Daryl Tan-Yeung Lau

Mao et al suggest that DR2-DR1 enrichment reflects biological selection and a ‘survivor signature’. This interpretation is consistent with our original conclusions. In the Abstract, …

Background: Hepatitis B surface antigen (HBsAg) can be derived from intrahepatic covalently closed circular DNA (cccDNA) and integrated hepatitis B virus (HBV) DNA (iDNA). Objective: We evaluated the cccDNA and iDNA from liver tissues of 24 hepatitis B e antigen (HBeAg)(+) and 32 HBeAg(−) treatment-naïve chronic hepatitis B (CHB) participants in the North American Hepatitis B Research Network. Design: For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove relaxed circular DNA and iDNA before real-time polymerase chain reaction. For iDNA detection, total DNA was subjected to HBV hybridisation-targeted next generation sequencing assay for identification of the HBV-host junction sequences. Comparisons of HBV cccDNA and iDNA with other virological biomarkers were assessed. Results: Intrahepatic cccDNA, serum HBV DNA, HBV RNA, hepatitis B core related antigen and quantitative hepatitis B surface antigen were higher in HBeAg(+) CHB. Intrahepatic hepatitis B core antigen staining was present in 87% HBeAg(+) but only 13% HBeAg(−) samples (p50% iDNA of total HBV DNA in their livers. For HBeAg(+) CHB, the iDNA integration sites were random with only 15.9% localised to the direct repeat 2 (DR2)-DR1 region. For HBeAg(−) CHB, 52.4% of the iDNA integrations were clustered at DR2-DR1. Microhomology-mediated end joining patterns of double-stranded linear DNA HBV integration was more frequent in HBeAg(+) livers. Conclusion: HBeAg(−) CHB was associated with high HBsAg staining concentration despite low cccDNA levels suggesting that iDNA was the primary source of HBsAg. The high frequency of DR2-DR1 iDNA distribution in HBeAg(−) CHB suggests the selection advantage and clonal expansion of this integrant in the natural history of CHB.

Background and aims: Hepatitis B virus (HBV) replication generates a double-stranded linear DNA (dslDNA) byproduct. This dslDNA can undergo intermolecular and intramolecular nonhomologous end-joining (NHEJ) recombination, resulting in viral integration and dslDNA-derived covalently closed circular DNAs (dsl-cccDNAs), respectively. The insertions and deletions (INDELs) at the end-joining site have been used to differentiate dsl-cccDNA from the authentic cccDNA. The prevalence and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear. Approach and results: HBV-targeted next-generation sequencing (NGS) was used to identify 32 dsl-cccDNA-positive candidates, 22 HBeAg(+) and 10 HBeAg(-), from 56 liver biopsies of antiviral treatment-naïve CHB patients for dsl-cccDNA confirmation and characterization by PSAD-cccDNA PCR NGS. INDELs within the DR2–1 region (nt 1600–1840) of the cccDNA were analyzed. Various clonally expanded, heterogenous ~22-nt deletions in the X gene region around nt 1760 were discovered in all 32 samples. The dsl-cccDNA species were then defined and characterized by the INDELs clustered at the DR1 surrounding region (nt 1800–1840). The proportion of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples. The diversity of dsl-cccDNA species positively correlated with cccDNA levels and serum viral load, and was higher in HBeAg(+) CHB. Conclusions: dsl-cccDNA is more abundant and diverse among the HBeAg(+) CHB subjects. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis.

