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.
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ABSTRACT: BACKGROUND: A point mutation (V600E) in the BRAF oncogene is a prognostic biomarker and may predict for nonresponse to anti-EGFR antibody therapy in patients with colorectal carcinoma. BRAF(V600E) mutations are frequently detected in tumors with microsatellite instability and indicate a sporadic origin. We used a mutation-specific antibody to examine mutant BRAF(V600E) protein expression and its concordance with BRAF(V600E) mutation data. METHODS: Primary stage III colon carcinomas were analyzed for BRAF(V600E) mutations in exon 15, and 50 BRAF(V600E) mutation carriers and 25 wild-type tumors were selected for analysis of BRAF proteins by immunohistochemistry (IHC). IHC was performed in archival tissue specimens using a pan-BRAF antibody and a mutation-specific antibody against BRAF(V600E) proteins. Staining was scored by 2 pathologists who were blinded to clinical and mutation data. RESULTS: Using a pan-BRAF antibody, total BRAF protein expression was observed in the tumor cell cytoplasm in 74 of 75 colon carcinomas. A mutation-specific antibody identified diffuse cytoplasmic staining of mutant BRAF(V600E) proteins in 49 of 74 cancers. Analysis using a polymerase chain reaction-based assay revealed that all 49 of these cancers carried BRAF(V600E) mutations. In contrast, BRAF(V600E) staining was absent in all 25 tumors that carried wild-type copies of BRAF. CONCLUSIONS: A BRAF mutation-specific (V600E) antibody detected tumors with BRAF(V600E) mutations and exhibited complete concordance with a DNA-based method. These results support the use of IHC as a simplified strategy to screen colorectal cancers for BRAF(V600E) mutations in clinical practice. Cancer 2013; © 2013 American Cancer Society.Cancer 05/2013; · 5.20 Impact Factor
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ABSTRACT: Background:Inhibitors of the epidermal growth factor (EGFR) signaling pathway have a major role in the treatment of KRAS wild-type colorectal cancer patients. The EGFR pathway has been shown to be activated in gastric cancer (GC). However, published data on KRAS and BRAF mutation status is limited in GC and has not been compared between GC from different geographic regions.Methods:The prevalence of KRAS and BRAF mutations was established in 712 GC: 278 GC from the United Kingdom, 230 GC from Japan and 204 GC from Singapore. The relationship between KRAS/BRAF mutation status, DNA mismatch repair (MMR) status, clinicopathological variables and overall survival was analysed.Results:Overall, 30 (4.2%) GC carried a KRAS mutation. In total, 5.8% of the UK GC, 4% of Japan GC and 1.5% of Singapore GC were KRAS mutant. KRAS mutant GC had fewer lymph node metastases in the UK cohort (P=0.005) and were more frequent in elderly patients in the Japan cohort (P=0.034). KRAS mutations were more frequent in MMR-deficient GC in the UK and the Japanese cohort (P<0.05). A BRAF mutation was only detected in a single Japanese GC.Conclusions:This large multicentre study demonstrated that KRAS mutations and DNA MMR deficiency have a role in a small subgroup of GC irrespective of country of origin, suggesting that this subgroup of GC may have developed along a common pathway. Further studies need to establish whether concomitant mutations or amplifications of other EGFR signalling pathway genes may contribute to the activation of this pathway in GC.British Journal of Cancer advance online publication, 19 March 2013; advance online publication, 19 March 2013; doi:10.1038/bjc.2013.109 www.bjcancer.com.British Journal of Cancer 03/2013; · 5.08 Impact Factor
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ABSTRACT: Background Non-small-cell lung carcinoma (NSCLC) patients with a BRAF(V600E) mutation benefit from targeted therapy. The usefulness of immunohistochemistry (IHC) as an alternative approach for the detection of BRAF(V600E) in NSCLC patients has not been evaluated until now. This study compared the specificity and sensitivity of IHC with other methods for the detection of BRAF(V600E) in primary lung adenocarcinoma.Patients and methodsBRAF mutations were analysed by DNA sequencing of a Caucasian subpopulation of selected 450 of 1509 (30%) EGFR, KRAS, PI3KA, Her2 and EML4-ALK wild-type (wt) primary lung adenocarcinomas. Detection of the BRAF(V600E) mutation was carried out by IHC using the VE1 clone antibody and compared with the results of other molecular methodologies.ResultsOf 450 (9%) of tumours, 40 harboured a BRAF mutation, which corresponded to either a BRAF(V600E) or a non-BRAF(V600E) mutation in 21 of 450 (5%) and 19 of 450 (4%) cases, respectively. The IHC VE1 assay was positive in 19 of 21 (90%) BRAF(V600E)-mutated tumours and negative in all BRAF(nonV600E)-mutated tumours.ConclusionIHC using the VE1 clone is a specific and sensitive method for the detection of BRAF(V600E) and may be an alternative to molecular biology for the detection of mutations in NSCLC.Annals of Oncology 11/2012; · 7.38 Impact Factor