Risk of de novo HCC in patients with MASLD following direct-acting antiviral-induced cure of HCV infection.

BACKGROUND & AIMS: Data are limited on the risk of de novo hepatocellular carcinoma (HCC) in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) after achieving sustained virologic response (SVR12) using direct-acting antivirals (DAAs) for hepatitis C virus (HCV). METHODS: 1598 eligible patients received biannual alpha-fetoprotein (AFP) and liver imaging surveillance to detect de novo HCC beyond achieving SVR12. MASLD was defined as presence of controlled attenuation parameter (CAP) ≥ 248 dB/m and ≥ one cardiometabolic risk factor (CMRF). Cumulative HCC incidence was compared between patients with/without MASLD. We built univariable and multivariable Cox proportional hazards models to evaluate factors associated with HCC. Sensitivity analysis was performed using the Fine-Gray subdistribution hazards model. Additionally, we evaluated the mediation effect of MASLD on CMRFs and of CMRFs on MASLD for HCC using mediation analysis with bootstrapping. RESULTS: The incidence rate of HCC was 1.44 per 100 person-years of follow-up (PYFU) [95% confidence interval (CI): 1.19-1.74]. Patients with MASLD had a higher cumulative HCC incidence than those without MASLD (log-rank test, p < 0.001). Multivariable Cox regression analysis revealed that in addition to age, sex, LSM, platelet count, and AFP, MASLD (adjusted hazard ratio (aHR): 2.07 [95% CI:1.36-3.16], p < 0.001) was independently associated with HCC. This finding was confirmed by the Fine-Gray model, which showed a subdistribution HR (sHR) of 2.07 (95% CI: 1.34-3.19, p < 0.001) for MASLD. MASLD significantly mediated CMRFs for HCC development. CONCLUSION: After achieving SVR12, patients with MASLD exhibited an increased HCC risk compared to those without MASLD. Vigilant HCC surveillance and control of CMRFs to mitigate the effect MASLD on HCC remain crucial for this population. IMPACT AND IMPLICATIONS: The risk of de novo HCC among patients with MASLD, a novel nomenclature of steatotic liver disease (SLD), after the attaining of SVR12 using DAAs remains to be confirmed. In this study recruiting 1598 patients in Taiwan, individuals with MASLD exhibited approximately a two-fold increased risk of de novo HCC, compared to those without MASLD after achieving SVR12. MASLD significantly mediated CMRFs for HCC development. Our findings underscore the critical importance of pharmacological interventions and proactive lifestyle modifications to control CMRFs in patients with MASLD, as well as the need for vigilant HCC surveillance to ensure favorable outcomes following HCV eradication.

Outcomes of radiofrequency ablation for hepatocellular carcinoma with concurrent steatotic liver disease.

BACKGROUND: Steatotic liver disease (SLD) is an emerging liver disease that has been associated with an increased risk for hepatocellular carcinoma (HCC). The impact of concurrent SLD on the prognosis of HCC remains unknown. This study investigates how concurrent SLD affects the outcomes of patients with HCC undergoing curative radiofrequency ablation (RFA) therapy. METHODS: A retrospective analysis of patients with early-stage HCC receiving curative RFA at a tertiary medical center was conducted. Laboratory data and HCC characteristics were recorded and analyzed by a Cox proportional hazards regression model to predict recurrence and all-cause mortality after RFA. RESULTS: A total of 598 patients with HCC were included between 2005 and 2015, with 139 and 459 classified in SLD and non-SLD groups, respectively. The SLD group exhibited a significantly better liver reserve and a lower cumulative incidence of HCC recurrence and liver-related and all-cause mortality after a median follow-up of 51 months. After adjusting for metabolic dysfunction, liver reserve, and HCC characteristics, the presence of SLD reduced all-cause mortality (adjusted hazard ratio [aHR], 0.67; 95% confidence interval [CI], 0.45-0.996; p = .048), which was supported by inverse probability weighting analysis (aHR, 0.65; 95% CI, 0.42-1.00; p = .049). Poor liver functional reserve (high albumin-bilirubin grades) increased all-cause mortality dose dependently. Barcelona Clinic Liver Cancer staging and a higher Fibrosis-4 index were predictors for HCC recurrence, whereas SLD was not. CONCLUSIONS: Among patients with HCC undergoing curative RFA, those with concurrent SLD had a lower risk of all-cause mortality compared to those with poor liver functional reserve. PLAIN LANGUAGE SUMMARY: The present research demonstrated that patients with both liver cancer and steatotic liver disease who received curative radiofrequency ablation for liver cancer survived longer compared to those without steatotic liver disease. Maintaining good liver function is an important prognostic factor for survival.

Unraveling the role of hepatitis B virus DNA integration in B-cell lymphomagenesis.

BACKGROUND: Studies have shown that hepatitis B virus (HBV)-associated B-cell non-Hodgkin lymphoma (NHL) constitutes a unique subgroup with distinct clinical features. It still leaves open the question of whether the integration of HBV DNA into the B-cell genome is a causal mechanism in the development of lymphoma. METHODS: Using the hybridisation capture-based next generation sequencing and RNA sequencing, we characterised the HBV integration pattern in 45 HBV-associated B-cell NHL tumour tissues. RESULTS: A total of 354 HBV integration sites were identified in 13 (28.9%) samples, indicating the relatively low integration frequency in B-cell NHLs. High plasma HBV DNA loads were not associated with the existence of HBV integration. The insertion sites distributed randomly across all the lymphoma genome without any preferential hotspot neither at the chromosomal level nor at the genetic level. Intriguingly, most HBV integrations were nonclonal in B-cell NHLs, implying that they did not confer a survival advantage. Analysis of the paired diagnosis-relapse samples showed the unstable status of HBV integrations during disease progression. Furthermore, transcriptomic analysis revealed the limited biological impact of HBV integration. CONCLUSION: Our study provides an unbiased HBV integration map in B-cell NHLs, revealing the insignificant role of HBV DNA integration in B-cell lymphomagenesis.

Hepatitis B virus DNA integration: Implications for diagnostics, therapy, and outcome.

Hepatitis B virus (HBV) DNA integration - originally recognised as a non-functional byproduct of the HBV life cycle - has now been accepted as a significant contributor to HBV pathogenesis and hepatitis D virus (HDV) persistence. Integrated HBV DNA is derived from linear genomic DNA present in viral particles or produced from aberrantly processed relaxed circular genomic DNA following an infection, and can drive expression of hepatitis B surface antigen (HBsAg) and HBx. DNA integration events accumulate over the course of viral infection, ranging from a few percent during early phases to nearly 100 percent of infected cells after prolonged chronic infections. HBV DNA integration events have primarily been investigated in the context of hepatocellular carcinoma development as they can activate known oncogenes and other growth promoting genes, cause chromosomal instability and, presumably, induce epigenetic alterations, promoting tumour growth. More recent evidence suggests that HBsAg expression from integrated DNA might contribute to HBV pathogenesis by attenuating the immune response. Integrated DNA provides a source for envelope proteins required for HDV replication and hence represents a means for HDV persistence. Because integrated DNA is responsible for persistence of HBsAg in the absence of viral replication it impacts established criteria for the resolution of HBV infection, which rely on HBsAg as a diagnostic marker. Integrated HBV DNA has been useful in assessing the turnover of infected hepatocytes which occurs during all phases of chronic hepatitis B including the initial phase of infection historically termed immune tolerant. HBV DNA integration has also been shown to impact the development of novel therapies targeting viral RNAs.

Data Independent Acquisition Mass Spectrometry Enhanced Personalized Glycosylation Profiling of Haptoglobin in Hepatocellular Carcinoma.

Aberrant glycosylation has gained significant interest for biomarker discovery. However, low detectability, complex glycan structures, and heterogeneity present challenges in glycoprotein assay development. Using haptoglobin (Hp) as a model, we developed an integrated platform combining functionalized magnetic nanoparticles and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) for highly specific glycopeptide enrichment, followed by a data-independent acquisition (DIA) strategy to establish a deep cancer-specific Hp-glycosylation profile in hepatitis B virus (HBV, n = 5) and hepatocellular carcinoma (HCC, n = 5) patients. The DIA strategy established one of the deepest Hp-glycosylation landscapes (1029 glycopeptides, 130 glycans) across serum samples, including 54 glycopeptides exclusively detected in HCC patients. Additionally, single-shot DIA searches against a DIA-based spectral library outperformed the DDA approach by 2-3-fold glycopeptide coverage across patients. Among the four N-glycan sites on Hp (N-184, N-207, N-211, N-241), the total glycan type distribution revealed significantly enhanced detection of combined fucosylated-sialylated glycans, which were the most dominant glycoforms identified in HCC patients. Quantitation analysis revealed 48 glycopeptides significantly enriched in HCC (p < 0.05), including a hybrid monosialylated triantennary glycopeptide on the N-184 site with nearly none-to-all elevation to differentiate HCC from the HBV group (HCC/HBV ratio: 2462 ± 766, p < 0.05). In summary, DIA-MS presents an unbiased and comprehensive alternative for targeted glycoproteomics to guide discovery and validation of glyco-biomarkers.

Fibrosis-4 index stratifies risks of hepatocellular carcinoma in patients with chronic hepatitis C.

BACKGROUND AND AIMS: Risk stratification for patients with a higher risk of hepatocellular carcinoma (HCC) is crucial. We aimed to investigate the role of the Fibrosis-4 (FIB-4) index in predicting chronic hepatitis C (CHC)-related HCC. METHODS: A retrospective cohort study consecutively included treatment-naive CHC patients receiving longitudinal follow-up at the National Taiwan University Hospital from 1986 to 2014. The clinical data were collected and traced for HCC development. Multivariable Cox proportional hazard regression analysis was used to investigate the predictors for HCC. RESULTS: A total of 1285 patients in the ERADICATE-C cohort were included. The median age was 54, 56% were females, and 933 had HCV viremia. There were 33%, 38%, and 29% of patients having FIB-4 index <1.45, 1.45-3.25, and ≥3.25, respectively. After a median of 9-year follow-up, 186 patients developed HCC. Multivariable analysis revealed that older age, AFP≥20 ng/mL, cirrhosis, and a higher FIB-4 index were independent predictors for HCC. Compared with patients with FIB-4 index <1.45, those with FIB-4 1.45-3.25 had a 5.51-fold risk (95% confidence interval [CI]: 2.65-11.46), and those with FIB-4 ≥ 3.25 had 7.45-fold risk (95% CI: 3.46-16.05) of HCC. In CHC patients without viremia, FIB-4 index 1.45-3.25 and FIB-4 ≥ 3.25 increased 6.78-fold and 16.77-fold risk of HCC, respectively, compared with those with FIB-4 < 1.45. CONCLUSION: The baseline FIB-4 index can stratify the risks of HCC in untreated CHC patients, even those without viremia. The FIB-4 index should thus be included in the management of CHC.

Dynamic change of metabolic dysfunction-associated steatotic liver disease in patients with hepatitis C virus infection after achieving sustained virologic response with direct-acting antivirals.

BACKGROUND: Information on the dynamics of metabolic dysfunction-associated steatotic liver disease (MASLD) among hepatitis C virus patients achieving sustained virologic response (SVR12) with direct-acting antivirals (DAAs) is limited. METHODS: We enrolled 1512 eligible participants in this prospective study. MASLD was defined by a controlled attenuation parameter (CAP) of ≥248 dB/m utilizing vibration-controlled transient elastography in conjunction with presence of ≥1 cardiometabolic risk factor. The distribution of MASLD and the changes in CAP were evaluated before treatment and at SVR12. Forward stepwise logistic regression analyses were performed to determine factors significantly associated with the regression or emergence of MASLD. RESULTS: The prevalence of MASLD decreased from 45.0% before treatment to 36.1% at SVR12. Among 681 participants with MASLD before treatment, 144 (21%) exhibited MASLD regression at SVR12. Conversely, among 831 participants without MASLD before treatment, 9 (1.1%) developed MASLD at SVR12. Absence of type 2 diabetes (T2D) [odds ratio (OR): 1.73, 95% confidence interval (CI): 1.13-2.65, p = 0.011], age > 50 years (OR: 1.73, 95% CI: 1.11-2.68, p = 0.015), and alanine transaminase (ALT) ≤ 2 times the upper limit of normal (ULN) (OR: 1.56; 95% CI: 1.03-2.37, p = 0.035) were associated with the regression of MASLD. Presence of T2D was associated with the emergence of MASLD (OR: 5.83, 95% CI: 1.51-22.56, p = 0.011). CONCLUSIONS: The prevalence of MASLD decreased after achieving SVR12 with DAAs. Patients with pre-existing T2D showed a diminished probability of MASLD regression and a heightened risk of MASLD emergence post-SVR12.

HBV DNA Integration into Telomerase or MLL4 Genes and TERT Promoter Point Mutation as Three Independent Signatures in Subgrouping HBV-Related HCC with Distinct Features.

Introduction: A set of genetic mutations to classify hepatocellular carcinoma (HCC) useful to clinical studies is an unmet need. Hepatitis B virus-related HCC (HBV-HCC) harbors a unique genetic mutation, namely, the HBV integration, among other somatic endogenous gene mutations. We explored a combination of HBV DNA integrations and common somatic mutations to classify HBV-HCC by using a capture-sequencing platform. Methods: A total of 153 HBV-HCCs after surgical resection were subjected to capture sequencing to identify HBV integrations and three common somatic mutations in genomes. Three mutually exclusive mutations, HBV DNA integration into the TERT promoter, HBV DNA integration into MLL4, or TERT promoter point mutation, were identified in HBV-HCC. Results: They were used to classify HBV-HCCs into four groups: G1 with HBV-TERT integration (25.5%); G2 with HBV-MLL4 integration (10.5%); G3 with TERT promoter mutation (30.1%); and G4 without these three mutations (34.0%). Clinically, G3 has the highest male-to-female ratio, cirrhosis rate, and associated with higher early recurrence and mortality after resection, but G4 has the best outcome. Transcriptomic analysis revealed a grouping different from the published ones and G2 with an active immune profile related to immune checkpoint inhibitor response. Analysis of integrated HBV DNA provided clues for HBV genotype and variants in carcinogenesis of different HCC subgroup. This new classification was also validated in another independent cohort. Conclusion: A simple and robust genetic classification was developed to aid in understanding HBV-HCC and in harmonizing clinical studies.

Major HBV splice variant encoding a novel protein important for infection.

BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.

Serum MYCN as a predictive biomarker of prognosis and therapeutic response in the prevention of hepatocellular carcinoma recurrence.

The proto-oncogene MYCN expression marked a cancer stem-like cell population in hepatocellular carcinoma (HCC) and served as a therapeutic target of acyclic retinoid (ACR), an orally administered vitamin A derivative that has demonstrated promising efficacy and safety in reducing HCC recurrence. This study investigated the role of MYCN as a predictive biomarker for therapeutic response to ACR and prognosis of HCC. MYCN gene expression in HCC was analyzed in the Cancer Genome Atlas and a Taiwanese cohort (N = 118). Serum MYCN protein levels were assessed in healthy controls (N = 15), patients with HCC (N = 116), pre- and post-surgical patients with HCC (N = 20), and a subset of patients from a phase 3 clinical trial of ACR (N = 68, NCT01640808). The results showed increased MYCN gene expression in HCC tumors, which positively correlated with HCC recurrence in non-cirrhotic or single-tumor patients. Serum MYCN protein levels were higher in patients with HCC, decreased after surgical resection of HCC, and were associated with liver functional reserve and fibrosis markers, as well as long-term HCC prognosis (>4 years). Subgroup analysis of a phase 3 clinical trial of ACR identified serum MYCN as the risk factor most strongly associated with HCC recurrence. Patients with HCC with higher serum MYCN levels after a 4-week treatment of ACR exhibited a significantly higher risk of recurrence (hazard ratio 3.27; p = .022). In conclusion, serum MYCN holds promise for biomarker-based precision medicine for the prevention of HCC, long-term prognosis of early-stage HCC, and identification of high-response subgroups for ACR-based treatment.

