HBV-related Hepatocellular Carcinoma Cases Research Articles

Progression of chronic liver disease to hepatocellular carcinoma: implications for surveillance and management

BackgroundCurrent opinion holds that hepatocellular carcinoma (HCC) arises as a stepwise progression from chronic liver disease (CLD) to cirrhosis and then to HCC. However, some HCCs may develop in a non-cirrhotic liver, raising uncertainty about their origin.MethodsWe analysed a prospectively accrued cohort of 2592 CLD patients (median follow-up = 13 years) with no prior evidence of liver cirrhosis. To track the progression of liver fibrosis prior to HCC diagnosis, we examined serial measurements of Fib-4 (an index of liver fibrosis). We also evaluated fibrosis progression in response to antiviral treatment in patients with hepatitis C (HCV) and hepatitis B (HBV). Recognising the limitations of serologic fibrosis assessment, we correlated Fib-4 and fibrosis histology within this cohort.ResultsAmong HCC patients, 28% had no indication of cirrhosis prior to HCC diagnosis. Only 31% of HBV-related HCC cases followed the cirrhotic pathway. HCV patients who achieved sustained virological response (SVR) developed cirrhosis approximately 7 years before HCC diagnosis.ConclusionsOur analysis challenges the notion of cirrhosis as an obligatory stage of HCC development in CLD patients. We affirm HBV’s direct oncogenic potential and find that achieving SVR does not universally prevent HCC development. Our findings have major implications for HCC surveillance.

SOCS5-RBMX stimulates SREBP1-mediated lipogenesis to promote metastasis in steatotic HCC with HBV-related cirrhosis

Abnormal lipid metabolism promotes hepatocellular carcinoma (HCC) progression, which engenders therapeutic difficulties owing to unclear mechanisms of the phenomenon. We precisely described a special steatotic HCC subtype with HBV-related cirrhosis and probed its drivers. Hematoxylin-eosin (HE) staining of 245 HCC samples revealed a special HCC subtype (41 cases) characterized by HBV-related cirrhosis and intratumoral steatosis without fatty liver background, defined as steatotic HCC with HBV-related cirrhosis (SBC-HCC). SBC-HCC exhibits a larger tumor volume and worse prognosis than non-SBC-HCC. Screening for driver genes promoting fatty acid (FA) biosynthesis in the Gao’s HBV-related cirrhosis HCC cases and GSE121248’ HBV-related HCC cases revealed that high expression of SOCS5 predicts increased FA synthesis and that SOCS5 is upregulated in SBC-HCC. Through proteomics, metabolomics, and both in vivo and in vitro experiments, we demonstrated that SOCS5 induces lipid accumulation to promote HCC metastasis. Mechanistically, through co-IP and GST-pulldown experiments, we found that the SOCS5-SH2 domain, especially the amino acids Y413 and D443, act as critical binding sites for the RBMX-RRM domain. SOCS5-RBMX costimulates the promoter of SREBP1, inducing de novo lipogenesis, while mutations in the SH2 domain, Y413, and D443 reverse this effect. These findings precisely identified SBC-HCC as a special steatotic HCC subtype and highlighted a new mechanism by which SOCS5 promotes SBC-HCC metastasis.

Abstract 3013: The immunogenetic basis of hepatitis B virus-related hepatocellular carcinoma

