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Background Microsatellite instability (MSI) occurs in several cancer types and is commonly used for prognosis and as a predictive biomarker for immune checkpoint therapy. Methods We analyzed n = 263 formalin‐fixed paraffin‐embedded (FFPE) tumor specimens (127 colorectal cancer (CRC), 55 endometrial cancer (EC), 33 stomach adenocarcinoma (STAD), and 48 solid tumor specimens of other tumor types) with a capillary electrophoresis based multiplex monomorphic marker MSI‐PCR panel and an amplicon‐based NGS assay for microsatellite instability (MSI+). In total, n = 103 (39.2%) cases with a known defect of the DNA mismatch repair system (dMMR), determined by a loss in protein expression of MSH2/MSH6 (n = 48, 46.6%) or MLH1/PMS2 (n = 55, 53.4%), were selected. Cases with an isolated loss of MSH6 or PMS2 were excluded. Results The overall sensitivity and specificity of the NGS assay in comparison with the MSI‐PCR were 92.2% and 98.8%. With CRC cases a nearly optimal concordance was reached (sensitivity 98.1% and specificity 100.0%). Whereas EC cases only show a sensitivity of 88.6% and a specificity of 95.2%, caused by several cases with instability in less than five monomorphic markers, which could be difficult to analyze by NGS (subtle MSI+ phenotype). Conclusions MSI analysis of FFPE DNA by NGS is feasible and the results show a high concordance in comparison with the monomorphic marker MSI‐PCR. However, cases with a subtle MSI+ phenotype, most frequently manifest in EC, have a risk of a false‐negative result by NGS and should be preferentially analyzed by capillary electrophoresis. Microsatellite instability (MSI) analysis of formalin‐fixed paraffin‐embedded (FFPE) DNA by NGS is feasible and the results show a high concordance in comparison with the monomorphic marker MSI‐PCR (overall sensitivity and specificity of the NGS assay in comparison with the MMR‐IHC/MSI‐PCR were 92.2% and 98.8%). However, cases with a subtle MSI+ phenotype, most frequently manifest in endometrial cancer (EC), have a risk of a false‐negative result by NGS and should be preferentially analyzed by capillary electrophoresis.

Background The tumor-agnostic indication of immune checkpoint inhibitors to treat cancers with mismatch repair deficiency (dMMR)/microsatellite instability (MSI) increased the demand for such tests beyond Lynch syndrome. International guideline recommendations accept immunohistochemistry (IHC) for dMMR or molecular techniques (PCR or NGS) for MSI status determinations considering the two tests are equal, although there are scattered reports contradicting to this presumption. Materials and methods Here we have directly compared four protein MMR immunohistochemistry (IHC) to MSI Pentaplex PCR test in a large cancer patient cohort (n = 1306) of our diagnostic center where the two tests have been run parallel in 703 cases. Results In this study we have found a high discrepancy rate (19.3%) of the two tests which was independent of the tumor types. The MSI PCR sensitivity for MMR IHC status was found to be very low resulting in a relatively low positive and negative predicting values. As a consequence, the correlation of the two tests was low (kappa < 0.7). During analysis of the possible contributing factors of this poor performance, we have excluded low tumor percentage of the samples, but identified dMMR phenotypes (classic versus non-classic or unusual) as possible contributors. Conclusion Although our cohort did not include samples with identified technical errors, our data strongly support previous reports that unidentified preanalytical factors might have the major influence on the poor performance of the MSI PCR and MMR IHC. Furthermore, the case is open whether the two test types are equally powerful predictive markers of immunotherapies.

Gastric carcinomas (GC) are heterogeneous malignancies characterised by distinct histological and molecular subtypes. The microsatellite instability (MSI) molecular subtype, resulting from deficient DNA mismatch repair (dMMR), accounts for approximately 22% of global GC cases. Empirical evidence indicates differences in clinicopathological features, demographics, and treatment response in MSI GC compared to microsatellite stable (MSS) GC. MSI status has emerged as a potential biomarker for advanced GC, and this study aimed to determine the MSI prevalence of histopathologically confirmed GC cases at our centre. This was a retrospective cross-sectional analysis of GC cases from 2018 to 2022, which were retrieved from the laboratory information system. DNA from these cases was isolated and assessed for MSI using a pentaplex PCR panel and confirmatory IHC on MSI-H was performed. Samples with no allelic size variation in the 5 microsatellite markers were classified as microsatellite stable (MSS), variation in 1 marker as microsatellite instability low (MSI-L), and variation in 2 or more microsatellite markers as MSI-H. The study consisted of 64 cases with a MSI prevalence of 21.9% (n = 14) displaying a male predominance (n = 10; 71.4%) and a mean age of 62.7 years. Among these 14 MSI cases, 42.9% (n = 6) were classified as MSI-H with a mean age of 59.3 years. Half (n = 3) of these cases presented with upper gastrointestinal bleeding, with a majority of them diagnosed with moderately differentiated adenocarcinomas (66.7%). Microsatellite instability low was seen in 57.1% (n = 8) of the cases with a mean age of 65.3 years, and of these, patients presented with vomiting, epigastric pain and dysphagia with equal frequency at 25% (n = 2 respectively). The frequency of MSI cases in this study is congruent with global trends, highlighting the importance of microsatellite status in GC for understanding clinicopathological differences between MSI and MSS patients. These findings support the potential of MSI status as a biomarker.

To investigate mononucleotide markers: BAT-25, BAT-26, NR-21, NR-22 and NR-24 in patients with colorectal cancer (CRC), and the status of , , and the promoter mutations in microsatellite unstable CRC. Genetic assessments were performed on samples obtained following resection of CRC in 200 patients. Allelic variations of were found in all 18 patients with microsatellite instabilities (MSIs) in at least three markers (high-frequency MSI). By contrast, mutations of were absent in all 20 patients with no MSI frequency. Eight out of 182 patients with low (instability in one marker) or no frequency MSI had allelic shifts due to polymorphisms of BAT-25 (1.5%), NR-21 (1.75%) and NR-24 (1.5%). mutations were associated with >5 bp shortening of . Patients with high-frequency MSI CRC had allelic variations of . mutations occur along with greater shortening in during oncogenesis via the MSI pathway.

To investigate mononucleotide markers: BAT-25, BAT-26, NR-21, NR-22 and NR-24 in patients with colorectal cancer (CRC), and the status of HSP110T17, KRAS, BRAF and the MLH1 promoter mutations in microsatellite unstable CRC. Genetic assessments were performed on samples obtained following resection of CRC in 200 patients. Allelic variations of HSP110T17 were found in all 18 patients with microsatellite instabilities (MSIs) in at least three markers (high-frequency MSI). By contrast, mutations of HSP110T17 were absent in all 20 patients with no MSI frequency. Eight out of 182 patients with low (instability in one marker) or no frequency MSI had allelic shifts due to polymorphisms of BAT-25 (1.5%), NR-21 (1.75%) and NR-24 (1.5%). BRAF mutations were associated with >5 bp shortening of HSP110T17. Patients with high-frequency MSI CRC had allelic variations of HSP110T17. BRAF mutations occur along with greater shortening in HSP110T17 during oncogenesis via the MSI pathway.

A pentaplex PCR was developed and optimised to detect the genes that encode the five most important putative virulence factors in Aeromonas isolates. It seems to be more efficient than previously reported techniques and promises to be a powerful tool for more accurate risk assessments and for monitoring pathogenic strains. [PUBLICATION ABSTRACT]