KRAS and BRAF Mutation Analysis in Routine Molecular Diagnostics Comparison of Three Testing Methods on Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA

DAM Heideman, Irene Lurkin, M Doeleman, EF Smit, HM Verheul, GA Meijer, PJF Snijders, E Thunnissen, Ellen Zwarthoff

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Abstract

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 (kappa, 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 (K 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. (J Mol Diagn 2012, 14:247-255; DOI: 10.1016/j.jmoldx.2012.01.011)
Original languageUndefined/Unknown
Pages (from-to)247-255
Number of pages9
JournalJournal of Molecular Diagnostics
Volume14
Issue number3
DOIs
Publication statusPublished - 2012

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