Study On The Relationship Between Hepatocellular Carcinoma And Hepatitis B Virus Genotypes Or Viral Mutations

Clinical and virological characteristics show that the most relevant oncogenicagent for hepatocellular carcinoma (HCC) development is chronic hepatitis B viralinfection (HBV). In most cases, HCC development complicates underlying longperiod and persistent HBV infection, and HBV infection may be persistent indifferent stages of liver diseases, such as chronic hepatitis, liver cirrhosis, HCC andthe stage of HCC recurrence or metastasis. Thus, HBV plays an important role inclinical manifestations and outcomes of HBV associated hepatic diseases. However,the oncogenic mechanisms of HBV are not fully understood.To date, eight different HBV genotypes (A-H) have been reported according toviral genomic heterogeneity. Based on a divergence greater than 4ï¼…but less than 8ï¼…in the complete nucleotide sequence, HBV genotype has been divided intosubgenotypes. In China, the most prevalent HBV strains are genotype B and C. Andviral mutations are very common in the course of chronic HBV infection. Datalinking HBV genotypes, viral mutations, HBV DNA levels with HCC are raisedrecently, however, the exact role of HBV on the development of HCC is stillcontroversial.To investigate the correlation between HBV genotypes, subgenotypes, viral mutations and HCC development, 112 HCC patients and 289 controls, whichincluding 70 cases of liver cirrhosis (LC), 129 asymptomatic HBV carriers (ASC) and90 patients of chronic hepatitis B (CH), were recruited from six medical centers inGuangdong Province of China. HBV genotypes were determined by PCR restrictionfragment length polymorphism (PCR-RFLP), BCP and PC mutations weredetermined by PCR and direct sequencing of amplified products, and subgenotypeswere determined with nucleotide sequence analysis or PCR-RFLP. Among 112 HCCpatients studied, 34(30.4ï¼…) genotype B and 78(69.6ï¼…) genotype C were identified,respectively. Out of 70 patients with LC, 34(49ï¼…) B and 36(51ï¼…) C were found,respectively. 129 cases with ASC, 64(49.6ï¼…) B and 65 (50.4ï¼…) C genotypes werefound, respectively. Out of 90 patients with CH, 49(54.4ï¼…)B and 41(45.6ï¼…) C wereidentified, respectively. These data showed that the distribution of genotype C wasstatistically higher in HCC patients than that in non HCC patients with chronic liverdiseases (x²=14.405, p=0.002).On the study of viral mutataions, we can find the ratios of BCP mutations were71.4ï¼…, 50.0ï¼…, 35.0ï¼…and 22.4ï¼…in cases with HCC, LC, CH and ASC, respectively,and it suggested that BCP mutations had significantly correlated with HCC. Out of401 subjects of chronic HBV infection (112 HCC patients and 289 controls), 220patients were infected with genotype C and 181 were infected with genotype B. Onanalysis of viral mutations, we found 130 patients with BCP mutations and 29patients with PC mutation infected with genotype C, respectively, and 46 BCPmutations and 49 PC mutations with genotype B, respectively. The results indicatedthat the incidence of BCP mutations was higher than PC mutation in genotype C,while PC mutation was statistically increased in genotype B. Out of 272 cases ofHCC and LC and CH, 155 patients infected with genotype C, in which 105(67.9ï¼…)cases were C1 and others C2. In summary, we concluded that genotype C and BCP mutations had relevantly correlation with HCC development. C1 and C2 hadsignificantly different tendancy to develop PC and BCP mutations, C1 wasassociated with the highest tendency to develop BCP mutations and PC mutationwas more frequently associated with genotype B. However, there was no correlationbetween genotype B and C on sex, age, liver function and HBsAg.In the second chapter, we further investigate tumor recurrence or metastasis ofthese 112 HCC patients for median 104 (10-130) weeks of follow up. After surgery ortransarterial chemoembolization (TACE), all patients were followed up by monthlyAFP and abdominal ultrasonography as well as 3 monthly helical computedtomographic (CT) scan for 1 year. Afterwards, we performed surveillance AFP andultrasound every 2 months and helical CT every 6 months. Hepatic angiography wasperformed whenever metastasis or recurrence was suspected. Diagnosis of Metastasisor recurrence was based on the combined findings of imaging results.Time-to-metastasis or recurrence was defined as the period between surgery or TACEand the diagnosis of metastasis or recurrence. Patients who