Fung-Yu Huang

Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos(t)ide analogues of different potency.

BACKGROUND & AIMS Few studies have investigated the effects of different nucleos(t)ide analogues against hepatitis B virus (HBV) on levels of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg) in patients. We measured the magnitude of reduction of cccDNA and HBsAg by nucleos(t)ide analogue therapy and assessed the correlation between their reductions. METHODS We recruited 124 patients who were treated with 1 of the 5 nucleos(t)ide analogues (lamivudine, adefovir, entecavir, telbivudine, or clevudine). All patients had undergone liver biopsy when treatment began (baseline) and 1 year later. The cccDNA and HBsAg levels were measured by real-time polymerase chain reaction and the Elecsys II HBsAg Assay, respectively. RESULTS After 1 year of treatment, HBV in 7 patients had become resistant to the nucleos(t)ide analogue. The remaining 117 patients had an average reduction of approximately 0.2 log10 IU/mL in HBsAg, 5 log10 IU/mL in serum level of HBV DNA, 2 log10 copies/cell in intrahepatic total HBV DNA, and 1 log10 copies/cell in cccDNA. Although 88 of 117 patients (75%) had undetectable serum levels of HBV DNA (<12 IU/mL), all had detectable levels of HBsAg, and only 5 (4%) had undetectable levels of cccDNA. On treatment with nucleos(t)ide analogues, patients with greater reductions in levels of cccDNA had greater reductions in HBsAg, but these reductions did not reach statistically significant correlations. CONCLUSIONS Although nucleos(t)ide analogues potently reduced serum levels of HBV DNA, the reduction of HBsAg and cccDNA was small. In short-term therapy, the magnitude of HBsAg reduction does not correlate with that of cccDNA reduction.

Reduction of hepatitis B surface antigen levels and hepatitis B surface antigen seroclearance in chronic hepatitis B patients receiving 10 years of nucleoside analogue therapy.

The profile and clinical significance of serum hepatitis B surface antigen (HBsAg) levels during long‐term nucleoside analogue (NA) therapy in chronic hepatitis B (CHB) is undetermined. From 1994 to 2002, 322 Chinese CHB patients were started on lamivudine in our center. Patients were recruited if they were continuously treated with lamivudine for at least 10 years and maintained favorable virologic responses throughout therapy (HBV DNA <2,000 IU/mL). HBsAg and HBV DNA levels were measured serially, and the predictability of HBsAg kinetics in determining NA‐related HBsAg seroclearance was determined. Seventy patients were recruited, of which 43 (61.4%) were hepatitis B e antigen (HBeAg)‐positive. Fifty‐two (74.3%) patients had undetectable viremia (HBV DNA <20 IU/mL) during therapy. Fifteen (21.4%) patients were followed up for 15 years. The median rate of HBsAg reduction was 0.104 log IU/mL/year, with no significant difference found when comparing patients who were HBeAg‐positive versus HBeAg‐negative, were genotype B versus C, and had detectable versus undetectable viremia during therapy (all P > 0.05). Seven (10%) patients achieved HBsAg seroclearance, and when compared with the remaining 63 patients, had significantly lower median baseline HBsAg levels (P = 0.012) and a greater median rate of HBsAg reduction (P < 0.001). Baseline HBsAg levels and the rate of HBsAg reduction achieved an area under the receiver operating characteristic curve of 0.860 (P = 0.004; 95% confidence interval [CI], 0.742‐0.978) and 0.794 (P = 0.018; 95% CI, 0.608‐0.979), respectively. Baseline HBsAg <1,000 IU/mL and on‐treatment reduction of HBsAg >0.166 log IU/mL/year were optimal cutoff levels in predicting subsequent HBsAg seroclearance (negative predictive values, 98.1% and 97.8%, respectively). Conclusion: Low baseline HBsAg levels and greater rate of HBsAg reduction achieved high predictive values for predicting HBsAg seroclearance, strengthening the prognostic role of HBsAg measurements during NA therapy. (Hepatology 2013;53:923–931)

Patterns of hepatitis B surface antigen decline and HBV DNA suppression in Asian treatment-experienced chronic hepatitis B patients after three years of tenofovir treatment

BACKGROUND & AIMS Patterns of serum hepatitis B surface antigen (HBsAg) decline during nucleos(t)ide analogue (NA) therapy have not been well investigated. METHODS We determined the cumulative serologic, virologic, and biochemical outcomes of 142 Asian CHB patients, with at least 6 months exposure to other NAs, receiving tenofovir with or without lamivudine for up to 3 years. Liver biochemistry, serum HBV DNA, and HBsAg levels were determined at baseline, 6 months and yearly from years 1 to 3. RESULTS 142, 123 (86.6%), and 70 (49.3%) CHB patients were followed up for 1, 2, and 3 years, respectively. Two phases of HBsAg decline were observed. Patients with baseline HBsAg ≥3 log IU/ml, when compared to patients with baseline HBsAg < 3 log IU/ml, had a greater median rate of HBsAg reduction through 3 years of treatment (0.155 and 0.039 log IU/ml/year respectively, p < 0.001). Among patients with 3 years of follow-up, there was a significantly greater median rate of HBsAg reduction during the first year when compared to the second and third years (0.220, 0.136, and 0.081 log IU/ml/year respectively, p < 0.001). HBeAg status, HBV genotype, and concomitant lamivudine therapy were not important determinants of HBsAg kinetics (all p > 0.05). The 3-year cumulative virologic suppression rate was 93.3%, with no cases of resistance detected. CONCLUSIONS Serum HBsAg levels in NA-experienced patients receiving tenofovir demonstrated a variable pattern of decline, with slower rates of reduction noted in patients with lower baseline HBsAg levels, and could explain the rarity of HBsAg seroclearance during NA therapy.

