A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

Sci Rep. 2013:3:1659. doi: 10.1038/srep01659.

Abstract

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3-4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.

MeSH terms

  • Australia
  • DNA Mutational Analysis / methods*
  • Female
  • Formaldehyde
  • Humans
  • Male
  • Melanoma / genetics*
  • Paraffin Embedding / methods*
  • Polymorphism, Single Nucleotide / genetics*
  • Proto-Oncogene Proteins B-raf / genetics*
  • Sequence Analysis, DNA / methods*
  • Single-Blind Method
  • Tissue Fixation / methods

Substances

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