometry, an MSI-H CRC tumor directly from a patient was stained for a variety of immunologic markers and analyzed by Vectra. The tumors contained both CD4- and CD8-positive cells, indicating T-cell infiltration. These data are consistent with what was observed by flow cytometry. Similar results were observed in an MSI-H CRC tumor grown as a xenograft in humanized mice both with and without nivolumab. However, in the MSS CRC PDX model, very few CD4 and CD8 cells were observed in the tumor or in the surrounding stroma. Conclusions: We have successfully established in vivo MSI-H and MSS humanized CRC PDXs. All mice were highly chimeric and the MSI-H model demonstrated high TILs, and responding tumors exhibited IFNγ production, high CD8%, higher effector memory % (HLADR+, CD45RO+), and decreased PD-L1 expression by flow cytometry. Interestingly, we also observed greater numbers of T cells in the lymph nodes of the MSI-H PDX. Moreover, immune infiltrates were observed in the MSI-H PDX compared to the MSS by immunohistochemistry. These data suggest that humanized PDX models may be useful in the development of rational combinations of immunotherapy. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, Christopher H. Lieu, Scott Kopetz, Sarah L. Davis, Kimberly Jordan, Stacey M. Bagby, Wells A. Messersmith, Roberta Pelanda, S. Gail Eckhardt. Humanized patient-derived xenografts (PDXs) recapitulate clinical responses in microsatellite stable (MSS) and unstable (MSI-H) colorectal cancer (CRC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A012.

Immunotherapy is reportedly effective in colorectal cancers (CRCs) with high microsatellite instability (MSI-H); however, the specific cell types that respond to immune checkpoint therapy are unclear. Herein, we aimed to examine the expression of programmed cell death-ligand 1 (PD-L1) and related proteins in MSI-H and microsatellite-stable (MSS) CRCs to investigate the immune microenvironment at the tumor's invasive front. The MSI status was retrospectively assessed in 499 patients undergoing surgical resection of primary CRC; of these, 48 were classified as MSI-H. Propensity score matching was performed, and tissues from 36 and 37 patients with MSI-H and MSS CRCs, respectively, were immunohistochemically evaluated for PD-L1, PD-1, CD8 and CD68. PD-L1 expression was evaluated separately for tumor cells (PD-L1 [T]) and tumor-infiltrating myeloid cells in the stroma (PD-L1 [I]). PD-L1 (T) was positive in only 5.4% and 36.1% of MSS and MSI-H CRCs, while PD-L1 (I) was positive in 27% and 72.2% of these CRCs, respectively. The PD-L1 (T) and PD-L1 (I) expression levels in MSI-H CRCs significantly correlated with poor differentiation, lymphatic invasion and vascular invasion (p < 0.05), and with early-stage adenocarcinoma and high budding grade (p < 0.05), respectively. Significantly more PD-L1 (I), CD8-positive cells and CD68-positive macrophages were present at the invasive front than in the central tumor in MSI-H CRCs (p < 0.005). PD-L1 was expressed on both tumor cells and CD68/CD163-positive (M2) macrophages at the invasive front of MSI-H CRCs. In conclusion, PD-L1-positive tumor cells and M2-type tumor-associated macrophages may contribute to tumor invasion and immune escape at the invasive front.

Patients with microsatellite instability-high (MSI-H) colorectal cancer (CRC) have high tumor mutation burden and tumor immunogenicity, exhibiting a higher response rate to immunotherapy and better survival. However, a portion of MSI-H CRC patients still experience adverse disease outcomes. We aimed to identify the tumor-autonomous regulators determining these heterogeneous clinical outcomes. The Cancer Genome Atlas (TCGA) dataset was used to identify regulators in MSI-H CRC patients with unfavorable outcomes. Stable CRC tumor clones expressing targeted regulators were established to evaluate migratory and stemness properties, immune cell vulnerability, and cell-in-cell (CIC) structure formation. RNA-sequencing (RNA-seq) was used to identify enriched biological pathways in stable CRC tumor clones. Clinicopathological characterization of formalin-fixed paraffin-embedded (FFPE) MSI-H CRC specimens was performed to explore the underlying mechanisms involved. We showed that cancer/testis antigen family 45 member A1 (CT45A1) expression was upregulated in MSI-H CRC patients with poor survival outcomes. CT45A1-expressing microsatellite stable (MSS) CRC cells showed enhanced migratory ability. However, CT45A1-expressing MSI-H CRC cells, but not MSS CRC cells, showed higher resistance to natural killer (NK) cell cytotoxicity and served as outer cells in homotypic CIC structures, preventing exogenous or therapeutic antibody access to inner CRCcells. Inactivating RHO-ROCK/MLCK-MLC2 signaling with small-molecule inhibitors or short-hairpin RNAs (shRNAs) targeting myosin light chain kinase (MYLK) abolished NK cell resistance and reduced the outer cell fate of CT45A1-expressing MSI-H CRC cells. In MSI-H CRC patients, CT45A1-positive tumors exhibited increased MLC2 phosphorylation, increased outer cell fate, and decreased survival. We demonstrated that CT45A1 potentiates the advanced progression of MSI-H CRC, and targeting MLC2 phosphorylation may enhance immunotherapy efficacy in CT45A1-positive MSI-H CRC patients.

