MSI-H colorectal cancers preferentially retain and expand intraepithelial lymphocytes rather than peripherally derived CD8+ T cells.

Colorectal cancer is a heterogeneous tumor type with regard to molecular pathogenesis and genetic instability. The majority of colorectal cancers display chromosomal instability and follow the classical adenoma-carcinoma sequence of tumor progression. A subset of about 15% of colorectal cancers, however, displays DNA mismatch repair (MMR) deficiency and the high-level microsatellite instability (MSI-H) phenotype. MSI-H colorectal cancers can occur as sporadic tumors or in the context of hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome. The MSI-H phenotype is a hallmark of Lynch syndrome-associated cancers, which is of diagnostic relevance for the identification of Lynch syndrome mutation carriers. MSI-H colorectal cancers are characterized by a distinct clinical behavior, which results from their particular molecular pathogenesis and gives microsatellite instability testing its clinical significance. The MSI-H phenotype shows association with proximal tumor localization, a dense local lymphocyte infiltration, and a low frequency of distant organ metastasis. Moreover, MSI-H colorectal cancers have a better prognosis than their microsatellite-stable counterparts. A distinct responsiveness of MSI-H colorectal cancer patients towards chemotherapy has been shown in several studies. The clinical characteristics of MSI-H cancers are closely linked to their molecular pathogenesis, and research on the molecular alteration characteristic of MSI-H cancers may provide the basis for novel diagnostic or therapeutic approaches.

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.

High-level microsatellite instability (MSI-H) in colorectal cancer accounts for about 12% of colorectal cancers and is typically associated with a dense infiltration with cytotoxic CD8-positive lymphocytes. The role of regulatory T cells that may interfere with the host's antitumoural immune response in MSI-H colorectal cancers has not been analysed yet. Using an antibody directed against the regulatory T-cell marker transcription factor forkhead box P3 (FOXP3), regulatory T cells were examined in 70 colorectal cancers with known MSI status (MSI-H, n=37; microsatellite stable, n=33). In MSI-H colorectal cancers, we found a significantly higher intraepithelial infiltration with FOXP3-positive cells (median: 8.5 cells per 0.25 mm2vs 3.1 cells per 0.25 mm2 in microsatellite stable, P<0.001), and a significantly elevated ratio of intraepithelial to stromal infiltration (0.05 vs 0.01 in microsatellite stable, P<0.001). CD8-positive cell counts were related positively to the number of FOXP3-positive cells (Spearman's ρ=0.56 and 0.55, respectively). Our results show that the elevated number of CD8-positive lymphocytes found in MSI-H colorectal cancers is paralleled by an enhanced infiltration with CD8-negative FOXP3-positive cells. These data suggest that FOXP3-positive cells may play a role in the regulation of the immune response directed against MSI-H colorectal cancers at the primary tumour site.

High level microsatellite instability (MSI-H) is a hallmark of Lynch syndrome-associated colorectal cancer (CRC). MSI-H CRC express immunogenic tumour antigens as a consequence of DNA mismatch repair deficiency-induced frameshift mutations. Consequently, frameshift antigen-specific immune responses are commonly observed in patients with Lynch syndrome-associated MSI-H CRC. Dendritic cells (DC) and macrophages play a crucial role in the induction and modulation of immune responses. We here analysed DC and macrophage infiltration in MSI-H and microsatellite-stable CRC. Sixty-nine CRC (MSI-H, n=33; microsatellite-stable, n=36) were examined for the density of tumour-infiltrating DC, Foxp3-positive regulatory T cells, and CD163-positive macrophages. In MSI-H lesions, S100-positive and CD163-positive cell counts were significantly higher compared to microsatellite-stable lesions (S100: epithelium P=0.018, stroma P=0.042; CD163: epithelium P<0.001, stroma P=0.046). Additionally, numbers of CD208-positive mature DC were significantly elevated in the epithelial compartment of MSI-H CRC (P=0.027). High numbers of tumour-infiltrating Foxp3-positive T cells were detected in tumours showing a low proportion of CD208-positive, mature DC among the total number of S100-positive cells. Our study demonstrates that infiltration with DC, mature DC, and macrophages is elevated in MSI-H compared to microsatellite-stable CRC. The positive correlation of Foxp3-positive Treg cell density with a low proportion of mature DC suggests that impaired DC maturation may contribute to local immune evasion in CRC. Our results demonstrate that DC and macrophages in the tumour environment likely play an important role in the induction of antigen-specific immune responses in Lynch syndrome. Moreover, impaired DC maturation might contribute to local immune evasion in CRC.

