Impact of Pre-Analytical Factors on MSI Test Accuracy in Mucinous Colorectal Adenocarcinoma: A Multi-Assay Concordance Study

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.

Evaluation of Micro Satellite Instability and Mismatch Repair Status in Different Solid Tumors: A Multicenter Analysis in a Real World Setting

“…However, evaluation and interpretation of immunohistochemical results may at times be challenging because of intra-and inter-observer variability and pre-analytical and analytical issues [1]. In this scenario, evaluation of microsatellite instability (MSI) through molecular approaches, such as polymerase chain reaction (PCR), genomic sequencing, and capillary electrophoresis, has been proposed as a valuable alternative to overcome some of the issues inherent to immunohistochemistry (IHC) [1,11].…”

“…Generally, the most widely adopted procedure for MSI evaluation relies on the Sanger sequencing-based Bethesda panel, which covers two mononucleotide (BAT-25 and BAT-26) and three dinucleotide (D5S346, D2S123, and D17S250) repetitions [13] or, alternatively, a panel covering five poly-A mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27) [14]. However, recent studies have demonstrated that the fully automated PCR high-resolution melt curve analysis (Idylla TM , Biocartis, Mechelen, Belgium) [11,[15][16][17][18][19][20][21][22][23][24][25][26] and the automated microfluidic electrophoretic run chip-based assay (TapeStation 4200, Agilent Technologies, Santa Clara, CA, USA) are easier to use, faster, and less expensive than Sanger sequencing [11,27,28].…”

“…Thus, this evidence strongly underlines the importance of expanding the evaluation of MSI-H/dMMR status to different types of solid tumors. Although our research group has demonstrated the feasibility of adopting the fully automated PCR high-resolution melt curve analysis (Idylla TM ) and the automated microfluidic electrophoretic run chipbased assay (TapeStation 4200) to assess MSI-H/dMMR status in CRC patients [11], little is yet known about their performance in other types of tumors. In an attempt to fill this knowledge gap, we compared the concordance rates between these two molecular approaches and IHC analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients.…”

Risk factors for recurrence of radically resected mucinous colorectal adenocarcinoma

“…22.64% of patients in our study group had tumors with high microsatellite instability (MSI-H) (12/53, only 53 patients were tested for MSI status). Recent work showed that MSI detection of mucinous adenocarcinoma requires caution [35]. Second, it warrants close observation of the progress of the disease and the level of tumor markers after operation, and close follow-ups should be scheduled for all these patients.…”

Circulating Tumor DNA Testing for Homology Recombination Repair Genes in Prostate Cancer: From the Lab to the Clinic

“…DNA quality is high in blood and variable in FFPE tissue [ 17 ]. Factors that can lower DNA quality are in common with other molecular tissue tests, i.e., presence of necrosis, high infiltration of inflammatory cells, aging-related degradation, poor fixation in formalin, cauterized tissue [ 18 ]. Nonetheless, the reliability of tumor testing in diagnosing or excluding germline mutations is questionable.…”