Abstract Background: The reported associations of genetic variants within the human leukocyte antigen (HLA) region with hepatis B virus (HBV)-related hepatocellular carcinoma (HCC) are not consistent, and the structure-function patterns involved in this disease remain unclear. Methods: Utilizing genotype data from 706 HBV-related HCC cases and 6197 chronic HBV carriers without HCC collected from three resources in Taiwan, we imputed the classical HLA alleles at 2-field resolution and analyzed the molecular landscape of HLA complexes by deeply dissecting their genotypic configurations, functional divergence, and HBV-antigen binding capabilities in associations with the HCC risk. Logistic regression models were used, and all models were adjusted for sex, age, and population structure using the top 10 principal components. We also tested the underlying biological implications of our findings by investigating the HLA binding affinities and HLA effect on control of viral replication (i.e, HBV DNA load), viral population diversity (i.e, quasispecies), and inflammation (i.e, chemokines, cytokines, and soluble programmed cell death 1 [sPD-1]-associated T cell exhaustion). Results: We found high accuracy (≥95% concordance) of HLA imputation at all HLA class I and class II alleles. The strongest evidence for an association with HCC was observed for HLA-DQB1*03:01 (adjusted OR= 1.35 in an additive genetic model, P=1.3×10-6). Three amino acids at specific positions within or near the peptide-binding groove of DQB1 molecules might explain the observed allele association, which leads to loss of binding HBV nucleocapsid protein (i.e., low binding affinity predicted by NetMHCIIpan 4.0 for HLA-DQB1*03:01). HLA-DQB1*03:01 was significantly associated with a higher HBV DNA load (P= 4.3×10-4) and an increased level of serum sPD-1 (P=6.8×10-4), but not with increased levels of chemokines, cytokines, and viral complexity (i.e., Shannon entropy for HBV quasispecies, all P values>0.05). Analysis of HLA zygosity confirmed that HLA-DQB1 heterozygosity was associated with a decreased risk of HCC (adjusted OR=0.77, P= 0.015). Analysis of HLA evolutionary divergence (HED) showed that high HED at HLA-DRB1 loci was marginally associated with a decreased risk of HCC among HLA-DQB1*03:01 non-carriers (P=0.05). Conclusion: Our data shed light on the immunogenetic risk associated with HBV-related HCC. The effect of HLA-DQB1*03:01 could be partly explained by its low binding affinity with HBV nucleocapsid antigen, and/or its effect on viral load control and sPD-1, but not by its control on other inflammation markers (chemokines and cytokines) and viral population diversity. The association between HLA-DQB1 heterozygosity and HCC risk could be independent of the HLA-DQB1*03:01 effect, which indicates underlying mechanisms related to increased immunosurveillance in clearing potential viral antigens or neoantigens. Citation Format: Zhiwei Liu, Chih-Jen Huang, Yu-Han Huang, Mei-Hung Pan, Mei-Hsuan Lee, Mathias Viard, Allan Hildesheim, Ruth M. Pfeiffer, Mary Carrington, Chien-Jen Chen, Tobias L. Lenz, Hwai-I Yang. The immunogenetic basis of hepatitis B virus-related hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3013.

Identification of Key Genes in the HBV-Related HCC Immune Microenvironment Using Integrated Bioinformatics Analysis.

Purpose Hepatocellular carcinoma (HCC) has poor prognosis and high mortality among gastrointestinal tumors because of its insidious onset and strong invasiveness. However, there was little understanding of their pathogenesis. The purpose of this study was to use bioinformatics analysis to identify genes associated with the immune microenvironment in HBV-related HCC and to develop new therapeutic targets to prevent and treat cancer. Methods RNA-seq data of HBV-related HCC cases were downloaded from TCGA-LIHC database. ESTIMATE and Deseq2 algorithms were used to screen out differentially expressed genes (DEGs). WGCNA was used to construct gene coexpression networks. In key modules, functional enrichment analysis was performed. Protein-protein interaction (PPI) was used to screen hub genes, and survival analysis was conducted to assess their prognostic significance. Following, we search for key genes differentially expressed between cancerous and paracancerous tissues in GSE136247 and GSE121248 datasets. Reveal the potential links between key genes in immune infiltration by using TIMER. Finally, in TCGA-LIHC database, integration of key genes with clinical data were used to further validate their correlation with prognosis. Results In the cohort of HBV-related HCC patients, immune/stromal/ESTIMATE scores were not significantly associated with patient prognosis. After bioinformatics analysis, screening out five key genes was significantly related to the prognosis of HBV-related HCC. Downregulation of SLAMF1 and TRAF3IP3 suggested poor prognosis and was related to a variety of immune cell infiltration. Furthermore, compared with adjacent nontumor tissues, TRAF3IP3 and SLAMF1 were highly expressed in tumor tissues and were linked to tumor recurrences. Conclusion In conclusion, SLAMF1 and TRAF3IP3 were identified with higher expression in tumor tissues and associated with tumor recurrence. It will be a new research direction of tumor progress and treatment.

Fine Mapping of the MHC Region Identifies Novel Variants Associated with HBV-Related Hepatocellular Carcinoma in Han Chinese.

IntroductionGenome-wide association studies identified susceptibility loci in the major histocompatibility complex region for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). However, the causal variants underlying HBV-related HCC pathogenesis remain elusive.MethodsWith a total of 1,161 HBV-related HCC cases and 1,353 chronic HBV carriers without HCC, we imputed human leukocyte antigen (HLA) variants based on a Chinese HLA reference panel and evaluated the associations of these variants with the risk of HBV-related HCC. Conditional analyses were used to identify independent signals associated with the risk of HBV-related HCC (P false-discovery rate (FDR) <0.20). A total of 14,930 variants within the MHC region were genotyped or imputed.ResultsWe identified two variants, rs114401688 (P = 1.05 × 10−6, PFDR = 2.43 × 10−3) and rs115126566 (P = 9.04 × 10−5, PFDR = 1.77 × 10−1), that are independently associated with the risk of HBV-related HCC. Single nucleotide polymorphism (SNP) rs114401688 is in linkage disequilibrium with a previously reported SNP rs9275319. In the current study, we found that its association with HCC could be explained by HLA-DQB1*04 and HLA-DRB1*04. SNP rs115126566 is a novel risk variant and may function by regulating transcriptions of HLA-DPA1/DPB1 through enhancer-mediated mechanisms. HLA zygosity analysis showed that homozygosity at HLA-DQB1 gene is suggestively associated with a higher risk of HCC (P = 0.10) and the risk was more pronounced in the older age group (age ≥50, P = 0.03).DiscussionOur findings further the understanding of the genetic basis for HBV-related HCC predisposition in chronic HBV carriers.

Effects of genetic polymorphisms in INTS10 and their interaction with environmental factors on progression from persistent HBV infection to hepatocellular carcinoma.

Genome-wide association study recently identified a novel antiviral gene INTS10 (index rs7000921) in suppression of hepatitis B virus (HBV) replication. However, data were lacking on single nucleotide polymorphisms (SNPs) of INTS10 in the context of hepatocellular carcinoma (HCC) induced by HBV infection. Herein, we conducted a case-control study, including 737 HBV-related HCC cases and 750 persistently HBV-infected controls, to investigate the effect of INTS10 SNPs and their gene-environment interactions on HBV-related HCC. In multivariate analysis, the CT genotype of rs7000921 conferred a decreased risk of HBV-related HCC compared to the TT genotype (adjusted odds ratio [OR] = 0.79, 95% confidence interval [CI] = 0.64-0.98, p for permutation test = .038). Among the 12 tagSNPs, the rs4268139 yielded a borderline significant association with disease risk under the additive model (adjusted OR = 0.80, 95% CI = 0.63-1.00, p for permutation test = .061). Random forest model further suggested the rs7000921 and rs7822495 as the two-top ranked important SNPs, and thus a weighted genetic risk score (wGRS) was generated from these two SNPs plus rs4268139. The highest tertile of wGRS was associated with an increased risk, with an adjusted OR of 1.36 (95% CI = 1.05-1.75, p for permutation test = .016) compared to the lowest wGRS. Furthermore, an additive interaction was seen between wGRS and drinking (attributable proportion due to interaction [AP] = 0.21, 95% CI = 0.02-0.43, p = .016). The additive interaction between wGRS and smoking approached near significance (AP = 0.15, 95% CI = 0.00-0.32, p = .045). INTS10 polymorphisms may contribute to the progression from HBV infection to HCC. More importantly, INTS10 polymorphisms interact with drinking and smoking to affect the progression.

Urine miR-93-5p is a promising biomarker for early detection of HBV-related hepatocellular carcinoma

IntroductionThe mortality rate of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC)continues to increase because sensitive, early and readily available diagnostic tools are lacking. To address this problem, we aimed to identify diagnosticbio markers to be used for early detection of HCC. Materials and methodsmiR-93–5p was selected as a candidate biomarker based on the analyses of relevant Gene Expression Omnibus (GEO) datasets; it was validated using qPCR to quantify its expression levels in tissue, plasma and saliva sample sets. ResultsmiR-93–5p was significantly upregulated in HBV-related HCC tissue. Notably, miR-93–5p in plasma and urine was also significantly increased in patients with early HBV-related HCC. The expression of miR-93–5p was significantly and positively correlated in pairwise comparisons of samples (tissue vs. plasma, tissue vs. urine, plasma vs. urine). Moreover, after curative hepatectomy,miR-93–5p in plasma and urine decreased significantly over one month after the curative hepatectomy and returned to normal levels. Furthermore, receiver operating characteristic (ROC) analysis indicated that both plasma and urine miR-39–5p could detect be used to early, advanced and overall HBV-related HCC cases with more than 85% sensitivities and 93% of specificities. Finally, urine miR-93–5p could be used to predict progress-free survival for early HCC patients who received curative hepatectomy and overall survival for advanced HCC patients without curative treatments. ConclusionsPlasma and urine miR-93–5p show great promise as potential novel biomarkers for early detection of HBV-related HCC. Moreover, urine miR-93–5p could be used to predict the prognosis of patients with HBV-related HCC.

