7. Rechsteiner et al. reported in a cohort of 81 colorectal carcinoma samples a coverage rate from 5139 to 17156. As the threshold of coverage was set to 100 all samples could be analyzed. The whole mutational spectrum could be detected by NGS and click here all cases were analyzed successfully. The cut off value defined for reliable mutation detection was set as a frequency of 5% mutant alleles. With this cut off all but one mutation were analyzed correctly. Case 30 showed only a 2% mutant allele frequency in the Integrative Genomic Viewer. Coverage rate using NGS was very low with 181 which may have influenced the results obtained. Inhibitors,Modulators,Libraries In the whole cohort the lowest frequency of mutant alleles detected with NGS was 7%. This makes a specificity of 100% for NGS but a sensitivity of 98. 6%.

NGS is characterized by a high working load with a lot of hands on time and high costs. These disadvantages are compensated by the multiplexing possibilities, the broad spectrum of mutations detected and the high sen sitivity. Recent publications state that almost 75% of can cer gene variations may be missed by an approach analyzing only hotspot mutations. The establishment of this Inhibitors,Modulators,Libraries rather new method for rou tine diagnostic is an ongoing process. The expertise in computational biology required to perform clinical NGS is significantly higher than for any other of the estab lished methods. Especially, the result interpretation is challenging, Where to define the cut off value for a reli able mutation, which spectrum of mutations to report, how to validate and to report the results, how to handle the massive data generated Standardization and valid ation of the test procedure and the data interpretation, cost reduction and getting to know the pitfalls of this method are the challenges of the future.

Immunohistochemistry Immunohistochemistry is characterized by a fast and cheap performance and allows the detection of even small amounts of tumor cells harboring the spe cific antigen. 49 of the 82 Inhibitors,Modulators,Libraries samples were subjected to immunohistochemistry. Staining Inhibitors,Modulators,Libraries was homogenous within the tumor cells as shown by other groups before. Figure 1 shows representative immunohistochemical stainings of p. V600E mutation and p. V600K mutation both in a melanoma sample and p. V600R mutation of a colorectal tumor. Staining with the p. V600E specific monoclonal antibody detected all evaluated p.

V600E mutations. 22 of these p. V600E mutated samples were melanoma and two were colorectal tumors. Colomba et al. described in con trast a IHC failure rate of 7. 2% in a cohort Inhibitors,Modulators,Libraries of 111 cases due to equivocal staining. Furthermore, case 25, showing Y-27632 chemical structure a double mutation in codon 600 and codon 601 of the BRAF gene was scored negative in IHC. This is in concordance with the study of Skorokhod et al. who could not detect the double mutation with the monoclonal VE1 antibody either. Eight cases with non p.