title: KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA.
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 (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.
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. dam.heideman@vumc.nl
authors.forename: Daniëlle A M
authors.initials: DA
authors.lastname: Heideman
authors.forename: Irene
authors.initials: I
authors.lastname: Lurkin
authors.forename: Marije
authors.initials: M
authors.lastname: Doeleman
authors.forename: Egbert F
authors.initials: EF
authors.lastname: Smit
authors.forename: Henk M
authors.initials: HM
authors.lastname: Verheul
authors.forename: Gerrit A
authors.initials: GA
authors.lastname: Meijer
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Snijders
authors.forename: Erik
authors.initials: E
authors.lastname: Thunnissen
authors.forename: Ellen C
authors.initials: EC
authors.lastname: Zwarthoff
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis; D011905:Genes, ras; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D017422:Sequence Analysis, DNA / Q000379:methods; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins