Abstract
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.
Highlights
The DNA mismatch repair (MMR) complex is highly conserved and performs an essential role in maintaining genomic stability by recognizing and repairing short insertions, short deletions, and single-base mismatches that can arise during DNA replication and recombination [1]
MMR/microsatellite instability (MSI) testing has acquired a key role in colorectal cancer (CRC) patient management [3,23,24]
It has been proved that patients with deficient MMR (dMMR)/MSI tumors may benefit from anti-PD-1 therapy; similar results were reported in a refractory MSI metastatic CRC patient setting, with 31.3% of the patients achieving an investigator-assessed objective response and 69% of the patients having disease for 12 weeks or longer [23]
Summary
The DNA mismatch repair (MMR) complex is highly conserved and performs an essential role in maintaining genomic stability by recognizing and repairing short insertions, short deletions, and single-base mismatches that can arise during DNA replication and recombination [1]. Germline mutation(s) of the MMR genes is the hallmark of Lynch syndrome and constitutional mismatch repair deficiency (CMMRD) [3,4]. Immunohistochemical (IHC) analysis of four of the seven MMR proteins (i.e., MLH1, PMS2, MSH2, and MSH6) represents the most widely used approach for the evaluation of microsatellite instability (MSI) status, but has some disadvantages [3,7]. The original approach used to determine MSI involves the direct determination of replication errors in DNA microsatellite sequences. Microsatellites may be affected by errors in the duplication process generally restored to by the MMR complex
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