Comparison of PCR-based high-resolution melting curve analysis and immunohistochemistry in detecting microsatellite...

Microsatellite instability (MS I) is an important biomarker for the prognosis and immunotherapy indications of colorectal cancer (CRC). Traditional detection methods such as immunohistochemistry (I HC) and polymerase chain reaction (PCR)-based capillary electrophoresis (PCR-CE) are widely used, but there are limitations such as strong subjectivity and time-consuming result judgment. This study aims to evaluate the polymerase chain reaction-high-resolution melting curve (PCR-HRM) as a fast, high-throughput MS I detection method.

Tumor samples of 251 CRC patients were analyzed. PCR-HRM and IHC methods are used to evaluate the status of MSI. If the results of PCR-HRM and IHC are inconsistent, the gold standard PCR-CE method is used for confirmation testing.

In this study cohort, 20.3% (51/251) of patients had MSI-H or mismatch repair defect (dMMR). PCR-HRM and IHC have excellent consistency, with an overall consistency rate of 99.20% and a Kappa coefficient of 0.971 (P < 0.01). Compared with IHC, the sensitivity of PCR-HRM is 98.04% and the specificity is 99.50%. In the case of inconsistency of PCR-HRM and IHC results, PCR-CE confirms the accuracy of PCR-HRM. These differences are attributed to biological phenomena, such as the presence of mutant proteins but functional inactivation (leading to false negative results in IHC) and human factors before analysis (leading to false positive results in IHC).

PCR-HRM is a high-precision, low-cost and efficient MSI screening method. It does not require the PCR post-processing required by the traditional method, thus simplifying the workflow. In addition, it also avoids potential problems related to IHC results. Our research shows that PCR-HRM can be a powerful tool to guide the decision-making of colorectal cancer treatment.