title: Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors.
abstract: 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.
authors.affiliation: Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pichler
authors.forename: Marija
authors.initials: M
authors.lastname: Balic
authors.forename: Elke
authors.initials: E
authors.lastname: Stadelmeyer
authors.forename: Christoph
authors.initials: C
authors.lastname: Ausch
authors.forename: Martina
authors.initials: M
authors.lastname: Wild
authors.forename: Christian
authors.initials: C
authors.lastname: Guelly
authors.forename: Thomas
authors.initials: T
authors.lastname: Bauernhofer
authors.forename: Hellmut
authors.initials: H
authors.lastname: Samonigg
authors.forename: Gerald
authors.initials: G
authors.lastname: Hoefler
authors.forename: Nadia
authors.initials: N
authors.lastname: Dandachi
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D044366:Transition Temperature
chemical_list: D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf