ViroISDC: a method for calling integration sites of hepatitis B virus based on feature encoding.

BACKGROUND: Hepatitis B virus (HBV) integrates into human chromosomes and can lead to genomic instability and hepatocarcinogenesis. Current tools for HBV integration site detection lack accuracy and stability. RESULTS: This study proposes a deep learning-based method, named ViroISDC, for detecting integration sites. ViroISDC generates corresponding grammar rules and encodes the characteristics of the language data to predict integration sites accurately. Compared with Lumpy, Pindel, Seeksv, and SurVirus, ViroISDC exhibits better overall performance and is less sensitive to sequencing depth and integration sequence length, displaying good reliability, stability, and generality. Further downstream analysis of integrated sites detected by ViroISDC reveals the integration patterns and features of HBV. It is observed that HBV integration exhibits specific chromosomal preferences and tends to integrate into cancerous tissue. Moreover, HBV integration frequency was higher in males than females, and high-frequency integration sites were more likely to be present on hepatocarcinogenesis- and anti-cancer-related genes, validating the reliability of the ViroISDC. CONCLUSIONS: ViroISDC pipeline exhibits superior precision, stability, and reliability across various datasets when compared to similar software. It is invaluable in exploring HBV infection in the human body, holding significant implications for the diagnosis, treatment, and prognosis assessment of HCC.

Long-term antiviral therapy is associated with changes in the profile of transcriptionally active HBV integration in the livers of patients with CHB.

Hepatitis B virus (HBV) integration exists throughout the clinical course of chronic hepatitis B (CHB). This study investigated the effects of long-term antiviral therapy on the level and profiles of transcriptionally active HBV integration. Serial liver biopsies and paired blood samples were obtained from 16, 16, and 22 patients with CHB at baseline, 78, and 260 weeks of entecavir monotherapy or combined with pegylated interferon alfa, respectively. Serum HBV biomarkers were longitudinally assessed. RNA-seq and HIVID2 program was used to identify HBV-host chimeric RNAs transcribed from integrated DNA. The counts of HBV integration reads were positively related to both serum HBV DNA levels (r = 0.695, p = 0.004) and HBeAg titers (r = 0.724, p = 0.021) at baseline, but the positive correlation exited only to the serum HBsAg levels after 260 weeks of antiviral therapy (r = 0.662, p = 0.001). After 78 weeks of antiviral therapy, the levels of HBV integration expression decreased by 12.25 folds from baseline. The viral junction points were enriched at the S and HBx genes after the long-term antiviral therapy. HBs-FN1 became one of the main transcripts, with the mean proportion of HBs-FN1 in all integrated expression increased from 2.79% at baseline to 10.54% at Week 260 of antiviral treatment. Antiviral therapy may reduce but not eliminate the HBV integration events and integration expression. Certain integration events, such as HBs-FN1 can persist in long-term antiviral treatment.

Multiomics analyses reveal pathological mechanisms of HBV infection and integration in liver cancer.

Hepatitis B virus (HBV) infection and integration are important for hepatocellular carcinoma (HCC) initiation and progression, while disease mechanisms are still largely elusive. Here, we combined bulk and single-cell sequencing technologies to tackle the disease mechanisms of HBV-related HCC. We observed high HBV mutation rate and diversity only in tumors without HBV integration. We identified human somatic risk loci for HBV integration (VIMs). Transcription factors (TFs) enriched in VIMs were involved in DNA repair and androgen receptor (AR) signaling. Aberration of AR signaling was further observed by single-cell regulon analysis in HBV-infected hepatocytes, which showed remarkable interactions between AR and the complement system that, together with the X-linked ZXDB regulon that contains albumin (ALB), probably contribute to HCC male predominance. Complement system dysregulation caused by HBV infection was further confirmed by analyses of single-cell copy numbers and cell-cell communications. Finally, HBV infection-associated immune cells presented critical defects, including TXNIP in T cells, TYROBP in NK cells, and the X-linked TIMP1 in monocytes. We further experimentally validated our findings in multiple independent patient cohorts. Collectively, our work shed light on the pathogenesis of HBV-related HCC and other liver diseases that affect billions of people worldwide.

