Identification Of Hepatitis B Virus Integration Sites In Hepatocellular Carcinoma Tissues From Patients And Its Relationship With Clinicopathological Parameters

Objective To indentify the integration of hepatitis B virus (HBV) in HBV-relatedhepatocellular carcinoma （HCC）, and to investigate their relations with theclinicopathological parameters.Methods Collection of40cases with HBV-related HCC during perioperativeperiod in patients with clinical and pathological information and follow-up. ExtractedDNA from40HCC liver tissue samples. According to the HBV X sequence andhuman Alu repeat, primers were designed respectively, and then amplify the nestedpolymerase chain reaction (PCR) with specially designed U-base primers.Integratedclones combined target HBV X gene and the adjacent cell gene sequences wereestablished by PCR.The PCR product was purified and subject to direct sequencingby ABI3700XL Auto sequencer. BCBI （national center for biotechnologyinformation）BLAST and MapViewer research were used for identification of HBVlocation on human genomes. Analysis of the association between the integration ofHBV with clinicopathological parameters. Recurrence free and overall survival rateswere analyzed by the Kaplan Meier method． Cox regression univariate andmultivariate analysis of factors affecting survival.Results In40HBsAg positive HCC samples,18cases showed the integration,andthe other22samples didn’t show any evidence of integration. Among18samples withintegration, forward insertions of HBV DNA into the host chromosomal DNA werefound in15samples, and reverse insertions were found in7samples while both forward and reverse in sertions were found in4samples. Four genes, homo sapienstelomerase reverse transcriptase, mixed-lineage leukemia4, cyclin E1and SUMOspecific protease5were found to be recurrently targed by HBV. Analysis fromviral-cellular junctions suggested that the integrations were all happened withtruncated viral DNA and could be in any locus of X gene. The HBV DNA intergrationwas closely related with HBeAg, HBV DNA level, tumor size and differentiationdegree (P<0.05), while nothing to do with the sex, age, AFP level, microscopictumor thrombus, vascular invasion, envelop and stove and recurrence（P>0.05）.39patients were followed．Up from3to35months (average17.2±7.6months)，thepatients in HBV DNA intergration group had a greater total and tumor free survivaltime than in non-intergration group, but no statistical difference (P>0.05).Conclusons HBV DNA intergration is not distributed evenly throughout the hostgenome. The HBV DNA intergration is closely related with HBeAg, HBV DNA level,tumor size and differentiation degree. HCC postoperative recurrence wereindependent risk factors for survival.