Defects of DNA mismatch repair (MMR) cause the high level microsatellite instability (MSI-H) phenotype. MSI-H cancers may develop either sporadically or in the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome that is caused by germline mutations of MMR genes. In colorectal cancer (CRC), MSI-H is characterized by a dense lymphocytic infiltration, reflecting a high immunogenicity of these cancers. As a consequence of immunoselection, MSI-H CRCs frequently display a loss of human leukocyte antigen (HLA) class I antigen presentation caused by mutations of the beta2-microglobulin (beta2m) gene. To examine the implications of beta2m mutations during MSI-H colorectal tumor development, we analyzed the prevalence of beta2m mutations in MSI-H colorectal adenomas (n=38) and carcinomas (n=104) of different stages. Mutations were observed in 6/38 (15.8%) MSI-H adenomas and 29/104 (27.9%) MSI-H CRCs. A higher frequency of beta2m mutations was observed in MSI-H CRC patients with germline mutations of MMR genes MLH1 or MSH2 (36.4%) compared with patients without germline mutations (15.4%). The high frequency of beta2m mutations in HNPCC-associated MSI-H CRCs is in line with the hypothesis that immunoselection may be particularly pronounced in HNPCC patients with inherited predisposition to develop MSI-H cancers. beta2m mutations were positively related to stage in tumors without distant metastases (UICC I-III), suggesting that loss of beta2m expression may promote local progression of colorectal MSI-H tumors. However, no beta2m mutations were observed in metastasized CRCs (UICC stage IV, p=0.04). These results suggest that functional beta2m may be necessary for distant metastasis formation in CRC patients.

Expression status of wild-type HSP110 correlates with HSP110 T17 deletion size and patient prognosis in microsatellite-unstable colorectal cancer

BACKGROUNDRecent studies in non-colorectal malignancy have associated T resident memory (TRM) cells with improved patient survival. It is unknown if TRM plays a role in colorectal cancer (CRC).AIMTo examine the potential role of TRM cells in providing immunogenicity in CRC stratified by microsatellite instability (MSI) and BRAF status.METHODSPatients with known MSI and BRAF mutation status were eligible for inclusion in this study. CRC tumour sections stained with haematoxylin and eosin were microscopically reviewed and the images scanned prior to assessment for location of invading edge and core of tumour. Sequential sections were prepared for quantitative multiplex immunohistochemistry (IHC) staining. Opal Multiplex IHC staining was performed with appropriate positive and negative controls and imaged using a standard fluorescent microscope fitted with a spectral scanning camera (Mantra) in conjunction with Mantra snap software. Images were unmixed and annotated in inForm 2.2.0. Statistical analysis was performed using Graphpad Prism Version 7 and Stata Version 15.RESULTSSeventy-two patients with known MSI and BRAF status were included in the study. All patients were assessed for MSI by IHC and high resolution capillary electrophoresis testing and 44 of these patients successfully underwent quantitative multiplex IHC staining. Overall, there was a statistically significant increase in CD8+ TRM cells in the MSI (BRAF mutant and wild type) group over the microsatellite stable (MSS) group. There was a statistically significant difference in CD8+ TRM between high level MSI (MSI-H):BRAF mutant [22.57, 95% confidence interval (CI): 14.31-30.84] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0076 andMSI-H:BRAF wild type [16.18 (95%CI: 10.44-21.93)] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0279. There was no statistically significant difference in CD8 T cells (both CD8+CD103- and CD8+CD103+TRM) between MSI-H: BRAF mutant and wild type CRC.CONCLUSIONThis study has shown that CD8+ TRM are found in greater abundance in MSI-H CRC, both BRAF mutant and MSI-H:BRAF wild type, when compared with their MSS counterpart. CD8+ TRM may play a role in the immunogenicity in MSI-H CRC (BRAF mutant and BRAF wild type). Further studies should focus on the potential immunogenic qualities of TRM cells and investigate potential immunotherapeutic approaches to improve treatment and survival associated with CRC.

Features of Colorectal Cancers with High-Level Microsatellite Instability Occurring in Familial and Sporadic Settings: Parallel Pathways of Tumorigenesis

Background: Colorectal cancers (CRC) with high level of microsatellite instability (MSI-H) are characterized by lower metastasis propensity and better prognosis than their stable microsatellite (MSS) counterpart. It was hypothesized that...

