Wen-Chun Liu

Association of intrahepatic cccDNA reduction with the improvement of liver histology in chronic hepatitis B patients receiving oral antiviral agents.

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is difficult to eradicate using current antiviral therapy. This study compares cccDNA reduction with relation to liver histology in nucleoside/nucleotide‐naïve chronic hepatitis B patients receiving oral antiviral monotherapy (n = 35), including entecavir (ETV, n = 13), adefovir dipivoxil (ADV, n = 22) or placebo (n = 14). Serum HBV DNA, intrahepatic total HBV DNA and cccDNA are quantified. Histological hepatic examination is performed at baseline and at 48 weeks of treatment. Treatment with ETV or ADV shows significant median reduction in serum HBV DNA (−6.21 and −4.27 log10 copies/mL) and intrahepatic total HBV DNA (−1.69 and −1.23 log10 copies/cell). Intrahepatic cccDNA levels are reduced slightly in the ETV and the ADV groups, but do not differ statistically from the placebo group (−0.17 vs. −0.01 vs. 0.02 copies/cell). Only the level of intrahepatic cccDNA correlates with Knodell necroinflammation activity (r = 0.527, P < 0.001) and Ishak fibrosis severity (r = 0.348, P = 0.015) before treatment. Multivariate logistic regression analysis indicates that treatment‐induced cccDNA reduction is associated with improved necroinflammation (P = 0.041) and fibrosis (P = 0.026). In conclusion, baseline intrahepatic cccDNA loads correlate with histologic activity. Although one‐year ETV or ADV treatment is insufficient for cccDNA eradication, oral antiviral therapies may improve liver histology, probably by suppressing intrahepatic cccDNA. J. Med. Virol. 83:602–607, 2011.

Genotyping of Hepatitis B Virus – Genotypes A to G by Multiplex Polymerase Chain Reaction

Objectives: Eight genotypes (A–H) of hepatitis B virus (HBV) are known with variations in nucleotide sequences greater than 8%. Several recent publications found that the clinical course and outcome of antiviral therapy depended on the genotype of the infecting HBV strain. Large epidemiological studies will require the availability of a system which is rapid, reliable and can be performed on a large number of samples. Methods: To establish a simple and accurate genotyping method, the study collected 369 HBV complete genomic sequences from the GenBank database. Type-specific primers were also designed that separated HBV genotypes A to G by multiplex polymerase chain reaction. Results: By comparison with the traditional restriction fragment length polymorphism method, over 93% of 441 samples were accurately genotyped by current assay, with a higher detection rate and sensitivity to detect mixed HBV infections. Conclusions: This methodology can be applied only to areas prevalent with HBV genotypes A to G. However, it provides an efficient alternative for clinical diagnosis and large-scale studies.

Aberrant Serum Immunoglobulin G Glycosylation in Chronic Hepatitis B Is Associated With Histological Liver Damage and Reversible by Antiviral Therapy

BACKGROUND Aberrant serum immunoglobulin G (IgG) glycosylation and its immunomodulatory effect are rarely addressed in chronic hepatitis B virus (HBV) infection. METHODS Serum IgG-Fc glycosylation profiles in 76 patients with HBV-related liver cirrhosis and 115 patients with chronic hepatitis B (CHB) before and after 48 weeks of anti-HBV nucleos(t)ide analogue treatment were analyzed using high-throughput liquid chromatography-mass spectrometry and were compared to profiles in 108 healthy controls. RESULTS The level of aberrant serum IgG-Fc glycosylation ,: particularly galactose deficiency, was higher in patients with CHB and those with cirrhosis (P < .001 for both) than in healthy controls. IgG galactose deficiency was correlated with the severity of liver necroinflammation and fibrosis in CHB. Multivariate logistic regression analyses showed that the IgG-Fc glycoform with fucosylation and fully galactosylation was an independent factor for a total Knodell necroinflammation score of ≥ 7 (odds ratio, 0.74; 95% confidence interval, .56-.97) and an Ishak fibrosis score of ≥ 3 (odds ratio, 0.69; 95% confidence interval, .49-.97). Administration of antiviral therapy for 48 weeks reversed aberrant IgG-Fc glycosylation in patients with CHB from week 12 onward but did not reverse glycosylation in patients with cirrhosis. Attenuated IgG opsonization in patients with CHB, which was correlated with aberrant Fc-glycosylation, was reversed after treatment as well. CONCLUSIONS Aberrant serum IgG-Fc glycosylation in CHB, which is highly associated with histological liver damage, affects IgG opsonizing activity and can be reversed by antiviral therapy.

