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.

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.

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).

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.

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.

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.

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.

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.

Tumor suppressive role of mitochondrial sirtuin 4 in induction of G2/M cell cycle arrest and apoptosis in hepatitis B virus-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is developed from uncontrolled cell growth after the malignant transformation of hepatocytes. The hepatitis B virus (HBV) X protein (HBx) has shown to induce cell cycle progression and hepatocarcinogenesis. A sub-fraction of HBx is localized in the mitochondria. Sirtuin 4 (SIRT4), a mitochondrial protein, has been demonstrated to play a tumor-suppressive role in many cancers, including HCC. However, little is known about the association between mitochondrial HBx and SIRT4 during hepatocarcinogenesis. We aimed to investigate the clinical significance and functional role of SIRT4 in HBV-related HCC. SIRT4 expression was significantly lower in the HCC tissues collected from 30 patients with HBV-related HCC than in normal liver tissues from control patients (p < 0.0001). TCGA data analysis indicated that SIRT4 expression was also lower in patients with HBV infection than in those without, and SIRT4 levels were positively associated with better patient survival. Similarly, HCC cell lines had lower SIRT4 expression than normal liver cell lines (all p < 0.01). Among the HCC cell lines, those harbored HBV had a lower SIRT4 expression than those without HBV (p < 0.0001). In vitro experiments revealed that stable HBx transfection suppressed SIRT4 expression in both HepG2 and Huh7 cells (both p < 0.001). Ectopic SIRT4 overexpression alone could induce cellular senescence through arresting cell-cycle progression at G2/M, and inducing cell apoptosis in HCC cells. Mechanistically, SIRT4 upregulated cell-cycle governing genes p16 and p21 protein expression, suppressed CyclinB1/Cdc2 and Cdc25c which normally induce cell-cycle progression, and suppressed survivin to induce apoptosis. Our findings demonstrate the interaction between HBV and SIRT4 in the context of HCC. SIRT4 involves in G2/M DNA damage checkpoint control and genomic stability in hepatocarcinogenesis, which could be targeted for future anticancer strategies.

Residual HBV DNA and pgRNA viraemia is associated with hepatocellular carcinoma in chronic hepatitis B patients on antiviral therapy.

BACKGROUND: We aimed to assess whether residual hepatitis B virus (HBV) viraemia is associated with HCC development. METHODS: This is a case-control study of 104 patients [52 HCC and 52 non-HCC (matched with age, gender, cirrhosis and treatment duration)] on ≥ 3 years entecavir (ETV) with unquantifiable HBV DNA by Cobas Taqman assay v2.0 (Roche Diagnostics; lower limit of quantification [LLOQ] 20 IU/mL). Serial sera within 1, 1-2, and > 2 years prior to HCC diagnosis or last follow-up (LFU) were measured for HBV DNA and pre-genomic (pg) RNA using a highly sensitive semi-quantitative PCR assay with lower limit of detection of 10 IU/mL and LLOQ of 51.5 IU/mL, respectively. RESULTS: Among the 104 patients (80.8% male, median age 61.2 years old, 38.5% cirrhosis, median duration of ETV 45.5 months), 38.5% and 9.6% HCC patients had undetectable serum DNA and pgRNA, respectively, compared to 65.4% and 36.5% in non-HCC patients; P = 0.005 & 0.001, respectively, at the time of HCC diagnosis/LFU. Detectable HBV DNA and pgRNA were associated with a higher 2-year risk of HCC development (HR 2.79, 95% CI 1.424-5.468 & HR 4.544, 95% CI 1.07-19.289, respectively). No significant differences were observed for qHBsAg levels between HCC and non-HCC patients. CONCLUSIONS: More than 50% CHB patients on ETV with HBV DNA < LLOQ by standard assay had persistent viraemia as determined by a more sensitive assay. Detectable HBV DNA or pgRNA by more sensitive assays was associated with HCC development. More potent viral suppression is required to further reduce the risk of HCC.

Nidogen 1-Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1.