Vertical transmission of hepatitis B virus from father to child: what can be concluded about this possibility?

Vertical hepatitis B virus (HBV) transmission is defined as transmission that occurs during pregnancy or postpartum from an HBV-infected mother to her fetus or child. It is an efficient route for the spread of HBV and is responsible for most of the cases of chronic HBV infection in adults. During pregnancy, vertical transmission can occur in the intrauterine phase, by placental infection via peripheral blood mononuclear cells, by placental leakage, or through female germ cells.The detection of HBV DNA in semen and spermatids from HBV-infected men has provided strong evidence that the male genital tract may act as a reservoir of the virus in HBV-infected men, supporting the possibility that vertical HBV transmission from an HBV-infected father to his child may also occur via the germ line at the time of fertilization, as occurs in HBV transmission from mother to child. Furthermore, it has been shown that integration of the HBV genome into the sperm cell genome can compromise sperm morphology and function and even cause hereditary or congenital biological effects in the offspring when an HBV-infected sperm fuses with an ovum.Since vertical HBV transmission from father to child can be a topic of interest and of global importance for controlling the spread of HBV, this article addresses the evidence supporting its occurrence via germ cells, the biological impact of integration of the HBV genome into the male germ cell genome, and the role of maternal immunoprophylaxis in vertical HBV transmission from father to child.

The integration model of hepatitis B virus genome in hepatocellular carcinoma cells based on high-throughput long-read sequencing.

HBV integration and function has gradually been expanding. However, the exact mode of HBV integration remains unclear. In our research, the high-throughput long-read sequencing was combined with bioinformatics to study the complete mode of HBV integration in hepatocellular carcinoma (HCC) cells. The results demonstrated that: 1) The HBV insertion sequences of HBV integration events accounted for 49.5% of the total HBV sequences. 2) Short insertion segments with the length of 0-1 kbp accounted for 50% and the long insertion segments (>3 kbp) accounted for 25% of HBV insertion events. 3)There were different HBV insertion length in the breakpoints formed within different regions. 4) The occurrence of HBV integration events was accompanied by more frequent structural variations. 5)Furthermore, multiple HBV integration patterns were confirmed based on complete HBV insertion sequences. Our research not only clarified a variety of perfect HBV integration models but also determined multiple specific features of HBV integration.

Hypomethylation in HBV integration regions aids non-invasive surveillance to hepatocellular carcinoma by low-pass genome-wide bisulfite sequencing.

BACKGROUND: Circulating cell-free DNA (cfDNA) methylation has been demonstrated to be a promising approach for non-invasive cancer diagnosis. However, the high cost of whole genome bisulfite sequencing (WGBS) hinders the clinical implementation of a methylation-based cfDNA early detection biomarker. We proposed a novel strategy in low-pass WGBS (~ 5 million reads) to detect methylation changes in circulating cell-free DNA (cfDNA) from patients with liver diseases and hepatocellular carcinoma (HCC). METHODS: The effective small sequencing depth were determined by 5 pilot cfDNA samples with relative high-depth WGBS. CfDNA of 51 patients with hepatitis, cirrhosis, and HCC were conducted using low-pass WGBS. The strategy was validated in an independent WGBS cohort of 32 healthy individuals and 26 early-stage HCC patients. Fifteen paired tumor tissue and buffy coat samples were used to characterize the methylation of hepatitis B virus (HBV) integration regions and genome distribution of cfDNA. RESULTS: A significant enrichment of cfDNA in intergenic and repeat regions, especially in previously reported HBV integration sites were observed, as a feature of cfDNA and the bias of cfDNA release. Methylation profiles nearby HBV integration sites were a better indicator for hypomethylation of tumor genome comparing to Alu and LINE (long interspersed nuclear element) repeats, and were able to facilitate the cfDNA-based HCC prediction. Hypomethylation nearby HBV integration sites (5 kb flanking) was detected in HCC patients, but not in patients with hepatitis and cirrhosis (MethylHBV5k, median:0.61 vs 0.72, P = 0.0003). Methylation levels of integration sites certain candidate regions exhibited an area under the receiver operation curve (AUC) value > 0.85 to discriminate HCC from non-HCC samples. The validation cohort achieved the prediction performance with an AUC of 0.954. CONCLUSIONS: Hypomethylation around viral integration sites aids low-pass cfDNA WGBS to serve as a non-invasive approach for early HCC detection, and inspire future efforts on tumor surveillance for oncovirus with integration activity.

Integration of hepatitis B virus DNA into p21-activated kinase 3 (PAK3) gene in HepG2.2.15 cells.

Integration of HBV DNA into host chromosomes was found in most of the patients with chronic hepatitis B (CHB). In this study, using inverse nested PCR (invPCR), we found the integration site chrX: 111,009,033, which inserted into the p21-activated kinase 3 (PAK3) gene in HepG2.2.15 cells. The viral-human chimeric transcripts were also observed and, significant differences of the copy numbers of integration site chrX: 111,009,033 (P = 0.012) and intra-cell HBV DNA levels (P = 0.027) were found between the cells with and without H2O2 treatment, respectively. This study may provide a novel insight into the elucidation of etiology of HBV integration.

Comprehensive analysis of transcriptome profiles in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC. METHODS: Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed. RESULTS: Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis. CONCLUSIONS: Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

Profile of HBV Integration in the Plasma DNA of Hepatocellular Carcinoma Patients.

BACKGROUND: Hepatitis B Viral (HBV) infection is one of the major causes of Hepatocellular Carcinoma (HCC). Mounting evidence had provided that the HBV integration might be a critical con-tributor of HCC carcinogenesis. OBJECTIVE AND METHODS: To explore the profile of HBV integration in the plasma DNA, the method of next-generation sequencing, HBV capture and bioinformatics had been employed to screen for HBV in-tegration sites in the plasma samples. RESULTS: In the initial experiment, a total of 87 breakpoints were detected in the 20 plasma samples. The distribution of breakpoints showed that there was significant enrichment of breakpoints in the region of intron. Furthermore, the HBV breakpoints were prone to occur in the region of X protein (1,700-2,000bp) in the plasma samples. The pathway analysis had revealed that the HBV integrations sites were specifically enriched in the cancer pathway. CONCLUSION: Altogether, our results had provided direct evidence for the HBV integration in plasma DNA, and they might be potentially useful for future HCC prognosis and diagnosis.

A pilot systematic genomic comparison of recurrence risks of hepatitis B virus-associated hepatocellular carcinoma with low- and high-degree liver fibrosis.

