Accurate mutation detection assays are strongly needed for use in routine molecular pathology analyses to aid in the selection of patients with cancer for targeted therapy. The high-resolution melting (HRM) assay is an ideal prescreening tool, and SNaPshot analysis offers a straightforward genotyping system. Our present study was determined to compare these mutation testing methods on formalin-fixed, paraffin-embedded (FFPE) tumor-derived DNA. We compared the performance of HRM, followed by cycle sequencing (HRM-sequencing); multiplex PCR assay, followed by SNaPshot analysis (multiplex mutation assay); and a successor assay using HRM, followed by SNaPshot (HRM-SNaPshot) for mutation analysis of both KRAS (codon 12/13/61) and BRAF (codon 600/601). In a series of 195 FFPE-derived DNA specimens, a high genotypic concordance between HRM-sequencing and multiplex mutation assay was found (κ, 0.98; 95% CI, 0.94 to 1), underlining the potential of a combined HRM-SNaPshot approach. In reconstruction experiments, the analytical sensitivity of HRM-SNaPshot was twofold to fourfold higher than HRM-sequencing and multiplex mutation assay, respectively. In addition, HRM-SNaPshot had a good performance rate (99%) on FFPE tumor-derived DNA, and mutation detection was highly concordant with the predecessor assays (κ for both, 0.98). The occurrence of BRAF and KRAS mutations is mutually exclusive. HRM-SNaPshot is an attractive method for mutation analysis in pathology, given its good performance rate on FFPE-derived DNA, high analytical sensitivity, and prescreening approach.

dx.doi.org/10.1016/j.jmoldx.2012.01.011, hdl.handle.net/1765/91322
The Journal of Molecular Diagnostics
Department of Pathology

Heideman, D.A.M, Lurkin, I, Doeleman, M, Smit, E.F, Verheul, H.M.W, Meijer, C.J.L.M, … Zwarthoff, E.C. (2012). KRAS and BRAF mutation analysis in routine molecular diagnostics: Comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA. The Journal of Molecular Diagnostics, 14(3), 247–255. doi:10.1016/j.jmoldx.2012.01.011