Analysis of precore/core covariances associated with viral kinetics and genotypes in hepatitis B e antigen-positive chronic hepatitis B patients

Hepatitis B virus (HBV) is one of the most common DNA viruses that can cause aggressive hepatitis, cirrhosis and hepatocellular carcinoma. Although many people are persistently infected with HBV, the kinetics in serum levels of viral loads and the host immune responses vary from person to person. HBV precore/core open reading frame (ORF) encoding proteins, hepatitis B e antigen (HBeAg) and core antigen (HBcAg), are two indicators of active viral replication. The aim of this study was to discover a variety of amino acid covariances in responses to viral kinetics, seroconversion and genotypes during the course of HBV infection. A one year follow-up study was conducted with a total number of 1,694 clones from 23 HBeAg-positive chronic hepatitis B patients. Serum alanine aminotransferase, HBV DNA and HBeAg levels were measured monthly as criteria for clustering patients into several different subgroups. Monthly derived multiple precore/core ORFs were directly sequenced and translated into amino acid sequences. For each subgroup, time-dependent covariances were identified from their time-varying sequences over the entire follow-up period. The fluctuating, wavering, HBeAg-nonseroconversion and genotype C subgroups showed greater degrees of covariances than the stationary, declining, HBeAg-seroconversion and genotype B. Referring to literature, mutation hotspots within our identified covariances were associated with the infection process. Remarkably, hotspots were predominant in genotype C. Moreover, covariances were also identified at early stage (spanning from baseline to a peak of serum HBV DNA) in order to determine the intersections with aforementioned time-dependent covariances. Preserved covariances, namely representative covariances, of each subgroup are visually presented using a tree-based structure. Our results suggested that identified covariances were strongly associated with viral kinetics, seroconversion and genotypes. Moreover, representative covariances may benefit clinicians to prescribe a suitable treatment for patients even if they have no obvious symptoms at the early stage of HBV infection.

Association of serum IgG N-glycome and transforming growth factor-β1 with hepatitis B virus e antigen seroconversion during entecavir therapy.

Aberrant serum IgG N-glycome has been demonstrated in various autoimmune diseases and viral infections. However, the correlation between serum IgG N-glycome and cytokine is unclear. In addition, the clinical relevance of IgG glycosylation and cytokine changes in the treatment outcome of chronic hepatitis B (CHB) has never been assessed. One hundred and three treatment-naive patients with CHB and 101 healthy controls were enrolled in this retrospective cohort study. Serum samples in patients before and after 48weeks of entecavir treatment were collected. In-gel trypsinized serum IgG heavy chain was analyzed using liquid chromatography-tandem mass spectrometry. Selected ion chromatograms corresponding to 10 N-glycoforms on asparagine 297 were individually extracted to calculate the percentage of each glycoforms. Serum cytokine profiles were examined using enzyme-linked immunosorbent assay. Forty-eight weeks of entecavir treatment resulted in decreases in galactose-deficient (total G0) IgG and transforming growth factor (TGF)-β1 levels (both P<0.001) in patients with CHB. The changes in TGF-β1 (ΔTGF-β1) and IgG total G0 (Δtotal G0) levels during treatment were significantly correlated (r=0.403, P<0.001). Furthermore, higher levels of Δtotal G0 (P<0.01) and ΔTGF-β1 (P<0.001) were found in hepatitis B virus e antigen (HBeAg)-positive patients than in HBeAg-negative patients and were also found in patients with HBeAg seroconversion at week 48. The area under the receiver operating characteristic (ROC) curves for Δtotal G0 and ΔTGF-β1 to discriminate a week-48 HBeAg seroconversion were 0.835 and 0.830, respectively. These results suggested a correlation between serum cytokine and IgG N-glycome and its effect on the outcome of entecavir treatment in patients with CHB.

