Virological response to nucleos(t)ide analogues treatment in chronic hepatitis B patients is associated with Bacteroides-dominant gut microbiome.

BACKGROUND: Gut dysbiosis is present in chronic hepatitis B virus (HBV) infection. In this study, we integrated microbiome and metabolome analysis to investigate the role of gut microbiome in virological response to nucleos(t)ide analogues (NAs) treatment. METHODS: Chronic HBV patients were prospectively recruited for steatosis and fibrosis assessments via liver elastography, with full-length 16S sequencing performed to identify the compositional gut microbiota differences. Fasting plasma bile acids were quantified by liquid chromatography-tandem mass spectrometry. FINDINGS: All patients (n = 110) were characterized into three distinct microbial clusters by their dominant genus: c-Bacteroides, c-Blautia, and c-Prevotella. Patients with c-Bacteroides had a higher plasma ursodeoxycholic acids (UDCA) level and an increase in 7-alpha-hydroxysteroid dehydrogenase (secondary bile acid biotransformation) than other clusters. In NAs-treated patients (n = 84), c-Bacteroides was associated with higher odds of plasma HBV-DNA undetectability when compared with non-c-Bacteroides clusters (OR 3.49, 95% CI 1.43-8.96, p = 0.01). c-Blautia was positively associated with advanced fibrosis (OR 2.74, 95% CI 1.09-7.31, p = 0.04). No such associations were found in treatment-naïve patients. Increased Escherichia coli relative abundance (0.21% vs. 0.03%, p = 0.035) was found in on-treatment patients (median treatment duration 98.1 months) with advanced fibrosis despite HBV DNA undetectability. An enrichment in l-tryptophan biosynthesis was observed in patients with advanced fibrosis, which exhibited a positive correlation with Escherichia coli. INTERPRETATION: Collectively, unique bacterial signatures, including c-Bacteroides and c-Blautia, were associated with virological undetectability and fibrosis evolution during NAs therapy in chronic HBV, setting up intriguing possibilities in optimizing HBV treatment. FUNDING: This study was supported by the Guangdong Natural Science Fund (2019A1515012003).

ALT to qHBsAg ratio predicts long-term HBsAg seroclearance after entecavir cessation in Chinese patients with chronic hepatitis B.

BACKGROUND & AIMS: Factors predicting HBsAg seroclearance after treatment cessation, irrespective of nucleos(t)ide analogue (NA) resumption, have important clinical implications. We evaluated predictors of long-term HBsAg seroclearance after entecavir cessation. METHODS: This study followed-up Chinese patients with chronic hepatitis B from two previous studies of entecavir cessation. All patients were non-cirrhotic, HBeAg-negative, with undetectable HBV DNA (<20 IU/ml) at end-of-treatment (EOT). They were monitored closely for 48 weeks with regular HBV DNA, quantitative HBsAg (qHBsAg) and alanine aminotransferase (ALT) measurements. Entecavir was resumed at HBV DNA >2,000 IU/ml, irrespective of ALT levels. After the initial 48 weeks, patients were assessed every 6 months, regardless of entecavir resumption, to monitor for HBsAg seroclearance. RESULTS: A total of 194 patients (63.4% male, mean age 49.9 years, on entecavir for a median of 47.2 months) were recruited; 94 (48.5%) and 158 (81.4%) patients had EOT qHBsAg <100 IU/ml and <1,000 IU/ml, respectively; 151 (77.8%) patients were eventually resumed on entecavir. After follow-up for a median of 70.7 (51.0-118.2) months, 28 (14.4%) patients had HBsAg seroclearance. qHBsAg levels at weeks 36 and 48 after EOT independently predicted HBsAg seroclearance (both p <0.01), whereas qHBsAg from EOT to week 24 only trended towards statistical significance. The ratio of ALT/qHBsAg at all time points from EOT to week 48 independently predicted HBsAg seroclearance (hazard ratios ranging from 1.003-1.028, all p <0.01) with excellent diagnostic performance (area under the receiver-operating characteristic curve 0.799-0.933, negative predictive value >90% at different time points), regardless of whether entecavir was resumed. CONCLUSIONS: The ALT/qHBsAg ratio after entecavir cessation predicts HBsAg seroclearance, even in patients who were resumed on treatment. Its use may mitigate the risk of severe hepatitis flares in patients managed by observation without treatment resumption. IMPACT AND IMPLICATIONS: Current predictors of HBsAg seroclearance after finite nucleos(t)ide analogue (NA) therapy have suboptimal predictive value. We demonstrated that the ALT/qHBsAg ratio may be able to reflect the balance between host control and virological activity. The ALT/qHBsAg ratio at different time points from end-of-treatment till week 48 independently and accurately predicted HBsAg seroclearance in patients who have stopped entecavir. The ALT/qHBsAg ratio may be utilized by clinicians for patient selection and retreatment decisions in finite NA therapy.

Plasma interferon-gamma-inducible-protein 10 level as a predictive factor of spontaneous hepatitis B surface antigen seroclearance in chronic hepatitis B patients.

