Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, NO.1 Beichen West Road, Chaoyang, Beijing, 100101, China.
Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing, 100083, China.
Hepatol Int. 2020 May;14(3):326-337. doi: 10.1007/s12072-020-10016-2. Epub 2020 Feb 25.
Host genome integration of HBV sequence is considered to be significant in HBV antigen expression and the development of hepatocellular carcinoma (HCC).
We developed a probe-based capture strategy to enrich integrated HBV DNA for deep-sequencing analysis of integration sites in paired patient samples derived from tumor, liver tissue adjacent to tumor, saliva and plasma, as a platform for exploring the correlation, significance and utility of detecting integrations in these sample types.
Most significantly, alpha fetoprotein levels significantly correlated to the amounts of integrations detected in tumor. Viral-host chimeric DNA fragments were successfully detected at high sequencing coverage in plasma rather than saliva samples from HCC patients, and each fragment of this type was only seen once in plasma from chronic hepatitis B patients. Almost all plasma chimeric fragments were derived from integrations in tumor rather than in adjacent liver tissues. Over 50% of them may produce viral-host chimeric transcripts according to deep RNA sequencing in paired tissue samples. Particularly, in patients with low HBV DNA level (< 250 UI/ml), the seemingly normal HBsAg titers may be explained by larger amounts of integrations detected. Meanwhile, we developed a strategy to predict integrants by pairing breakpoints for each integration event. Among four resolved viral patterns, the majority of Pattern I events (81.2%) retained the complete opening reading frame for HBV surface proteins.
We achieve the efficient enrichment of plasma cell-free chimeric DNA from integration site, and demonstrate that chimeric DNA profiling in plasma is a promising noninvasive approach to monitor HBV integration in liver cancer development and to determine the ability of integrated sequences to express viral proteins that can be targeted, e.g. by immunotherapies.
HBV 序列的宿主基因组整合被认为在 HBV 抗原表达和肝细胞癌（HCC）的发展中具有重要意义。
我们开发了一种基于探针的捕获策略，用于富集整合 HBV DNA，以便对来自肿瘤、肿瘤相邻肝组织、唾液和血浆的配对患者样本中的整合位点进行深度测序分析，作为探索这些样本类型中检测整合的相关性、意义和用途的平台。
最显著的是，甲胎蛋白水平与肿瘤中检测到的整合数量显著相关。在 HCC 患者的血浆而不是唾液样本中，成功地以高测序覆盖度检测到病毒-宿主嵌合 DNA 片段，并且这种类型的每个片段仅在慢性乙型肝炎患者的血浆中出现一次。几乎所有的血浆嵌合片段都来自肿瘤中的整合，而不是来自相邻的肝组织。根据配对组织样本的深度 RNA 测序，超过 50%的嵌合片段可能产生病毒-宿主嵌合转录本。特别是在 HBV DNA 水平较低（<250 UI/ml）的患者中，似乎正常的 HBsAg 滴度可能是由于检测到更多的整合。同时，我们开发了一种通过为每个整合事件配对断点来预测整合子的策略。在四种解析的病毒模式中，大多数模式 I 事件（81.2%）保留了 HBV 表面蛋白的完整开放阅读框。
我们实现了从整合部位有效富集血浆无细胞嵌合 DNA，并证明了血浆中嵌合 DNA 谱分析是一种很有前途的非侵入性方法，可以监测 HBV 整合在肝癌发展中的作用，并确定整合序列表达可靶向的病毒蛋白的能力，例如通过免疫疗法。