BACKGROUND: Chronic hepatitis B virus (HBV) infection leads to liver fibrosis, which is a major risk factor in hepatocellular carcinoma (HCC) and an independent risk factor of recurrence after HCC tumor resection. The HBV genome can be inserted into the human genome, and chronic inflammation may trigger somatic mutations. However, how HBV integration and other genomic changes contribute to the risk of tumor recurrence with regards to the different degree of liver fibrosis is not clearly understood. METHODS: We sequenced mRNAs of 21 pairs of tumor and distant non-neoplastic liver tissues of HBV-HCC patients and performed comprehensive genomic analyses of our RNAseq data and public available HBV-HCC sequencing data. RESULTS: We developed a robust pipeline for sensitively identifying HBV integration sites based on sequencing data. Simulations showed that our method outperformed existing methods. Applying it to our data, 374 and 106 HBV host genes were identified in non-neoplastic liver and tumor tissues, respectively. When applying it to other RNA sequencing datasets, consistently more HBV integrations were identified in non-neoplastic liver than in tumor tissues. HBV host genes identified in non-neoplastic liver samples significantly overlapped with known tumor suppressor genes. More significant enrichment of tumor suppressor genes was observed among HBV host genes identified from patients with tumor recurrence, indicating the potential risk of tumor recurrence driven by HBV integration in non-neoplastic liver tissues. We also compared SNPs of each sample with SNPs in a cancer census database and inferred samples' pathogenic SNP loads. Pathogenic SNP loads in non-neoplastic liver tissues were consistently higher than those in normal liver tissues. Additionally, HBV host genes identified in non-neoplastic liver tissues significantly overlapped with pathogenic somatic mutations, suggesting that HBV integration and somatic mutations targeting the same set of genes are important to tumorigenesis. HBV integrations and pathogenic mutations showed distinct patterns between low and high liver fibrosis patients with regards to tumor recurrence. CONCLUSIONS: The results suggest that HBV integrations and pathogenic SNPs in non-neoplastic tissues are important for tumorigenesis and different recurrence risk models are needed for patients with low and high degrees of liver fibrosis.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. METHODS: Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. RESULTS: Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. CONCLUSIONS: A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. LAY SUMMARY: HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC.

The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma.

BACKGROUND & AIMS: Although hepatitis B virus (HBV) integration into the human genome has been considered as one of the major causative factors to hepatocarcinogenesis, the underlying mechanism(s) was still elusive. Here we investigate the essential difference(s) of HBV integration between HCC tumor and adjacent non-tumor tissues and explore the factor(s) that determine the oncogenicity of HBV integration. METHODS: 1115 HBV integration sites were collected from four recent studies. Functional annotation analysis of integration targeted host genes (ITGs) was performed using DAVID based on Gene Ontology and KEGG pathway databases. Array-based expression profiles, real-time qPCR and western blot were used to detect the expression of recurrent integration targeted genes (RTGs). The biological consequences of the overexpression of UBXN8 in 8 HCC cell lines were studied in vitro. RESULTS: HBV is prone to integrate in genic regions (exons, introns, and promoters) and gene-dense regions. Functional annotation analysis reveals that, compared to those in adjacent non-tumor tissues, ITGs in HCC tumor tissues were significantly enriched in functional terms related to negative regulation of cell death, transcription regulation, development and differentiation, and cancer related pathways. 32% of the 75 RTGs identified in this analysis expressed abnormally in HCC tissues. UBXN8, one of the RTGs, was identified as a new tumor suppressor candidate which functions in a TP53 dependent manner. CONCLUSIONS: The oncogenicity of HBV integration was determined, to some extend by the function of HBV integration targeted host genes in HCC.

Hepatitis B virus enhancer 1 activates preS1 and preS2 promoters of integrated HBV DNA impairing HBsAg secretion.