Background/Aim: Hepatitis D virus (HDV) infection is the most severe form of viral hepatitis but is under-recognized. We evaluated HDV screening frequency and clinical characteristics of HDV-positive patients in our liver center in the United States. Method: Patients with chronic hepatitis B (CHB) evaluated between January 2021 and December 2023 were included. For those tested anti-HDV(+), HDV RNA was performed and clinical data were recorded. Results: Among 1,337 (M=716, F=621) CHB patients, 93.3% were foreign-born. The cohort included 74.9% Asians, 12.3% Blacks, 9.1% Whites and 1.3% Hispanics. HDV screening significantly increased after 2011 reflecting an increase in HDV awareness. Patients aged 18-40 years had the highest screening rate of 58%. Overall, 13 (1.95%) were tested HDV positive and 11 were repeatedly HDV RNA(+). PWID was noted in 5 of 11. Despite low HBV DNA levels, all had ≥stage 2 fibrosis, and 7 had advanced stage 3-4 disease. After a median follow-up of 8.75 years, 8 (73%) developed hepatic decompensation and 4 had hepatocellular carcinoma. Conclusion: HDV screening reached 51.3% among our predominantly foreign-born CHB patients. The high prevalence of advanced fibrosis at diagnosis underscores the importance of HDV screening. Early diagnosis and effective therapy are urgently needed for this challenging disease.

Patients with chronic hepatitis B often require prolonged nucleos(t)ide analogue since functional cure is rarely achieved. Medication nonadherence can trigger life-threatening hepatitis B virus (HBV) reactivation. We report a 58-year-old Asian man with HBeAg-negative chronic hepatitis B and cirrhosis. After 10 years of entecavir therapy, he achieved undetectable HBV DNA and fibrosis regression, but quantitative hepatitis B surface antigen remained >1200 IU/mL. He self-discontinued medication and presented with severe hepatitis reactivation. Liver condition gradually improved on restarting nucleos(t)ide analogue. This case highlights that medication cessation with a high hepatitis B surface antigen titer can precipitate severe hepatitis and liver failure, even after long-term HBV DNA suppression.

Background/Objectives: Chronic hepatitis B and metabolic dysfunction-associated steatotic liver disease (MASLD) are associated with risk of advanced hepatic fibrosis and cirrhosis. Given the increased risk, the presence of insulin resistance, metabolic syndrome, and hepatic fibrosis should be assessed in individuals with chronic hepatitis B. The Lipoprotein Insulin Resistance Index (LP-IR) is a simple method of assessing insulin resistance in the general population, and lipoprotein profiles have been shown to reflect hepatic steatosis and fibrosis in patients with MASLD. However, LP-IR and lipoprotein profiles have not been evaluated in patients with chronic hepatitis B. Methods: Lipoprotein profiles and LP-IR scores were evaluated in a cohort of patients with chronic hepatitis B using NMR spectroscopy. Results: Serum samples from 50 patients with chronic hepatitis B and 44 with chronic hepatitis B and metabolic syndrome were analyzed. A cut-off value was defined that differentiates patients with and without insulin resistance. Increased large VLDL and reduced small VLDL particles were observed, suggesting the presence of hepatic steatosis and fibrosis. Conclusions: In this largely Asian cohort of chronic hepatitis B patients, where BMI and conventional markers are less reliable, LP-IR scores differentiated patients with and without insulin resistance and metabolic syndrome. In addition, distinctive lipoprotein profiles such as increased large VLDL and reduced small VLDL particles were observed in these chronic hepatitis B patients, suggesting the presence of hepatic steatosis and fibrosis. Future studies with larger sample sizes are necessary to confirm the utility of these NMR-measured lipoprotein parameters in predicting metabolic disease and fibrosis among patients with underlying liver disease.