Linearized hepatitis B surface antigen and hepatitis B core-related antigen in the natural history of chronic hepatitis B

Changes in two novel HBV serological markers, linearized hepatitis B surface antigen (HQ-HBsAg) and hepatitis B core-related antigen (HBcrAg), in the natural history of chronic hepatitis B (CHB) have not been well characterized. Serum HQ-HBsAg and HBcrAg levels of 404 Asian treatment-naïve CHB patients were analysed in a cross-sectional manner. Patients were categorized into five groups: immune tolerant (IT group, n=52), immune clearance (IC group, n=105), hepatitis B e antigen (HBeAg)-negative hepatitis (ENH group, n=97), HBeAg-negative quiescent group (ENQ group, n=95) and CHB with hepatitis B surface antigen (HBsAg) seroclearance (SC group, n=55). HQ-HBsAg and HBcrAg were measured and correlated with HBV DNA, HBsAg, HBV genotype and clinical parameters. HQ-HBsAg showed good correlation with HBsAg, especially in the ENQ group (r=0.874, p<0.001). Correlation of HQ-HBsAg with HBV DNA was less prominent and weakest in the ENH group (r=0.268, p 0.008). HBcrAg correlated best with HBV DNA in the ENQ group (r=0.537, p<0.001). In the ENQ group, 42.1% of patients had undetectable HBcrAg; this subgroup of patients, when compared with those with detectable HBcrAg, had significantly lower median HBV DNA (3.17/4.48 log IU/mL, p<0.001) and HBsAg (5.05/5.96 log mIU/mL, p<0.001) levels. Forty per cent of the SC group patients had detectable HQ-HBsAg and/or HBcrAg up to 42 months after HBsAg seroclearance. When comparing anti-HBs positivity and median time after HBsAg seroclearance in the SC group with and without detectable HQ-HBsAg/HBcrAg, there was no significant difference (22.7% and 36.4%, respectively, p 0.284, and 76.5 and 93.2 months, respectively, p 0.245). HQ-HBsAg and HBcrAg showed unique patterns of distribution throughout the five disease phases of CHB, including high detectability rates after HBsAg seroclearance, opening up different possibilities for their applicability.

Changes of HBsAg and HBV DNA levels in Chinese chronic hepatitis B patients after 5 years of entecavir treatment

Hepatitis B surface antigen (HBsAg) kinetics during long‐term entecavir therapy has not been well investigated.

Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B

BACKGROUND AND AIMS Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), a mini-chromosome essential for HBV replication, is supposed to be resistant to nucleos(t)ide analogue treatment. We investigated the effect of long-term nucleos(t)ide analogue treatment on cccDNA. METHODS Among 129 patients who had been enrolled in previous international nucleos(t)ide analogue clinical trials and had liver biopsies at baseline and one year after treatment, we recruited 43 patients on long-term continuous treatment for 72 to 145months for a third liver biopsy. Serum HBV DNA, hepatitis B surface antigen (HBsAg) levels, total intrahepatic HBV DNA (ihHBV DNA), cccDNA, HBV pregenomic RNA (pgRNA) as well as histologic changes were examined. RESULTS At the time of the third biopsy, serum HBV DNA levels were undetectable in all but one patient. The median levels of HBsAg, ihHBV DNA, and cccDNA were 2.88logIU/ml, 0.03copies/cell, and 0.01copies/cell, respectively. Compared to baseline levels, there was reduction of HBsAg levels by 0.54log (71.46%), ihHBV DNA levels by 2.81log (99.84%), and cccDNA levels by 2.94log (99.89%), with 49% having cccDNA levels below the detection limit. One patient had undetectable HBsAg. The median pgRNA level, measured only in the third biopsy, was 0.021copies/cell, with 40% of patients having undetectable pgRNA. CONCLUSIONS Long-term nucleos(t)ide analogue treatment induced marked depletion of cccDNA in the majority of patients while serum HBsAg levels, though reduced, were detectable in all but one patient. Whether cccDNA depletion is sustained and associated with better patient outcome requires further study. LAY SUMMARY It is generally presumed that a form of hepatitis B virus DNA, called covalently closed circular DNA (cccDNA), which hides inside the nuclei of liver cells of patients with chronic hepatitis B, cannot be reduced by antiviral treatment. The present study showed that with prolonged treatment (median period 126months), cccDNA can be markedly reduced, with 49% of liver biopsies having undetectable cccDNA. This suggests that viral replication capacity would be very low after prolonged antiviral treatment.