BACKGROUND AND AIM: Spontaneous seroclearance of hepatitis B surface antigen (HBsAg) is a rare event that occurs in patients that are chronically infected with the hepatitis B virus. As the functional cure and ultimate treatment endpoint of chronic hepatitis B (CHB), HBsAg seroclearance is an important milestone in the natural history of CHB and serves great clinical value. This study aims to identify host and viral factors associated with HBsAg seroclearance. METHODS: This is a retrospective study carried out in the Queen Mary Hospital, Hong Kong. By analyzing the plasma retrieved from the serum archive (collected during 2011-2021) of 100 CHB patients attending the hospital's liver clinic, the longitudinal cytokine profiles between the HBsAg-losers and the control groups were compared. RESULTS: Data revealed that plasma levels of IP-10 were significantly lower at 3-5 years prior to HBsAg seroclearance compared with patients who remained HBsAg positive (P < 0.05). Receiver operating characteristic curve analysis reveals that plasma IP-10 levels at multiple time points before HBsAg seroclearance return area under receivor-operating characteristic curve (AUC) greater than 0.7. Plasma IP-10 levels at 42.39 pg/mL produced an AUC = 0.723 with 74.0% sensitivity and 75.5% specificity to predict subsequent HBsAg seroclearance in the next 3-5 years. Low plasma IP-10 identified 91.4% patients with quantitative HBsAg < 100 IU/mL who would subsequently develop HBsAg seroclearance, compared with 37% with higher plasma IP-10 levels (P < 0.001). CONCLUSIONS: Low plasma levels of IP-10 are associated with subsequent HBsAg seroclearance, suggesting potential clinical utilities of measurement of IP-10 in predicting HBsAg seroclearance, especially among patients with low HBsAg.

FAT4 loss initiates hepatocarcinogenesis through the switching of canonical to noncanonical WNT signaling pathways.

BACKGROUND: Mutation and downregulation of FAT atypical cadherin 4 (FAT4) are frequently detected in HCC, suggesting a tumor suppressor role of FAT4. However, the underlying molecular mechanism remains elusive. METHODS: CRISPR-Cas9 system was used to knockout FAT4 (FAT4-KO) in a normal human hepatic cell line L02 to investigate the impact of FAT4 loss on the development of HCC. RNA-sequencing and xenograft mouse model were used to study gene expression and tumorigenesis, respectively. The mechanistic basis of FAT4 loss on hepatocarcinogenesis was elucidated using in vitro experiments. RESULTS: We found that FAT4-KO disrupted cell-cell adhesion, induced epithelial-mesenchymal transition, and increased expression of extracellular matrix components. FAT4-KO is sufficient for tumor initiation in a xenograft mouse model. RNA-sequencing of FAT4-KO cells identified PAK6-mediated WNT/ß-catenin signaling to promote tumor growth. Suppression of PAK6 led to ß-catenin shuttling out of the nucleus for ubiquitin-dependent degradation and constrained tumor growth. Further, RNA-sequencing of amassed FAT4-KO cells identified activation of WNT5A and ROR2. The noncanonical WNT5A/ROR2 signaling has no effect on ß-catenin and its target genes (CCND1 and c-Myc) expression. Instead, we observed downregulation of receptors for WNT/ß-catenin signaling, suggesting the shifting of ß-catenin-dependent to ß-catenin-independent pathways as tumor progression depends on its receptor expression. Both PAK6 and WNT5A could induce the expression of extracellular matrix glycoprotein, laminin subunit alpha 4. Laminin subunit alpha 4 upregulation in HCC correlated with poor patient survival. CONCLUSIONS: Our data show that FAT4 loss is sufficient to drive HCC development through the switching of canonical to noncanonical Wingless-type signaling pathways. The findings may provide a mechanistic basis for an in-depth study of the two pathways in the early and late stages of HCC for precise treatment.

Anti-HBc: a significant host predictor of spontaneous HBsAg seroclearance in chronic hepatitis B patients - a retrospective longitudinal study.

BACKGROUND AND AIM: In chronic hepatitis B infection (CHB), seroclearance of hepatitis B surface antigen (HBsAg) is associated with favourable clinical outcomes compared to those with persistent HBsAg seropositivity, and thus considered as a desired treatment endpoint. This current study explores the possibility of serum antibody to hepatitis B core antigen (anti-HBc) as a potential predictive factor of HBsAg seroclearance. METHODS: This is a retrospective study that analyzed the plasma samples of CHB patients using the LUMIPULSE® G1200 analyzer. The longitudinal anti-HBc level between patients who subsequently achieved HBsAg seroclearance (S-losers) and those with persistent HBsAg-positivity (controls) were compared at multiple time points before the event. RESULTS: A total of 240 subjects (120 S-losers and 120 controls; age- and gender-matched) were included (mean age 56.42 ± 10.81, 65% male). Compared to controls, S-losers had significantly lower plasma anti-HBc levels prior to HBsAg seroclearance, with a significant trend of declining plasma anti-HBc 8-5 years prior to HBsAg seroclearance (p < 0.01), while such trend was not observed in controls. ROC curve analysis revealed that plasma anti-HBc at multiple time points before HBsAg seroclearance return AUC greater than 0.7. Plasma anti-HBc level at the cut-off value of 82.50 COI was 68.3% sensitive and 90% specific for HBsAg seroclearance within 1 year. Combining with quantitative HBsAg < 100 IU/mL, anti-HBc < 82.5 COI identified 88.2% patients who would develop HBsAg seroclearance within 1 year. CONCLUSION: Plasma anti-HBc level began to decline 10 years prior to HBsAg seroclearance and can serve as a potential predictor for subsequent HBsAg seroclearance.

Small Extracellular Vesicle-Derived vWF Induces a Positive Feedback Loop between Tumor and Endothelial Cells to Promote Angiogenesis and Metastasis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a hypervascular malignancy by which its growth and dissemination are largely driven by the modulation of tumor-derived small extracellular vesicles (sEVs). Proteomic profiling of circulating sEVs of control individuals and HCC patients identifies von Willibrand factor (vWF) to be upregulated progressively along HCC stages. Elevated sEV-vWF levels are found in a larger cohort of HCC-sEV samples and metastatic HCC cell lines compared to their respective normal counterparts. Circulating sEVs of late-stage HCC patients markedly augment angiogenesis, tumor-endothelial adhesion, pulmonary vascular leakiness, and metastasis, which are significantly compromised by anti-vWF antibody. The role of vWF is further corroborated by the enhanced promoting effect of sEVs collected from vWF-overexpressing cells. sEV-vWF modulates endothelial cells through an elevated level of vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Mechanistically, secreted FGF2 elicits a positive feedback response in HCC via the FGFR4/ERK1 signaling pathway. The co-administration of anti-vWF antibody or FGFR inhibitor significantly improves the treatment outcome of sorafenib in a patient-derived xenograft mouse model. This study reveals mutual stimulation between HCC and endothelial cells by tumor-derived sEVs and endothelial angiogenic factors, facilitating angiogenesis and metastasis. It also provides insights into a new therapeutic strategy involving blocking tumor-endothelial intercellular communication.

Association of Diabetes Mellitus with All-Cause and Cause-Specific Mortality among Patients with Metabolic-Dysfunction-Associated Fatty Liver Disease: A Longitudinal Cohort Study.

BACKGROUND: Diabetes mellitus (DM) is a comorbidity commonly presenting with metabolic-dysfunction-associated fatty liver disease (MAFLD); however, few tests for interaction have been reported. Our target was to evaluate the prognostic implications of DM in patients with different forms of MAFLD. METHODS: Using data from the Third National Health and Nutrition Examination Survey (NHANES III) in the United States, we screened 14,797 participants aged 20-74 who received ultrasound examinations from 1988-1994. Among them, 4599 patients met the diagnosis of MAFLD, and we defined mortality as the outcome event. Survival analysis of competitive risk events was performed using Cox regression and sub-distributed risk ratio (SHR). RESULTS: During 21.1 years of follow-up, cardiovascular diseases seemed to be the most common cause of death among MAFLD patients. Of them, DM was present in 25.48% and was independently associated with increased risk of all-cause mortality (HRs: 1.427, 95% CIs: 1.256-1.621, p < 0.001) and cause-specific mortality (cardiovascular-related mortality (HRs: 1.458, 95% CIs: 1.117-1.902, p = 0.005), non-cardiovascular-related mortality (HRs: 1.423, 95% CIs: 1.229-1.647, p < 0.001), and non-cancer-related mortality (HRs: 1.584, 95% CIs: 1.368-1.835, p < 0.001), respectively). Surprisingly, this association was more significant for young patients (p-value for interaction <0.001). Moreover, DM had a greater risk of all-cause and cause-specific mortality among overweight and obese MAFLD patients (p-value for interaction <0.001). CONCLUSIONS: DM increased the risk of all-cause and cause-specific mortality (cardiovascular-related, non-cardiovascular-related, and non-cancer-related) in MAFLD patients, especially in younger patients with excess obesity.

A longitudinal study to detect hepatitis B surface and core-related antigens in chronic hepatitis B patients with hepatitis B surface antigen seroclearance using highly sensitive assays.