Nonreceptor protein tyrosine phosphatases (NRPTPs) gene family associates with the risk of hepatocellular carcinoma in a Chinese hepatitis B virus-related subjects.

Nonreceptor protein tyrosine phosphatases (NRPTPs) are reported to be associated with several human cancers, but their roles in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remain unclear. Here, we integrated bioinformatics tools, population association analyses, and biological assays to systematically screen for potentially functional single nucleotide polymorphisms (SNPs) within the 17 NRPTPs genes and evaluate the effects of candidate SNPs on the risk of HCC or persistent HBV infection. A total of 790 HBV-related HCC cases and 1454 cancer-free controls were enrolled. Controls included 711 HBV persistent carriers and 743 spontaneously recovered subjects. Results demonstrated that PTPN4 rs9308777 (odds ratio [OR] = 1.25, 95% confidence interval [CI] = 1.06-1.49, P = .009) and PTPN12 rs350050 (OR = 1.26, 95% CI = 1.10-1.45, P = .001), were significantly associated with HCC risk, but not with persistent HBV infection risk. The cumulative risk effect of these two SNPs was more significantly increased the susceptibility to HCC (OR = 1.27, 95% CI = 1.14-1.41, P = 2.40 × 10-5 ). Subsequent biological assays further revealed the potential pathogenesis that PTPN4 rs9308777 might decrease the gene expression, and PTPN12 rs3750050 might promote cell proliferation by attenuating PTPN12's inhibitory activity on EGFR/ERK pathway. In summary, our integrative study highlights that PTPN4 and PTPN12 are significantly associated with HBV-related HCC risk, but do not influence persistent HBV infection. These findings shed light on the importance of the synergistic effects of regulatory and missense variants on the risk for HCC, and provide data to support personalized cancer medicine in the future.

PNPLA3 rs738409 is not associated with the risk of hepatocellular carcinoma and persistent infection of hepatitis B virus (HBV) in HBV-related subjects: A case-control study and meta-analysis on Asians.

The association between rs738409 (C>G, I148M) with patatin-like phospholipase domain-containing 3 (PNPLA3) gene and the risk of hepatocellular carcinoma (HCC) was controversial in different ethnic populations. Our study aimed to explore the effect of PNPLA3 rs738409 on the risk of HCC and persistent infection of Hepatitis B virus (HBV) in a Chinese HBV-related population, and further evaluate its role in HCC risk among Asians. First, we performed a case-control study by recruiting 786 HBV-related HCC cases, 695 HBV persistent carriers and 719 HBV natural clearance subjects. PNPLA3 rs738409 was genotyped by MassARRAY platform. Second, we conducted a systematic review and meta-analysis on Asians to further validate our results. Our case-control study demonstrated that PNPLA3 rs738409 was not associated with HCC risk or persistent HBV infection (All P>0.05). The subsequent meta-analysis included 13 Asian studies with 9,802 subjects. Results showed that PNPLA3 rs738409 might increase HCC risk among healthy subjects (pooled odds ratio [OR]=1.47, 95% confidence interval [CI]=1.11-1.95), but it had no influence on the development of HCC among HBV-related subjects (pooled OR=1.07, 95%CI=0.89-1.30). Our case-control study highlights that PNPLA3 rs738409 is probably not associated with the risk of HCC or persistent HBV infection in a Chinese HBV-related population. Besides, our systematic review and meta-analysis on Asians further suggest that PNPLA3 rs738409 may confer an increased risk of HCC among healthy people, but contribute little to the development of HCC among HBV-related subjects. Future studies are required to confirm these results.

Significance of tumor-infiltrating immunocytes for predicting prognosis of hepatitis B virus-related hepatocellular carcinoma.

