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Is Hepatocellular Carcinoma Associated With Hbv

Hbv Proteins With Pro

Evaluating new CAR constructs for chronic hepatitis B and HBV-associated HCC in the clinic

X protein.

Most HBV-related HCCs show the integration of viral genomic sequences including the HBV X gene . The integrated forms of HBx are frequently rearranged and may show numerous point mutations, deletions or truncation with fusion to cellular DNA but despite this integrated HBx might encode functionally active proteins with trans-activating ability . Although characterization of HBx expression in malignant hepatocytes and infected liver tissues has been often hampered by the difficulty in obtaining valid high-affinity anti-HBX antibodies for immunodetection , there is strong evidence demonstrating that the expression of HBx is maintained through the multistage process of hepatocarcinogenesis from preneoplastic nodules or foci of transformed hepatocytes to HCC .

Through the use of hepatic laser microdissection, it has been shown that HBx sequences deleted in the C-terminal portion are frequently and specifically detectable in HCC tumoral cells and in vitro analysis has demonstrated that these HBX mutants are able to induce hepatocellular transformation. Other HBX genetic variants frequently isolated from HCCs are those showing the amino acid substitutions at positions 130 and 131 of the protein and a recent study has indicated that the selection of these mutations precedes HCC development . However, the possible HBX functional modifications induced by the 130 and 131 amino acidic substitutions have not yet been investigated.

Surface proteins.

Integration Of Hbv Dna Into Host Dna

HBx truncation occurs with HBV integration into host DNA. The 3-end of HBx is the preferred region of HBV genome involved in integration. When HBV integrates, the 3-end of HBx is often deleted. Therefore, HBV integration is an important step in HCC development . The C-terminal region produced by HBx truncation also contributes to HCC development. The C-terminal region has been suggested to be required for ROS production and 8-oxoguanine formation, biomarkers of oxidative stress . The 24 amino acids truncated at the C-terminal end play a role in increasing cell invasiveness and metastasis in HCC through activation of MMP10 by C-Jun signaling . Lastly, C-terminal truncated HBx has been reported to directly regulate miRNA transcription and promote hepatocellular proliferation .

Landscape Of Fusion Events In Hcc

Transcriptome analysis has been widely used to identify gene fusions in human cancers . To gain insight into fusion events in HCC, we aligned transcriptome reads to the hg19 reference genome by using star software . An average of ~ 86 million reads per sample were uniquely mapped to the reference genome . We found that 80% of expressed genes were proteinâcoding genes. We also found that 14% of expressed genes were long noncoding RNA and 5% were pseudogenes annotated in GENCODE . We then used the parameters to identify fusion events.

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Differential Expression Of Mirnas In Hcc

There were 86 deregulated miRNAs in total between HCC tumor and non-tumor tissues by TLDA analysis. In order to select the most significant candidates, miRNAs altered by at least 3-fold in all three pairs of the samples were selected. Under these strict criteria, 11 up-regulated miRNAs and 13 down-regulated miRNAs were identified .2). To validate the miRNA array data, qRT-PCR was performed in 18 pairs of HCC tissues. Four up-regulated 1B) and 4 down-regulated miRNAs showed consistent changes in more than 70% tumorous tissues .1C). Six up-regulated and 7 down-regulated miRNAs showed consistent changes in more than 50% tumorous tissues . No significant difference was found between one up-regulated and two down-regulated miRNAs in paired tumorous tissues .

MiRNA profiles differentiate hepatitis B virus-associated hepatocellular carcinoma from adjacent non-tumor tissues. A: The cluster analysis of down-regulated and up-regulated miRNAs identified in hepatocellular carcinoma . Samples consist of paired samples from three patients B: Validation of Taqman array data using quantitative reverse transcription polymerase chain reaction for up-regulated miRNA C: Validation of Taqman array data using quantitative RT-PCR for down-regulated miRNA. Triplicate assays were done for each RNA sample and the relative amount of each miRNA was normalized to U6 snRNA. aP< 0.01 vs control group.

Hepatitis B Core Related Antigen

Mechanisms of hepatocellular carcinoma progression

Description

The 3.5-kb precore RNA derived from the HBV Pre-C/C gene can act as the template for three viral proteins: HBcAg, HBeAg and a truncated 22 kDa precore protein . The so-called hepatitis B core related antigen consists of these three proteins which share an identical 149 long amino acid sequence. HBcAg forms the viral capsid subunits. HBeAg is synthesized by removing the C-terminal region of p22 and is secreted from infected cells . p22Cr is the pre-core protein with additional post-translational processing at both the N- and C-termini .

