Background:The detection of V600E BRAF mutations has fundamental clinical consequences as the treatment option with BRAF inhibitors such as vemurafenib or dabrafenib yields response rates of ∼48%. Heterogeneity with respect to BRAF mutation in different metastases has been described in single cases. As this has important implications for the determination of BRAF status and treatment of patients, it is essential to acquire more data.Methods:A total of 300 tumour samples from 187 melanoma patients were analysed for BRAF mutations by pyrosequencing. Equivocal results were confirmed by capillary sequencing. Clinical data with respect to melanoma type, tumour site and survival were summarised for 53 patients with multiple analysed tumour samples (2–13 per patient).Results:BRAF mutations were found in 84 patients (44.9%) and 144 tumour samples (48%) with BRAF mutations in 45.5% of primary tumours and 51.3% of metastases, respectively. In 10 out of 53 patients (18.9%) where multiple samples were analysed results were discordant with respect to mutation findings with wild-type and mutated tumours in the same patient. Mutations did not appear more frequently over the course of disease nor was its occurrence associated with a specific localisation of metastases.Conclusion:As heterogeneity with respect to BRAF mutation status is detected in melanoma patients, subsequent testing of initially wild-type patients can yield different results and thus make BRAF inhibitor therapy accessible. The role of heterogeneity in testing and for clinical response to therapy with a BRAF inhibitor needs to be further investigated.

Background:The detection of V600E BRAF mutation in melanoma is fundamental since here BRAF inhibitors represent an effective treatment. Non-V600E BRAF mutations that may also respond are not detected by certain screening methods. Thus, knowledge about detection of these mutations is needed.Methods:A total of 276 tumour samples from 174 melanoma patients were investigated for BRAF mutations by pyrosequencing. Rare mutations were confirmed by capillary sequencing and compared with findings from COBAS test and immunohistochemistry using a novel BRAF antibody. Melanoma type, localisation, and survival were summarised.Results:BRAF mutations were found in 43% of patients (124 tumours in 75 patients). Among those, 14 patients (18.7%) exhibited rare mutations. The V600EK601del and V600DK601del mutations have never been described before in melanoma. Furthermore, V600K, V600E2, and V600D, V600G, V600R, and L597S mutations were detected. Mutations were not detected by COBAS test in 7 out of these 14 patients and immunohistochemistry only reliably detected patients with the V600E2 and V600EK601del mutation.Conclusion:Accurate diagnosis of rare BRAF mutations is crucial. We show that pyrosequencing is accurate, highly sensitive, reliable, and time saving to detect rare BRAF mutations. Missing these rare variant mutations would exclude a subset of patients from available effective BRAF-targeting therapy.

Background: BRAF mutation frequencies in melanoma subtypes have clinical implications and offer pathogenetic clues. Objectives: To characterize BRAF mutation status in melanoma of unknown primary (MUP) patients, in histological melanoma subtypes and by localization of primary tumors. Methods: In 179 patients with stage IV metastatic melanoma, BRAF mutation status, histological subtype and localization of primary (except for 29 MUP patients) were analyzed. Results: BRAF mutations were found in 44.3%, of which 80.5% were BRAF V600E and 19.5% showed non-V600E BRAF mutations. BRAF mutation frequency depended on histological subtype (57.4% superficial spreading melanoma, 54.7% nodular melanoma, 11.1% mucosal melanoma, 28.6% acral lentiginous melanoma) and concerning non-V600E BRAF mutations on localization of primary. In MUP the BRAF mutation pattern resembled superficial spreading and nodular melanomas. Conclusion: BRAF mutation frequencies depend on histological subtype and localization of primary melanoma. Non-V600E BRAF mutations mostly occur in patients with primaries on ‘head and neck' as well as ‘trunk' but not on ‘extremities'.