Identifying colorectal cancers (CRCs) with high levels of microsatellite instability (MSI-H) is clinically important. MSI-H is a positive prognostic marker for CRC, a predictive marker for resistance to standard 5-fluorouracil-based adjuvant chemotherapy, and an important feature for identifying individuals and families with Lynch syndrome. Our aim was to compare and improve upon the existing predictive pathology models for MSI-H CRCs. We tested 2 existing models used to predict MSI-H tumors, (1) Revised Bethesda Guidelines and (2) MsPath, in our population-based cohort of CRCs diagnosed less than 75 years from Newfoundland (N=710). We also scored additional histologic features not described in the other models. From this analysis, we developed a model for the prediction of MSI-H CRCs; Pathologic Role in Determination of Instability in Colorectal Tumors (PREDICT). An independent pathologist validated this model in a second cohort of all CRCs (N=276). Tumor histology was a better predictor of MSI status than was personal and family history of cancer. MsPath identified MSI-H CRCs with a sensitivity of 92.1% and a specificity of 37.8%, whereas the Revised Bethesda Guidelines had a sensitivity of 81.3% and a specificity of 39.5%. PREDICT included some new histology features, including peritumoral lymphocytic reaction and increased proportion of plasma cells in the tumor stroma. PREDICT was superior to both existing models in the development cohort with a sensitivity of 97.4% and a specificity of 53.9%. In the validation cohort, sensitivity was 96.9% and specificity 76.6%. We conclude that PREDICT is a good predictor of MSI-H CRC.

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.

High level microsatellite instability (MSI-H) occurs in about 15% of colorectal cancer (CRCs), either as sporadic cancers or in the context of hereditary non-polyposis cancer or Lynch syndrome. In MSI-H CRC, mismatch repair deficiency leads to insertion/deletion mutations at coding microsatellites and thus to the translation of frameshift peptides (FSPs). FSPs are potent inductors of T cell responses in vitro and in vivo. The present study aims at the identification of FSP-specific humoral immune responses in MSI-H CRC and Lynch syndrome. Sera from patients with history of MSI-H CRC (n = 69), healthy Lynch syndrome mutation carriers (n = 31) and healthy controls (n = 52) were analyzed for antibodies against FSPs using peptide ELISA. Reactivities were measured against FSPs derived from genes frequently mutated in MSI-H CRCs, AIM2, TGFBR2, CASP5, TAF1B, ZNF294, and MARCKS. Antibody reactivity against FSPs was significantly higher in MSI-H CRC patients than in healthy controls (P = 0.036, Mann-Whitney) and highest in patients with shortest interval between tumor resection and serum sampling. Humoral immune responses in patients were most frequently directed against FSPs derived from mutated TAF1B (11.6%, 8/69) and TGFBR2 (10.1%, 7/69). Low level FSP-specific antibodies were also detected in healthy mutation carriers. Our results show that antibody responses against FSPs are detectable in MSI-H CRC patients and healthy Lynch syndrome mutation carriers. Based on the high number of defined FSP antigens, measuring FSP-specific humoral immune responses is a highly promising tool for future diagnostic application in MSI-H cancer patients.

Several previous studies have demonstrated that the CDX2-negative (CDX2) and/or CK20-negative (CK20) phenotypes of colorectal cancers (CRCs) might be associated with high levels of microsatellite instability (MSI-H). The aim of this study was to investigate the clinicopathologic and molecular features of MSI-H CRCs with different CDX2/CK20 expression statuses. The CDX2 and CK20 expression statuses were immunohistochemically evaluated in 109 MSI-H CRC tissue samples, and the correlations of these statuses with clinicopathologic, molecular, and survival data were statistically analyzed. Of the 109 MSI-H CRCs, 15 were CDX2 (13.8%), and 19 were CK20 (17.4%). The simultaneous loss of CDX2 and CK20 expression (CDX2/CK20) was observed in 9 cases (8.3%). CDX2 loss was correlated with lymph node metastasis, poor differentiation, MLH1 loss, the mutation of BRAF, and CpG island methylator phenotype-high (CIMP-H) status. Right-sided tumor location, nodal metastasis, poor differentiation, and CIMP-H status were significant characteristics of CK20 tumors. The CDX2/CK20 phenotype was associated with older age (above 56 y), higher stage (stage III or IV), deep invasion (pT3 or pT4), lymph node metastasis (pN1 or pN2), poor differentiation (nonmedullary/non-signet ring cell type), the mutation of BRAF, and CIMP-H status among MSI-H CRCs. Patients with CDX2/CK20 tumors exhibited worse overall and disease-free survival compared with the patients with CDX2 and/or CK20 tumors (P<0.001). In the multivariate analysis for disease-free survival, the CDX2/CK20 phenotype was an independent prognostic factor for MSI-H CRC (P=0.030, hazard ratio=3.288). The CDX2/CK20 phenotype defines a distinct subgroup of MSI-H CRCs with poor differentiation, CIMP-H status, and unfavorable prognosis.

In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.