Quantification

Hepatitis B core related antigen was first measured by a sensitive enzyme immunoassay that denatures antibodies prior to analysis and therefore can detect HBcAg and HBeAg in anti-HBc or anti-HBe antibody-positive patients . Currently, a newly chemiluminescence enzyme immunoassay with monoclonal antibodies to HBeAg and HBcAg was developed for the detection of HBcrAg. This assay showed the HBcrAg concentration correlates strongly with the HBV DNA concentration over a 5-log range. Moreover, the correlation between HBV load and circulating HBcrAg was not affected in HBeAg-negative patients nor those with pre-core mutations . Particularly for patients under NA treatment, the HBcrAg assay could be a sensitive and clinically useful surrogate marker of intrahepatic HBV cccDNA levels .

Molecular Association With HCC

Performance as a Predictor of HCC

HCC occurrence
HCC recurrence

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Hcc And Ifn Maintenance Therapy

Because IFN has anti-fibrotic and anti-carcinogenic properties the use of IFN as maintenance therapy in patients who were non-responders to prior treatment provided the rationale for several clinical trials. In a randomized trial of 53 patients Shiffman et al. found that maintenance IFN therapy with 3 MU three times a week reduced fibrosis stage with a decrease in stage from 2.5 at baseline to 1.7 after 30 months of therapy compared to essentially no change in fibrosis score in the group that stopped IFN . The study was not designed to detect significant differences in rates of HCC between the groups. In a clinical trial that included 90 patients randomized to IFN-alpha 6 million units for 12 to 24 weeks or symptomatic treatment , followed for 2 to 7 years, 2 patients in the IFN group and 17 patients in the control group developed HCC, P=0.002 . Data from these studies provided rationale to consider prescribing IFN maintenance therapy in HCV patients with advanced fibrosis who were non-responders.

Patients And Clinical Samples

Two typical multiâfocal hepatitis B virus âHCC patients were enrolled for the study, which was previously reported . The raw RNAâseq data were deposited at the European Genomeâphenome Archive with accession number EGAS00001000372.

The distribution of fusion events in two multifocal HCC patients*. Ideograph of two multifocal HCC patients. The number of fusion events identified in each sample. The ratio of fusion events between different chromosomes and in the same chromosome. The type of two fusion genes involving fusion events. The clusters of fusions appear at least two samples. *PI, Patient I PIâP, primary HCC of Patient I PIâM, intrahepatic metastases of Patient I PIâV, portal vein tumor thrombus of Patient I PIâN, noncancerous liver of Patient I PII, Patient II PIIâR, right lobes HCC of Patient II PIIâL, left lobes HCC of Patient II PIIâN, noncancerous liver of Patient II.

To identify reliable recurrent fusions, we collected publicly available RNAâseq data of HCC using the keywords âhepatocellular carcinomaâ, âHCCâ, âliverâ, âRNAâseqâ, ânextâgeneration sequencingâ, and âhigh through outputâ in the GEO database and NCBI SRA database. Finally, we obtained four available RNAâseq datasets .

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Demographic And Clinical Characteristics

A total of 107 HCC patients were recruited between December 2012 and August 2015. The mean age was 46years , range 1890years. The majority were male 78% and 55% were Black African. Twenty two percent of cases were HIV infected, of whom 91% had evidence of current or past HBV infection. There was insufficient specimen for HIV testing for seven cases. Table shows the demographic and clinical characteristics of the 100 patients with known HIV status.

Table 1 Demographic and clinical characteristics of HCC cases by known HIV status

The median serum AFP was 6176g/L . Of 98 cases, 69 had serum AFP greater than 400g/L. The majority of cases were diagnosed using imaging modalities in combination with clinical laboratory data. Contrast-enhanced computed tomography scan was done in 77 of 107. Liver histological examination was done on 31/100 of cases on whom information was provided .

Multivariable Binary Logistic Regression Analysis Of Independent Influencing Factors Of Mvi In Patients With Positive Seral Hbsag

CAR-T therapy, a novel promising treatment option for chronic hepatitis B and HBV-associated HCC

The multivariable logistic regression method was adopted to analyze predictive factors of MVI in patients with positive seral HBsAg. As shown in Table 2, detectable serum HBV DNA load was an independent risk factor for microvascular invasion of hepatocellular carcinoma in multivariable regression analysis. Antiâhepatitis B virus treatment for more than six months was an independent protective factor in microvascular invasion of hepatocellular carcinoma. In addition, tumor-related factors, including male patients , AFP levels > 7.02ng/ml , tumor size > 3cm , Edmondson-Steiner histological grade III/IV , tumor encapsulation incompleteness , and presence of satellite nodules were significantly and independently related to the formation of microvessel invasion of cell carcinoma.

