Investigation of BRAF mutation analysis with different technical platforms in metastatic melanoma

Pathol Res Pract. 2017 May;213(5):522-530. doi: 10.1016/j.prp.2017.01.010. Epub 2017 Jan 21.


In metastatic melanoma, the detection of somatic mutations in the BRAF gene is crucial regarding patient selection for targeted therapy. Several screening methods have been developed to identify BRAF gene mutations. In this study, our objective was to evaluate the detection of the BRAF V600 mutations using two molecular methods, real-time polymerase chain (real-time PCR) assay and pyrosequencing, and immunohistochemistry (IHC), and to compare the results of these different technical platforms. This study included 98 patients diagnosed with metastatic melanoma at the Hacettepe University, Department of Pathology between 2002 and 2014. BRAF mutation analysis was tested with real-time PCR, pyrosequencing and IHC methods. The results of all three tests were compared with a reference test, and the sensitivity, specificity rates and kappa coefficient values were analysed for each test. We successfully analysed BRAF mutations using all three methods in 92 patients. According to our findings, the pyrosequencing method had the highest kappa value regarding the determination of BRAF V600 mutations. The kappa values were at almost perfect agreement levels in pyrosequencing and real-time PCR assay (kappa coefficient for pyrosequencing=0.895 (95% CI: 0.795-0.995); kappa coefficient for real-time PCR=0.871 (95% CI: 0.761-0.981). The kappa value was at a substantial agreement level in the IHC analysis (kappa coefficient=0.776 (95% CI: 0.629-0.923). According to our results, we found that real-time PCR and pyrosequencing methods were equally excellent in determination of BRAF V600 mutations. The IHC method, which is commonly used in routine pathology practice, can also be safely used as a screening test for determination of BRAF V600 mutations.

Keywords: BRAF mutation; Immunohistochemistry; Pyrosequencing; Real-time PCR.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers, Tumor / genetics*
  • DNA Mutational Analysis
  • Female
  • Humans
  • Immunohistochemistry
  • Male
  • Melanoma / enzymology
  • Melanoma / genetics*
  • Melanoma / pathology
  • Melanoma / secondary
  • Middle Aged
  • Mutation
  • Proto-Oncogene Proteins B-raf / genetics*
  • Real-Time Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Skin Neoplasms / enzymology
  • Skin Neoplasms / genetics*
  • Skin Neoplasms / pathology
  • Skin Neoplasms / secondary
  • Young Adult


  • Biomarkers, Tumor
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf