Phase 1 Trial of the Safety, Pharmacokinetics, and Antiviral Activity of EDP-514 in Untreated Viremic Chronic Hepatitis B Patients.

Background & Aims: Oral EDP-514 is a potent core protein inhibitor of hepatitis B virus (HBV) replication, which produced a >4-log viral load reduction in HBV-infected chimeric mice with human liver cells. This study evaluated the safety, pharmacokinetics (PK), and antiviral activity of three doses of EDP-514 in treatment naïve viremic patients with HBeAg-positive or -negative chronic HBV infection. Methods: Patients with HBsAg detectable at screening and at least 6 months previously were eligible. HBeAg-positive and -negative patients had a serum/plasma HBV DNA level ≥20,000 and ≥2,000 IU/mL, respectively. Twenty-five patients were randomized to EDP-514 200 (n=6), 400 (n=6) or 800 mg (n=7) or placebo (n=6) once daily for 28 days. Results: A dose-related increase in EDP-514 exposure (AUClast and Cmax) was observed across doses. At Day 28, mean reductions in HBV DNA were 2.9, 3.3, 3.5 and 0.2 log10 IU/mL with EDP-514 200 mg, 400 mg, 800 mg, and placebo groups, respectively. The corresponding mean change from baseline for HBV RNA levels was 2.9, 2.4, 2.0, and 0.02 log10 U/mL. No virologic failures were observed. No clinically meaningful changes from baseline were observed for HBsAg, HBeAg or HBcrAg. Nine patients reported treatment emergent adverse events (TEAEs) of mild or moderate severity with no discontinuations, serious AEs or deaths. Conclusions: In treatment-naïve viremic patients, oral EDP-514 was generally safe and well-tolerated, displayed PK profile supportive of once-daily dosing, and markedly reduced HBV DNA and HBV RNA.

Efficacy, safety, and pharmacokinetics of capsid assembly modulator linvencorvir plus standard of care in chronic hepatitis B patients.

BACKGROUND/AIMS: Four-week treatment of linvencorvir (RO7049389) was generally safe and well tolerated, and showed anti-viral activity in chronic hepatitis B (CHB) patients. This study evaluated the efficacy, safety, and pharmacokinetics of 48-week treatment with linvencorvir plus standard of care (SoC) in CHB patients. METHODS: This was a multicentre, non-randomized, non-controlled, open-label phase 2 study enrolling three cohorts: nucleos(t)ide analogue (NUC)-suppressed patients received linvencorvir plus NUC (Cohort A, n=32); treatment-naïve patients received linvencorvir plus NUC without (Cohort B, n=10) or with (Cohort C, n=30) pegylated interferon-α (Peg-IFN-α). Treatment duration was 48 weeks, followed by NUC alone for 24 weeks. RESULTS: 68 patients completed the study. No patient achieved functional cure (sustained HBsAg loss and unquantifiable HBV DNA). By Week 48, 89% of treatment-naïve patients (10/10 Cohort B; 24/28 Cohort C) reached unquantifiable HBV DNA. Unquantifiable HBV RNA was achieved in 92% of patients with quantifiable baseline HBV RNA (14/15 Cohort A, 8/8 Cohort B, 22/25 Cohort C) at Week 48 along with partially sustained HBV RNA responses in treatment-naïve patients during follow-up period. Pronounced reductions in HBeAg and HBcrAg were observed in treatment-naïve patients, while HBsAg decline was only observed in Cohort C. Most adverse events were grade 1-2, and no linvencorvir-related serious adverse events were reported. CONCLUSION: 48-week linvencorvir plus SoC was generally safe and well tolerated, and resulted in potent HBV DNA and RNA suppression. However, 48-week linvencorvir plus NUC with or without Peg-IFN did not result in the achievement of functional cure in any patient.

Novel urine cell-free DNA methylation markers for hepatocellular carcinoma.

