Re-evaluation of the Carcinogenic Significance of Hepatitis B Virus Integration in Hepatocarcinogenesis

To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820–1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis.为探究乙型肝炎病毒（hepatitis B virus, HBV）整合在肝细胞癌变过程中的作用，本研究针对60例HBV相关肝细胞癌（hepatocellular carcinoma, HCC）患者队列开展了系统性对比研究。研究采用Alu聚合酶链反应（Alu-polymerase chain reaction）与连接介导PCR技术，共获得233个随机分布于人类所有染色体的病毒-宿主连接位点；结果显示，除脆性位点外（P=0.0070），肿瘤与非肿瘤组织间未观察到显著差异。本研究中发现部分HBV整合位点紧邻癌症相关基因（如hTERT），但整体而言此类整合事件较为罕见。通过高灵敏度的基于阵列的比较基因组杂交（array-based comparative genomic hybridization, aCGH）检测，未发现染色体畸变数量与HBV整合事件数目间存在直接关联；但多个肿瘤抑制基因（TP53、RB1、CDNK2A及TP73）的突变状态与染色体畸变数目呈显著正相关（r=0.6625, P=0.0003）。对病毒基因组的分析显示，43%的整合片段位于前C/C区，57%位于HBV X基因区。值得注意的是，约24%的整合事件的断裂点位于一段仅15 nt的病毒基因组区域（1820–1834 nt）。由此，所有已确认的X基因整合均发生C端截短，丢失了其生长抑制结构域；但该类X基因C端截短的模式在肿瘤与非肿瘤来源样本中均有检出。此外，两组样本中的整合病毒序列均以较低频率携带C1653T、T1753V及A1762T/G1764A突变。肿瘤组织与对应癌旁非肿瘤样本的HBV整合频率与模式均相似，提示大多数HBV DNA整合事件与肝细胞癌变并无关联。