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Comparison of microsatellite instability detection by immunohistochemistry and molecular techniques in colorectal and endometrial cancer.

ABSTRACT: DNA mismatch repair deficiency (dMMR) testing is crucial for diagnosing Lynch syndrome and detection of microsatellite unstable (MSI) tumors eligible for immunotherapy. The aim of this study was to compare the relative diagnostic performance of three molecular MSI assays: polymerase chain reaction (PCR), MSI testing by Idylla and next-generation-sequencing (NGS) on 49 tumor samples (28 colorectal and 21 endometrial adenocarcinomas) versus immunohistochemistry (IHC). Discrepancies were investigated by MLH1 methylation analysis and integrated with germline results if available. Overall, the molecular assays achieved equivalent diagnostic performance for MSI detection with area under the ROC curves (AUC) of respectively 0.91 for Idylla and PCR, and 0.93 for NGS. In colorectal cancers with tumor cell percentages ≥ 30% all three molecular assays achieved 100% sensitivity and specificity (AUC = 1) versus IHC. Also, in endometrial cancers, all three molecular assays showed equivalent diagnostic performance, albeit at a clearly lower sensitivity ranging from 58% for Idylla to 75% for NGS, corresponding to negative predictive values from 78 to 86%. PCR, Idylla and NGS show similar diagnostic performance for dMMR detection in colorectal and endometrial cancers. Molecular MSI analysis has lower sensitivity for dMMR detection in endometrial cancer indicating that combined use of both IHC and molecular methods is recommended.Clinical Trial Number/IRB: B1172020000040, Ethical Committee, AZ Delta General Hospital.

SUBMITTER: Dedeurwaerdere F

PROVIDER: S-EPMC8213758 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectivesIdentification of endometrial cancers (EC) with mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H) is essential for Lynch syndrome screening and treatment stratification. We aimed to assess the utility of immunohistochemistry (IHC) staining for MMR protein expression and polymerase chain reaction (PCR)-based MSI assays in EC and the correlation between MMR/MSI status and various clinicopathological parameters.MethodsWe reviewed the clinical and pathological information of 333 patients with EC. MMR protein expression was assessed as retained or lost to determine MMR status by IHC staining, and MSI status was identified by PCR capillary electrophoresis (PCR-CE) testing with a National Cancer Institute (NCI) panel. The correlation of MMR/MSI status with clinicopathological features was determined by statistical analysis. Discrepant results were further analyzed using an alternative PCR-CE MSI (Promega panel) method, MLH1 promoter methylation assays, and next-generation sequencing (NGS).ResultsAmong the EC patients, the overall percentage of dMMR was 25.2%, and the overall percentage of MSI-H was 24%. Among the dMMR patients, 50 (59.5%) showed loss of MLH1 and PMS2 expression, 19 (22.6%) loss of MSH2 and MSH6 expression, and seven (8.3%) and eight (9.5%) loss of PMS2 and MSH6 expression, respectively. The dMMR subgroup was significantly younger than the pMMR subgroup, especially for <60-years-old patients (p = 0.038). In addition, we identified a strong correlation between MMR/MSI status and high-grade endometrioid or nonendometrioid components (p = 0.004 or p = 0.003). IHC staining and PCR-CE assay results showed a high level of overall concordance (98.8%, Cohen's κ = 0.98). Four patients were found to have dMRR/MSS in both examinations. We reanalyzed them with additional methods. One case showed MLH1 promotor methylation, and the other three cases harbored MSH6 germline pathogenic variations. One of the cases with MSH6 deficiency was reanalyzed as MSI-H by alternative PCR-CE assay or NGS testing.ConclusionsThis study indicates that the combined use of MMR-IHC and PCR-CE MSI analyses may effectively avoid misdiagnoses of EC patients with dMMR/MSI-H. However, use of PCR-CE alone to evaluate MMR/MSI status may lead to missed diagnosis, especially for EC patients with MSH6 deficiency and presenting MSS.

Project description:BackgroundMicrosatellite instability (MSI) is a key biomarker for cancer immunotherapy and prognosis. Integration of MSI testing into a next-generation-sequencing (NGS) panel could save tissue sample, reduce turn-around time and cost, and provide MSI status and comprehensive genomic profiling in single test. We aimed to develop an MSI calling model to detect MSI status along with the NGS panel-based profiling test using tumor-only samples.MethodsFrom January 2019 to December 2020, a total of 174 colorectal cancer (CRC) patients were enrolled, including 31 MSI-high (MSI-H) and 143 microsatellite stability (MSS) cases. Among them, 56 paired tumor and normal samples (10 MSI-H and 46 MSS) were used for modeling, and another 118 tumor-only samples were used for validation. MSI polymerase chain reaction (MSI-PCR) was performed as the gold standard. A baseline was built for the selected microsatellite loci using the NGS data of 56 normal blood samples. An MSI detection model was constructed by analyzing the NGS data of tissue samples. The performance of the model was compared with the results of MSI-PCR.ResultsWe first intersected the target genomic regions of the NGS panels used in this study to select common microsatellite loci. A total of 42 loci including 23 mononucleotide repeat sites and 19 longer repeat sites were candidates for modeling. As mononucleotide repeat sites are more sensitive and specific for detecting MSI status than sites with longer length motif and the mononucleotide repeat sites performed even better than the total sites, a model containing 23 mononucleotide repeat sites was constructed and named Colorectal Cancer Microsatellite Instability test (CRC-MSI). The model achieved 100% sensitivity and 100% specificity when compared with MSI-PCR in both training and validation sets. Furthermore, the CRC-MSI model was robust with the tumor content as low as 6%. In addition, 8 out of 10 MSI-H samples showed alternations in the four mismatch repair genes ( MLH1 , MSH2 , MSH6 , and PMS2 ).ConclusionMSI status can be accurately determined along the targeted NGS panels using only tumor samples. The performance of mononucleotide repeat sites surpasses loci with longer repeat motif in MSI calling.

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Comparison of microsatellite instability detection by immunohistochemistry and molecular techniques in colorectal and endometrial cancer.

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