An optimized hepatocellular carcinoma (HCC)-targeted methylation next generation sequencing assay was developed to discover HCC-associated methylation markers directly from urine for HCC screening. Urine cell-free DNA (ucfDNA) isolated from a discovery cohort of 31 non-HCC and 30 HCC was used for biomarker discovery, identifying 29 genes with differentially methylated regions (DMRs). Methylation-specific qPCR (MSqPCR) assays were developed to verify the selected DMRs corresponding to 8 genes (GRASP, CCND2, HOXA9, BMP4, VIM, EMX1, SFRP1, and ECE). Using archived ucfDNA, methylation of GRASP, HOXA9, BMP4, and ECE1, were found to be significantly different (p < 0.05) between HCC and non-HCC patients. The four markers together with previously reported GSTP1 and RASSF1A markers were assessed as a 6-marker panel in an independent training cohort of 87 non-HCC and 78 HCC using logistic regression modeling. AUROC of 0.908 (95% CI, 0.8656-0.9252) was identified for the 6-marker panel with AFP, which was significantly higher than AFP-alone (AUROC 0.841 (95% CI, 0.778-0.904), p = 0.0026). Applying backward selection method, a 4-marker panel was found to exhibit similar performance to the 6-marker panel with AFP having 80% sensitivity compared to 29.5% by AFP-alone at a specificity of 85%. This study supports the potential use of methylated transrenal ucfDNA for HCC screening.

Tenofovir alafenamide and tenofovir disoproxil fumarate reduce incidence of hepatocellular carcinoma in patients with chronic hepatitis B.

Background & Aims: Antiviral therapy may attenuate the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). We aimed to explore how tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF) affect HCC risk in patients with CHB. Methods: The REACH-B, aMAP, and mPAGE-B models were utilized to assess HCC risk in patients with CHB from two global randomized-controlled trials evaluating the impact of TAF vs. TDF treatment. Standard incidence ratios (SIRs) were calculated using data from the REACH-B model as a ratio of observed HCC cases in the TAF- or TDF-treated patients vs. predicted HCC cases for untreated historical controls. Proportions of treated patients shifting aMAP and mPAGE-B risk categories between baseline and Week 240 were calculated. Results: Of the 1,632 patients (TAF, n = 1,093; TDF, n = 539) followed for up to 300 weeks, 22 HCC cases developed. Those receiving TAF had an SIR that was lower compared to the SIR of individuals receiving TDF: 0.32 (p <0.001) vs. 0.56 (p = 0.06). In the general study population, individuals without cirrhosis at baseline had an SIR that was lower compared to the SIR of individuals with cirrhosis at baseline: 0.37 (p <0.001) vs. 0.58 (p = 0.15). Of the patients at low risk of HCC at baseline, the majority (97%) remained low risk by mPAGE-B and aMAP scoring at Week 240. Among those at medium or high risk at baseline, substantial portions shifted to a lower risk category by Week 240 (mPAGE-B: 22% and 42%; aMAP: 39% and 63%, respectively). Conclusions: This evaluation provides evidence that treatment with TAF or TDF can reduce HCC risk in patients with CHB, particularly in patients without cirrhosis. Impact and implications: Despite the substantial impact of HCC on long-term outcomes of patients with CHB, the differential risk of HCC development among those receiving treatment with TAF vs. TDF has not been well elucidated. Using three validated risk prediction models, we found that TAF is at least as effective as TDF in reducing HCC risk in patients with CHB. While TDF is well-studied in the context of HCC risk reduction, our novel findings underscore the effectiveness of TAF as a treatment option for patients with CHB. Clinical trial numbers: NCT01940341; NCT02836249; NCT01940471; NCT02836236.

The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). METHODS: Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein-protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. RESULTS: We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. CONCLUSIONS: Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract.

Hepatic arterial infusion chemotherapy and immune checkpoint inhibitors, alone or in combination, in advanced hepatocellular carcinoma with macrovascular invasion: a single-centre experience in Taiwan.