Background & Aims: The expression of HBsAg from integrated HBV DNA limits the achievement of functional cure for chronic hepatitis B. Thus, characterising the unique expression and secretion of HBsAg derived from integrated HBV DNA is of clinical significance. Methods: A total of 563 treatment-naive patients and 62 functionally cured patients were enrolled, and HBsAg and HBcAg immunohistochemistry of their liver biopsy tissues was conducted followed by semi-quantitative analysis. Then, based on stratified analysis of HBeAg-positive and -negative patients, long-read RNA sequencing analysis, as well as an in vitro HBV integration model, we explored the HBsAg secretion characteristics of integrated HBV DNA and underlying mechanisms. Results: In contrast to the significantly lower serum HBsAg levels, no significant decrease of intrahepatic HBsAg protein was observed in HBeAg-negative patients, as compared with HBeAg-positive patients. The results of long-read RNA sequencing of liver tissues from patients with chronic HBV infection and in vitro studies using integrated HBV DNA mimicking dslDNA plasmid revealed that, the lower HBsAg secretion efficiency seen in HBeAg-negative patients might be attributed to an increased proportion of preS1 mRNA derived from integrated HBV DNA instead of covalently closed circular DNA. The latter resulted in an increased L-HBsAg proportion and impaired HBsAg secretion. Enhancer 1 (EnhI) in integrated HBV DNA could retarget preS1 (SP1) and preS2 (SP2) promoters to disrupt their transcriptional activity balance. Conclusions: The secretion of HBsAg originating from integrated HBV DNA was impaired. Mechanistically, functional deficiency of core promoter leads to retargeting of EnhI and thus uneven activation of the SP1 over the SP2 promoter, resulting in an increase in the proportion of L-HBsAg. Impact and implications: Integrated hepatitis B virus (HBV) DNA can serve as an important reservoir for HBV surface antigen (HBsAg) expression, and this limits the achievement of a functional cure. This study revealed that secretion efficiency is lower for HBsAg derived from integrated HBV DNA than HBsAg derived from covalently closed circular DNA, as determined by the unique sequence features of integrated HBV DNA. This study can broaden our understanding of the role of HBV integration and shed new light on antiviral strategies to facilitate a functional cure. We believe our results are of great general interest to a broad audience, including patients and patient organisations, the medical community, academia, the life science industry and the public.

Persistently Elevated HBV Viral-Host Junction DNA in Urine as a Biomarker for Hepatocellular Carcinoma Minimum Residual Disease and Recurrence: A Pilot Study.

Hepatitis B virus (HBV)-host junction sequences (HBV-JSs) has been detected in the urine of patients with HBV infection. This study evaluated HBV-JSs as a marker of minimum residual disease (MRD) and tumor recurrence after treatment in HBV-hepatocellular carcinoma (HCC) patients. Archived serial urine DNA from two HBV-HCC with recurrence as confirmed by MRI and four HBV-related cirrhosis (LC) patients were used. Urinary HBV-JSs were identified by an HBV-targeted NGS assay. Quantitative junction-specific PCR assays were developed to investigate dynamic changes of the most abundant urinary HBV-JS. Abundant urinary HBV-JSs were identified in two cases of tumor recurrence. In case 1, a 78-year-old female with HBV- HCC underwent a follow-up MRI following microwave ablation. While MRI results were variable, the unique HBV-JS DNA, HBV-Chr17, steadily increased from initial diagnosis to HCC recurrence. In case 2, a 74-year-old male with HBV-HCC contained two HBV-JS DNA, HBV-Chr11 and HBV-TERT, that steadily increased after initial HCC diagnosis till recurrence. One LC examined had HBV-TERT DNA detected, but transiently in 3.5 years during HCC surveillance. HBV-JS DNA was persistently elevated prior to the diagnosis of recurrent HCC, suggesting the potential of urinary HBV-JS DNA to detect MRD and HCC recurrence after treatment.

Analysis of viral integration reveals new insights of oncogenic mechanism in HBV-infected intrahepatic cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma.

BACKGROUND: Integration of HBV DNA into the human genome could progressively contribute to hepatocarcinogenesis. Both intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC) are known to be associated with HBV infection. However, the integration of HBV and mechanism of HBV-induced carcinogenesis in ICC and CHC remains unclear. METHODS: 41 patients with ICC and 20 patients with CHC were recruited in the study. We conducted HIVID analysis on these 61 samples to identify HBV integration sites in both the tumor tissues and adjacent non-tumor liver tissues. To further explore the effect of HBV integration on gene alteration, we selected paired tumors and adjacent non-tumor liver tissues from 3 ICC and 4 CHC patients for RNA-seq and WGS. RESULTS: We detected 493 HBV integration sites in ICC patients, of which 417 were from tumor samples and 76 were from non-tumor samples. And 246 HBV integration sites were detected in CHC patients, of which 156 were located in the genome of tumor samples and 90 were in non-tumor samples. Recurrent HBV integration events were detected in ICC including TERT, ZMAT4, MET, ANKFN1, PLXNB2, and in CHC like TERT, ALKBH5. Together with our established data of HBV-infected hepatocellular carcinoma, we found that HBV preferentially integrates into the specific regions which may affect the gene expression and regulation in cells and involved in carcinogenesis. We further performed genomic and transcriptomic sequencing of three ICC and four CHC patients, and found that HBV fragments could integrate near some important oncogene like TERT, causing large-scale genome variations on nearby genomic sequences, and at the same time changing the expression level of the oncogenes. CONCLUSION: Comparative analysis demonstrates numerous newly discovered mutational events in ICC and CHC resulting from HBV insertions in the host genome. Our study provides an in-depth biological and clinical insights into HBV-induced ICC and CHC.

