The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic things melanoma. Vemurafenib exhibits an approxi mately 30 fold selectivity for p. V600E mutated compared to wildtype BRAF melanomas. In addition, patients car rying a p. V600K mutation included in the BRIM 3 study showed response to this inhibitor. Inhibitors,Modulators,Libraries In a phase I trial, a 70% response rate to vemurafenib among 32 genotype selected metastatic melanoma patients was observed. Recent in vitro and in vivo experiments indicate that vemurafenib might have an effect in patients with rare mutations in codon 600 of the BRAF gene as for instance p. V600D or p. V600R. Furthermore, dab rafenib, another selective BRAF inhibitor shows good clinical response rates not only for patients with p. V600E or p.

V600K mutations but also in patients carrying a p. V600R, p. V600M or a double p. mutation giving new therapy options for melanoma patients with rare BRAF mutations. The FDA approved vemurafenib with the cobas BRAF V600 test as companion diagnostic tool. The Euro pean Inhibitors,Modulators,Libraries Medicine Agency`s Committee for Human Medicinal Products approved vemurafenib in February 2012 with two main differences to the FDA approval a companion diagnostic test was not defined and treatment option is Inhibitors,Modulators,Libraries given for patients with melanomas carrying any mutation in codon 600 of the BRAF gene. Because a mutation in codon 600 determines eligibility for BRAF inhibitor treatment, several molecular screening methods have been developed. However, the level of validation and characterization of the performance features is not defined.

The aim of this study was to evaluate several parame ters such as sensitivity and feasibility of different methods for the BRAF mutation analysis. Here, we compare the allele specific PCR done by the cobas Inhibitors,Modulators,Libraries BRAF V600 test, the pyrosequencing using the therascreen BRAF Pyro Kit, the high resolution melting analysis, the immunohistochemistry, the next generation sequencing approach and the bidirectional Sanger sequencing with regard to their sensitivity, specifi city, costs, amount of work, feasibility and limitations. To our knowledge, this is the only study comparing these five PCR based methods with IHC. Methods Samples A total of 82 tumor samples were collected in the years 2010 until 2013 under approved ethical protocols com plied with the Ethics Committee Inhibitors,Modulators,Libraries of the University of Cologne and with informed consent from each patient.

Of these, 63 samples were melanomas, 11 were lung adenocarcinomas and eight were colorectal carcinomas. Tumors were diagnosed by an http://www.selleckchem.com/products/MLN8237.html experienced pathologist and tumor content and pigmentation were defined. All samples were analyzed with Sanger sequencing as gold standard and the in house method high resolution melting analysis. The other methods were evaluated with a smaller number of samples due to the limited amount of tumor tissue available.