Background: The presence of vascular invasion is associated with poor survival in advanced hepatocellular carcinoma (HCC). We compared the effectiveness of hepatic arterial infusion chemotherapy (HAIC) and immune checkpoint inhibitors (ICIs), alone or in combination, in patients with advanced HCC. Methods: We retrospectively reviewed medical records of adult patients with unresectable HCC and macrovascular invasion (MVI) who were treated with HAIC or ICIs alone or in combination at a single centre in Taiwan. Overall tumour response, vascular thrombi response, overall survival (OS) and progression-free survival (PFS) in 130 patients were analysed. Results: The treatment group showed no significant effect on the overall tumour response [objective response rate (ORR), 22.86% for HAIC, 26.09% for ICI, 50.00% for HAIC+ICI; P=0.111], but showed a significant effect on vessel response (objective response rate of tumour thrombi (ORRT), 38.57% for HAIC, 45.65% for ICI, 78.57% for HAIC+ICI; P=0.023). Post-hoc comparisons followed by Bonferroni correction revealed that vessel ORRT was significantly different between the HAIC+ICI and HAIC groups (P=0.014). A significant effect of treatment group on portal vein tumour thrombus (PVTT) was also detected (ORRT, 40.00% for HAIC, 50.00% for ICI, 90.00% for HAIC; P=0.013), with significant difference between the HAIC+ICI and HAIC groups (P=0.005). Patients treated with HAIC, ICI, and HAIC+ICI respectively had 12-month OS rates of 44.9%, 31.4%, and 67.5% (P=0.127) and 12-month PFS rates of 21.2%, 24.6%, and 33.2% (P=0.091). In multivariate analysis of PFS, HAIC+ICI was associated with reduced risk of progression or death compared with HAIC alone (adjusted hazard ratio: 0.46; 95% confidence interval: 0.23-0.94; P=0.032). Conclusions: HAIC combined with ICIs had a superior response of PVTT compared to HAIC alone, and was associated with reduced risk of progression or death. Future studies are needed to address the survival benefit of the combination therapy in advanced HCC with MVI.

Real-World Effectiveness of Sorafenib versus Lenvatinib Combined with PD-1 Inhibitors in Unresectable Hepatocellular Carcinoma.

Immune checkpoint inhibitors (ICIs) combined with multitarget tyrosine kinase inhibitors (MTKIs) exert a synergistic effect and are effective in unresectable hepatocellular carcinoma (uHCC). However, precise data regarding the real-world clinical applications of these combination therapies in uHCC are lacking. This study compared the treatment efficacy of sorafenib versus lenvatinib in combination with programmed cell death protein-1 (PD-1) inhibitors in patients with uHCC in a clinical setting. Among 208 patients with uHCC treated with PD-1 inhibitors, 88 were administered with ICIs in combination with sorafenib or lenvatinib. The treatment response and survival outcomes were evaluated. Predictors of survival were assessed by multivariate analysis. A total of 49 patients were treated with PD-1 inhibitors combined with sorafenib, and 39 patients were treated with PD-1 inhibitors combined with lenvatinib. The lenvatinib group exhibited a stronger objective response rate (ORR) (20.51% vs. 16.33%) and had a higher disease control rate (41.03% vs. 28.57%) than did the sorafenib group. The median overall survival was longer in the lenvatinib group than the sorafenib group (13.1 vs. 7.8 months; hazard ratio = 0.39, p = 0.017). The incidence of treatment-related adverse events was similar. PD-1 inhibitors combined with lenvatinib can be a feasible treatment strategy for HCC patients receiving MTKI-based combination therapy. PD-1 inhibitors combined with lenvatinib resulted in more favorable survival outcomes without increased toxic effects compared with PD-1 inhibitors with sorafenib. Additional larger-scale and prospective studies should be conducted to verify the study results.

Real-World Comparative Effectiveness of Nivolumab versus Pembrolizumab in Patients with Unresectable Hepatocellular Carcinoma.

