To further confirm the previous RT-PCR and western blot findings, we used immunohistochemical staining to assess the correlation between the expression levels of

thrombin see more and OPN in HCC tumor tissues from 230 patients. We also analyzed the association of thrombin and OPN levels with HCC prognosis in the same 230 HCC patients. Positive staining for thrombin and OPN was found in 33% (77/230) and 39% (90/230) of patients, respectively. HCC tissue from 36 (15.7%) patients was positive for both thrombin and OPN (Fig. 3A). As shown in Table 1, thrombin-positive expression in tumor tissue was significantly correlated with tumor size (P = 0.0438), vascular invasion (P = 0.0317), and TNM stage (P = 0.0352) of HCC. However, no statistically significant association was found between the thrombin expression and other clinical characteristics. In the patients with positive OPN (OPN+), positive thrombin staining in the tumor tissue was significantly correlated with preoperative serum alpha-fetoprotein (AFP) (P = 0.0304), tumor size (P = 0.0024), vascular RG7204 purchase invasion (P = 0.0018), TNM stage (P = 0.0080), tumor differentiation (P = 0.0373), and tumor encapsulation (P = 0.0477). However, no statistically significant correlation was found between thrombin expression and these characteristics in the patients with undetectable OPN expression (OPN−)

(Table 2). The 1-, 3-, and 5-year tumor recurrence rates of those thrombin-positive (thrombin+) patients were 41.6, 67.5, and 68.8%, respectively; these tumor recurrence rates were

much higher than those of thrombin-negative (thrombin−) patients (24.8, 43.1, and 47.1%, respectively; P = 0.0001). The 1-, 3-, and 5-year OS rates of thrombin+ patients (75.3, 42.9, and 40.2%, respectively) were significantly lower than those of thrombin− patients (85.6, 59.5, and 57.5%, respectively; P = 0.005) (Fig. 3B). To further evaluate the prognostic value of thrombin for HCC patients, univariate and multivariate analyses were performed with the clinicopathological characteristics and 上海皓元医药股份有限公司 expression of thrombin and OPN (Supporting Information Tables S3 and S4). In the univariate analysis, tumor size, vascular invasion, TNM stage, and tumor differentiation were revealed to associate with OS and TTR of HCC patients. Thrombin expression was also significantly associated with both OS and TTR and, particularly, this association was much stronger in OPN+ patients (OS, P = 0.001; TTR, P < 0.0001) compared with OPN− patients (OS, P = 0.596; TTR, P = 0.728). No significant prognostic significance was found in the other characteristics including sex, age, and hepatitis B surface antigen (HBsAg) positivity of patients for OS or TTR (Supporting Information Table S3). Individual features that showed significance by univariate analysis were adopted as covariates in a multivariate Cox proportional hazards model and then combined variables were further analyzed.