Validation Of Signatures For Predicting Recurrence In Hepatocellular Carcinoma And Analyses Of Prognostic Values Of Important Molecules

Liver cancer is one of the most common malignant tumors, The global burden of which continues to increase.According to the latest data,liver cancer in men is the fifth most frequently diagnosed cancer worldwide but the second most frequent cause of cancer death. In women, it is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer death. An estimated 748,300 new liver cancer cases and 695,900 cancer deaths occurred worldwide in 2008.Half of these cases and deaths were estimated to occur in China. Among primary liver cancers, hepatocellular carcinoma (HCC) represents the major histological subtype, accounting for 70% to 85% of the total liver cancer burden worldwide.HBV infection accounts for about 60% of the total liver cancer in developing countries and for about 23% of cancer in developed countries.Although radical resection is still the most effective method, 5-year survival rate is only about 50%. This extremely poor prognosis, mainly attributed to the high frequency of metastatic recurrence. It is a challenge to identify patients who are at a greater risk for tumor recurrence after curative treatment for HCC. Traditional prediction focuses on macro indicators, such as tumor size, number, vascular invasion and so on. Criteria are too subjective and the prediction is not precise enough. Identification of molecular markers could provide supplemental and useful information for predicting clinical outcome in HCC patients. In our previous studies of gene expression profiling via cDNA array, we found that some gene signatures are associated with metastases and recurrence of HCC. However, these signatures need external validation. The current study was based on previous findings. We confirmed and refined gene candidates with real-time PCR, and further evaluated the prognostic values of protein levels of key signatures. PARTâ… Validation of gene signatures for predicting metastases and recurrence in hepatocellular carcinoma with real-time PCRObjective:Choose the predictive gene expression signatures for survival and recurrence of hepatocellular carcinoma in early-stage to fit large sample validation.Methods:Choose 80 HCC after curative resection randomly by strict standards. Real-time PCR (TaqMan(?)MGB) was used to detect mRNA expression of 68 genes in the tumors and matched peritumoral samples of HCC in early stage. Select genes that can be used for prognosis of hepatocellular carcinoma in early-stage with follow-up data.Results:In the 80 pares of HCC samples, expression levels of 46 genes were significantly different between tumor tissues and matched peritumoral tissues. In tumor tissues, there were significant correlation between expression levels and OS in RTP4(P=0.005), DDIT4(P=0.015) and CMPK2(P=0.050), and significant correlation between expression levels and TTR in RTP4(P=0.016), FLJ34(P=0.018), GAK(P=0.020); In peritumoral tissues, there were significant correlation between expression levels and OS in PAR1 (P=0.001), FRAT1(P=0.030), DDIT4(P=0.043), and significant correlation between expression levels and TTR in DDIT4 (P=0.003), SNAI2(P=0.003), ADM (P=0.011) and PAR1 (P=0.018)Conclusion:In tumor tissues, RTP4 mRNA expression was significantly correlated with both OS and TTR. In peritumoral tissues, the same phenomena were found in PAR1 and DDIT4. The three genes can be scrolled for large sample validation predictive signature for prognosis of hepatocellular carcinoma. PARTâ…¡The association of protein level of thrombin receptors with prognosis of hepatocellular carcinoma Patients after curative resectionObjective:This study aimed to evaluate the relationship between PAR1 (Protease-Activated Receptor 1) and PAR4 protein levels and clinicopathologic features, and investigate the prognostic value of PAR1 and PAR4 protein expression levels in hepatocellular carcinoma (HCC) after curative resection.Methods:Real-time PCR was used to detect PAR1 expression in 153 pairs of tumors and matched peritumoral samples of HCC. The protein expression levels of PAR1 and PAR4 was evaluated with immunohistochemistry(Envision) and prognostic value of PAR1 and PAR4 protein levels were evaluated by Cox proportional hazards regression model and Kaplan-Meier analysis.Results:Peritumoral PAR1 protein expression was related to tumor differentiation (P=0.018). Kaplan-Meier analysis showed that Peritumoral PAR1 protein expression was associated with overall survival (OS) (P=0.021) and time to recurrence (TTR) (P=0.030) of HCC patients. The 1,3,5-year overall survival time in the high PAR1 protein expression group and the cumulative recurrence time were significantly lower than the low expression group in the peritumoral liver tissue. Kaplan-Meier analysis showed that Peritumoral PAR4 protein expression was associated with overall survival (OS) (P=0.007) of HCC patients. The 1,3,5-year overall survival time in the high PAR4 protein expression group were significantly higher than the low expression group in the peritumoral liver tissue.Conclusion:Peritumoral PAR1 and PAR4 expression is closely associated with prognosis for HCC patients after curable surgery. PAR1 and PAR4 may be involved in thrombin-mediated invasion process and serve as prognostic markers for HCC. PART IIIThe prognostic value of plasma abnormal prothrombin (DCP) for hepatocellular carcinoma patients after curative resectionObjective:PARs, which mainly express in stromal cells of peritumor liver tissue, are more difficult to detect in peripheral blood. PARs ligand-thrombin is generated from prothrombin. In addition to normal prothrombin, abnormal prothrombin (APT),also known as DCP (Des-gamma-carboxy prothrombin) can be found in patients with hepatocellular carcinoma (HCC).DCP can be applied to early diagnosis of liver cancer. So we evalued role of plasma DCP as an predictor in recurrence and survival of HCC.Methods:The plasma samples from 78 HCC patients were determined with ELISA and prognostic values of DCP were evaluated by Cox proportional hazards regression model,Kaplan-Meier analysis and Log-rank tests.Results:The protein levels of plasma DCP were significantly associated with tumor sizes (P=0.002), tumor encapsulation (P=0.008), vascular invasion (P=0.005) and serum AFP (P< 0.001). Kaplan-Meier analysis showed that plasma DCP levels were correlated to recurrence (P=0.005) and overall survival (P=0.003) of HCC patients.Conclusion:The protein levels of plasma DCP are closely associated with prognosis for HCC patients after curative resection. The plasma DCP could provide useful information for predicting clinical outcome in HCC patients.