BACKGROUNDHepatitis B virus (HBV) has been recognized as a leading cause of hepatocellular carcinoma (HCC). Numerous reports suggest that immune infiltration can predict the prognosis of HCC. Nonetheless, no creditable markers for prognosis of HBV-related HCC have been established by systematically assessing the immune-related markers based on tumor transcriptomes.AIMTo establish an immune-related marker based on the cell compositions of immune infiltrate obtained based on tumor transcriptomes, so as to enhance the prediction accuracy of HBV-related HCC prognosis.METHODSRNA expression patterns as well as the relevant clinical data of HCC patients were obtained from The Cancer Genome Atlas. Twenty-two immunocyte fraction types were estimated by cell type identification by estimating relative subsets of RNA transcripts. Subsequently, the least absolute shrinkage and selection operator (LASSO) Cox regression model was employed to construct an immunoscore based on the immunocyte fraction types. Afterwards, the receiver operating characteristic (ROC) curve, Kaplan-Meier, and multivariate Cox analyses were performed. Additionally, a nomogram for prognosis that integrated the immunoscore as well as the clinical features was established. Meanwhile, the correlation of immunoscore with immune genes was also detected, and gene set enrichment analysis (GSEA) of the immunoscore was conducted.RESULTSA total of 22 immunocyte fraction types were predicted and compared among the tumor as well as non-tumor samples. An immunoscore was constructed through adopting the LASSO model, which contained eight immunocyte fraction types. Meanwhile, the areas under the ROC curves for the immunoscore biomarker prognostic model were 0.971, 0.912, and 0.975 for 1-, 3-, and 5-year overall survival (OS), respectively. Difference in OS between the high-immunoscore group and the low-immunoscore group was statistically significant [hazard ratio (HR) = 66.007, 95% confidence interval (CI): 8.361-521.105; P < 0.0001]. Moreover, multivariable analysis showed that the immunoscore was an independent factor for predicting the prognosis (HR = 2.997, 95%CI: 1.737-5.170). A nomogram was established, and the C-index was 0.757 (95%CI: 0.648-0.866). The immunoscore showed a significant negative correlation with the expression of PD-1 (P = 0.024), PD-L1 (P = 0.026), PD-L2 (P = 0.029), and CD27 (P = 0.033). Eight pathways were confirmed by GSEA.CONCLUSIONThe established immunoscore can potentially serve as a candidate marker to estimate the OS for HBV-related HCC cases.

Decreasing role of HCV and HBV infections as aetiological factors of hepatocellular carcinoma in Italy.

The epidemiology of hepatocellular carcinoma (HCC) is characterized by a dynamical temporal trend of well-established and emerging risk factors. We evaluated the temporal trend of aetiological factors of HCC over the last two decades in Italy. HCC cases were recruited from two previously published national studies in 1996 and in 2008 and HCC cases were also enlisted from two national surveys in 2001 and in 2014 enrolling consecutive subjects with chronic liver disease (CLD) referring to more than 80 liver units scattered all over the country for a 6-month period. Out of the 9997 subjects with CLD recruited in 2001 and the 2408 recruited in 2014, 3.3% and 5.7% (P < 0.001), respectively, had HCC. The temporal trend of HBsAg -/HCV + HCC cases significantly linearly decreased from 71.1% in 1996 to 57.2% in 2014 (P < 0.001). Conversely, that of virus-negative cases significantly linearly increased from 12.1% to 28.3% (P < 0.001). The proportion of HBV-related HCC cases showed a steady low rate, reflecting the reduced endemicity of the infection in Italy. The proportion of HCC with compensated cirrhosis (i.e., Child-Pugh A) linearly increased over time from 55.6% in 1996 to 76.0% in 2014 (P < 0.001) reflecting the growing effectiveness of semi-annual ultrasound surveillance for early detection of HCC. In conclusion, with decreasing viral aetiology, an overall decrease in the incidence of HCC might be expected in the future. The proportion of metabolic diseases is conversely increasing being considered as an aetiology. The growing prevalence of metabolic disorders in the general population may further increase this trend in the years to come.

Vitamin D Receptor Fok I Polymorphism and Risk of Hepatocellular Carcinoma in HBV-Infected Patients

Context: We conducted a meta-analysis to evaluate the role of the vitamin D receptor (VDR) polymorphism in the risk of hepatocellular carcinoma (HCC) in patients infected with hepatitis B virus (HBV). Objective: To develop new preventions, mechanisms, and therapies for HCC, it is important to investigate the underlying causes of HCC. Recently, there has been increasing attention to the relationship between VDR polymorphism and the risk of HBV-related HCC, but no specific conclusion has been made. Data Sources: PubMed/Medline, EMBASE, Cochrane Library, Chinese National Knowledge Infrastructure, VIP Database for Chinese Technical Periodicals, Wanfang Data, and SINOMED databases were searched until August 2018. Studies reporting VDR polymorphism and HBV-related HCC risks were included and the quality was assessed by Newcastle-Ottawa scale (NOS). Odds ratio (OR) and 95% confidence interval (CI) were calculated to compare the pooled data between HCC risks and different genotypes, such as FF, Ff, F, f, and ff, with each other. Results: Three case-control studies with totally 728 HBV-related HCC cases and 920 HBV controls were included. When comparing HBV-related HCC cases with HBV controls, our data showed that all genotypes of Fok I polymorphism significantly increased the risk of HCC in the overall population (ff vs. FF: OR = 1.816, 95% CI = 1.161 - 2.841, P = 0.0009; Ff vs. FF: OR = 1.315, 95% CI = 1.037 - 1.667, P = 0.024; ff/Ff vs. FF, OR = 1.504, 95% CI = 1.206 - 1.876, P < 0.001; ff vs. FF/Ff: OR = 1.591, 95% CI = 1.270 - 1.992, P < 0.001; ff vs. Ff: OR = 1.435, 95% CI = 1.127 - 1.827, P = 0.003; f vs. F: OR = 1.375, 95% CI = 1.075 - 1.759). Conclusion: The results of our analysis suggest the “f” allele of Fok I polymorphism might be a risk factor for HCC in HBV-infected patients and it could be a predictive factor to screen HCC in HBV-infected patients. However, more accurate analyses and larger studies are needed.

