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: Martin Pichler, Marija Balic, Elke Stadelmeyer, Christoph Ausch, Martina Wild, Christian Guelly, Thomas Bauernhofer, Hellmut Samonigg, Gerald Hoefler, Nadia Dandachi
• Journal: The Journal of molecular diagnostics : JMD (J Mol Diagn) Vol. 11 Issue 2 Pg. 140-7 (Mar 2009) ISSN: 1525-1578 [Print] United States
• PMID: 19213871 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, Validation Study)

Chemical References

• Formaldehyde
• BRAF protein, human
• Proto-Oncogene Proteins B-raf

Topics

• Adult
• Aged
• Aged, 80 and over
• Cell Line, Tumor
• Colorectal Neoplasms (diagnosis, genetics)
• DNA Mutational Analysis (methods)
• Formaldehyde (chemistry)
• Humans
• Middle Aged
• Molecular Diagnostic Techniques (methods)
• Proto-Oncogene Proteins B-raf (genetics)
• Reproducibility of Results
• Sensitivity and Specificity
• Tissue Fixation
• Transition Temperature