Abstract Background Multiple mutation (MM) within a single gene has recently been reported as a mechanism involved in carcinogenesis. The present study investigated the clinical significance of MMs in hepatocellular carcinoma (HCC). Methods Two hundred twenty-three surgically resected HCCs were subjected to gene expression profiling and whole-exome sequencing. Results MMs in individual genes were detected in 178 samples (MM tumors: 79.8%). The remaining samples all carried a single mutation (SM tumors: 20.2%). Recurrence-free survival in the MM group was significantly worse in comparison to the SM group (P = 0.012). A Cox proportional hazard analysis revealed that MM tumor was an independent predictor for worse a prognosis (hazard ratio, 1.72; 95% confidence interval, 1.01–3.17; P = 0.045). MMs were frequently observed across in various genes, especially MUC16 (15% of samples had at least one mutation in the gene) and CTNNB1 (14%). Although the MUC16 mRNA expression of MUC16 wild-type and MUC16 SM tumors did not differ to a statistically significant extent, the expression in MUC16 MM tumors was significantly enhanced in comparison to MUC16 SM tumors (P < 0.001). In MUC16, MMs were associated with viral hepatitis, higher tumor marker levels and vascular invasion. The MUC16 MMs group showed significantly worse recurrence-free survival in comparison to the MUC16 SM group (P = 0.022), while no significant difference was observed between the MUC16 SM group and the MUC16 wild-type group (P = 0.324). Conclusions MM was a relatively common event that may occur selectively in specific oncogenes and is involved in aggressive malignant behavior.
