Microsatellite Instability Detection by High-Resolution Melting Analysis

Abstract

BACKGROUNDMicrosatellite instability (MSI) is an important marker for screening for hereditary nonpolyposis colorectal cancer (Lynch syndrome) as well as a prognostic and predictive marker for sporadic colorectal cancer (CRC). The mononucleotide microsatellite marker panel is a well-established and superior alternative to the traditional Bethesda MSI analysis panel, and does not require testing for corresponding normal DNA. The most common MSI detection techniques—fluorescent capillary electrophoresis and denaturing HPLC (DHPLC)—both have advantages and drawbacks. A new high-resolution melting (HRM) analysis method enables rapid identification of heteroduplexes in amplicons by their lower thermal stability, a technique that overcomes the main shortcomings of capillary electrophoresis and DHPLC.METHODSWe investigated the straightforward application of HRM for the detection of MSI in 70 archival CRC samples. HRM analysis for 2 MSI markers (BAT25 and BAT26) was evaluated, and 2 different HRM-enabled instruments were compared—the LightCycler® 480 (Roche Diagnostics) and the LightScannerTM (Idaho Technology). We also determined the analytical sensitivity and specificity of the HRM assay on both instruments using 11 known MSI-positive and 54 microsatellite-stable CRC samples.RESULTSAll MSI-positive samples were detected on both instruments (100% analytical sensitivity). The LightScanner performed better for analytical specificity, giving a combined specificity value of 99.1% compared with 92.3% on the LightCycler 480.CONCLUSIONSWe expanded the application of the HRM analysis method as an effective MSI detection technique for clinical samples.