DeepHBV: a deep learning model to predict hepatitis B virus (HBV) integration sites.

BACKGROUND: The hepatitis B virus (HBV) is one of the main causes of viral hepatitis and liver cancer. HBV integration is one of the key steps in the virus-promoted malignant transformation. RESULTS: An attention-based deep learning model, DeepHBV, was developed to predict HBV integration sites. By learning local genomic features automatically, DeepHBV was trained and tested using HBV integration site data from the dsVIS database. Initially, DeepHBV showed an AUROC of 0.6363 and an AUPR of 0.5471 for the dataset. The integration of genomic features of repeat peaks and TCGA Pan-Cancer peaks significantly improved model performance, with AUROCs of 0.8378 and 0.9430 and AUPRs of 0.7535 and 0.9310, respectively. The transcription factor binding sites (TFBS) were significantly enriched near the genomic positions that were considered. The binding sites of the AR-halfsite, Arnt, Atf1, bHLHE40, bHLHE41, BMAL1, CLOCK, c-Myc, COUP-TFII, E2A, EBF1, Erra, and Foxo3 were highlighted by DeepHBV in both the dsVIS and VISDB datasets, revealing a novel integration preference for HBV. CONCLUSIONS: DeepHBV is a useful tool for predicting HBV integration sites, revealing novel insights into HBV integration-related carcinogenesis.

Recurrent HBV Integration Targets as Potential Drivers in Hepatocellular Carcinoma.

Chronic hepatitis B virus (HBV) infection is the major etiology of hepatocellular carcinoma (HCC), frequently with HBV integrating into the host genome. HBV integration, found in 85% of HBV-associated HCC (HBV-HCC) tissue samples, has been suggested to be oncogenic. Here, we investigated the potential of HBV-HCC driver identification via the characterization of recurrently targeted genes (RTGs). A total of 18,596 HBV integration sites from our in-house study and others were analyzed. RTGs were identified by applying three criteria: at least two HCC subjects, reported by at least two studies, and the number of reporting studies. A total of 396 RTGs were identified. Among the 28 most frequent RTGs, defined as affected in at least 10 HCC patients, 23 (82%) were associated with carcinogenesis and 5 (18%) had no known function. Available breakpoint positions from the three most frequent RTGs, TERT, MLL4/KMT2B, and PLEKHG4B, were analyzed. Mutual exclusivity of TERT promoter mutation and HBV integration into TERT was observed. We present an RTG consensus through comprehensive analysis to enable the potential identification and discovery of HCC drivers for drug development and disease management.

Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System.

