RESUMO
PURPOSE: Mutation testing of the key genes involved in melanoma oncogenesis is now mandatory for the application of targeted therapeutics. However, knowledge of the mutational profile of melanoma remains largely unknown in Brazil. PATIENTS AND METHODS: In this study, we assessed the mutation status of melanoma driver genes BRAF, NRAS, TERT, KIT, and PDGFRA in a cohort of 459 patients attended at Barretos Cancer Hospital between 2001 and 2012. We used polymerase chain reaction followed by Sanger sequencing to analyze the hot spot mutations of BRAF exon 15 (V600E), NRAS (codons 12/13 and 61), TERT (promoter region), KIT (exons 9, 11, 13, and 17), and PDGFRA (exons 12, 14, and 18) in tumors. The mutational profile was investigated for associations with demographic, histopathologic, and clinical features of the disease. RESULTS: The nodular subtype was most frequent (38.9%) followed by the superficial spreading subtype (34.4%). The most frequent tumor location was in the limbs (50.0%). The mutation rates were 34.3% for TERT and 34.1% for BRAF followed by NRAS (7.9%), KIT (6.2%), and PDGFRA (2.9%). The BRAF (P = .014) and TERT (P = .006) mutations were associated with younger patients and with different anatomic locations, particularly in the trunk, for the superficial spreading and nodular subtypes, respectively (P = .0001 for both). PDGFRA mutations were associated with black skin color (P = .023) and TERT promoter mutations with an absence of ulceration (P = .037) and lower levels of lactate dehydrogenase. There was no association between patient survival rates and mutational status. CONCLUSION: The similar mutational profile we observe in melanomas in Brazil compared with other populations will help to guide precision medicine in this country.
Assuntos
GTP Fosfo-Hidrolases/genética , Melanoma/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Neoplasias Cutâneas/genética , Telomerase/genética , Adulto , Idoso , Brasil , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Regiões Promotoras GenéticasRESUMO
Melanin is produced by melanocytes and protects against DNA damage by ultraviolet light. Unfortunately, the melanin protein present in melanoma tumor cells is often co-purified during DNA extraction, and this contamination may inhibit subsequent PCR methods, which directly impacts research applications and the molecular diagnostic tests needed for targeted therapeutics. There are presently no described purification protocols that efficiently remove melanin from genomic DNA. In this study, we compare six different methods for melanin removal from genomic DNA: Agarose Gel Electrophoresis, 1mg Chelex®-100, Chelex®-100 5%, centrifugation, OneStep™ PCR Inhibitor Removal Kit and centrifugation plus OneStep™ PCR Inhibitor Removal Kit. Each comparison was made using 16 formalin-fixed paraffin-embedded (FFPE) and 11 fresh cell line samples. All samples were initially tested using the multiplex PCR reaction for GAPDH gene that generates different sized amplified products: 100, 200, 300 and 400 base pairs, which could be inhibited by the addition of exogenous melanin. Six purification protocols were then applied, and all samples that amplified at least one GAPDH fragment were sequenced to analyze the presence of the BRAF V600E mutation. The efficiencies of amplification decreased for larger sized fragments in all methods. Our comparisons showed that centrifugation combined with the OneStep™ PCR Inhibitor Removal Kit was superior to all other methods for successful BRAF sequencing with 100% (100bp), 75% (200bp), 50% (300bp), and 31.3% (400bp) amplification efficiencies for the different amplicon sizes. In conclusion, this genomic DNA extraction method is highly efficient for successful PCR when tumor samples are contaminated with melanin.
