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Evaluation of High-Resolution Melting Analysis as a Diagnostic Tool to Detect the BRAF V600E Mutation in Colorectal Tumors

https://doi.org/10.2353/jmoldx.2009.080100Get rights and content

BRAF V600E is the predominantly occurring mutation of the cytoplasmic kinase BRAF, and, in colorectal cancer, its determination provides a diagnostic exclusion criterion for hereditary nonpolyposis colorectal cancer. The aim of our study was to develop a sensitive BRAF V600E high resolution melting (HRM) assay. We first established and optimized the BRAF HRM assay using a cell line dilution model, enabling us to detect 1% mutant DNA in a background of wild-type DNA. In a comparison, DNA sequencing and real-time allele-specific PCR in the cell line dilution model HRM assay proved to be more sensitive than DNA sequencing and denaturing high performance liquid chromatography, retaining the same sensitivity as real-time allele-specific PCR. In a learning set of 13 patients with known BRAF V600 status, the mutation was detected with high concordance by all four methods. Finally, we validated the HRM assay on 60 formalin-fixed, paraffin-embedded colorectal cancer samples. Although all mutated samples were correctly identified by HRM, the detection limit of the HRM assay decreased when using low-quality DNA derived from formalin-fixed, paraffin-embedded samples. In conclusion, HRM analysis is a powerful diagnostic tool for detection of BRAF V600E mutation with a high sensitivity and high-throughput capability. Despite the expected decrease in sensitivity, HRM can reliably be applied in archival formalin-fixed, paraffin-embedded samples tissues.

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Supported by the Austrian National Bank Fund (grant 12680 to N.D.) and by the Austrian Ministry for Health, Family and Youth (project GZ 70420/0134-IV/B/12/2006 to G.H.).

M.P. and M.B. contributed equally to this work.

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