BACKGROUND: This study aimed to evaluate the usefulness of two novel assays, namely the iTACT-hepatitis B surface antigen (iTACT-HBsAg) and iTACT-hepatitis B core-related antigen (iTACT-HBcrAg) assays, in chronic hepatitis B (CHB) patients with HBsAg seroclearance (SC) documented by standard assays. METHODS: HBsAg and HBcrAg were measured by the two iTACT-assays in 556 serial sera collected from 96 CHB patients at 7 different time points spanning from 5 years before to 10 years after SC and 120 HBsAg-negative, anti-HBc-positive individuals. As controls, 60 seronegative individuals, who were negative for HBsAg, anti-HBc and anti-HBs, were tested. RESULTS: Using the iTACT-assays, HBsAg was detectable in 154/418 (36.8%) samples collected after SC. HBcrAg was detectable in 78.3% and 65.9% of samples collected before and after SC, respectively. The detectability rates of both HBsAg and HBcrAg progressively decreased over time after SC. At 10 years after SC, 20.4% and 64.5% of the patients still had detectable HBsAg and HBcrAg, respectively. 66 (71%) patients had detectable HBsAg and/or HBcrAg. Among the 120 HBsAg-negative, anti-HBc-positive individuals, 11 (9.2%) and 4 (3.3%) had detectable HBsAg and HBcrAg respectively. Both HBsAg and HBcrAg were undetectable in the controls. CONCLUSION: The iTACT assays detected a low level of HBsAg and/or HBcrAg in >70% of patients even at 10 years after SC, suggesting that CHB patients with SC still harbour a low level of HBV protein expression. The clinical significance of detectable viral proteins after SC with regard to disease progression and HBV reactivation deserves further investigations.

Glycemic burden and the risk of adverse hepatic outcomes in patients with chronic hepatitis B with type 2 diabetes.

BACKGROUND AND AIMS: Type 2 diabetes (T2D) is common among patients with chronic hepatitis B infection (CHB) and has been associated with increased risk of carcinogenesis, including HCC. We investigated factors associated with HCC and fibrosis progression among patients with CHB with T2D (CHB+T2D). APPROACH AND RESULTS: Chinese patients with CHB were prospectively recruited for the incidence of HCC and fibrosis progression defined by transient elastography. Among patients with CHB+T2D, glycemic control was assessed by mean glycated hemoglobin (HbA1c) and HbA1c variability determined using HbA1c measurements in the 5 years preceding recruitment. A total of 2330 patients with CHB were recruited (mean age 54.6 ±11.8 years old, 55.5% male, 57.9% antiviral-treated), with 671 (28.8%) having CHB+T2D (mean T2D duration 7.2 ± 4.6 years, mean HbA1c 7.2 ± 0.9%). T2D was independently associated with HCC (HR 2.080, 95% CI 1.343-3.222) and fibrosis progression (OR 4.305, 95% CI 3.416-5.424) in the overall cohort. In patients with CHB+T2D, factors reflecting glycemic burden (T2D duration [HR 1.107, 95% CI 1.023-1.198]), mean HbA1c (HR 1.851, 95% CI 1.026-3.339), time reaching target HbA1c (HbA1c-TRT; HR 0.978, 95% CI 0.957-0.999), liver stiffness (HR 1.041-1.043), and smoking (HR 2.726-3.344) were independently associated with HCC (all p < 0.05), but not HbA1c variability or controlled attenuation parameter. The same glycemic burden-related factors (T2D duration, mean HbA1c, and HbA1c-TRT), in addition to baseline fasting glucose, baseline HbA1c, AST and antiviral therapy, were independently associated with fibrosis progression at 3 years. CONCLUSIONS: High glycemic burden was associated with HCC development and fibrosis progression among patients with CHB+T2D, highlighting the importance of glycemic control in reducing liver-related complications.

Sparse logistic regression revealed the associations between HBV PreS quasispecies and hepatocellular carcinoma.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) has been proved highly associated with the development of hepatocellular carcinoma (HCC). AIMS: The purpose of the study is to investigate the association between HBV preS region quasispecies and HCC development, as well as to develop HCC diagnosis model using HBV preS region quasispecies. METHODS: A total of 104 chronic hepatitis B (CHB) patients and 117 HBV-related HCC patients were enrolled. HBV preS region was sequenced using next generation sequencing (NGS) and the nucleotide entropy was calculated for quasispecies evaluation. Sparse logistic regression (SLR) was used to predict HCC development and prediction performances were evaluated using receiver operating characteristic curves. RESULTS: Entropy of HBV preS1, preS2 regions and several nucleotide points showed significant divergence between CHB and HCC patients. Using SLR, the classification of HCC/CHB groups achieved a mean area under the receiver operating characteristic curve (AUC) of 0.883 in the training data and 0.795 in the test data. The prediction model was also validated by a completely independent dataset from Hong Kong. The 10 selected nucleotide positions showed significantly different entropy between CHB and HCC patients. The HBV quasispecies also classified three clinical parameters, including HBeAg, HBVDNA, and Alkaline phosphatase (ALP) with the AUC value greater than 0.6 in the test data. CONCLUSIONS: Using NGS and SLR, the association between HBV preS region nucleotide entropy and HCC development was validated in our study and this could promote the understanding of HCC progression mechanism.

Enhanced Liver Fibrosis Score Stratifies Hepatocellular Carcinoma Risk in Patients With Hepatitis B Surface Antigen Seroclearance.

