Comparison of Allelic Discrimination by dHPLC, HRM, and TaqMan in the Detection of BRAF Mutation V600E

Abstract

The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffinembedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent ( ‫؍‬ 0.9411 and ‫؍‬ 0.8988, respectively); for HRM, the concordances were good ( ‫؍‬ 0.7973 and ‫؍‬ 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in Recent advances in DNA sequencing technology have made it possible to use high-throughput sequencing on the entire human colorectal cancer (CRC) spectrum, finding 848 genes that may be somatically mutated. 1,2 The subsequent study of these mutations in CRC specimens worldwide would be facilitated by the use of less expensive and more efficient screening methods for mutation detection. Because most of these mutations are found in heterozygosis, screening techniques based on heteroduplex detection, such as denaturing high-performance liquid chromatography (dHPLC) or high-resolution melting (HRM) analysis, are strongly recommended. The V600E mutation in the BRAF oncogene has been found in CRCs with mismatch-repair gene deficiency. Familial CRCs with mismatch-repair gene deficiency do not harbor this mutation; rather, V600E is associated with the sporadic form of CRC associated with mismatch-repair gene deficiency. In addition to the diagnostic value of the BRAF mutation assessment, recent evidence 3,4 has demonstrated that stage IV CRCs with the BRAF V600E mutation do not respond to epidermal growth factor receptor inhibitor therapy, thus extending its value to predictive grounds. In previous articles, 5 V600E detection using TaqMan chemistry (Applied Biosystem, Foster City, CA) saved cost, time, and manual labor over direct sequencing, with the former showing 100% sensitivity and 100% specificity when the latter is considered as the reference method. Also, HRM has proved to be a reliable technique for V600E detection...

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