The study was approved by the institutional review board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Book Title: Viral Integration Signature in Multifocal Hepatocellular Carcinoma During Occult Hepatitis B Virus Infection: a Single-cell Sequencing Analysis
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Published on 2015 by
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Book is About: Carcinoma
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Abstract: Background: Patients with hepatocellular carcinoma frequently develop multiple anatomically separate liver tumours even at early stages of the disease, thus severely limiting the benefit of surgery. Hepatitis B virus is one of the most well recognised carcinogens causing hepatocellular carcinoma in man, and the integration of viral DNA into the host genome might sufficiently induce hepatocyte transformation. The question of whether virus integration has a role in development of multiple hepatocellular carcinomas cannot be answered until the pattern of hepatitis B virus DNA integration is better understood. We aimed to search for a potential link between viral integration and development of multiple hepatocellular carcinoma. Methods: We studied a Chinese man (aged 47 years) with multiple hepatocellular carcinomas that were potentially caused by an occult hepatitis B virus infection. We combined advanced liquid-phase hybridisation and capture techniques with single-cell sequencing to analyse viral integration sites in individual cells taken from the patient. The pattern of integration of hepatitis B virus was used as a genetic marker to investigate clonal evolution in hepatocellular carcinomas and hypervascular liver metastases. The study was approved by the institutional review board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Written informed consent was obtained from the patient before recruitment, according to the regulations of the institutional review board. Findings: Hepatitis serology testing showed that the patient was HBsAg negative and HBsAb positive, and hepatitis B virus DNA was undetectable in his serum samples; all of these findings are consistent with the definition of occult hepatitis B virus infection. However, we detected integration of hepatitis B virus into hepatocellular carcinoma, adjacent non-hepatocellular carcinoma tissues, and tumour thrombi from the inflow and outflow hepatic vasculature. By sequencing 270 single cells from six tumour tissues, we identified two integration sites that are recurrent among almost all HBV-integrated cells—the intergenic region of MED30–EXT1 on chromosome 8 and the intragenic region of CSMD2 on chromosome 1. The preferred integration hot spots were located in the core protein-coding gene (for MED30–EXT1 ) and the DR1 region on the HBX gene (for CSMD2 ). Additionally, the DR1 region of HBX has a preference of integrating into the intronic region of CSMD2, resulting in a truncated HBX gene, which might alter the oncogenic transforming ability of hepatocytes. Interpretation: Our findings lend support to the hypothesis that multifocal hepatocellular carcinomas are monoclonal in origin and are formed in the absence of hepatitis B virus replication. Furthermore, the sites of oncogenic viral integration are not random and occur in genes that might have important roles in carcinogenesis and metastasis. Funding: Major and Special Program of National Science and Technology in Twelfth Five-year Plan of China (2012ZX10002016-004 to X-PC), Major Science Foundation of the Ministry of Health of China (201302009 to X-PC), the National Natural Science Foundation of China (31200666 and 81471612 to QC; 81202300 to H-FL, and 81372495 to X-PC), the National Basic Research Program of China (973 Program; No.2011CB809203), Chinese 863 Program (2012AA02A502, 2012AA02A201), Guangdong Innovative Research Team Program (2009010016), the Guangdong Enterprise Key Laboratory of Human Disease Genomics, and the Shenzhen Municipal Government of China (CXB201108250094A, CXB201108250096A).
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