| Objective: The four reading frames which constitute HBV genome are overlapping and open, and each of them encodes a protein. X gene overlap with the the basal core promoter and Enhancer2, and the transcription of precore and core regions are controlled by the basal core promoter and Enhancer 2, which are overlapped with X gene. As the proofreading defects of HBV polymerase, HBV genome is much more likely to mutation. The HBV genotype and HBV DNA copy are both associated with HBV-HCC risk. Besides that, mutations in the pre S1, pre S2, BCP, and Enh2 regions have been extensively investigated in case–control studies for HBV-HCC risk; several mutations in these regions were shown to be associated with HCC development. However, few studies focus on the prognostic value of these mutations in HBV-HCC patients have been conducted especially in X gene region. By detecting HBV DNA in liver tissue samples, Yeh et al. discover that the A1762T/G1764 A mutation as a basal core promoter component is an independently predictive factor of postoperative survival in HCC patients. In this research, we sequence the HBV X(HBx) regions using HBV DNA from HCC tissue specimens from HBV-HCC patients to identify and recognize mutations which are related to postoperative survival.Methods:1 Sample collection and patients following-up: tumor tissue samples for experimental group were collected from HCC patients who were diagnosed of hepatocellular carcinoma by imaging tests and histopathological method at Fourth Hospital of Hebei Medical University. Surgical specimens had been collected from patients received resection of liver cancer in the Department of hepatobiliary Surgery from 2007 to 2009. The patients’ clinical characteristics(gender, age, et al) were accurate recorded. We followed up for 3-years survival status of post-operative patients by outpatient clinic, phones and letters.2 DNA extraction, amplification and sequencing: Using DNA extraction kit to extract the genomic DNA from tumor tissue samples. The target genes of qualified DNA samples were then amplified by using PCR technique. In this study, we designed two pairs of primers as follows:HBV1F:5’-ATGCGTGGAACCTTTGTG-3’,HBV1R:5’-TCGGTCGTTGACATTGCT-3’;HBV2F:5’-CACCTCTGCACGTAGCAT-3’,HBV2R:5’-TCCCATACAGAGCAGAG-3’. The DNA products were confirmed by agarose gel electrophoresis, and then sent them to sequencing facility.3 Statistical analyses of data: Among univariate analysis, survival curves of each factor were composed by the Kaplan-Meier method, and then using the Log-rank test to evaluate the significant differences between these survival curves. The multi-factor analysis was completed by Cox regression model. SPSS software 13.0 was used to perform all the analyses, P<0.05 was considered statistically significant.Results:1 The clinical characteristic of HBV-HCC patients. This study included 106 HBV-HCC patients in all. A review of the patients was performed every 3 months for 3 years. Seven cases lost follow-up, and the lost rate of follow up was lower than 20%, which did not impact the results. HBV DNA was extracted from HCC tissue for virological analysis; the mean HBV DNA level was 5×109±2.616×1010 copies/μg DNA with a maximum copy number of 1.97×1011 and a minimum copy number of 1.39×104. There were 114 mutations in the HBx region by DNA sequencing. The highest mutation rate was found at the 1386 nucleotide; 74 cases carried this mutation. Statistical analysis revealed that the HBV DNA expressional levels were not associated with increased mutation quantities(P=0.146). 2 This study chooses 94 cases of postoperative patients carrying the B or C HBV genotype for survival analysis. The clinical characteristics which may affect the hepatocellular carcinoma operation prognosis were chosen for simple factor analysis, including sex, age, tumor number, Child-Pugh grade, HBV DNA copy, tumor size, TNM classification, portal vein thrombosis. Univariate analysis showed that 3 years of survival rate is no difference between male and female(P=0.415). Clinical characteristics such as age, tumor number, Child-Pugh grade, TNM classification were not associated with the postoperative survival rate of hepatocellular carcinoma patients, but tumor size, portal vein thrombosis were associated with the postoperative survival rate of hepatocellular carcinoma patients.(P<0.05).Among the mutational sites, we chose 21 mutational sites with mutational rates higher than 5% in HCC patients for survival analysis. Among these 21 sites, the following 7 sites were associated with postoperative survival at statistically significant levels in HCC tissue based on the log-rank test with Kaplan-Meier method: nucleotides 1383, 1461, 1485, 1544, 1613, 1653 and 1762.Based on the multi-factor analysis with the Cox proportional hazards model, portal vein thrombosis was confirmed as an independent predictor of postoperative survival in HBV-HCC patients. Among the above 7 sites, the following 4 mutational sites were confirmed as independent predictors of HBV-HCC survival at statistically significant levels: 1461(RR 3.162, 95%CI 1.319-7.582, P=0.010), 1485(RR 1.989, 95%CI 1.054-3.751, P=0.034), 1544(RR 2.602, 95%CI 1.040-6.513, P=0.041), 1653(RR 2.652, 95%CI 1.184-5.938, P=0.018).3 The genotype, DNA copy level, E antigen status and mutation number of patients were compared using Kaplan-Meier methods for survival analysis. The results show that DNA copy level(<6.0E+08 and ≥6.0E+08,P=0.061) was associated with survival at a border line significance level, and the other three factors were not associated with HCC survival.Conclusions:Three mutational sites: 1461, 1485, 1544, 1653 were confirmed as independent predictors of postoperative survival in HBV-HCC patients. Among the clinical characteristics, portal vein thrombosis was confirmed as an independent predictor of postoperative survival in HBV-HCC patients. |
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