Role of HLA-DP Polymorphisms on Chronicity and Disease Activity of Hepatitis B Infection in Southern Chinese

Background and Aims The association between HLA-DP single nucleotide polymorphisms (SNPs) and chronic hepatitis B virus (HBV) infection varies between different populations. We aimed to study the association between HLA-DP SNPs and HBV infection and disease activity in the Chinese population of Hong Kong. Methods We genotyped SNPs rs3077 (near HLA-DPA1) and rs9277378 and rs3128917 (both near HLA-DPB1) in 500 HBV carriers (hepatitis B surface antigen [HBsAg]-positive), 245 non-HBV infected controls (HBsAg- and antibody to hepatitis B core protein [anti-HBc]-negative), and 259 subjects with natural HBV clearance (HBsAg-negative, anti-HBc-positive). Inactive HBV carriers state was defined by HBV DNA levels <2,000 IU/ml and persistently normal alanine aminotransferase level for least 12 months. Results Compared to the non-HBV infected subjects, the HBV carriers had a significantly lower frequency of the rs3077 T allele (p = 0.0040), rs9277378 A allele (p = 0.0068) and a trend for lower frequency of rs3128917 T allele (p = 0.054). These alleles were associated with an increased chance of HBV clearance (rs3077: OR = 1.41, p = 0.0083; rs9277378: OR = 1.61, p = 0.00011; rs3128917: OR = 1.54, p = 0.00017). Significant associations between HLA-DP genotypes and HBV clearance were also found under different genetic models. Haplotype TAT was associated with an increased chance of HBV clearance (OR = 1.64, p = 0.0013). No association was found between these SNPs and HBV disease activity. Conclusion HLA-DP SNPs rs3077, rs9277378 and rs3128917 were associated with chronicity of HBV disease in the Chinese. Further studies are required to determine whether these SNPs influence the disease endemicity in different ethnic populations.

Estradiol induces apoptosis via activation of miRNA-23a and p53: implication for gender difference in liver cancer development.

Estrogen (E2) has been suggested to have a protective role in attenuating hepatocellular carcinoma (HCC) development. miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control gene expression. However, little is known about the mechanism underlying the protective role of E2 in hepatocarcinogenesis and the effects of E2 on apoptotic miRNAs expression. Using miRNA PCR array, we found more than 2-fold alteration was observed in 25 upregulated and 10 downregulated apoptotic miRNAs in E2-treated cells. Among these miRNAs, we found expression of miR-23a was related to p53 functional status in the male-derived liver cell-lines. We demonstrated that E2 via ERα transcriptionally activated miR-23a and p53 expression, and thus enhanced p53 activation of miR-23a expression. Moreover, miR-23a expression correlated inversely with the expression of target gene X-linked inhibitor of apoptosis protein (XIAP), but positively with the caspase-3/7 activity. Decreasing of XIAP might contribute to caspase-3 activity and cell apoptosis. Taken together, our findings reveal a novel E2-signaling mechanism in regulating miRNAs expression for controlling apoptosis in liver cells. Delineating the role of E2 in regulating the activation of p53 and miR-23a, expression in HCC is crucial to the understanding of the sex difference observed in HCC.

Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a key to viral persistence in chronic hepatitis B infection. Serum hepatitis B core‐related antigen (HBcrAg) is a novel marker for HBV disease. We aimed to determine whether HBcrAg could be a surrogate marker for intrahepatic cccDNA.

Effect of Hepatitis B Virus Reverse Transcriptase Variations on Entecavir Treatment Response

BACKGROUND Entecavir therapy often reduces hepatitis B virus (HBV) DNA to an undetectable level, but HBV DNA remain detectable in some patients. We investigated whether baseline HBV reverse transcriptase (rt) polymorphism and quasispecies complexity and diversity were associated with treatment response. METHODS Pretreatment HBV DNA levels, HBV rt sequence, serology, and quasispecies complexity and diversity from 305 entecavir-treated patients were determined. These data were tested for their association with year 1 virological outcome, defined by optimal response (undetectable HBV DNA; lower limit of detection, ≤12 IU/mL) or partial response (detectable HBV DNA). RESULTS Four rt variants were more frequently detected in the 64 partial responders than in the 241 optimal responders (all P < .05). Multivariate analysis revealed that high baseline HBV DNA level (P < .0001; odds ratio [OR], 2.32), HBV e antigen (HBeAg) positivity (P < .001; OR, 3.70), and rt124N (P = .002; OR, 3.06) were associated with a partial entecavir response. Compared with the optimal responders, the partial responders had a lower quasispecies complexity and diversity. CONCLUSIONS Apart from the known factors (high baseline HBV DNA level and HBeAg positivity), a novel single nucleotide polymorphism (rt124N) and lower quasispecies complexity and diversity were associated with partial entecavir response at year 1.