PURPOSE: Immune checkpoint inhibitors are effective therapies for advanced hepatocellular carcinoma (HCC); however, comparisons of the clinical efficacy and safety profile for these drugs are still scarce. Thus, the aims of this study were to investigate the differences in efficacy and safety between nivolumab and pembrolizumab in unresectable HCC patients in a real-world setting. PATIENTS AND METHODS: A total of 115 patients who received treatment with nivolumab (n = 73) or pembrolizumab (n = 42) in combination with or without tyrosine kinase inhibitors was enrolled. Therapeutic response, survival outcomes, and safety profiles were compared among these groups. Multivariate analysis of survival response was performed using Cox proportional hazards regression. RESULTS: Patients treated with pembrolizumab demonstrated a significantly higher objective response rate than those with nivolumab (38.1% vs. 15.1%; odds ratio 4.18, p = 0.005) regardless of the combination strategies. In addition, pembrolizumab performed a better overall survival (OS) than nivolumab, (34.9 vs. 9.5 months; hazard ratio (HR) = 0.39, p = 0.004). In subgroup analysis, pembrolizumab exhibited favorable OS than nivolumab for monotherapy (HR = 0.16, p = 0.001) or combination therapy (HR = 0.33, p = 0.006) as second-line or later-line (HR = 0.19, p = 0.001) therapy and those with (HR = 0.31, p = 0.006) or without (HR = 0.15, p = 0.004) well-compensated liver disease. The incidence of adverse events was comparable for both treatments. CONCLUSION: Both pembrolizumab and nivolumab had significant effects for HCC therapy, and pembrolizumab had a significant survival benefit as compared with nivolumab.

The Predictive Role of Hepatitis B Biomarkers on HBV Reactivation following Direct-Acting Antiviral Therapy in HBV/HCV Coinfected Patients.

Hepatitis B and C (HBV/HCV) coinfected patients have a potential risk of hepatitis B reactivation (HBVr) after direct-acting antivirals (DAAs) treatment. The study intends to investigate the predictive role of HBV biomarkers in HBVr. Forty-six HBV/HCV coinfected patients receiving DAAs were enrolled. All patients completed treatment and follow-up to the 12th-week post-DAA treatment (P12). Blood samples were measured for HBV biomarkers, including hepatitis B surface antigen (HBsAg), hepatitis B core-related antigen (HBcrAg), and HBV pregenomic RNA (HBV pgRNA). The predictive factors for HBVr after DAA treatment were analyzed. Among 31 patients without nucleot(s)ide analogue (NA) treatment, seven (22.5%, 7/31) developed HBVr without hepatitis flare-up. Patients with HBVr had higher HBsAg titers than those without HBVr from baseline to P12 (p = 0.008, 0.009, 0.004, and 0.006 at baseline, week 4, end of treatment, and P12, respectively). The baseline HBsAg level was the only predictive factor associated with HBVr (HR, 2.303; 95% CI, 1.086−4.882; p = 0.030). In predicting HBVr, a baseline HBsAg titer > 20 IU/mL had a sensitivity, specificity, positive predictive value, and negative predictive value of 85.7%, 75.0%, 50%, and 94.7%, respectively. No patient had HBVr if the baseline HBsAg titer was <8 IU/mL. Serum HBcrAg and HBV pgRNA levels had no role in predicting HBVr. In conclusion, HBV/HCV coinfected patients are at risk of HBVr after DAA treatment. The baseline HBsAg level was the predictive factor associated with HBVr. Patients with a baseline HBsAg titer < 8 IU/mL can be considered as not having HBVr.

Impact of HCV viremia on HBV biomarkers in patients coinfected with HBV and HCV.

