engleski

Melanin as preanalytical factor responsible for unsuccessful PCR analysis of BRAF mutations

Background: BRAF mutations are predictive marker for targeted therapy with BRAF-inhibitors in patients with metastatic melanoma. BRAF mutations are most often determined by real-time PCR. Melanoma specimens may be highly pigmented with abundance of melanin which is known inhibitor of PCR reaction. Melanin inhibits PCR by binding to thermostable DNA polymerases and reducing its ability to extend newly synthesized DNA. The aim of this study was to determine if melanin is responsible for unsuccessful PCR analysis of BRAF mutations. Materials and Methods: DNA was isolated (QIAamp DNA FFPE Tissue, Qiagen), from archived formalin-fixed paraffin-embedded (FFPE) melanoma tissue and assayed by real-time PCR (7500 Real Time PCR System, Applied Biosystems) for BRAF V600E and BRAF V600K mutations using specific TaqMan probes. For statistical analysis Mann-Whitney U test or χ2 test were used, depending on type of variable. Results: A total of 218 samples were analysed and among them, 20 were highly pigmented with abundant melanin. Analysis was considered unsuccessful if gene reference assay Ct was >30 and Ct for BRAF mutation was >9, 96. Highly pigmented samples had higher gene reference assay Ct values compared to non-highly pigmented samples (median Ct 30.17 and 27.29, p<0, 001). Frequency of samples with unsuccessful BRAF analysis was significantly higher among highly pigmented samples compared to non-highly pigmented samples (30% vs 10%, p=0, 009). Conclusions: Our results show that presence of melanin can be responsible for unsuccessful PCR analysis of BRAF mutations in melanoma tissue samples. Possible solution to overcome melanin inhibition is to dilute sample DNA added to PCR reaction which should be tested in further studies.

melanin ; real-time PCR ; inhibition ; BRAF

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