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Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors.

Immunohistochemistry (IHC) and polymerase chain reaction (PCR) and fragment separation by capillary electrophoresis represent the current clinical laboratory standard for the evaluation of microsatellite instability (MSI) status. The importance of reporting MSI status in colorectal cancer is based on its potential for guiding treatment and as a prognostic indicator. It is also used to identify patients for Lynch syndrome testing. Our aim was to evaluate pre-analytical factors, such as age of formalin-fixed and paraffin-embedded (FFPE) block, neoplastic cell percentage, mucinous component, and DNA integrity, that may influence the accuracy of MSI testing and assess the concordance between three different MSI evaluation approaches. We selected the mucinous colorectal cancer (CRC) histotype for this study as it may possibly represent an intrinsic diagnostic issue due to its low tumor cellularity. Seventy-five cases of mucinous CRC and corresponding normal colon tissue samples were retrospectively selected. MMR proteins were evaluated by IHC. After DNA quality and quantity evaluation, the Idylla™ and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Seventy-three (97.3%) cases were successfully analyzed by the three methodologies. Overall, the Idylla™ platform showed a concordance rate with IHC of 98.0% for microsatellite stable (MSS)/proficient MMR (pMMR) cases and 81.8% for MSI/deficient MMR (dMMR) cases. The TapeStation 4200 system showed a concordance rate with IHC of 96.0% for MSS/pMMR cases and 45.4% for MSI/dMMR cases. The concordance rates of the TapeStation 4200 system with respect to the Idylla™ platform were 98.1% for MSS profile and 57.8% for MSI profile. Discordant cases were analyzed using the Titano MSI kit. Considering pre-analytical factors, no significant variation in concordance rate among IHC analyses and molecular systems was observed by considering the presence of an acellular mucus cut-off >50% of the tumor area, FFPE year preparation, and DNA concentration. Conversely, the Idylla™ platform showed a significant variation in concordance rate with the IHC approach by considering a neoplastic cell percentage >50% (p-value = 0.002), and the TapeStation 4200 system showed a significant variation in concordance rate with the IHC approach by considering a DNA integrity number (DIN) ≥4 as cut-off (p-value = 0.009). Our data pinpoint a central role of the pre-analytical phase in the diagnostic outcome of MSI testing in CRC.