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

• Autores: Daniëlle A. M. Heideman, Irene Lurkin, Marije Doeleman, Egbert F. Smit, Henk M. Verheul, Gerrit A. Meijer, Peter J. F. Snijders, Erik Thunnissen, Ellen C. Zwarthoff
• Localización: The Journal of molecular diagnostics, ISSN 1525-1578, Vol. 14, Nº. 3, 2012, págs. 247-255
• Idioma: inglés
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• Resumen

  • 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 (?, 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 (? 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