In this prospective study involving 337 chronic hepatitis B patients who achieved spontaneous hepatitis B surface antigen (HBsAg) seroclearance (SC), serum enhanced liver fibrosis (ELF) before SC was associated with hepatocellular carcinoma (HCC) (hazard ratio 2.588), and ELF <10.8 was associated with >97% reduction in risk of HCC development in patients with age SC ≥ 50 (n = 190).

Hepatitis B virus X protein promotes hepatocarcinogenesis via the activation of HMGA2/STC2 signaling to counteract oxidative stress-induced cell death.

Chronic hepatitis B virus (HBV) infection can cause oxidative stress and induce cell death. The mechanisms by which cells overcome oxidative stress to survive remain largely unknown. Here, we used human sera, liver tissues and cell lines to study how HBV modulates cellular pathways to counteract oxidative stress-induced cell death. We found high-mobility group AT-hook 2 (HMGA2), an architectural transcription factor is upregulated in hepatocellular carcinoma (HCC) tissues and cell lines. Elevated serum HMGA2 is significantly associated with viral load in HBV carriers, and HBV-related HCC. We showed that HBV X protein (HBx) encoded by HBV-induced cell growth via HMGA2 activation. The growth-promoting effect is abolished when HMGA2 is suppressed. Ectopic HBx expression induced DNA damage and oxidative stress. HMGA2 silencing reduced oxidative stress in HBx-expressing cells. Cytoprotective stanniocalcin 2 (STC2) protein is a downstream target of HMGA2. Consistent with the findings in HMGA2, STC2 mRNA and protein expression are upregulated in HCC tissues. Elevated serum STC2 is also associated with viral load in HBV carriers, and HCC. STC2 is transcriptionally upregulated by HBx and HMGA2 to elicit cytoprotection against apoptosis. STC2 knockdown disrupted Bax/Bcl-2 balance that increased cytochrome c release, caspase 3/7 activity and apoptosis, and thus abolished the growth-promoting effect of HMGA2. Clinical relevance of HBx/HMGA2/STC2 signaling is evidenced by the significant correlation of serum HMGA2/STC2 in active HBV infection and HCC. These findings reveal a novel HBx regulatory HMGA2/STC2 pathway in counteracting reactive oxygen species-induced cell death. HMGA2 and STC2 may be therapeutic targets for prevention of hepatocarcinogenesis in chronic HBV infection.

Alleviation of Hepatic Steatosis: Dithizone-Related Gut Microbiome Restoration During Paneth Cell Dysfunction.

Non-alcoholic fatty liver disease (NAFLD), the world's most common chronic liver disease, is increasingly linked to gut dysbiosis. Paneth cells secrete antimicrobial peptides that regulate the gut microbiome, but their role in the pathogenesis of NAFLD remains unclear. Here, we determine the changes in NAFLD development and gut microbial composition and function via the injection of dithizone that can pharmacologically deplete the granules of Paneth cells. Eight-week-old C57BL/6J male mice (n = 31) were given a high-fat diet (HFD) or standard control diet for 12 weeks. Dithizone (10 mg/kg) was intravenously injected every 3 weeks during the period of diet feeding. Metagenomic DNA was extracted from fecal samples for PacBio Single-Molecule Real-Time sequencing to identify changes in microbial composition and predicted function. We observed dithizone-treated HFD mice, when compared to non-treated HFD mice, to have significant reductions in hepatic triglyceride content (28.98 vs. 53.52 mg/g, p = 0.0419); plasma insulin level (2.18 vs. 6.63 ng/ml, p = 0.0079); and relative mRNA levels of fatty acid synthase (0.52 vs. 1.57, p = 0.0428) and stearoyl-CoA desaturase-1 (0.43 vs. 1.20, p = 0.0121). Bacterial taxonomic profiling found dithizone-treated HFD mice, when compared to non-treated HFD mice, had a lower Firmicutes/Bacteroidetes ratio (2.53 vs. 5.26, p = 0.0541); a higher relative abundance of Bacteroides ASV21 and ASV42 (1.04 vs. 0.22%, p = 0.0277 and 0.96 vs. 0.09%, p = 0.0213); and a reduction in microbes belonging to Firmicutes (all p < 0.05). Bacteroides species correlated positively with predicted microbial functions such as L-methionine (r = 0.54, p = 0.0019) and tetrahydrofolate (r = 0.52, p = 0.0029) biosynthesis. Collectively, dithizone treatment was associated with alleviation in the severity of liver steatosis in HFD mice, possibly through gut microbiome modulation involving the increase in Bacteroides, suggesting microbiome-targeted therapies may have a role in the treatment of NAFLD.

Role of Serum M2BPGi Levels in Predicting Persistence of Advanced Fibrosis in Chronic Hepatitis B Virus Infection.

BACKGROUND: Serum mac-2-binding protein glycosylation isomer (M2BPGi) is a novel marker for liver fibrosis assessment in patients with different liver diseases. For chronic hepatitis B infection (CHB), advanced fibrosis or cirrhosis is a risk factor for liver cancer and hepatic decompensation. We aimed to assess the role of serum M2BPGi in prediction of persistence of advanced fibrosis in CHB patients despite potent antiviral therapy. METHODS: CHB patients with advanced fibrosis or cirrhosis who were put on nucleos(t)ide analogs for ≥ 3 years with normal alanine aminotransferase and undetectable serum HBV DNA were prospectively recruited. Assessment of liver fibrosis with transient elastography (TE) and M2BPGi measurements were performed at baseline and repeated at 3 years. Advanced fibrosis and cirrhosis were defined by liver stiffness (LS) ≥ 9.0 kPa and ≥ 12.0 kPa, respectively. RESULTS: A total of 143 patients (M:F = 101:42; median age 58.7 years; 53.8% cirrhotic) were recruited and completed paired assessment. The median value of baseline LS and M2BPGi were 12.0 (IQR: 10.5-18.2) kPa and 0.99 cut-off-index (IQR: 0.75-1.74) (COI), respectively, with 96% concordance for diagnosing F3/F4. Ninety-six (67.1%) patients had persistent advanced fibrosis or cirrhosis at 3 years despite continuation of long-term antiviral treatment. Upon multivariate analysis, baseline M2BPGi (OR 2.128, 95% CI 1.037-4.366) and presence of central obesity (OR 4.648, 95% CI 1.742-12.402) were significantly associated with persistent advanced fibrosis or cirrhosis at 3 years. Baseline M2BPGi ≥ 1.265 COI has 50.6% sensitivity and 79.4% specificity for predicting persistent advanced fibrosis or cirrhosis at 3 years (area under the receiver-operating characteristic curve: 0.695). The presence of central obesity in combination with baseline M2BPGi ≥ 1.265 COI was associated with 95.7% patients having persistent advanced fibrosis or cirrhosis at 3 years. HCC development was observed in five patients during follow-up and was associated with bigger median increase in the level of serum M2BPGi compared to patients without HCC (46% vs 6.2%, P = 0.038). CONCLUSION: Persistent advanced fibrosis or cirrhosis was observed in two-thirds of CHB patients despite potent antiviral therapy. High serum M2BPGi and central obesity were associated with more than twofold and fourfold increase in risk of persistent advanced fibrosis or cirrhosis, respectively.

Stanniocalcin 1 is a serum biomarker and potential therapeutic target for HBV-associated liver fibrosis.

Stanniocalcin 1 (STC1), a secreted protein, is upregulated in human cancers including hepatocellular carcinoma (HCC). While most HCCs develop from chronic liver disease, which involves progressive parenchymal injury and fibrosis, the role of STC1 in this preneoplastic stage remains poorly understood. In this study we investigated the clinical relevance and functional significance of secreted STC1 in liver fibrosis. To this end, the STC1 level was determined in the serum samples of chronic hepatitis B patients and correlated with the degree of liver fibrosis. Diagnostic performance of STC1 was analysed by area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, positive predictive value, and negative predictive value. The results were compared with other well-characterised serum biomarkers for liver fibrosis: Aspartate transaminase to Platelet Ratio Index (APRI) and Fibrosis-4 (FIB-4). The functional role of STC1 was interrogated by in vitro experiments using cell line models. Expression of fibrogenic markers was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Our results showed that the serum STC1 level in chronic hepatitis B patients was positively correlated with the degree of liver fibrosis and showed a stepwise increase in accordance with the severity of fibrosis. The AUROCs for detecting significant fibrosis (>9.0 kPa) and cirrhosis (>12.0 kPa) was 0.911 and 0.880, respectively. STC1 demonstrated a superior specificity and positive predictive value when compared to APRI and FIB-4. Consistent with this, STC1 was elevated in the liver tissues and sera of CCl4 -treated mice showing marked liver fibrosis. In vitro, STC1 was secreted by the human hepatic stellate cell line LX2. Human recombinant STC1 (rhSTC1) induced expression of fibrogenic markers in LX2 cells. The profibrogenic phenotype conferred by rhSTC1 or TGF-ß1 in LX2 cells could be attenuated using anti-STC1 antibody. Taken together, STC1 is a specific serum biomarker for HBV-associated liver fibrosis. STC1 functionally promotes liver fibrogenesis and is a potential actionable target. © 2022 The Pathological Society of Great Britain and Ireland.

Phenotypic Changes of PD-1 and GITR in T Cells Are Associated With Hepatitis B Surface Antigen Seroclearance.

BACKGROUND: Regulatory T cells (Tregs) possess hepatitis B virus (HBV)-specific immunoregulatory effects in chronic HBV infection. The role of Tregs in spontaneous seroclearance of hepatitis B surface antigen (HBsAg) remains to be determined. METHODS: We recruited treatment-naive chronic HBV patients achieving spontaneous HBsAg seroclearance (experimental group) and matched HBsAg-positive controls. Peripheral blood mononuclear cells were isolated using the Ficoll-Paque density gradient centrifugation method. The frequency of Tregs and inhibitory phenotypes and immunoregulatory cytokines of Tregs were detected by flow cytometry. RESULTS: Twenty-seven patients with HBsAg seroclearance (mean age: 52.40±6.00 y, 55.6% male) and 27 matched controls were recruited. Median HBsAg and HBV DNA levels in the control group were 2.80 (1.24 to 3.43) and 3.16 (1.68 to 3.85) log IU/mL, respectively. Mean frequencies of Tregs and expressions of FoxP3+ Tregs were comparable in both groups (both P>0.05). The mean expression of programmed death 1 (PD-1) and glucocorticoid-induced TNFR family-related gene (GITR) in total CD4+ T cells were significantly downregulated in the experimental group when compared with the control group (10.62% vs. 13.85%, P=0.003; 16.20% vs. 27.02%, P=0.002, respectively). When compared with the control group, PD-1+CD4+ Tregs expression in the experimental group was significantly downregulated (13.85% vs. 10.62%, P=0.003). A similar phenomenon was noted for GITR+CD8+ Tregs (20.16% vs. 14.08%, P=0.049). Intracellular cytokine productions showed no significant differences (all P>0.05). CONCLUSIONS: The reduced expression of PD-1 and GITR might attenuate the immunosuppressive capability of Tregs. Decreased expression on CD4+ T cells might represent an enhanced antiviral function, playing a role in initiating the "functional cure" of chronic HBV infection.

Long-term serological, virological and histological responses to RNA inhibition by ARC-520 in Chinese chronic hepatitis B patients on entecavir treatment.

OBJECTIVE: We examined the serological, virological (in serum and liver) and histological profiles in chronic hepatitis B virus (HBV) patients during and after completion of multiple dose (MD) ARC-520. DESIGN: The present phase 1b study was a multidose, open-label extension cohort of patients that had received single dose ARC-520 in our previous study. Eight patients received 4-9 4 weekly doses of MD ARC-520 and entecavir. Liver biopsies were performed in six patients. Intrahepatic and serum HBV DNA, HBV RNA and viral antigens were measured. RESULTS: All patients had 28.9-30.4 months of follow-up after the last MD. All three hepatitis B e antigen (HBeAg)-positive patients had profound reductions in hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen and HBV RNA with two undergoing HBeAg seroconversion. One further achieved HBsAg seroconversion (anti-HBs level of 25.1 IU/L) and the remaining two had HBsAg reductions of -1.7 and -3.5 log IU/mL >30 months after MD. Among the five HBeAg-negative patients, four had modest HBsAg reduction >29 months after completion of MD and one achieved HBsAg seroconversion (anti-HBs level of 152.5 IU/L) and was negative for liver HBsAg staining. Entecavir was successfully stopped in this patient 12 months after HBsAg seroconversion. Temporally related alanine aminotransferase elevations preceded by HBsAg reductions were observed in three patients suggesting immune activation. HBcAg staining was negative in all six biopsied patients. Two patients with <10% HBsAg positive staining of hepatocytes had correspondingly low serum HBsAg levels of 1.5 and 11.5 IU/mL. CONCLUSIONS: MD ARC-520 therapy achieved sustained and profound reductions of viral antigens and HBV RNA. HBsAg seroclearance was achievable. TRIAL REGISTRATION NUMBER: NCT02065336.

Patient pIgR-enriched extracellular vesicles drive cancer stemness, tumorigenesis and metastasis in hepatocellular carcinoma.

BACKGROUND & AIMS: Extracellular vesicles (EVs) play a pivotal role in connecting tumor cells with their local and distant microenvironments. Herein, we aimed to understand the role (on a molecular basis) patient-derived EVs play in modulating cancer stemness and tumorigenesis in the context of hepatocellular carcinoma (HCC). METHODS: EVs from patient sera were isolated, quantified and characterized. The EVs were vigorously tested, both in vitro and in vivo, through various functional assays. Proteomic analysis was performed to identify the functional components of EVs. The presence and level of polymeric immunoglobulin receptor (pIgR) in circulating EVs and tumor and non-tumorous tissues of patients with HCC were determined by ELISA, immunoblotting, immunohistochemistry and quantitative PCR. The functional role and underlying mechanism of EVs with enhanced pIgR expression were elucidated. Blockade of EV-pIgR with neutralizing antibody was performed in nude mice implanted with patient-derived tumor xenografts (PDTXs). RESULTS: Circulating EVs from patients with late-stage HCC (L-HCC) had significantly elevated pIgR expression compared to the EVs released by control individuals. The augmenting effect of L-HCC-EVs on cancer stemness and tumorigenesis was hindered by an anti-pIgR antibody. EVs enriched with pIgR consistently promoted cancer stemness and cancerous phenotypes in recipient cells. Mechanistically, EV-pIgR-induced cancer aggressiveness was abrogated by Akt and ß-catenin inhibitors, confirming that the role of EV-pIgR depends on the activation of the PDK1/Akt/GSK3ß/ß-catenin signaling axis. Furthermore, an anti-pIgR neutralizing antibody attenuated tumor growth in mice implanted with PDTXs. CONCLUSIONS: This study illustrates a previously unknown role of EV-pIgR in regulating cancer stemness and aggressiveness: EV-pIgR activates PDK1/Akt/GSK3ß/ß-catenin signaling cascades. The blockade of the intercellular communication mediated by EV-pIgR in the tumor microenvironment may provide a new therapeutic strategy for patients with cancer. LAY SUMMARY: The World Health Organization estimates that more than 1 million patients will die from liver cancer, mostly hepatocellular carcinoma (HCC), in 2030. Understanding the underlying mechanism by which HCC acquires aggressive attributes is crucial to improving the diagnosis and treatment of patients. Herein, we demonstrated that nanometer-sized extracellular vesicles released by tumors promote cancer stemness and tumorigenesis. Within these oncogenic vesicles, we identified a key component that functions as a potent modulator of cancer aggressiveness. By inhibiting this functional component of EVs using a neutralizing antibody, tumor growth was profoundly attenuated in mice. This hints at a potentially effective therapeutic alternative for patients with cancer.