Background: Chronic hepatitis B is associated with virus-specific and global T-cell dysfunction. We hypothesized that therapeutic reduction in serum HBV DNA, ALT, and HBsAg would restore HBV-specific T-cell function and modify T-cell regulatory phenotype, with associated posttreatment ALT flare. Methods: HBV-specific T-cell lymphoproliferative responses and global T-cell phenotype were prospectively examined at baseline, weeks 24, 48, 192, 216, and 240 in 34 adults with immune-active chronic hepatitis B treated with 192 weeks of tenofovir alone (n=21) or combined with pegylated interferon (PegIFN) in the first 24 weeks (n=13). HBV-specific T-cell IFNγ responses at weeks 0, 24, and 48 were examined by ELISpot assay ex vivo in 24 patients. Posttreatment flare was defined by serum ALT >5 times the upper limit of normal. Results: Tenofovir therapy did not promote sustained induction of HBV-specific T-cell proliferative responses, regardless of PegIFN therapy or decreased serum HBsAg, HBV DNA, or ALT levels. Instead, HBV-specific T-cell IFNγ responses declined significantly by 48 weeks of therapy (p=0.008). Posttreatment ALT flare was associated with higher baseline %PD1+/CD8 (p=0.019), %PD1+/CD4 (p=0.039), and %CTLA4+/CD4 (p=0.003) T cells compared to non-flares, but without associated HBsAg loss or increased HBV-specific T-cell responsiveness. Conclusion: HBV-specific T-cell function was not restored after 192 weeks of tenofovir therapy and did not correlate with HBsAg levels before, during, or after therapy. Baseline global T-cell regulatory phenotype was a predictor for ALT flare post-therapy without associated HBsAg decline. These findings support the need for more novel immune-modulatory approaches to enhance HBV-specific T-cell responsiveness.

Background HBsAg can be derived from intrahepatic cccDNA and integrated HBV DNA (iDNA). We examined the iDNA from liver tissues of 24 HBeAg(+) and 32 HBeAg(−) treatment-naive CHB participants. Methods Liver tissues were obtained from the North American Hepatitis B Research Network (HBRN). For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove rcDNA and iDNA prior to qPCR. For iDNA detection, total DNA was subjected to HBV hybridization-targeted next generation sequencing (HBV-NGS) assay. The HBV-host junction sequences were identified by ChimericSeq. Comparison of HBV cccDNA and iDNA with serum and intrahepatic virological parameters were assessed. Results Intrahepatic cccDNA, serum HBV DNA, HBV RNA, HBcrAg and qHBsAg were higher among the HBeAg(+) participants. Among the HBeAg(+) samples, 87% had positive intrahepatic HBcAg staining compared to 13% of HBeAg(−) samples (p50% iDNA in their livers. The iDNA junction-breakpoint distributions for the HBeAg(+) group were random with 15.9% localized to the DR2-DR1 region. In contrast, 52.4% of the iDNA were clustered at DR2-DR1 region among the HBeAg(−) participants. Microhomology-mediated end joining (MMEJ) patterns of dslDNA HBV integration was more frequent in HBeAg (+) livers. Conclusion Serum RNA and HBcrAg reflect the intrahepatic cccDNA concentrations. HBeAg(−) CHB participants had high levels of intrahepatic iDNA and HBsAg despite lower cccDNA levels suggesting that iDNA is the primary source of HBsAg in HBeAg(−) CHB.

Gastrointestinal stromal tumors (GISTs) originate from mesenchymal cells and account for ∼1% of primary malignant tumors in the digestive system. They are diagnosed based on characteristic immunohistochemical staining pattern, including CD117 and DOG1, as well as genetic analysis for mutations in the KIT and platelet-derived growth factor receptor α genes. Extragastrointestinal stromal tumors (EGISTs) share very similar morphology with GISTs but arise outside the gastrointestinal tract. The most common locations for EGISTs are the omentum, mesentery, retroperitoneum, and pancreas, followed by the liver, vagina, and prostate. The mean age of presentation of these tumors is in the sixth decade of life and tumor dimensions at different locations typically range from 7 to 15.8 cm. Most of these tumors are unifocal and of the spindle cell type. GISTs generally have a better prognosis than EGISTs, with cumulative 5-year survival rates of 85% for GISTs and 38%–60.9% for EGISTs. Among EGISTs, omental tumors have higher overall survival than mesenteric or retroperitoneal tumors. Additionally, age of >60 years, male sex, larger tumor size, higher mitotic rate, and nuclear pleomorphism are associated with worse prognosis in EGISTs.