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Background: Microsatellite instability (MSI) due to mismatch repair deficiency (dMMR) is common in colorectal cancer (CRC). These cancers are associated with somatic coding events, but the noncoding pathophysiological impact of this genomic instability is yet poorly understood. Here, we perform an analysis of coding and noncoding MSI events at the different steps of colorectal tumorigenesis using whole exome sequencing and search for associated splicing events via RNA sequencing at the bulk-tumor and single-cell levels., Results: Our results demonstrate that MSI leads to hundreds of noncoding DNA mutations, notably at polypyrimidine U2AF RNA-binding sites which are endowed with cis-activity in splicing, while higher frequency of exon skipping events are observed in the mRNAs of MSI compared to non-MSI CRC. At the DNA level, these noncoding MSI mutations occur very early prior to cell transformation in the dMMR colonic crypt, accounting for only a fraction of the exon skipping in MSI CRC. At the RNA level, the aberrant exon skipping signature is likely to impair colonic cell differentiation in MSI CRC affecting the expression of alternative exons encoding protein isoforms governing cell fate, while also targeting constitutive exons, making dMMR cells immunogenic in early stage before the onset of coding mutations. This signature is characterized by its similarity to the oncogenic U2AF1-S34F splicing mutation observed in several other non-MSI cancer., Conclusions: Overall, these findings provide evidence that a very early RNA splicing signature partly driven by MSI impairs cell differentiation and promotes MSI CRC initiation, far before coding mutations which accumulate later during MSI tumorigenesis., (© 2024. The Author(s).)

Background & Aims: Next-generation sequencing (NGS) was recently approved by the United States Food and Drug Administration to detect microsatellite instability (MSI) arising from defective mismatch repair (dMMR) in patients with metastatic colorectal cancer (mCRC) before treatment with immune checkpoint inhibitors (ICI). In this study, we aimed to evaluate and improve the performance of NGS to identify MSI in CRC, especially dMMR mCRC treated with ICI., Methods: CRC samples used in this post hoc study were reassessed centrally for MSI and dMMR status using the reference methods of pentaplex polymerase chain reaction and immunohistochemistry. Whole-exome sequencing (WES) was used to evaluate MSISensor, the Food and Drug Administration-approved and NGS-based method for assessment of MSI. This was performed in (1) a prospective, multicenter cohort of 102 patients with mCRC (C1; 25 dMMR/MSI, 24 treated with ICI) from clinical trials NCT02840604 and NCT033501260, (2) an independent retrospective, multicenter cohort of 113 patients (C2; 25 mCRC, 88 non-mCRC, all dMMR/MSI untreated with ICI), and (3) a publicly available series of 118 patients with CRC from The Cancer Genome Atlas (C3; 51 dMMR/MSI). A new NGS-based algorithm, namely MSICare, was developed. Its performance for assessment of MSI was compared with MSISensor in C1, C2, and C3 at the exome level or after downsampling sequencing data to the MSK-IMPACT gene panel. MSICare was validated in an additional retrospective, multicenter cohort (C4) of 152 patients with new CRC (137 dMMR/MSI) enriched in tumors deficient in MSH6 (n = 35) and PMS2 (n = 9) after targeted sequencing of samples with an optimized set of microsatellite markers (MSIDIAG)., Results: At the exome level, MSISensor was highly specific but failed to diagnose MSI in 16% of MSI/dMMR mCRC from C1 (4 of 25; sensitivity, 84%; 95% confidence interval [CI], 63.9%-95.5%), 32% of mCRC (8 of 25; sensitivity, 68%; 95% CI, 46.5%-85.1%), and 9.1% of non-mCRC from C2 (8 of 88; sensitivity, 90.9%; 95% CI, 82.9%-96%), and 9.8% of CRC from C3 (5 of 51; sensitivity, 90.2%; 95% CI, 78.6%-96.7%). Misdiagnosis included 4 mCRCs treated with ICI, of which 3 showed an overall response rate without progression at this date. At the exome level, reevaluation of the MSI genomic signal using MSICare detected 100% of cases with true MSI status among C1 and C2. Further validation of MSICare was obtained in CRC tumors from C3, with 96.1% concordance for MSI status. Whereas misdiagnosis with MSISensor even increased when analyzing downsampled WES data from C1 and C2 with microsatellite markers restricted to the MSK-IMPACT gene panel (sensitivity, 72.5%; 95% CI, 64.2%-79.7%), particularly in the MSH6-deficient setting, MSICare sensitivity and specificity remained optimal (100%). Similar results were obtained with MSICare after targeted NGS of tumors from C4 with the optimized microsatellite panel MSIDIAG (sensitivity, 99.3%; 95% CI, 96%-100%; specificity, 100%)., Conclusions: In contrast to MSISensor, the new MSICare test we propose performs at least as efficiently as the reference method, MSI polymerase chain reaction, to detect MSI in CRC regardless of the defective MMR protein under both WES and targeted NGS conditions. We suggest MSICare may rapidly become a reference method for NGS-based testing of MSI in CRC, especially in mCRC, where accurate MSI status is required before the prescription of ICI., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Importance: Primary resistance to immune checkpoint inhibitors is observed in 10% to 40% of patients with metastatic colorectal cancer (mCRC) displaying microsatellite instability (MSI) or defective mismatch repair (dMMR)., Objective: To investigate possible mechanisms underlying primary resistance to immune checkpoint inhibitors of mCRC displaying MSI or dMMR., Design, Setting, and Participants: This post hoc analysis of a single-center, prospective cohort included 38 patients with mCRC diagnosed as MSI or dMMR by local laboratories and entered into trials of immune checkpoint inhibitors between January 1, 2015, and December 31, 2016. The accuracy of MSI or dMMR status was also assessed in a retrospective cohort comprising 93 cases of mCRC that were diagnosed as MSI or dMMR between January 1, 1998, and December 31, 2016, in 6 French hospitals. Primary resistance of mCRC was defined as progressive disease according to Response Evaluation Criteria in Solid Tumors criteria, 6 to 8 weeks after initiation of immune checkpoint inhibitors, without pseudo-progression. All tumor samples were reassessed for dMMR status using immunohistochemistry with antibodies directed against MLH1, MSH2, MSH6, and PMS2, and for MSI using polymerase chain reaction with pentaplex markers and with the HSP110 T17 (HT17) repeat., Main Outcomes and Measures: The primary outcome was positive predictive value., Results: Among the 38 patients (15 women and 23 men; mean [SD] age, 55.6 [13.7] years) in the study with mCRC displaying MSI or dMMR, primary resistance to immune checkpoint inhibitors was observed in 5 individuals (13%). Reassessment of the status of MSI or dMMR revealed that 3 (60%) of these 5 resistant tumors were microsatellite stable or displayed proficient mismatch repair. The positive predictive value of MSI or dMMR status assessed by local laboratories was therefore 92.1% (95% CI, 78.5%-98.0%). In the retrospective cohort of 93 patients (44 women and 49 men; mean [SD] age, 56.8 [18.3] years) without immune checkpoint inhibitor treatment, misdiagnosis of the MSI or dMMR status by local assessment was 10% (n = 9), with a positive predictive value of 90.3% (95% CI, 82.4%-95.0%). Testing for MSI with the HT17 assay confirmed the MSI or dMMR status in 2 of 4 cases showing discrepant results between immunohistochemistry and pentaplex polymerase chain reaction (ie, dMMR but microsatellite stable)., Conclusions and Relevance: Primary resistance of mCRC displaying MSI or dMMR to immune checkpoint inhibitors is due mainly to misdiagnosis of their MSI or dMMR status. Larger studies are required to confirm these findings. Microsatellite instability or dMMR status should be tested routinely using both immunohistochemistry and polymerase chain reaction methods prior to treatment with immune checkpoint inhibitors.

