All samples had an RNA Integrity Number greater than 5.0. Contaminant DNA was removed by digestion with RNase-free DNase (Qiagen). Using 2 μg of total RNA, complementary RNA was prepared using one-cycle target labeling and a control reagents kit (Affymetrix, Santa Clara, CA). Hybridization and signal detection of HG-U133 Plus 2.0 arrays (Affymetrix) was performed after the manufacturer’s instruction. A total of 127 microarray datasets were normalized using robust multiarray average method under R statistical software (version 2.12.0),
together with the BioConductor package. Estimated gene-expression levels were obtained in log2-transformed values, and 62 control probe sets were removed for further analysis. To identify candidate LY294002 research buy genes for prediction of recurrence in early-stage HCC, we applied the combination of criteria for selection of gene probe sets (Fig. 1). First, probe sets corresponding to known genes were selected based on the NetAffx annotation file, version 31 (available at: http://www.affymetrix.com/analysis/index.affx). Then, we selected probe sets marked as “present” by Gene Expression Console software version 1.1 (version 1.1; Affymetrix) for more than 70% of patients. Next, RXDX-106 molecular weight the univariate Cox proportional hazards regression model was used to estimate the relationship between a gene-expression pattern and tumor recurrence
rate for each probe set. Separate analyses were conducted for the cancer tissues, and the adjacent noncancerous tissues. Probe sets that satisfy P < 0.01 by the likelihood ratio test and more than 2-fold change in mean expression values between recurrence and nonrecurrence groups were selected. Furthermore, probe sets that satisfied
P < 0.01 by the Wilcoxon signed-rank test and more than 2-fold change between the paired cancer and adjacent noncancerous tissues were selected. To identify the set of genes that best explain the recurrence of HCC, a multivariate Cox regression analysis with a forward variable-selection MCE procedure, based on Akaike information criterion (AIC), was performed as, essentially, described by Lu et al.13 At each step, a variable showing the lowest AIC value was added. This procedure was started with a null model (i.e., a model with only the intercept parameter) and terminated if there was no improvement in the AIC value. Clinicopathological factors associated with recurrence were examined by a univariate Cox regression analysis. Factors that satisfied P < 0.05 were subjected to further analysis. A multivariate Cox regression analysis with a forward variable-selection procedure was then performed in an identical manner to the gene-selection method described above using the candidate factors. To establish the optimal predictive model for HCC recurrence, expression levels of the candidate genes and clinicopathological factors were combined.