engleski

Comparison of different methods for BRAF mutation detection in melanoma patients

Background: Introduction of personalized approach into treatment of metastatic melanoma with BRAF and KIT inhibitors has increased the demand for genetic testing of melanoma patients. Currently, routine testing in Croatia is mostly limited to detection of BRAF mutations using different techniques. Nevertheless, conventional methods of mutation detection are prone to generating false positive or negative results due to problems with variable fraction of tumor cells harboring the mutation and normal cells in the analyzed clinical sample, DNA quality and efficiency of PCR and following reactions. The reason for this is that most of the analyzed DNA samples are extracted form formalin-fixed paraffin-embedded tissue (FFPE) which can create problems in the performance of this analysis. Methods: Therefore in this study we have aimed at finding the most optimal method to detect mutations in exon 15 of the BRAF gene and to implement it into routine practice in our laboratory. Genomic DNA was extracted using the conventional (proteinase K-digestion) extraction and commercial kits for automated DNA isolation. Furthermore, in addition to two stages enriched PCR-RFLP method, a high resolution melting fluorescent detection of heteroduplexes (HRM) method followed by DNA sequencing was introduced for mutation detection in our laboratory. Results: Some of the samples designated as BRAF V600E positive by the PCR-RFLP method were found to be negative when HRM method followed by sequencing was applied. The quality of the mutation detection was further increased by usage of commercial kits for DNA isolation. In addition, microdissection applied to avoid high percentage of normal cells in the analyzed sample could further improve the sensitivity and specificity of this analysis. Conclusion: Therefore we can conclude that in addition to being more accurate, the HRM method has proved itself to be the simplest and faster alternative to conventional methods of mutation detection.

BRAF; HRM; melanoma

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