Detection of the Hepatitis B Surface Antigen in Patients with Occult Hepatitis B by Use of an Assay with Enhanced Sensitivity.

Patients with occult hepatitis B infection (OBI) have undetectable hepatitis B surface antigen (HBsAg) by conventional assays but detectable hepatitis B virus (HBV) DNA in blood/liver. We evaluated the key performance characteristics of a sensitive HBsAg assay (Architect HBsAg Next qualitative assay, referred to as NEXT) with respect to HBsAg detection. Assay precision, sample carryover, and seroconversion sensitivity of NEXT were evaluated. HBsAg was measured by NEXT in 1,138 individuals, including 1,038 patients who attended liver clinics in a tertiary hospital (200 HBV DNA-positive blood donors whose HBsAg was undetectable by conventional assays, 38 patients receiving immunosuppressive therapy, and 800 chronic hepatitis B patients with HBsAg seroclearance) and 100 HBsAg-negative subjects recruited from a community project. The within-run and within-laboratory coefficients of variation were <6% for the positive sample pools. In 9 seroconversion panels tested, NEXT allowed an earlier HBsAg detection than conventional assays. NEXT detected HBsAg in 10/200 (5%) HBsAg-negative blood donors, 1/20 (5%) and 0/18 HBsAg-negative patients with and without HBV reactivation, respectively, and 59/800 (7.3%) patients with HBsAg seroclearance. HBsAg was detectable by NEXT in 27.8%, 8.2%, 6.9%, 3.8%, and 1.9% samples at <3, 3 to 5, >5 to 8, >8 to 11, and >11 years after HBsAg seroclearance, respectively. Seven out of 100 HBsAg-negative community-identified subjects were tested positive by NEXT. Compared with conventional HBsAg assays, NEXT demonstrated a higher sensitivity and conferred an increment of 5 to 7% detection rate in patients with OBI, thereby helping in identifying HBV carriers and prevention of OBI-associated HBV transmission and reactivation.

Reduced hepatic steatosis is associated with higher risk of hepatocellular carcinoma in chronic hepatitis B infection.

BACKGROUND: Concomitant chronic hepatitis B infection (CHB) and non-alcoholic fatty liver disease (NAFLD) is common, but the implications of NAFLD on clinical outcomes of CHB, including hepatocellular carcinoma (HCC), are not well-investigated. METHODS: CHB patients were recruited for transient elastography assessment for liver stiffness (LS), and controlled attenuation parameter (CAP), a non-invasive quantification of hepatic steatosis, and were prospectively followed up for development of HCC. Steatosis and severe steatosis were diagnosed by CAP ≥ 248 dB/m and ≥ 280 dB/m respectively, and advanced fibrosis/cirrhosis was diagnosed by LS ≥ 9 kPa. The independent effect of hepatic steatosis on HCC was examined via propensity score matching (PSM) of LS and other significant clinical variables. RESULTS: Forty-eight patients developed HCC among 2403 CHB patients (55.6% male, median age 55.6 years, 57.1% antiviral-treated, median ALT 26 U/L) during a median follow-up of 46.4 months. Multivariate Cox regression analysis showed age (HR 1.063), male (HR 2.032), Albumin-Bilirubin score (HR 2.393) and CAP (HR 0.993) were associated with HCC development. The cumulative probability of HCC was 2.88%, 1.56% and 0.71%, respectively for patients with no steatosis, mild-to-moderate steatosis, and severe steatosis, respectively (p = 0.01). The risk of HCC increased from 1.56 to 8.89% in patients without severe steatosis if advanced fibrosis/cirrhosis was present (p < 0.001). PSM yielded 957 pairs of CHB patients and hepatic steatosis was independently associated with HCC (HR 0.41). CONCLUSION: Reduced hepatic steatosis was significantly associated with a higher risk of incident HCC in CHB infection. Routine CAP and LS measurements are important for risk stratification.