KRAS and BRAF Mutation Analysis in Routine Molecular Diagnostics

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.

Mutation‐specific antibody detects mutant BRAFV600E protein expression in human colon carcinomas

“…However, limitations of this approach include the potential for suboptimal quality of extracted DNA from FFPE tissue, although the use of microdissection and more sensitive methods have significantly improved detection rates with an ability to detect 1% to 5% of the BRAF V600E mutation in a pool of wild-type. 16, 25, 26 However, the assay time, expense, and the requirement for a molecular diagnostic laboratory remain limitations. In contrast, IHC offers the advantage of a relatively rapid, easy to perform, and cost effective assay that can be readily performed by most hospital pathology laboratories.…”

Shifted termination assay (STA) fragment analysis to detect BRAF V600 mutations in papillary thyroid carcinomas

“…A variety of methods have been applied for BRAF mutations. PCR-based screening methods such as SNaPshot assays, AS-PCR, COLD-PCR, Taqman® SNP assay, pyrosequencing and HRM analysis have been applied and the commonly used methods in pathology laboratories are summarized in Table 3[13-18]. Although direct sequencing of PCR products is the gold standard for BRAF mutation detection in routine diagnostics, it remains laborious, time consuming and requires rather expensive equipment [5,6].…”

Development of [11C]vemurafenib employing a carbon-11 carbonylative Stille coupling and preliminary evaluation in mice bearing melanoma tumor xenografts

“…For Colo829 and MeWO, the mutational status of BRAF exon 15 was assessed by HRM (high-resolution melting) assay followed by Sanger sequencing of HRM-PCR products with an aberrant melting curve, essentially as described previously [36]. …”