Introduction: A BRAFV600E mutation is an unfavorable prognostic biomarker for CRC and is associated with short-lived treatment response to BRAF and EGFR blockade. Anti-PD-1 therapies are ineffective in MSS CRC but demonstrate efficacy in combination with BRAF + EGFR inhibition for MSS BRAFV600E CRC. A comprehensive characterization of MSS BRAFV600E CRC as an immunologically distinct subpopulation of MSS CRC has not been performed. Here, we characterize the clinicopathological and transcriptomic features of MSS BRAFV600E CRC patients, relative to BRAFWT MSS CRC patients.Methods: De-identified records of 12,009 MSS CRC patients were retrospectively analyzed from the Tempus clinicogenomic database. CMS subtypes were derived using the CMScaller algorithm. Survival was assessed using Kaplan-Meier survival analysis with risk set adjustment (RSA) method; log-rank test was used to compare survival. Categorical and continuous variables were compared using chi-squared test and Wilcoxon rank sum test, respectively. Results: BRAFV600E was found in 5% (n=630) of the patients. Demographics and clinicopathological features varied significantly between the BRAFWT and BRAFV600E cohorts (Table 1). Relative to the BRAFWT cohort, BRAFV600E tumors were enriched for the immune-activated CMS1 signature (53% vs 11%, p&amp;lt; .001), PD-L1 detection (9.5% vs 3%, p&amp;lt;0.001), and CD8+ T-cell infiltration (4.6% vs. 2.5%, p&amp;lt;0.001). CMS4 was linked to an unfavorable prognosis in the BRAFWT cohort (HR=0.58 compared to CMS1, p=0.026), yet this was not observed in the BRAFV600E cohort (HR=2.60 compared to CMS1, p=0.083). Conclusions: BRAFV600E MSS CRC exhibited immune activation characteristics that were not observed in the BRAFWT group, including greater CMS1 status, PD-L1 expression, and CD8-T cell infiltration. Our findings support investigation of novel immune-based therapeutic strategies of MSS BRAFV600E CRC as an immunologically distinct subpopulation of MSS CRC. Cohort characteristics BRAFWT BRAFV600E P-value Demographics Age (median) 60 (51,69) 65 (55,74) &amp;lt;0.001 Ethnicity Not Hispanic or Latino 3,444 (83%) 200 (93%) 0.001 Hispanic or Latino 699 (17%) 16 (7.4%) Gender Female 4,771 (42%) 345 (55%) &amp;lt;0.001 Male 6,608 (58%) 285 (45%) Race White 5,050 (75%) 318 (87%) &amp;lt;0.001 Black or African American 888 (13%) 19 (5.2%) Other 517 (7.7%) 20 (5.4%) Asian 297 (4.4%) 10 (2.7%) Clinicopathology TNM stage I 61 (0.7%) 0 (0%) 0.1 II 388 (4.6%) 17 (3.2%) III 1,130 (13%) 88 (17%) IV 6,941 (81%) 424 (80%) Laterality Right 777 (15%) 89 (39%) &amp;lt;0.001 Transverse 193 (3.8%) 27 (12%) Left 2,326 (46%) 73 (32%) Rectum 1,761 (35%) 37 (16%) CMS CMS1 1,077 (11%) 307 (53%) &amp;lt;0.001 CMS2 3,067 (31%) 6 (1%) CMS3 1,777 (18%) 71 (12%) CMS4 3,920 (40%) 199 (34%) Immunology CD8 T-cell infiltration (median, IQR) 2.5% (0.0, 7.7) 4.6% (0.0, 9.4) &amp;lt;0.001 PD-L1 127 (3%) 22 (9.5%) &amp;lt;0.001 Citation Format: Ymke van der Pol, Binyam Yilma, Van K. Morris, Calvin Chao, Michelle Harris, Justin Guinney, Scott Kopetz. Molecular characterization of microsatellite stable (MSS) colorectal cancer (CRC) patients with a BRAF V600E mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5056.

Immune checkpoint inhibitors have resulted in treatment paradigm changes for the management of patients with solid tumors, including microsatellite instability-high (MSI-H) colorectal cancer (CRC). Although the benefit of these agents appears to be limited for microsatellite stable (MSS) CRC, recent studies suggest that the immune microenvironment of the early-stage MSS CRC and perhaps those with advanced-stage disease without active liver metastasis may be more immune permissive where relatively more promising responses were noted. At this time, biomarkers of immunotherapy for patients with CRC have not been well-defined. Except for the loss of mismatch repair protein (MMR) function and POLE/POLD1 mutations, most of the biomarkers of response are largely investigational. In this review article, we summarize recent research and drug development with immune checkpoint inhibitors for patients with MSS and MSI-H CRC and elaborate on investigational biomarkers, including but not limited to tumor mutation burden and immunoscore. We also discuss the relevance and potential applicability of these biomarkers to clinical practice for the use of immune checkpoint inhibitors and provided further perspective on future biomarker development.