If the concentration of the extracted DNA was too low, the maximum DNA volume of 25 ul was used. The results were displayed selleck chem inhibitor automatically as report by the cobas z 480 software. Immunohistochemistry Anti BRAF p. V600E immunohistochemical staining was performed using the specific monoclonal mouse anti body VE1. Dewaxing, heat induced epitope retrieval with citrate buffer, antibody incubation and counter staining were carried out on a BOND Max immunostai ner by using Bond Epitope Retrieval Solution 1 and the Bond Polymer Refine Detection kit. Immunohistochemical staining was carried out within 2 weeks after cutting the 4 um sections. Staining results were scored from 0 to 3 by a senior pathologist blinded to the results of molecular analysis. The staining was considered as posi tive for p.
V600E staining when the majority of viable tumor cells showed clear cytoplasmic staining. Negative staining results were interpreted when there was no or only slight staining, Inhibitors,Modulators,Libraries staining of only single cells or of monocytes and macrophages. Results and discussion Inhibitors,Modulators,Libraries Precise identification of genomic alterations is essential for personalized therapy in cancer. Concerning melan oma, particularly patients carrying a mutation in codon 600 of the BRAF gene respond to vemurafenib. As no companion diagnostic test for this drug is prescribed in Europe, we aimed at evaluating a sensitive and specific molecular method for BRAF mutation analysis by compar ing high resolution melting analysis, pyrosequenc ing allele specific PCR Sanger sequencing, next generation sequencing and immunohisto chemistry.
82 tumor samples Inhibitors,Modulators,Libraries evaluated during routine diagnostics from 2010 2013 and covering a wide range of different mutations as well as wildtype samples were subjected to analysis. Because of limited tumor tissue available we were not able to analyze all samples with each method but we paid attention to the fact that each mutation type was once analyzed with each method. At least, 40 samples were analyzed with all six evaluated methods. Lung adenocarcinomas as well as colorectal carcinomas were included into this study to get a broader spectrum of mutations. Hereby, Inhibitors,Modulators,Libraries the frequency of mutations other than p. V600E is significantly higher than in melanoma. BRAF mutations were mainly found in codon 600, codon 469 and codon 594 of non small cell lung cancer samples.
Inhibitors,Modulators,Libraries Furthermore, therapies targeting BRAF mutant tumors have recently been identified selleckchem Brefeldin A in NSCLC. Tumor content and pigmentation was assessed by an experienced pathologist. The proportion of tumor cells ranged from 15 100% and pigmentation was scored as no, low and high pigmentation. High resolution melting analysis and Sanger sequencing Using the high resolution melting method and Sanger sequencing, 81 of 82 samples could be amplified and analyzed using the same PCR products.