In hepatocellular carcinoma (HCC) patients with extrahepatic metastasis, the lung is the most frequent site of metastasis. However, how the lung microenvironment favors disseminated cells remains unclear. Here, it is found that nidogen 1 (NID1) in metastatic HCC cell-derived extracellular vesicles (EVs) promotes pre-metastatic niche formation in the lung by enhancing angiogenesis and pulmonary endothelial permeability to facilitate colonization of tumor cells and extrahepatic metastasis. EV-NID1 also activates fibroblasts, which secrete tumor necrosis factor receptor 1 (TNFR1), facilitate lung colonization of tumor cells, and augment HCC cell growth and motility. Administration of anti-TNFR1 antibody effectively diminishes lung metastasis induced by the metastatic HCC cell-derived EVs in mice. In the clinical perspective, analysis of serum EV-NID1 and TNFR1 in HCC patients reveals their positive correlation and association with tumor stages suggesting the potential of these molecules as noninvasive biomarkers for the early detection of HCC. In conclusion, these results demonstrate the interplay of HCC EVs and activated fibroblasts in pre-metastatic niche formation and how blockage of their functions inhibits distant metastasis to the lungs. This study offers promise for the new direction of HCC treatment by targeting oncogenic EV components and their mediated pathways.

Occult hepatitis B infection and hepatocellular carcinoma: Epidemiology, virology, hepatocarcinogenesis and clinical significance.

Occult hepatitis B infection (OBI) refers to a condition where replication-competent HBV DNA is present in the liver, with or without HBV DNA in the blood, in individuals with serum HBsAg negativity assessed by currently available assays. The episomal covalently closed circular DNA (cccDNA) in OBI is in a low replicative state. Viral gene expression is mediated by epigenetic control of HBV transcription, including the HBV CpG island methylation pathway and post-translational modification of cccDNA-bound histone, with a different pattern from patients with chronic HBV infection. The prevalence of OBI varies tremendously across patient populations owing to numerous factors, such as geographic location, assay characteristics, host immune response, coinfection with other viruses, and vaccination status. Apart from the risk of viral reactivation upon immunosuppression and the risk of transmission of HBV, OBI has been implicated in hepatocellular carcinoma (HCC) development in patients with chronic HCV infection, those with cryptogenic or known liver disease, and in patients with HBsAg seroclearance after chronic HBV infection. An increasing number of prospective studies and meta-analyses have reported a higher incidence of HCC in patients with HCV and OBI, as well as more advanced tumour histological grades and earlier age of HCC diagnosis, compared with patients without OBI. The proposed pathogenetic mechanisms of OBI-related HCC include the influence of HBV DNA integration on the hepatocyte cell cycle, the production of pro-oncogenic proteins (HBx protein and mutated surface proteins), and persistent low-grade necroinflammation (contributing to the development of fibrosis and cirrhosis). There remain uncertainties about exactly how, and in what order, these mechanisms drive the development of tumours in patients with OBI.

Rebound of HBV DNA after cessation of nucleos/tide analogues in chronic hepatitis B patients with undetectable covalently closed.

BACKGROUND & AIMS: Nucleos(t)ide analogues (NUCs) effectively suppress serum HBV DNA. Previously, we have identified 21 patients with undetectable covalently closed circular DNA (cccDNA) upon long-term NUC therapy. This study investigated the effect of NUC withdrawal in patients with undetectable cccDNA. METHODS: Nineteen patients on long term NUCs (median 13.4 years) were recruited: 13 were randomized to discontinue NUCs; 6 to continue taking NUCs. All had undetectable cccDNA at the time of last liver biopsy (median time 2.9 years prior to randomization). Serum HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B core-related antigen (HBcrAg), liver biochemistry, and serum HBV RNA were monitored. RESULTS: At the time of randomization, all patients had undetectable serum HBV DNA and HBV RNA. Twelve of the 13 patients had HBV DNA rebound to 100 IU/ml within 20 weeks of NUC discontinuation. The thirteenth patient had HBV DNA rebound at week 70. Three patients experienced biochemical flares after re-treatment which subsequently resolved. There was no significant association between the time of HBV DNA rebound and baseline HBsAg, HBcrAg and alanine aminotransferase, duration of treatment, and age at which treatment was stopped (all p >0.05). At the time of HBV DNA rebound, HBV DNA levels correlated with HBcrAg levels (p = 0.003), but not with HBsAg levels (p = 0.262). CONCLUSIONS: In patients with undetectable intrahepatic cccDNA, virologic rebound still occurred after NUC cessation. At the rebound of HBV DNA, the kinetics of HBsAg production were independent of those of viral DNA replication. Additional studies are required to determine the factors that may predict virologic rebound and when NUCs can be discontinued in HBsAg-positive patients with chronic hepatitis B. LAY SUMMARY: It has been shown that following long-term nucleos(t)ide analogue treatment for chronic hepatitis B, some patients have undetectable levels of viral DNA in their livers. We tested the results of withdrawing nucleos(t)ide analogue treatment in these patients and found that viral relapse could occur in patients with undetectable viral DNA. Further research is required to determine whether nucleos(t)ide analogue treatment can be discontinued in specific patients with chronic hepatitis B.