died for reasons notrelated to HCC were censored at the time of death and were treated as controls.Absence of evidence of tumor metastasis or recurrence within 2 years was regardedas successful tumor clearance. Serum HBV DNA levels were detected by LightCycler-based real-time fluorescence quantitative polymerase chainreaction-restriction (PCR) system. HBV genotypes were determined by using PCRrestriction- fragment length polymorphism. Among the 112 patients with HCC, 6patients were excluded from analysis because of the lack of follow-up data and theremaining 106 patients gained follow up for two years and finally entered into theanalyses. Results showed that 69 patients out of 106 HCC cases had tumor metastasisor recurrence during the follow-up and the cumulative probability of HCC metastasisor recurrence was 3.8ï¼…, 9.4ï¼…, 18.9ï¼…, 42.5ï¼…, 59.4ï¼…and 65.1ï¼…in 20, 40, 60,80, 100 and 130 weeks of follow-up, respectively. On multivariate analysis, genotype Cand HBV DNA levels were the exact risk factors for HCC recurrence or metastasis.The incidence of HCC recurrence or metastasis increased with baseline serum HBVDNA level in a dose-response relationship ranging from (22%) for HBV DNA levelof less than 3 log₁₀ copies/ml, 60% for HBV DNA level of between 3 log₁₀ and 5log₁₀copies/ml, to 80% for an HBV DNA level of 5 log₁₀ copies/ml or greater. Afteradjusted for potential confounders, serum HBV DNA level was still independentlyassociated with HCC metastasis or recurrence. Seventy-seven patients were infectedby HBV genotype C and twenty-nine patients by HBV genotype B. Fifty-seven(74.0%) and twelve (41.4%) patients had metastasis or recurrence in HCC patientswith genotype C and B, respectively. The adjusted odd ratio for patients infected withgenotype C HBV compared with patients infected with genotype B was 9.755(95%CI=1.787-53.253, p=0.009).Thus, HBV genotype C and high HBV DNA levels arethe independent risk factors for the development of HCC metastasis or recurrenceafter surgery or TACE. Based on these findings, viral genotypes should be determinedamong patients with HCC for risk stratification of future tumor metastasis orrecurrence. The exact reason for the higher risk of HCC metastasis or recurrenceassociated with HBV genotype C needs further investigation. The associationbetween higher level of HBV DNA and HCC recurrence or metastasis might hint arole of antiviral treatment after treatment of HCC.The previous studies suggested that HBV genotype C and BCP mutations weresignificantly associated with the development of HCC, HBV DNA level and genotypeC were independent risk predictors of HCC recurrence or metastasis. As BCPmutations may induce not only amino acid change in HBV X protein but also analteration of HBV gene expression. And the alteration of HBV X protein might play arole in hepatocarcinogenesis, because its coding sequence overlaps regions of crucial importance for increasing the proto-oncogene. So we tried to define the influences ofHBx protein on the regulation of cycline-dependent kinase inhibitor p21^WAF1/CIP1expression in order to understand the mechanism of HBV associated with HCC. Atfirst, four HBx-expressing plasmids were constructed, including plasmids withpositive BCP mutations (pCMV-Tag2B-HBx1 and pCMV-Tag2B-HBx2) and withnegative BCP mutations (pCMV-Tag2B-HBx3 and pCMV-Tag2B-HBx4). ThenHepG2 cells were transiently transfected with constructed plasmids throughLipofectamine^TM2000. Finally, each cell extract was separated by SDS-PAGE andtransferred onto a nitrocellulose membrane. Western blotting was performed witheither mouse monoclonal p21^WAF1/CIP1 antibody-11, anti-Flag protein. Our datashowed that HBV X with negative BCP mutation enhanced the expression ofp21^WAF1/CIP1, while HBV X with positive BCP mutation decreased the expression ofp21^WAF1/CIP1. So we can speculate that HBV X with negative BCP is more commonduring an early stage of viral infection, up-regulating of p21^WAF1/CIP1 by HBV X maycause prolonged arrest in G1, lead to apoptosis and thus contribute to thedevelopment of hepatitis. As advancement of chronic HBV infection, BCP mutationsoccur very frequently, effect of HBV X protein on the regulation of cell-cycle controlshows suppression of p21^WAF1/CIP1 expression. Lower level or absence of p21^WAF1/CIP1may induce rapid and uncontrolled cell proliferation and thus make it easy toaccumulate more mutations on other oncogenes or tumor suppressor genes, ultimatelyleading to HCC.