BACKGROUNDS: Hepatitis B virus (HBV) biomarkers reflect the status of HBV infection; however, their role in patients with chronic hepatitis B and C (HBV/HCV) coinfection remains unknown. This study evaluated the characteristics of HBV biomarkers in patients with chronic HBV/HCV coinfection. METHODS: One hundred untreated HBV/HCV coinfected patients were enrolled. Active viral infection was defined as viral load above 2000 U/L and 15 U/L for HBV and HCV, respectively. Blood samples were analyzed for HBV biomarkers, including hepatitis B surface antigen (HBsAg), hepatitis B core-related antigen (HBcrAg), HBV DNA, and HBV pregenomic RNA (HBV pgRNA). The impact of HCV viremia was also studied. RESULTS: A total of 15 patients were HBV-inactive/HCV-inactive, 63 patients were HBV-inactive/HCV-active, 14 patients were HBV-active/HCV-inactive and 8 patients were HBV-active/HCV-active. A total of 71 (71%) patients were active HCV and 22 (22%) were active HBV. HBsAg, HBcrAg, and HBV DNA correlated with each other (P < 0.001). HBV pgRNA displayed no correlations with HBV DNA, HBsAg, or HBcrAg. Patients with HCV viremia had significantly lower HBV DNA, HBsAg, and HBcrAg levels as well as higher HBV pgRNA levels and lower HBV DNA:pgRNA ratio than those without viremia (HBV DNA, P < 0.001; HBsAg, P = 0.015; HBcrAg, P = 0.006; HBV pgRNA, P = 0.073; and HBV DNA:pgRNA ratio, P < 0.001). CONCLUSIONS: In patients coinfected with HBV and HCV, HBsAg, HBcrAg, and HBV DNA significantly correlated with each other. HBV and HCV coinfected patients with HCV viremia have lower HBV DNA, HBsAg, HBcrAg, and HBV DNA:pgRNA ratio as well as higher HBV pgRNA levels.

Urine DNA biomarkers for hepatocellular carcinoma screening.

BACKGROUND: Hepatocellular carcinoma (HCC) occurs in a well-defined high-risk patient population, but better screening tests are needed to improve sensitivity and efficacy. Therefore, we investigated the use of urine circulating tumour DNA (ctDNA) as a screening test. METHODS: Candidate markers in urine were selected from HCC and controls. We then enrolled 609 patients from five medical centres to test the selected urine panel. A two-stage model was developed to combine AFP and urine panel as a screening test. RESULTS: Mutated TP53, and methylated RASSF1a, and GSTP1 were selected as the urine panel markers. Serum AFP outperformed the urine panel among all cases of HCC, but the urine panel identified 49% of HCC cases with low AFP < 20 ng/ml. Using the two-stage model, the combined AFP and urine panel identified 148 of the 186 HCC cases (79.6% sensitivity at 90% specificity), which was 30% more than the cases detected with serum AFP alone. It also increased early-stage HCC detection from 62% to 92% (BCLC stage 0), and 40% to 77% (BCLC stage A). CONCLUSION: Urine ctDNA has promising diagnostic utility in patients in HCC, especially in those with low AFP and can be used as a potential non-invasive HCC screening test.

Clinical characteristics and outcomes of drug-induced liver injury in Taiwan: With emphasis on the impact of chronic hepatitis B infection.