Iron metabolism disorders in patients with hepatitis B-related liver diseases.

AIMTo investigate the relationship between levels of iron metabolism markers and hepatitis B virus (HBV)-related chronic liver diseases.METHODSThis case-control study with 318 participants included 78 cases of chronic hepatitis B, 85 cases of HBV-related liver cirrhosis, 77 cases of HBV-related hepatocellular carcinoma, and 78 healthy controls. Markers of iron metabolism were detected in participants. Hematological and biochemical parameters and HBV-DNA were assessed. Child-Pugh grade and Barcelona Clinic Liver Cancer stage were determined for each hepatocellular carcinoma patient. Perls’ staining was performed on liver sections. The SPSS program was used for all statistical analyses, and statistical significance was considered if a P-value < 0.05.RESULTSSignificantly higher serum ferritin and lower serum hepcidin levels were detected in all groups of HBV-infected patients compared with healthy controls. Serum iron, total iron binding capacity, and serum transferrin levels were significantly lower in patients with cirrhosis and hepatocellular carcinoma, whereas the hepcidin level was higher than that in chronic hepatitis B patients. Correlation analysis indicated that serum hepcidin was negatively correlated with HBV-DNA load (P < 0.01). Serum ferritin and transferrin saturation levels increased proportionally to the extent of liver cirrhosis and poorer Child-Pugh scores (P < 0.05). The decreased serum iron and transferrin saturation levels were significantly correlated with a smaller hepatocellular carcinoma tumor burden according to Barcelona Clinic Liver Cancer staging. Liver histology showed a clearly increasing trend in iron deposition in the liver tissues with increased fibrosis, which became prominent at stages 3 (severe liver fibrosis) and 4 (cirrhosis).CONCLUSIONIron metabolism disorders occur in patients with HBV-related liver diseases. The serum markers of iron metabolism disorders vary in different stages of HBV-related liver diseases.

Expression of Plasma hsa-miR122 in HBV-Related Hepatocellular Carcinoma (HCC) in Vietnamese Patients.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death in the world and considered as one of the most susceptible cancers in humans. The microRNA molecule, hsa-miR122, considered as a potential biological marker linked with the injury of hepatocellular tissue, is the most common microRNA in human liver cancer. Understanding the expression profile of hsa-miR122 plays an important role in the diagnosis of HCC. Identification and comparison of cut-off values of plasma hsa-miR122 expression were conducted in blood samples of healthy control, HBV infected and HBV-related HCC Vietnamese patients. Fifty-two blood samples of healthy control and HBV-related HCC cases, collected between 2015 and 2017 were obtained from Ho Chi Minh City Oncology Hospital, Vietnam. Written informed consent was attained from all patients and the Human Research Ethics Committee, Oncology Hospital (#08/BVUB-HDDD) approved the research protocol. Total RNA was isolated from blood samples with TrizolTM Reagent (Thermo Fisher Scientific, USA). To analyze the expression level of hsa-miR122, miRNA specific reverse transcription was performed using Sensi- FASTTM cDNA Synthesis Kit (Bioline, UK) as described by the manufacturer, followed by running RT-qPCR with SensiFASTTMSYBR No-ROX Kit (Bioline, UK). The housekeeping gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used for normalization. The presence of hsamiR122 and HBV-DNA was identified in human blood using RT-PCR and LAMP techniques. Downregulation of plasma hsa-miR122 was observed in HBV-related HCC patients with a .Ct value of 7.9 ± 2.1 which was significantly lower than found in healthy control (p<0.01). The loss of hsa-miR122 expression was observed in HBV infected patients. We also identified the difference of diagnostic values of this microRNA in different populations and provided a high diagnostic accuracy of HCC (AUC = 0.984 with sensitivity and specificity of 96% and 94%, respectively). hsa-miR122 was downregulated in HBV-related HCC patients and found to be lower by approximately 10 fold than in healthy control, resulting in a potential biomarker for microRNA based diagnosis of HCC in human blood.