HIV coinfection exacerbates HBV-induced liver fibrogenesis through a HIF-1α- and TGF-ß1-dependent pathway.

BACKGROUND & AIMS: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-ß1 signaling. METHODS: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. RESULTS: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-ß1 (TGF-ß1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-ß1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-ß1-induced fibrogenesis. CONCLUSIONS: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-ß1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. IMPACT AND IMPLICATIONS: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-ß1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection.

Codelivery of TGFß and Cox2 siRNA inhibits HCC by promoting T-cell penetration into the tumor and improves response to Immune Checkpoint Inhibitors.

Upregulation of TGFß and Cox2 in the tumor microenvironment results in blockade of T-cell penetration into the tumor. Without access to tumor antigens, the T-cell response will not benefit from administration of the immune checkpoint antibodies. We created an intravenous polypeptide nanoparticle that can deliver two siRNAs (silencing TGFß and Cox2). Systemic administration in mice, bearing a syngeneic orthotopic hepatocellular carcinoma (HCC), delivers the siRNAs to various cells in the liver, and significantly reduces the tumor. At 2 mg/kg (BIW) the nanoparticle demonstrated a single agent action and induced tumor growth inhibition to undetectable levels after five doses. Reducing the siRNAs to 1mg/kg BIW demonstrated greater inhibition in the presence of PD-L1 mAbs. After only three doses BIW, we could still recover a smaller tumor and, in tumor sections, showed an increase in penetration of CD4+ and CD8+ T-cells deeper into the remaining tumor that was not evident in animals treated with non-silencing siRNA. The combination of TGFß and Cox2 siRNA co-administered in a polypeptide nanoparticle can act as a novel therapeutic alone against HCC and may augment the activity of the immune checkpoint antibodies. Silencing TGFß and Cox2 converts an immune excluded (cold) tumor into a T-cell inflamed (hot) tumor.

Serum RNase L levels in patients with chronic hepatitis B virus infection.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection still poses a major threat to global health. Oligoadenylate synthetase-ribonuclease L (RNase L) antiviral pathway is one of interferon-induced antiviral effectors. The relationship between RNase L and HBV has never been investigated and we aim to examine the serum RNase L levels in patients with different stages of chronic HBV infection. METHODS: The patients were enrolled from 1985 to 2000, who had been HBsAg positive for longer than 6 months, at the National Taiwan University Hospital. In total, 426 patients with chronic HBV infection were included in this study, including 135 inactive carriers, 148 cirrhosis, and 143 hepatocellular carcinoma (HCC) cases. RESULTS: The RNase L levels increase as the disease severity increases. Higher RNase L levels were associated with higher HBV viral load, and the HBV-RNase L relationship was replaced by the disease severity status when adding disease status into the model. Compared with inactive carriers, the risk of liver cirrhosis was 60-fold (odds ratio = 60.8, 95% confidence interval = 3.49-1061) with the highest quintile of RNase L levels, after the adjustment of HBV DNA. The dose-response trend was statistically significant with quintiles and one increment of RNase L level in relation to liver cirrhosis. Similar results were found when HCC was compared with inactive carriers, while there was no association when compared between liver cirrhosis and HCC. CONCLUSIONS: A positive relationship between serum RNase L and HBV viral titers or advanced disease status is uncovered in this study. Further investigation in this area may provide more details of an innate immune response for HBV and opportunity for novel therapeutic strategy.

HBcrAg-based risk score performs better than the HBV DNA-based scores for HCC prediction in grey zone patients who are HBeAg-negative.

