We then applied three different prediction GDC-0980 purchase methods—diagonal linear discriminant analysis, support vector machines, and k-nearest neighbor—to determine the prediction accuracy of the selected panel (method B) using data on the remaining 22 pairs.32 The hierarchical clustering of the methylation data was performed with the top 1,000 most significantly differentially methylated sites and with the two selected panels of CpG sites using methods A and B. Gene-ontology analysis

was performed by the PANTHER classification system (http://www.pantherdb.org) to compare the significant methylated gene lists with the reference (National Center for Biotechnology Information, human genome build 36).33 The binomial test was used to identify significantly enriched pathways, biological processes, molecular functions, cellular components, and protein class terms after Bonferroni’s correction for

multiple comparisons, with a cutoff of p ≤ 0.05. To investigate whether methylation levels are affected by HCC risk factors, such as HBsAg status, HCV status, cigarette smoking (i.e., ever/never), alcohol consumption (i.e., ever/never), AFB1-DNA adduct level, and gender within tumor and adjacent nontumor tissues separately, Regorafenib solubility dmso we used a two-sample t test with Bonferroni’s correction for multiple testing. In the second-stage confirmatory analysis, Pearson’s correlations between methylation levels using Illumina arrays and pyrosequencing on selected sites were calculated. All analyses were conducted using the R language (http://www.r-project.org/). Clinical and pathological characteristics are described in Table 1. Almost 90% of cases were male and 79% were HBsAg positive. Approximately 31% of 上海皓元医药股份有限公司 subjects were positive for HCV. Seven subjects were negative for both HBV and HCV, 36 subjects were positive

for HBsAg and negative for HCV, 13 subjects were both HBV and HCV positive, and the remaining 6 subjects were negative for HBsAg and positive for HCV. Thus, viral infection, primarily HBV, was the major risk factor in this population. The average age at HCC diagnosis was 52.2 ± 14.2 years. Approximately 40% of cases smoked and 13% consumed alcohol, but data were missing for approximately 20% of subjects. AFB1-DNA adducts, measured previously in all tumor tissues and in approximately half of the adjacent tissues,34, 35 are also summarized in Table 1. Reproducibility of the Illumina platform was evaluated using replicates of four paired samples on a different day. High concordance was observed for all eight replicates, with coefficients of determination (R2) ranging from 0.96 to 0.98. A representative example of the concordance between two replicates for an adjacent tissue sample is given in Supporting Fig. 1 and is consistent with previous studies.