Microsatellite Instability Detection in Clinical Cancer Samples: A Multiplex qPCR Approach without Matching Normal Samples

BackgroundMicrosatellite instability (MSI) indicates DNA mismatch repair deficiency in cancers like colorectal cancer. The current gold standard technique, PCR/capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. Methods101 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared to the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical MSI-H samples. Analytical sensitivity, specificity, limit of detection and input requirements were assessed using synthetic commercial reference standards. ResultsVarTrace demonstrated 100% test success rate, 100% sensitivity and 98% specificity compared to OncoMate across 101 clinical FFPE samples. It detected MSI-H with 97% accuracy down to 10% tumor percentage. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. ConclusionsThis study validates VarTrace as a swift, accurate and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTraces capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical workflows and broadening the availability of this crucial test.