Integration of hepatitis B virus DNA in chronically infected patients assessed by Alu-PCR.

Hepatitis B virus (HBV) infection is the main risk factor for hepatocellular carcinoma (HCC) worldwide. Integration of HBV DNA into the human genome has been found in >80% of HBV-related HCC cases. Some studies have, however, found similar integration patterns in tumorous and nontumorous tissues. Thus, the role of integrations for the development of HCC as well as the rate of integration in different stages of infection remain unclear. The aim of this study was to investigate integrations in patients without HCC, representing different stages of chronic HBV (CHB) infection. Extracted DNA in liver biopsies from 74 patients (one with 2 available biopsies) with CHB infection was analyzed by Alu-PCR. Amplicons were further analyzed by Sanger sequencing. Integration was detected in 39 biopsies (52%) as an amplicon containing both human and HBV sequences by Alu-PCR with one primer targeting a region in the HBV genome. Integrations were found in patients representing the different stages of CHB infection. A majority of the HBV sequences were located upstream or downstream of nucleotide position 1820, which previously has been identified as a common breakpoint in the HBV genome in integrated sequences. Approximately 60% of the HBV integrations were found in noncoding regions of the human genome. Integrations of HBV DNA into the human genome is an event frequentlyfound in mild phases of chronic hepatitis.

Anti-viral therapy is associated with improved survival but is underutilised in patients with hepatitis B virus-related hepatocellular carcinoma: real-world east and west experience.

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. It remains incompletely understood in the real world how anti-viral therapy affects survival after HCC diagnosis. This was an international multicentre cohort study of 2518 HBV-related HCC cases diagnosed between 2000 and 2015. Cox proportional hazards models were utilised to estimate hazard ratios (HR) with 95% (CI) for anti-viral therapy and cirrhosis on patients' risk of death. Approximately, 48% of patients received anti-viral therapy at any time, but only 17% were on therapy at HCC diagnosis (38% at US centres, 11% at Asian centres). Anti-viral therapy would have been indicated for >60% of the patients not on anti-viral therapy based on American criteria. Patients with cirrhosis had lower 5-year survival (34% vs 46%; P<0.001) while patients receiving anti-viral therapy had increased 5-year survival compared to untreated patients (42% vs 25% with cirrhosis and 58% vs 36% without cirrhosis; P<0.001 for both). Similar findings were seen for other patient subgroups by cancer stages and cancer treatment types. Anti-viral therapy was associated with a decrease in risk of death, whether started before or after HCC diagnosis (adjusted HR 0.62 and 0.79, respectively; P<0.001). Anti-viral therapy improved overall survival in patients with HBV-related HCC across cancer stages and treatment types but was underutilised at both US and Asia centres. Expanded use of anti-viral therapy in HBV-related HCC and better linkage-to-care for HBV patients are needed.

Hepatitis B surface antigen levels in hepatitis B virus-related hepatocellular carcinoma: a retrospective cross-sectional study

Objective To investigate the distribution of serum hepatitis B surface antigen (HBsAg) levels in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Methods A total of 226 cases of HBV-related HCC were collected from June 2009 to December 2013. Demographic characteristics of patients with different barcelona clinica liver cancer (BCLC) stages, the status of cirrhosis and HBsAg levels with different virological indicators were compared. HBsAg quantification was tested by chemiluminescence. The statistical analysis was conducted by t test, χ2 test, Kruskal-Wallis H rank sum test and Mann-Whitney U rank sum test. Results A total of 226 cases were included with 201 male patients and 25 female patients. HBsAg levels in HBV-related HCC patients with different ages were significantly different (χ2=12.30, P=0.015), but with no statistical difference in those with different gender (Z=-0.35, P>0.05). The HBsAg levels were not significantly different between patients with or without liver cirrhosis (Z=-0.80, P=0.419). HBsAg levels in liver cirrhosis cases with different liver function stages were not significant different (χ2=2.15, P=0.341). HBsAg levels in HBeAg-positive group or HBV DNA positive group were significantly higher than those in HBeAg-negative group or HBV DNA negative group, respectively (Z=-3.67 and -4.80, respectively, both P 0.05). The constituent ratios of patients with different HBsAg levels in different BCLC stages were statistically different (χ2=28.17, P=0.005). Conclusions There are no significant differences of HBsAg levels in patients with different BCLC stages, indicating that HBsAg may not be a contributor for disease progression after emergence of HCC. Key words: Hepatitis B surface antigens; Carcinoma, hepatocellular; Liver cirrhosis; Barcelona clinical liver cancer

Hepatitis B virus X protein induces hepatic stem cell-like features in hepatocellular carcinoma by activating KDM5B.