Colorectal cancers (CRC) with microsatellite instability (MSI) display unique clinicopathologic features including a mucinous pattern with frequent expression of the secreted mucins MUC2 and MUC5AC. The mechanisms responsible for this altered pattern of expression remain largely unknown. We quantified DNA methylation of mucin genes (MUC2, MUC5AC, MUC4) in colonic cancers and examined the association with clinicopathological characteristics and molecular (MSI, KRAS, BRAF, and TP53 mutations) features. A control cohort was used for validation. We detected frequent hypomethylation of MUC2 and MUC5AC in CRC. MUC2 and MUC5AC hypomethylation was associated with MUC2 and MUC5AC protein expression (p = 0.004 and p < 0.001, respectively), poor differentiation (p = 0.001 and p = 0.007, respectively) and MSI status (p < 0.01 and p < 0.001, respectively). Interestingly, MUC5AC hypomethylation was specific to MSI cancers. Moreover, it was significantly associated with BRAF mutation and CpG island methylator phenotype (p < 0.001 and p < 0.001, respectively). All these results were confirmed in the control cohort. In the multivariate analysis, MUC5AC hypomethylation was a highly predictive biomarker for MSI cancers. MUC5AC demethylation appears to be a hallmark of MSI in CRC. Determination of MUC5AC methylation status may be useful for understanding and predicting the natural history of CRC., (© 2014 UICC.)

The use of neoadjuvant chemotherapy (NAC) in patients with mismatch repair (MMR) deficient (dMMR) localized gastric and oeso-gastric junction (OGJ) adenocarcinoma is subject of debate. Histological response assessment might help to better evaluate the impact of dMMR on response to NAC. Patients with localized gastric/OGJ adenocarcinoma resected after NAC were retrospectively identified. MMR protein expression status was assessed by immunohistochemistry. The primary objective was the frequency of histological responders to NAC defined by tumour regression grade (TRG) using Mandard's (TRG1-2) and Becker's (TRG1) classifications, according to the MMR status. In total, 247 patients with 43 dMMR and 204 pMMR gastric/OGJ adenocarcinoma were identified. Among dMMR tumours, 18 (42%) arose from the OGJ. Histological response (Becker TRG1-2) was observed for 28% and 35% of dMMR and pMMR tumours, respectively (p = 0.35). Similar results were observed with Mandard classification. With a median follow-up of 37.5 months, median disease-free and overall survival were not reached for the dMMR group. Histological response after NAC in patients with localized dMMR gastric/OGJ adenocarcinoma is not statistically different to those with pMMR tumours. This study provides additional data for the discussion about avoiding NAC in patients with dMMR gastric/OGJ adenocarcinomas. [ABSTRACT FROM AUTHOR]