cRNA was prepared from 100 ng total RNA using two-cycle target labeling and a control reagents kit (Affymetrix, Santa Clara, CA). Hybridization and signal detection of the Human Genome U133 Plus 2.0 arrays (Affymetrix) were carried out according to the manufacturer’s instructions. The gene expression data was deposited in the Gene Expression Omnibus (GEO) under accession ID GSE71222 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=izcnsaygjjkpped&acc=GSE71222 ).
The gene expression data were then analyzed to identify the candidate genes related to distant recurrence. We defined the patients with distant recurrence as the recurrence group, and the patients without any distant recurrences as the non-recurrence group. The criteria to select candidate genes were as follows; (i) a higher expression level in cancer tissues than in non-cancerous mucosa, (ii) a significantly higher expression level in cancer cells of the recurrence group than in the non-recurrence group. A higher expression was defined as a numerical value 1.5 times greater than those in another group.
The gene expression data were then analyzed to identify the candidate genes related to distant recurrence. We defined the patients with distant recurrence as the recurrence group, and the patients without any distant recurrences as the non-recurrence group. The criteria to select candidate genes were as follows; (i) a higher expression level in cancer tissues than in non-cancerous mucosa, (ii) a significantly higher expression level in cancer cells of the recurrence group than in the non-recurrence group. A higher expression was defined as a numerical value 1.5 times greater than those in another group.