| Hepatocellular carcinoma is one of the high incidence and poor prognosis of malignant tumors. High rate of recurrence after surgery is the ultimate cause of death, which seriously affect the prognosis. Searching risk factors for recurrence, and taking effective measures to prevent and treatment relapse is one of the most important strategy to improve the survival rate. Studies have shown that the main prognostic factors that affect HCC recurrence are tumor size.number,envelope,vascular invasion, preoperative AFP levels,intraoperative blood transfusion and HBV infection.AIM:Quantitation of hepatitis B virus cccDNA in liver tissues and serum by real time quantitative PCR. To investigate correlation between HBV covalently closed circular DNA (cccDNA), clinical factors and survival time after hepatectomy.METHODS:A retrospective analysis was carried out in60cases with Hepatitis B virus-related hepatocellular carcinoma (HCC) receiving radical operation from2003to2006. Child-Pugh score, whether with cirrhosis, AFP level, tumor number, Tumour diameter, growth pattern and other indicators before operation were recorded. Liver tissue and blood samples from patients who underwent resection were collected, rapidly frozen and stored in-80℃. In55patients with acquired complete follow-up, Liver tissue restriction enzyme with a restriction endonuclease Hind Ⅲ joint with Plasmid-safeTM ATP-Dependent DNase. The serum and liver tissue HBV DNA and cccDNA levels were detected by the real-time quantitative PCR. The HBV DNA, cccDNA and the clinical characteristics were retrospective analysis. Cumulative survival rate using the Life-Table Method, the overall survival(OS) and disease-free survival(DFS) rates were calculated with Kaplan-Meier survival curve and Cox regression model.RESULTS:60patients were choose with,51men,9women. The average age of patients is53.37±11.49. Among the60patients,5patients were excluded from analysis because of no complete follow-up. In55patients with acquired complete follow-up, the maximum follow-up time was88months. HBV cccDNA was positive in only one in serum samples (1/35), but positive in20%(11/55) of the tumor tissues. The liver tissue sample was digested with restrietion endonuclease Hind III and Plasmid-Safe-ATP-Dependent DNase.then was amplified by71F which could reduce 5orders of magnitude non-specifie amplifieation. There was a significant correlation between intrahepatic HBV DNA, cccDNA in the tumor tissues and non-tumor tissues (r=0.437、0.478,P<0.05).Total HBV DNA level in serum correlated with intrahepatic cccDNA in the tumor tissues (r=0.359, P<0.001),and did not correlated with intrahepatic cccDNA level in the non-tumor tissues(r=0.264, P=0.052). HBsAg negative in20%(11/55). In HBsAg-negative, HBV cccDNA positive rate in the tumor and non-tumor tissues were100%. The total HBV DNA level had significant difference between HBsAg-positive group and HBsAg-negative group (P=0.002、0.045), but the HBV cccDNA level had no significant difference(P=0.068、0.435). The1-,3-,5-year overall survival and RFS rates of55patients with HCC resection were73%、51%and38%,63%、29%and19%. respectively. Univariatea analysis indicates that there is a negative relationship with AFP level(P=0.039), growth pattern (P<0.001), tumor number (P<0.005), intrahepatic vessel invasion (P<0.001), TNM staging(P=0.007), tumour diameter (P=0.028) for OS, and intrahepatic cccDNA in tumor tissue (P=0.028). AFP level(P=0.021), tumor number(P=0.006), intrahepatic vessel invasion (P=0.048), TNM Staging(P=0.027), pathologic grades(P=0.030) for DFS. Multivariate analysis showed that tumor number(P=0.011). and intrahepatic vessel invasion(P=0.001)were important prognostic faetors for OS, and the independent prognostic factor influencing DFS were the level of intrahepatic cccDNA in tumor and growth pattern.CONCLUSIONS:The liver tissue sample was digested with restrietion endonuclease Hind Ⅲ and PS AD, then was amplified by71F with high specificity to investigate the quantitation of HBV cccDNA. With follow-up observation periods up to more than five years, the patients with solitary tumor, without vessel invasion showed a higher OS and with a level less than1000copies/μg of intrahepatic HBV cccDNA in tumor tissue, expansive growth showed a higher DFS. Liver tissue of HBV cccDNA can be used to assess the prognosis of hepatocellular carcinoma... |
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