Validation of a rapid pcr assay for microsatellite instability testing in colorectal cancer

Background: Detection of microsatellite instability (MSI) continues to gain clinical importance, particularly in light of targeted therapy approval of checkpoint inhibitors in patients whose solid tumors are MSI high. Several methods of testing already exist, including PCRbased capillary-gap electrophoresis and next generation sequencing. Most of these methods suffer a prolonged turnaround time, which may impact/delay treatment decisions. Design: We wished to evaluate and validate a relatively novel rapid PCR assay (Idylla; Biocartis) for MSI testing on a cohort of 72 colorectal cancers (CRC). The assay was validated against a clinically validated immunohistochemistry-based mismatch-repair protein assay that was previously clinically validated using outcome data from a large cohort of stage II colon cancer patients samples (QUASAR trial). The MMR assay included four antibodies against MLH1, MSH2, MSH6 and PMS2. We also wished to evaluate the east of use of the rapid molecular assay, failure rate and turnaround time. Following automated DNA extraction from a single, 5-micron section, the polymerase chain reaction was accompanying by simultaneous generation of melting curves via fluorescence detection. The multiplex assay detects tumor-specific mutations in 7 MSI loci which are ACVR2A (2p22.3-q23.1), BTBD7 (14q32.12), DIDO1 (20q13.33), MRE11 (11q21), RYR3 (15q13.3-q14), SEC31A (4q21.22), and SULF2 (20q13.12). Amplification of < 5 markers renders the test results invalid. Otherwise, the tumor is either classified as MSI-H (MSI-high) or MSS (MS - stable). Results: All but one of the 72 tumors yielded a valid result (failure rate 1.3%). The valid results only required a single tissue section, and the entire procedure from extraction to PCR and detection lasted only 2.5 hours. 12 tumors (16%) were classified as MSI High (MSI-H), all of which also show deficiency in either MLH1/PMS2 or MSH2/MSH6 protein complexes. All 60 MSI-stable (MSI-S) tumors also show MMR proficiency by immunohistochemistry. Conclusions: This study validate the clinical use of a relatively novel MSI PCR-based assay on a cohort of 72 CRC samples, by showing 100% correlation with MMR protein status in a clinically validated MMR assay. The extremely fast turnaround time, ease of use and very low failure rate are advantageous for adopting this platform for molecular MSI testing.