The integration of HBV DNA is one of the carcinogenic mechanisms of HBV. The clearance of HBV integration in hepatocyte is of great significance to cure chronic HBV infection and thereby prevent the occurrence of HBV-related hepatocellular carcinoma (HCC). However, the low throughput of traditional methods, such as Alu-PCR, results in low detecting sensitivity of HBV integration. Although the second-generation sequencing can obtain a large amount of sequencing data, but the sequencing fragments are extremely short, so it cannot fully explore the characteristics of HBV integration. In this study, we used the third-generation sequencing technology owning advantages both in sequencing length and in sequencing depth to analyze the HBV integration characteristics in PLC/PRF/5 cells comprehensively. A total of 4,142,311 cleaning reads was obtained, with an average length of 18,775.6 bp, of which 84 reads were fusion fragments of the HBV DNA and human genome. These 84 fragments located in seven chromosomes, including chr3, chr4, chr8, chr12, chr13, chr16, and chr17. We observed lots of DNA rearrangement both in the human genome and in HBV DNA fragments surrounding the HBV integration site, indicating the genome instability causing by HBV integration. By analyzing HBV integrated fragments of PLC/PRF/5 cells that can potentially express HBsAg, we selected three combinations of sgRNAs targeting the integrated fragments to knock them out with CRISPR/Cas9 system. We found that the sgRNA combinations could significantly decrease the level of HBsAg in the supernatant of PLC/PRF/5 cells, while accelerated cell proliferation. This study proved the effectiveness of third-generation sequencing to detect HBV integration, and provide a potential strategy to reach HBsAg clearance for chronic HBV infection patients, but the knock-out of HBV integration from human genome by CRISPR/Cas9 system may have a potential of carcinogenic risk.

Studies on the correlation between mutation and integration of HBV in hepatocellular carcinoma.

It is well known that both the mutation and integration of the Hepatitis B virus (HBV) are of great significance in liver cancer, however, the relationship between mutation and integration is still unclear. In the present study, sequencing data from 426 previously published samples were analyzed and 5374 specific HBV mutations in cancer tissues were discovered. By comparing integrated samples and non-integrated samples, we found that the integrated samples had higher sample single nucleotide variants (SNVs) positive rates and SNV numbers, as well as higher sample frequency of SNV in the X region of the HBV genome. Samples with HBV integration in the telomerase reverse transcriptase (TERT) region showed higher SNV positive rates and numbers than samples without integration. Moreover, the SNVs (209 [T>G] and 531 [T>C; T>G]) were seen with higher frequency in samples with integration in the TERT region. Our study showed that the occurrence of viral integration events is closely related to the occurrence of SNV, and SNV in the X region should be more directly associated with viral integration. The present study provides an initial exploration of the relationship between HBV mutation and integration to help improve our understanding of the relationship between viral integration and mutation.

HBV DNA integrates into upregulated ZBTB20 in patients with hepatocellular carcinoma.

Hepatitis B virus (HBV) affects the malignant phenotype of hepatocellular carcinoma (HCC). The aim of the present study was to investigate the integration sites of HBV DNA and the expression of the zinc finger protein, zinc finger and BTB domain containing 20 (ZBTB20) in patients with hepatocellular carcinoma. Integration of the HBV gene was detected using a high­throughput sequencing technique based on the HBV­Alu­PCR method. The expression of ZBTB20 was detected by western blotting. HBVX integration sites were detected in ~70% of the HCC tissue samples. HBV­integrated subgene X detection suggested that 67% of the integrated specimens were inserted into the host X gene in a forward direction, 57% in a reverse direction, 24% in both forward and reverse directions, and 38% had two HBV integration sites. A total of 3,320 HBV integration sites were identified, including 1,397 in HCC tissues, 1,205 in paracancerous tissues and 718 in normal liver tissues. HBV integration fragments displayed enrichment in the 200­800 bp region. Additionally, the results suggested that HBV was highly integrated into transmembrane phosphatase with tensin homology, long intergenic non­protein coding RNA (LINC)00618, LOC101929241, ACTR3 pseudogene 5, LINC00999, LOC101928775, deleted in oesophageal cancer 1, LINC00824, EBF transcription factor 2 and ZBTB20 in tumour tissues. Furthermore, the expression of ZBTB20 was upregulated in HCC tissues compared with normal control liver tissues, and was associated with HBV integration frequency. The present study suggested that HBV DNA integrated into upregulated ZBTB20 in patients with hepatocellular carcinoma, which might promote the occurrence and development of HCC. Furthermore, the results of the present study may provide a theoretical basis for the diagnosis and treatment of HCC.