Discovering Clinical Biomarkers of Chronic Hepatitis B by Mining Mutation Hotspots

Hepatitis B virus (HBV) is the most common DNA virus that may cause hepatitis, cirrhosis and hepatocellular carcinoma. Although many people are persistently infected with HBV, the serum viral load and host immune response varies from person to person. Because the high rate of mutations in HBV protein sequences will alter the protein expressions and even their functions, in this paper, we explore to discover clinical biomarkers of chronic hepatitis B by mining mutation hotspots. A one year follow-up study was conducted with a total number of 1,694 clones from 23 patients with HBeAg-positive chronic hepatitis B. Serum alanine aminotransferase, HBV DNA and HBeAg levels were monthly measured and used as the criteria for clustering the patients into different subgroups. Using monthly derived HBV precore/core protein sequences, we analyzed amino acid mutations responsible for serologic and clinical outcome of each patient subgroup. Using an integration of covariance network and point mutation rule methods, we identified several representative covariance networks of each patient subgroup. Validation with literature-curated mutation hotspots showed that the identified mutations were strongly associated with the viral loads, presence of HBeAg-seroconversion in sera, HBV genotypes and amino acid properties. We further used these identified networks containing mutation hotspots to develop a feature tree, which is applicable for clinicians to prescribe patients a suitable treatment at early stage of HBV infection even though the patients exhibit no obvious symptoms.

Applications of next-generation sequencing analysis for the detection of hepatocellular carcinoma-associated hepatitis B virus mutations

BackgroundNext-generation sequencing (NGS) is a powerful and high-throughput method for the detection of viral mutations. This article provides a brief overview about optimization of NGS analysis for hepatocellular carcinoma (HCC)-associated hepatitis B virus (HBV) mutations, and hepatocarcinogenesis of relevant mutations.Main bodyFor the application of NGS analysis in the genome of HBV, four noteworthy steps were discovered in testing. First, a sample-specific reference sequence was the most effective mapping reference for NGS. Second, elongating the end of reference sequence improved mapping performance at the end of the genome. Third, resetting the origin of mapping reference sequence could probed deletion mutations and variants at a certain location with common mutations. Fourth, using a platform-specific cut-off value to distinguish authentic minority variants from technical artifacts was found to be highly effective. One hundred and sixty-seven HBV single nucleotide variants (SNVs) were found to be studied previously through a systematic literature review, and 12 SNVs were determined to be associated with HCC by meta-analysis. From comprehensive research using a HBV genome-wide NGS analysis, 60 NGS-defined HCC-associated SNVs with their pathogenic frequencies were identified, with 19 reported previously. All the 12 HCC-associated SNVs proved by meta-analysis were confirmed by NGS analysis, except for C1766T and T1768A which were mainly expressed in genotypes A and D, but including the subgroup analysis of A1762T. In the 41 novel NGS-defined HCC-associated SNVs, 31.7% (13/41) had cut-off values of SNV frequency lower than 20%. This showed that NGS could be used to detect HCC-associated SNVs with low SNV frequency. Most SNV II (the minor strains in the majority of non-HCC patients) had either low (< 20%) or high (> 80%) SNV frequencies in HCC patients, a characteristic U-shaped distribution pattern. The cut-off values of SNV frequency for HCC-associated SNVs represent their pathogenic frequencies. The pathogenic frequencies of HCC-associated SNV II also showed a U-shaped distribution. Hepatocarcinogenesis induced by HBV mutated proteins through cellular pathways was reviewed.ConclusionNGS analysis is useful to discover novel HCC-associated HBV SNVs, especially those with low SNV frequency. The hepatocarcinogenetic mechanisms of novel HCC-associated HBV SNVs defined by NGS analysis deserve further investigation.

Hepatocellular carcinoma-associated single-nucleotide variants and deletions identified by the use of genome-wide high-throughput analysis of hepatitis B virus

