The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma.,Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response.,Therefore, the precise identification of the underlying somatic mutations is essential.,Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.,Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples.,DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction.,BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC).,All mutations were independently reassessed by Sanger sequencing.,Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).,There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%).,All mutations down to 6.6% allele frequency could be detected with 100% specificity.,In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity.,The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib.,NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity.,IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.,Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.
CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma.,Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype.,All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL).,Phenotypic data related to primary melanoma and pigmentation characteristics were collected.,The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced.,Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation.,The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years.,Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM).,A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects.,CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (p<0.001).,MPM subjects had a higher frequency of MC1R red hair colour variants compared with subjects with one tumour (p=0.010).,Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM.,In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation.