JMD GMP oligos for in vitro Diagnostics
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Originally published online as doi:10.2353/jmoldx.2009.080100 on February 12, 2009

Published online before print February 12, 2009
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Journal of Molecular Diagnostics 2009, Vol. 11, No. 2
Copyright © 2009 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2009.080100

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

Martin Pichler*, Marija Balic*, Elke Stadelmeyer*, Christoph Ausch{dagger}, Martina Wild{ddagger}, Christian Guelly§, Thomas Bauernhofer*, Hellmut Samonigg*, Gerald Hoefler{ddagger} and Nadia Dandachi*

From the Division of Oncology, * Department of Internal Medicine, Medical University of Graz, Graz, Austria; the Department of Surgery, {dagger} Ludwig Boltzmann Research Institute of Surgical Oncology, Danube Hospital, Vienna, Austria; the Institute of Pathology, {ddagger} Medical University of Graz, Graz, Austria; and the Center for Medical Research, § Medical University of Graz, Graz, Austria

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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Copyright © 2009 by the American Society for Investigative Pathology and the Association for Molecular Pathology.