This study investigated hepatitis B virus (HBV) single‐nucleotide variants (SNVs) and deletion mutations linked with hepatocellular carcinoma (HCC). Ninety‐three HCC patients and 108 non‐HCC patients were enrolled for HBV genome‐wide next‐generation sequencing (NGS) analysis. A systematic literature review and a meta‐analysis were performed to validate NGS‐defined HCC‐associated SNVs and deletions. The experimental results identified 60 NGS‐defined HCC‐associated SNVs, including 41 novel SNVs, and their pathogenic frequencies. Each SNV was specific for either genotype B (n = 24) or genotype C (n = 34), except for nt53C, which was present in both genotypes. The pathogenic frequencies of these HCC‐associated SNVs showed a distinct U‐shaped distribution pattern. According to the meta‐analysis and literature review, 167 HBV variants from 109 publications were categorized into four levels (A–D) of supporting evidence that they are associated with HCC. The proportion of NGS‐defined HCC‐associated SNVs among these HBV variants declined significantly from 75% of 12 HCC‐associated variants by meta‐analysis (Level A) to 0% of 10 HCC‐unassociated variants by meta‐analysis (Level D) (P < 0.0001). PreS deletions were significantly associated with HCC, in terms of deletion index, for both genotypes B (P = 0.030) and C (P = 0.049). For genotype C, preS deletions involving a specific fragment (nt2977–3013) were significantly associated with HCC (HCC versus non‐HCC, 6/34 versus 0/32, P = 0.025). Meta‐analysis of preS deletions showed significant association with HCC (summary odds ratio 3.0; 95% confidence interval 2.3–3.9). Transfection of Huh7 cells showed that all of the five novel NGS‐defined HCC‐associated SNVs in the small surface region influenced hepatocarcinogenesis pathways, including endoplasmic reticulum‐stress and DNA repair systems, as shown by microarray, real‐time polymerase chain reaction and western blot analysis. Their carcinogenic mechanisms are worthy of further research. Copyright

HCC-Associated Single Nucleotide Variants and Deletions Identified Using Genome-Wide High Throughput Analysis of HBV

This study investigated hepatitis B virus (HBV) single‐nucleotide variants (SNVs) and deletion mutations linked with hepatocellular carcinoma (HCC). Ninety‐three HCC patients and 108 non‐HCC patients were enrolled for HBV genome‐wide next‐generation sequencing (NGS) analysis. A systematic literature review and a meta‐analysis were performed to validate NGS‐defined HCC‐associated SNVs and deletions. The experimental results identified 60 NGS‐defined HCC‐associated SNVs, including 41 novel SNVs, and their pathogenic frequencies. Each SNV was specific for either genotype B (n = 24) or genotype C (n = 34), except for nt53C, which was present in both genotypes. The pathogenic frequencies of these HCC‐associated SNVs showed a distinct U‐shaped distribution pattern. According to the meta‐analysis and literature review, 167 HBV variants from 109 publications were categorized into four levels (A–D) of supporting evidence that they are associated with HCC. The proportion of NGS‐defined HCC‐associated SNVs among these HBV variants declined significantly from 75% of 12 HCC‐associated variants by meta‐analysis (Level A) to 0% of 10 HCC‐unassociated variants by meta‐analysis (Level D) (P < 0.0001). PreS deletions were significantly associated with HCC, in terms of deletion index, for both genotypes B (P = 0.030) and C (P = 0.049). For genotype C, preS deletions involving a specific fragment (nt2977–3013) were significantly associated with HCC (HCC versus non‐HCC, 6/34 versus 0/32, P = 0.025). Meta‐analysis of preS deletions showed significant association with HCC (summary odds ratio 3.0; 95% confidence interval 2.3–3.9). Transfection of Huh7 cells showed that all of the five novel NGS‐defined HCC‐associated SNVs in the small surface region influenced hepatocarcinogenesis pathways, including endoplasmic reticulum‐stress and DNA repair systems, as shown by microarray, real‐time polymerase chain reaction and western blot analysis. Their carcinogenic mechanisms are worthy of further research. Copyright

Prediction of early hepatocellular carcinoma recurrence using germinal center kinase-like kinase

Germinal center kinase-like kinase (GLK) is a key controller of autoimmunity. In this study, we assessed the clinical relevance and tumorigenic effects of GLK in hepatocellular carcinoma (HCC). Using immunohistochemistry, we showed that the GLK proportion score increased in both cancerous and adjacent non-cancerous liver tissue from patients with HCC recurrence. A Kaplan-Meier analysis revealed that patients with a wide distribution of GLK in non-cancerous liver tissue had a higher rate of HCC recurrence than those with very low or no GLK expression. Multivariate Cox regression analyses indicated that a high GLK proportion score in non-cancerous liver tissue was an independent predictor of early HCC recurrence after resection. Lentiviral vector-mediated overexpression of GLK activated the nuclear factor kappa B (NFκB) signaling cascade and accelerated cell cycle progression in primary human hepatocytes, thereby promoting proliferation. An increase in GLK expression coincided with NFκB activation and enhanced expression of proliferating cell nuclear antigen in HCC tissue. Our findings demonstrate a potential hepatocarcinogenic effect of GLK and the feasibility of using GLK to predict early HCC recurrence.