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Fluorescent Mirna Ish And Immunofluorescence Staining

The cells were fixed with 4% formaldehyde followed by ISH. The hybridization probes used were: 200 nM of 5â²-DIG LNA-modified-miR-224 and 5â²-DIG LNA-modified-let-7a . The miRNA signal was amplified by TSA Plus Cyanine 5 system . Anti-LC3 and anti-Lamp1 antibodies were incubated with miRNA solution for 1 hour. Alexa Flour 488 was added for another 1 hour. Fluorescent change of the cells was investigated under a confocal microscope .

Hbs/hbx Sequencing And Genotyping

Viral DNA was amplified by PCR techniques using the Expand High Fidelity PCR System , according to manufacturer’s instructions, and oligonucleotide primers specific for HBV DNA sequences flanking the S and X genomic regions. In several cases, HBV full-length genomes were amplified by a nested PCR approach using two different primer pairs to obtain the amplification of HBS and HBX fragment., Each purified PCR product was sequenced using Applied Biosystems BigDye terminator sequencing chemistry and run on an ABI Prism 3500Genetic Analyzer according to manufacturers instructions. The sequences were analysed with Sequencher V.4.7 software . HBV genotyping was then performed according to the sequence of HBS by multiple sequence alignments using the Clustal Omega .,

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Hepatitis B E Antigen

Hepatitis B e antigen is the major product of the pre C-C gene and is a secreted, non-particulate form of the viral nucleocapsid . The HBeAg is regarded as an accessory protein of HBV and is not required for viral replication or infection. The natural history of CHB is typically divided into two phases: HBeAg-positive and HBeAg-negative. Patients with HBeAg-positive CHB have significant downregulation of TLR2 on the surface of monocytes circulating in the peripheral blood and also on hepatocytes and Kupffer cells in their livers. In contrast, HBeAg-negative infection results in upregulation of TLR2 and cytokine expression. Exposure to HBeAg is able to downregulate TLR2 expression but not TLR4 expression on monocytes and results in downstream effects on cytokine production . This inhibition of cytokine production is mediated through TLR signaling pathways. The proposed interaction between the precore protein and the TLR2 pathway may broaden its immunological role in hepatitis B pathogenesis .

Inflammatory Factor And Ros

Natural history of HBV infections. HCC, hepatocellular carcinoma  NK ...

In chronic hepatitis B , liver injury is thought to be mediated by host immune responses, including HBV-specific T cells as well as infiltrating neutrophils, natural killer cells and non-antigen-specific lymphocytes that are attracted by the production of non-specific chemokines . The release of inflammatory cytokines and chemokines also favors hepatocyte proliferation. Although virus-specific lymphocytes are readily detectable in inflammatory lesions in the liver, they are often not sufficient to clear virus infection. Many virus non-specific NK cells, NK T cells and polymorphonuclear leukocytes are also found in these lesions, mediating hepatocellular damage, but they are not effective in virus clearance .

Moreover, oxidative stress induced by inflammation and by accumulation of viral surface proteins in the endoplasmic reticulum leads to increased levels of intracellular reactive oxygen species , in turn enhancing genomic instability and allowing the accumulation of genetic abnormalities and gene mutations. More specifically, ROS may trigger the activation of intracellular signaling pathways such as the mitogen-activated protein kinase and nuclear factor-B pathways that play an important role in hepatic carcinogenesis .

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Key Genes Identification Of The Selected Module

To identify hub/key genes of the selected module, the Cytoscape software was utilized to construct of the network of the module genes. The important nodes were predicted and explored by cytoHubba plugin . The topological algorithms of cytoHubba consist of Degree, Edge Percolated Component , Maximum Neighborhood Component , Density of Maximum Neighborhood Component , Maximal Clique Centrality and centralities based on shortest paths, such as Bottleneck , EcCentricity, Closeness, Radiality, Betweenness, and Stress, were applied to get respective top 20 ranked genes set. Then, the intersection of nine top 20 ranked genes sets were identified as the key genes.

Implementation Of Hcc Screening

In the United States, several potential barriers exist for effective HCC screening in patients with chronic HBV infection have been identified, including undiagnosed chronic HBV infection, unknown hepatic fibrosis stage, lack of clinician awareness of HCC screening guidelines, scheduling logistics, and cost of screening. Current guidelines recommend HCC screening in the context of chronic hepatitis B for patients with a variety of risk factors that include but are not exclusive to cirrhosis. It is up to the clinician to first identify these patients and then assess their risk based on these multiple features that can be challenging in non-expert settings.

