40 Background: Exploratory biomarker analysis was conducted to identify factors related to outcomes of patients enrolled in the ACTS-GC study, a randomized controlled trial comparing surgery followed by adjuvant S-1 therapy with surgery alone in 1,059 patients with stage II/III gastric cancer. Methods: Formalin-fixed paraffin-embedded specimens obtained from 105 patients, an institutional subset of this biomarker study of 829 patients, were extensively examined for EGFR and HER2 by immunohistochemistry (IHC), MET amplification by FISH, and K-ras mutation status (codon 12, 13) by PNA-enriched direct sequencing. The expressions of 63 genes involved in pyrimidine metabolic pathway, growth factor signaling pathway, apoptosis, DNA repair, etc., were measured by quantitative real-time RT-PCR. The relations of the expressions of these genes to clinicopathological features such as histological type, T grade, N grade, tumor stage, gender, and age were also investigated. Results: The rates of K-ras mutation, EGFR overexpression, HER2 overexpression, and MET amplification were 6.7% (7/105), 14.3% (15/105), 11.4% (12/105), and 21.0% (22/105), respectively. These factors were almost mutually exclusive. Co-expressions were seen in 2 (EGFR & HER2), 4 (HER2 & MET), 5 (MET and EGFR) and 1 (EGFR & HER2 & MET) of the 105 patients. MET amplification and EGFR overexpression were associated with worse outcomes. In contrast, KRAS and HER2 status were not associated with overall survival or relapse-free survival. Expression levels of PCAM1, ESR1, MUC2, CAV1, ITGB3, and APC mRNA were significantly higher in diffuse-type cancer than in intestinal-type cancer, whereas expression levels of HER2, E2F1, LDHA and TOP2A mRNA were significantly higher in intestinal-type than in diffuse-type cancer (FDR P<0.05). Associations of other clinically significant genes with clinicopathological features other than histologic type were not screened. Conclusions: Many clinical, pathological, molecular, and genetic factors have been investigated with a growing understanding of the molecular pathogenesis of gastric cancer. We review and discuss such prognostic markers in the ACTS-GC study.