We used 1267 colorectal cancer cases, diagnosed up to 2006, based on the availability of KRAS sequencing data. In order to examine the prognostic role of specific KRAS mutations, independent of BRAF mutation, BRAF mutated cancers, cases with missing BRAF mutation status, and tumors with KRAS mutations identified in two or more of codons 12, 13, 61 and 146 were ex cluded. As a result, www.selleckchem.com/products/AZD2281(Olaparib).html a final total of 1067 BRAF wild type cases were used for survival analyses. Informed consent was obtained from all Inhibitors,Modulators,Libraries study subjects. This study was approved by the Human Subjects Committees at Harvard School of Public Health and Brigham and Womens Hospital. All clinicopathologi cal and molecular analyses were performed blinded to other data, including patient Inhibitors,Modulators,Libraries outcome.

Histopathological evaluation Inhibitors,Modulators,Libraries Hematoxylin and eosin stained sections of all cases were examined by a pathologist Inhibitors,Modulators,Libraries unaware of other data. Tumor differentiation was categorized as well moderate or poor. Peritumoral lymphocytic reaction was examined as previously de scribed. Sequencing of KRAS codons 61 and 146 DNA was extracted from paraffin embedded tissue as previ ously described, and polymerase chain reaction and pyrosequencing, targeted for KRAS codons 61 and 146, were performed. Dispensation orders were designed such that all possible mutations would be de tected. All mutations were confirmed by replicate analysis. Sequencing of KRAS codons 12 and 13, BRAF, and PIK3CA, and MSI analysis We performed PCR and pyrosequencing targeted for KRAS. BRAF and PIK3CA as previously described. MSI analysis was performed using 10 microsatellite markers.

MSI high was defined as instability in Inhibitors,Modulators,Libraries 30% of the markers. MSI low tumors were grouped with microsatellite stable tumors because we have previously demonstrated that these two groups show similar features. Methylation analyses for CpG islands and LINE 1 Using validated bisulfite DNA treatment and real time PCR, we quantified DNA methylation selleck in eight CIMP specific promoters. CIMP high was defined as the presence of 6 8 methyl ated promoters, CIMP low as 1 5 8 methylated promoters, and CIMP negative as the absence of methylated pro moters, according to established criteria. In order to ac curately quantify LINE 1 methylation levels, we used bisulfite pyrosequencing as previously described. Statistical analysis All statistical analyses were performed using SAS. All P values were two sided. Univariate analyses were performed to inves tigate clinicopathological and molecular characteristics according to KRAS mutation status. a chi square test or Fishers exact test was used for categorical data, while a Wilcoxon or Kruskal Wallis test was applied to continuous data.