Background & Aims: Risk scores have been designed to predict the development of hepatocellular carcinoma (HCC) in treatment-naive patients with chronic hepatitis B (CHB). However, little is known about their predictive accuracy in HBeAg-negative patients in the grey zone (GZ). We aimed to develop a HBcrAg-based HCC risk score and explore whether it outperforms other risk scores in GZ patients. Methods: Two retrospective cohorts of HBeAg-negative patients with American Association for the Study of Liver Diseases-defined GZ were established for derivation and validation (Taiwanese, N = 911; Japanese, N = 806). All of them were non-cirrhotic at baseline and remained treatment-naive during the follow-up. The primary endpoint was HCC development. Results: In a median follow-up period of 15.5 years, 85 patients developed HCC in the derivation cohort. We found that age, sex, alanine aminotransferase, platelet count, and HBcrAg, but not HBV DNA levels, were independent predictors and a 20-point GZ-HCC score was developed accordingly. The 10-year and 15-year area under the ROC curve (AUROC) ranged from 0.83 to 0.86, which outperformed the HBV DNA-based HCC risk scores, including REACH-B and GAG-HCC scores (AUROC ranging from 0.66 to 0.74). The better performance was also validated in EASL- and Asian Pacific Association for the Study of the Liver-defined GZ patients. These findings remained consistent in the validation cohort. Finally, the low-risk and high-risk GZ patients (stratified by a score of 8) had an HCC risk close to inactive CHB and immune-active CHB patients, respectively, in both cohorts. Conclusions: The HBcrAg-based GZ-HCC score predicts HCC better than other HBV DNA-based risk scores in GZ patients who are HBeAg-negative patients, which may help optimise their clinical management. Impact and implications: We have developed a risk score based on HBcrAg, which has shown better predictive ability for HCC compared with other risk scores based on HBV DNA. Using a score of 8, GZ patients can be classified into low- and high-risk groups, which can guide follow up and early treatment, respectively. This validated risk score is a valuable tool for optimising the management of GZ patients who are HBeAg-negative.

Serial increase and high alpha-fetoprotein levels predict the development of hepatocellular carcinoma in 6 months.

AIM: Alpha-fetoprotein (AFP) checkup with abdominal ultrasonography for hepatocellular carcinoma (HCC) surveillance remains controversial. We evaluated a serial AFP-increase and high AFP levels in the prediction of HCC. METHODS: At-risk patients with chronic liver disease underwent HCC surveillance with trimonthly AFP measurement were included and categorized into HCC and non-HCC groups. Their AFP levels at 12, 9, and 6 months (-6M) before the outcome date were evaluated. Group-based trajectory analysis and multivariable regression analysis were performed to identify AFP trajectories as risk predictors for HCC. RESULTS: Overall, 2776 patients were included in the HCC (n = 326) and non-HCC (n = 2450) groups. Serial AFP levels were significantly higher in the HCC than the non-HCC groups. Trajectory analysis identified AFP-increase group (11%) increased 24-fold risks of HCC compared with the AFP-stable (89%) group. Compared with patients without the AFP-increase, a serial 3-month AFP-increase ≥10% elevated HCC risk by 12.1-fold (95% CI: 6.5-22.4) in 6 months, and the HCC risks increased 13-60 fold in patients with cirrhosis, hepatitis B, or C receiving antiviral therapy, or AFP levels <20 ng/ml. Combining serial AFP-increase ≥10% and AFP ≥20 ng/ml at -6M significantly increased 41.7-fold (95% CI: 13.8-126.2) HCC risks. In patients who underwent biannual AFP checkups, those with both 6-month AFP-increase ≥10% and AFP ≥20 ng/ml increased 22.1-fold (95% CI: 12.52-39.16) HCC risks in 6 months. Most HCCs were detected at an early stage. CONCLUSIONS: Serial 3-6-month AFP-increase of ≥10% previously and AFP level of ≥20 ng/ml significantly increased HCC risks in 6 months.

Distinct effects of hepatic steatosis and metabolic dysfunction on the risk of hepatocellular carcinoma in chronic hepatitis B.