Entecavir Reduced Serum Hepatitis B Core-Related Antigen in Chronic Hepatitis B Patients with Hepatocellular Carcinoma.

Serum hepatitis B core-related antigen (HBcrAg) was shown to predict the risk of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) patients undergoing treatment. We investigated the longitudinal profile of HBcrAg in entecavir (ETV)-treated CHB patients with subsequent HCC development. We identified HCC cases diagnosed at ≥1 year after ETV initiation. CHB patients without HCC (matched for age, sex, cirrhosis status, baseline hepatitis B virus [HBV] DNA level, and ETV treatment duration) were identified as controls at an HCC:non-HCC ratio of 1:2. Serum samples were retrieved at baseline (ETV initiation) and at 3 and 5 years of ETV therapy for HBcrAg measurement (log IU/mL). In total, 180 patients (60 HCC patients matched with 120 CHB patients without HCC; median age, 56.5 years; 80.6% male; baseline HBV DNA, 5.9 log IU/mL; median follow-up, 6.8 years) were recruited. The median time from ETV initiation to HCC development was 3.2 years. HBcrAg levels were higher in HCC cases than in controls at all three time points: 5.69 log IU/ mL versus 5.02 log IU/mL (p=0.025), 4.23 log IU/mL versus 3.36 log IU/mL (p=0.007), and 3.86 log IU/mL versus 3.36 log IU/mL (p=0.009), respectively. ETV led to similar rates of decline in HBcrAg from baseline to 3 years in both groups (0.34 log IU/mL/year vs 0.39 log IU/mL/year, p=0.774), although the decline from 3 to 5 years was slower in the non- HCC group (0.05 log IU/mL/year) than in the HCC group (0.09 log IU/mL/year, p=0.055). ETV time-dependently reduced HBcrAg in HCC and non-HCC patients. HBcrAg interpretation should consider the antiviral treatment duration.

HBV X protein mutations affect HBV transcription and association of histone-modifying enzymes with covalently closed circular DNA.

The hepatitis B X protein (HBx) plays a role in the epigenetic regulation of hepatitis B virus (HBV) replication. This study investigated the effects of HBx mutations on HBV transcription and the recruitment of HBx, histone acetyl-transferase P300 and histone deacetylase 1 (HDAC1) to circularized HBV DNA (which resembles covalently closed circular DNA [cccDNA]). Compared with wild type, majority of mutants had lower levels of intracellular HBV RNA (44-77% reduction) and secretory HBsAg (25-81% reduction), and 12 mutants had a reduction in intracellular encapsidated HBV DNA (33-64% reduction). Eight mutants with >70% reduction in HBV RNA and/or HBsAg were selected for chromatin immunoprecipitation analysis. Four HBx mutants with mutations in amino acid residues 55-60 and 121-126 had a lower degree of HBx-cccDNA association than wild type HBx (mean % input: 0.02-0.64% vs. 3.08% in wild type). A reduced association between cccDNA and P300 (mean % input: 0.69-1.81% vs. 3.48% in wild type) and an augmented association with HDAC1 (mean % input: 4.01-14.0% vs. 1.53% in wild type) were detected. HBx amino acid residues 55-60 and 121-126 may play an important role in HBV transcription regulation, via their impeded interaction with cccDNA and altered recruitment of histone modifying enzymes to cccDNA.