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Immunotherapeutic Interventions For Chb

The two therapeutic approaches available for the suppression of HBV replication include antiviral agents and immune-based therapies . However, the rates of HBsAg loss and seroconversion to HBsAb are very low , and drug resistance or poor response rate are accompanied by numerous side effects. The goal of therapy is to improve quality of life and survival by preventing progression to cirrhosis, decompensated cirrhosis, HCC and death through sustained suppression of HBV replication . Various therapeutic interventions have been tried as adjuvants to inhibit HBV replication, such as immunotherapeutic interventions, in the treatment of CHB .

Table 2

Strategies designed to increase the efficacy of immune-based therapy

A low frequency of T cell immune responses to HBsAg was found in Chinese subjects with HBsAg seroclearance following antiviral therapy, which indicated that HBsAg-specific immune responses were not responsible for HBsAg seroclearance . Robust anti-core T cell responses were found in patients with reduced HBsAg serum levels, suggesting that core-specific T cell responses mediated a protective effect in HBV control . These results suggested that expansion of HBV-specific T cells in vitro through HBcAg stimulation may be one immunotherapeutic intervention.

Mirna In Situ Hybridization

Antiviral Therapy Reduces Risk of Hepatocellular Carcinoma in Patients With Hepatitis C…

MiRNA ISH was performed as described.13 Briefly, slides were hybridized with 200 nM of 5â²-digoxigenin LNA-modified-miR-224 using the IsHyb In Situ Hybridization kit . After washing and blocking, slides were incubated with anti-DIG-horseradish peroxidase for 1 hour. MiR-224 signal was amplified by the TSA Plus DNP system and then incubated with anti-DNP-HRP for 1 hour. Slides were treated with an AEC solution and hematoxylin.

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Acute Immune Tolerant Phase

In the acute phase of infection with HBV, the immune tolerant phase is HBeAg with high viral loads, normal serum alanine aminotransferase , and near normal liver histology . When HBV is acquired in adulthood, this phase is very short however, perinatal and early childhood infection lead to a long immune-tolerant phase . The risk of progression to chronic carrier state differs greatly between those infected perinatally and as an adult . At the current time, antiviral treatment is not recommended during the immune-tolerant phase but rather for the immune clearance phase. Interestingly, some recent reports have shown evidence of immune reactivity during the immune-tolerant stage . As was presented by Zoulim and Mason, there is an argument to consider earlier treatment of CHB in order to prevent HCC .

Evidence Supporting Hcc Screening In Chronic Hepatitis B

The most well-known clinical study to support HCC screening is a cluster-randomized, controlled trial conducted in China that assessed the impact of HCC screening on HCC-related mortality. This study enrolled 18,816 HBV individuals with chronic HBV aged 35 to 59 from 300 factories, businesses and schools in urban Shanghai. Half of these 300 units were randomized to screening using serum alfa-fetoprotein , with cutoff value 20 ng/mL, and ultrasound every 6 months the other arm underwent usual care without screening and were not actively followed. The screening group had only a 58% compliance with screening, but notably had HCC diagnosed at an earlier stage and this group had a lower HCC-related 5-year mortality rate compared with the control group . This study, however, had some methodologic limitations, conferring a high risk for bias. A number of observational cohort studies have since suggested a survival benefit with screening but the quality of this evidence is limited by selection, lead time and length-time biases. There are also several observational trials and reviews involving patients with cirrhosis that have shown surveillance for HCC was associated with earlier-stage tumor detection and improved survival. Despite the suboptimal quality of evidence supporting HCC screening, it is currently the standard of practice and it is unlikely that a rigorously conducted randomized study would take place given the widespread acceptance of screening at this time.

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Significance Of This Study

What is already known on this subject?

  • Hepatocellular carcinoma is the fifth most common cancer and hepatitis B is the most common underlying cause of HCC worldwide.

  • HCC is a group of heterogeneous tumours with several molecular subclasses.

  • HBV infection synergises with exposure to aflatoxin B1 to promote HCC with R249S TP53 mutation in subtropical countries.

What are the new findings?

  • HBX inactivating mutations are selected in HCC tissues suggesting specific pressure of selection during hepatocarcinogenesis.

  • In patients with a low number of HBV DNA copies per cell in the liver, we identified additional risk factor suggesting a cooperative effect for HBV-induced carcinogenesis.

  • TP53 mutations demonstrate different prognostic consequences according to the viral status relation with poor prognosis was restricted to HBV-infected resected patients.

  • HBV-related tumours demonstrate more frequent progenitor phenotype than non-HBV HCC with an upregulation of genes involved in cell-cycle regulation and encoding oncofetal/progenitor proteins.

How might it impact on clinical practice in the foreseeable future?

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