OBJECTIVE: Chronic hepatitis B (CHB) and metabolic dysfunction-associated fatty liver disease (MAFLD) are the leading causes of hepatocellular carcinoma (HCC). We aim to explore the impact of concurrent MAFLD on the risk of HCC in CHB. METHODS: Patients with CHB were consecutively recruited from 2006 to 2021. MAFLD was defined by steatosis and either obesity, diabetes mellitus, or other metabolic abnormalities. The cumulative incidence of HCC and associated factors were compared between the MAFLD and non-MAFLD groups. RESULTS: 10,546 treatment-naïve CHB patients were included with a median follow-up of 5.1 years. CHB patients with MAFLD (n = 2212) had fewer hepatitis B e antigen (HBeAg)-positivity, lower HBV DNA levels, and Fibrosis-4 index compared with the non-MAFLD group (n = 8334). MAFLD was independently associated with a 58% reduced risk of HCC (adjusted hazard ratio [aHR] 0.42, 95% confidence interval [CI] 0.25-0.68, p < 0.001). Furthermore, steatosis and metabolic dysfunction had distinct effects on HCC. Steatosis was protective against HCC (aHR 0.45, 95% CI 0.30-0.67, p < 0.001), while a greater burden of metabolic dysfunction increased the risk (aHR 1.40 per dysfunction increase, 95% CI 1.19-1.66, p < 0.001). The protective effect of MAFLD was further confirmed in analysis with inverse probability of treatment weighting (IPTW), patients who had undergone antiviral therapy, those with probable MAFLD, and after multiple imputation for missing data. CONCLUSIONS: Concurrent hepatic steatosis is independently associated with a lower risk of HCC, whereas the increasing burden of metabolic dysfunction aggravates the risk of HCC in untreated CHB patients.

Pre-operative gamma-glutamyl transferase levels predict outcomes in hepatitis B-related hepatocellular carcinoma after curative resection.

BACKGROUND: Surgical resection is a curative therapy for early-stage hepatocellular carcinoma (HCC); however, HCC recurrence is not uncommon. Identifying outcome predictors helps to manage the disease. Gamma-glutamyl transferase (GGT) may predict the development of HCC, but its role to predict the outcomes after surgical resection of HCC was unclear. This study aimed to investigate pre-operative GGT levels for outcome prediction in patients with hepatitis B virus (HBV)-related HCC. METHODS: We conducted a retrospective cohort study to include patients with HBV-related HCC receiving surgical resection. Clinical information, HCC characteristics and usage of antiviral therapy were collected. A time-dependent Cox proportional hazard regression analysis were used to predict HCC recurrence and survival. RESULTS: A total of 699 consecutive patients with HBV-related HCC who received surgical resection with curative intent between 2004 and 2013 were included. After a median of 4.4 years, 266 (38%) patients had HCC recurrence. Pre-operative GGT positively correlated with cirrhosis, tumor burden and significantly increased in patients to develop HCC recurrence. Multivariable analysis demonstrated that pre-operative GGT ≥38 U/L increased 57% risk (hazard ratio [HR]: 1.57, 95% confidence interval [CI]: 1.20-2.06) of recurrent HCC after adjustment for confounding factors. Specifically, pre-operative GGT ≥38 U/L predicted early (<2 years) HCC recurrence (HR: 1.94, 95% CI: 1.30-2.89). Moreover, pre-operative GGT ≥38 U/L predicted all-cause mortality (HR: 1.73, 95% CI: 1.06-2.84) after surgery. CONCLUSION: Pre-operative GGT levels ≥38 U/L independently predict high risks of HCC recurrence and all-cause mortality in HBV-related HCC patients receiving surgical resection.

Safety and dose escalation of the targeted oncolytic adenovirus OBP-301 for refractory advanced liver cancer: Phase I clinical trial.

OBP-301 is an oncolytic adenovirus modified to replicate within cancer cells and lyse them. This open-label, non-comparative, phase I dose-escalation trial aimed to assess its safety and optimal dosage in 20 patients with advanced hepatocellular carcinoma. Good tolerance was shown with a maximum tolerated dose of 6 × 1012 viral particles. The most common treatment-emergent adverse events were influenza-like illness, pyrexia, fatigue, decreased platelet count, abdominal distension, and anemia. Cohorts 4 and 5 had approximately 50% higher levels of CD8+ T cells in the peripheral blood after injection. The best target response occurred in 14 patients, 4 of whom had progressive disease. Multiple intratumoral injections of OBP-301 were well tolerated in patients with advanced hepatocellular carcinoma. The stable disease rate for the injected tumors was greater than the overall response rate, even with no obvious tumor response. OBP-301 might have a greater impact on local response as histological examination revealed that the presence of OBP-301 was consistent with the necrotic area at the injection site. Increased infiltration of CD8+ T cells and <1% PD-L1 expression were observed in tumors after injection. Improved antitumor efficacy might be achieved in future studies via viral injection with volume adjustment and in combination with other immuno-therapeutics.