AIMTo determine the role of hepatitis B virus X protein (HBx), HBx in regulating hepatic progenitor cell (HPC)-like features in hepatocellular carcinoma (HCC) and the underlying molecular mechanisms.METHODSWe used a retrovirus vector to introduce wild type HBx or empty vector into HepG2 cells. We then used these cells to analyze cell proliferation, senescence, transformation, and stem-like features. Gene expression profiling was carried out on Affymetrix GeneChip Human U133A2.0 ver.2 arrays according to the manufacturer’s protocol. Unsupervised hierarchical clustering analysis and Class Comparison analysis were performed by BRB-Array Tools software Version 4.2.2. A total of 238 hepatitis B virus (HBV)-related HCC patients’ array data were used for analyzing clinical features.RESULTSThe histone demethylase KDM5B was significantly highly expressed in HBV-related HCC cases (P < 0.01). In HBV proteins, only HBx up-regulated KDM5B by activating c-myc. Hepatic stem cell (HpSC) markers (EpCAM, AFP, PROM1, and NANOG) were significantly highly expressed in KDM5B-high HCC cases (P < 0.01). KDM5B played an important role in maintaining HpSC-like features and was associated with a poor prognosis. Moreover, inhibition of KDM5B suppressed spheroid formation and cell invasion in vitro.CONCLUSIONHBx activates the histone demethylase KDM5B and induces HPC-like features in HCC. Histone demethylases KDM5B may be an important therapeutic target against HBV-related HCC cases.

Detection of Nucleoside/Nucleotide Drug Resistant Mutants in Liver Cancer Cases: An Experience from India

Use of nucleoside and nucleotide analogues to treat patients infected with hepatitis B virus (HBV) has been found to be associated with mutations in the polymerase gene of the virus. The current study was carried out in HBV-related hepatocellular carcinoma (HCC) cases to trace the presence of drug-related mutants. A total of 75 HBV-related HCC cases were included for the study as per Bruix et al., 2001 EASL guidelines. HBV viral DNA was isolated by the previously standardized manual phenol-chloroform methods. The 3.2 kb genome of HBV was amplified by six sets of overlapping primers. The amplicons were sequenced commercially [Macrogen, South Korea (ABI PRISM)]. Sequences for the polymerase gene were analyzed using commercial bioinformatics software ( http://www.hepseq.org/Public/Tool/annotator_tool.php ). The different drug-resistant mutations detected were confirmed twice, ahead of reporting. Four drug-resistant mutations were detected in total: L80I (lamivudine), N236T (adefovir), I169T (entecavir) and A181V (lamivudine/adefovir). Interestingly, all four of the drug-resistant mutants were found in genotype D of HBV. The low number (only four) of drug-resistant mutations detected in this study population can be attributed to the fact that most of the cases were not treated and presented late. This study’s findings confirm the presence of previously reported drug-resistant mutations in the HBV genome infecting Indian patients; however, its associations with late stage disease and with the virus genotype D, in particular, need to be further studied in a larger population.

Research advances in diagnostic and therapeutic application of long-chain non-coding RNAs in hepatocellular carcinoma

In recent years, hepatocellular carcinoma (HCC) has become one of the serious health-threatening malignancies worldwide, and its incidence and mortality rates continue to rise. Hepatitis B (HBV) is moderately endemic in China, with enormous numbers of HBV-related HCC cases. Although serum alpha-fetoprotein (AFP) and ultrasound are the major diagnostic methods for HCC, they have limited application for screening out early or small HCC. The current management of HCC is based on tumor size and location, not on suppressing tumorigenesis, and therefore patients are often faced with low 5-year survival and high relapse rates. Recent studies have shown that long-chain non-coding RNAs (lncRNAs) are closely associated with HCC tumorigenesis, which may have considerable utility as new diagnostic marker and treatment target for HCC. Here, we review the application of lncRNAs in the diagnosis, metastasis, treatment, recurrence, and prognosis of HCC.