Hepatitis D double reflex testing of all hepatitis B carriers in low-HBV- and high-HBV/HDV-prevalence countries.

Hepatitis D virus (HDV) infection occurs as a coinfection with hepatitis B and increases the risk of hepatocellular carcinoma, decompensated cirrhosis, and mortality compared to hepatitis B virus (HBV) monoinfection. Reliable estimates of the prevalence of HDV infection and disease burden are essential to formulate strategies to find coinfected individuals more effectively and efficiently. The global prevalence of HBV infections was estimated to be 262,240,000 in 2021. Only 1,994,000 of the HBV infections were newly diagnosed in 2021, with more than half of the new diagnoses made in China. Our initial estimates indicated a much lower prevalence of HDV antibody (anti-HDV) and HDV RNA positivity than previously reported in published studies. Accurate estimates of HDV prevalence are needed. The most effective method to generate estimates of the prevalence of anti-HDV and HDV RNA positivity and to find undiagnosed individuals at the national level is to implement double reflex testing. This requires anti-HDV testing of all hepatitis B surface antigen-positive individuals and HDV RNA testing of all anti-HDV-positive individuals. This strategy is manageable for healthcare systems since the number of newly diagnosed HBV cases is low. At the global level, a comprehensive HDV screening strategy would require only 1,994,000 HDV antibody tests and less than 89,000 HDV PCR tests. Double reflex testing is the preferred strategy in countries with a low prevalence of HBV and those with a high prevalence of both HBV and HDV. For example, in the European Union and North America only 35,000 and 22,000 cases, respectively, will require anti-HDV testing annually.

Serum cytokine profiles predict outcomes of chronic hepatitis B patients discontinuing entecavir or tenofovir therapy.

BACKGROUND/PURPOSE: Distinct hepatitis relapse has been observed after discontinuing entecavir (ETV) or tenofovir disoproxil fumarate (TDF) therapy in chronic hepatitis B (CHB) patients. End-of-therapy (EOT) serum cytokines were compared and used for outcome prediction. METHODS: A total of 80 non-cirrhotic CHB patients in a tertiary medical center in Taiwan who discontinued ETV (n = 51) or TDF (n = 29) therapy after fulfilling the APASL guidelines were prospectively enrolled. Serum cytokines were measured at EOT and 3rd month afterwards. Multivariable analysis was performed to predict virological relapse (VR, HBV DNA >2000 IU/mL), clinical relapse (CR, VR and alanine aminotransferase > 2-fold upper limit of normal) and hepatitis B surface antigen (HBsAg) seroclearance. RESULTS: Compared with TDF group, ETV stoppers had greater interleukin 5 (IL-5), IL-12 p70, IL-13, IL-17 A and tumor necrosis factor alpha (TNF-alpha) (all P < 0.05) at EOT. Older age, TDF use, higher EOT HBsAg and IL-18 (Hazard ratio [HR], 1.01; 95% CI, 1.00-1.02) levels at EOT predicted VR, while older age, higher EOT HBsAg and IL-7 (HR, 1.25; 95% CI, 1.00-1.56) levels predicted CR. In TDF stoppers, higher IL-7 (HR, 1.29; 95% CI, 1.05-1.60) and IL-18 (HR, 1.02; 95% CI, 1.00-1.04) levels predicted VR, while IL-7 (HR, 1.34; 95% CI, 1.08-1.65) and interferon-gamma (IFN-gamma) (HR, 1.08; 95% CI, 1.02-1.14) levels predicted CR. A lower EOT HBsAg level was associated with HBsAg seroclearance. CONCLUSION: Distinct cytokine profiles were observed after stopping ETV or TDF. Higher EOT IL-7, IL-18, and IFN-gamma could be probable predictors for VR and CR in patients discontinuing NA therapies.