BACKGROUND: Whether hepatitis B virus (HBV) infection can affect the outcomes of drug-induced liver injury (DILI) is controversial. This study aimed to evaluate the characteristics and outcomes of DILI in Taiwan, with an emphasis on the impact of HBV infection. METHODS: We prospectively recruited patients with DILI from multiple centers in Taiwan from 2010 to 2018. RESULTS: A total of 1,014 patients were enrolled. The leading culprit drug category was antimicrobials (481, 47.4%), followed by nonsteroidal anti-inflammatory drugs, anticonvulsants, and statins. Among the antimicrobials, antituberculosis agents were most likely to induce liver injury (257, 25.3%), followed by antibacterial, antifungal, and antiviral agents. The liver-related mortality rate was 8.2% (83/1,014). The patients who died had higher rates of hepatocellular-type liver injury, elevated liver biochemical tests, preexisting liver cirrhosis, jaundice, chronic HBV infection, and antituberculosis drug-induced liver injury (ATDILI) than the survivors. A total of 131 patients (12.9%) with DILI were HBV carriers, of whom 23 (17.6%) died of hepatic failure. The rate of HBV-DNA > 2000 IU/mL was higher in the patients who died (47.8% vs. 26.9%, p = 0.047) than in the survivors. After adjusting for possible risk factors, active HBV infection with HBV-DNA > 2000 IU/mL was the most significant risk factor for liver-related mortality (adjusted HR, 4.40, 95% CI, 2.31%-8.38%, p < 0.001). The other independent risk factors for mortality were ATDILI and albumin-bilirubin (ALBI) score (adjusted HR, 1.25 and 4.09, respectively, p < 0.003). CONCLUSION: Antituberculosis agents were the leading cause of DILI in Taiwanese, and they were associated with poorer outcomes than other drug categories. Active HBV infection, ATDILI and ALBI score were independent risk factors for fatal DILI. Close monitoring of liver tests and timely antiviral therapy should be implemented in HBV carriers during the administration of high-risk drugs, such as antituberculosis agents.

Ledipasvir/Sofosbuvir for Patients Coinfected With Chronic Hepatitis C and Hepatitis B in Taiwan: Follow-up at 108 Weeks Posttreatment.

BACKGROUND: For patients coinfected with hepatitis C virus (HCV) and hepatitis B virus (HBV), HCV treatment with direct-acting antivirals can lead to HBV reactivation. We evaluated HBV reactivation during ledipasvir/sofosbuvir treatment and 108-week follow-up. METHODS: In Taiwan, 111 patients with HCV genotype 1 or 2 and HBV received ledipasvir/sofosbuvir (90mg/400mg) once daily for 12 weeks. HBV virologic reactivation was defined as postbaseline increase in HBV DNA from either less than the lower limit of quantification (LLOQ, 20 IU/mL) to equal to or more than LLOQ or equal to or more than LLOQ to >1 log10 IU/mL. HBV clinical reactivation was HBV virologic reactivation with alanine aminotransferase (ALT) >2× upper limit of normal. Factors associated with development of HBV virologic or clinical reactivation were evaluated with logistic regression analysis. RESULTS: All patients (100%, 111/111) maintained HCV suppression through 108 weeks after treatment. HBV virologic reactivation occurred in 73% of patients (81/111). Clinical reactivation occurred in 9% (10/111). The majority of HBV virologic reactivations (86%, 70/81) occurred by follow-up week 12, whereas clinical reactivation was generally more delayed. Eight (7%, 8/111) initiated HBV therapy. In regression analyses, baseline HBV DNA and hepatitis B surface antigen (HBsAg) levels were associated with HBV virologic reactivation and baseline ALT and HBV DNA, and HBsAg levels were associated with HBV clinical reactivation. CONCLUSION: Among HCV/HBV coinfected patients treated with direct-acting antivirals for HCV, HBV virologic reactivation occurred in a majority of patients during treatment and follow-up. In most patients, HBV virologic reactivation was asymptomatic; only a small proportion initiated HBV treatment. Notably, clinical reactivation may still occur >3 months after end of therapy. CLINICAL TRIALS REGISTRATION: NCT02613871.

Risk scores to predict HCC and the benefits of antiviral therapy for CHB patients in gray zone of treatment guidelines.

BACKGROUNDS: ALT ≥ 80 U/L and HBV DNA ≥ 2000 IU/ml are treatment criteria of APASL guidelines for chronic hepatitis B (CHB) patients. The need of antiviral therapy for patients in gray zone (ALT < 80 U/L or HBV DNA < 2000 IU/ml) is controversial. This study aimed to develop a scoring system to predict hepatocellular carcinoma (HCC) and evaluate the benefit of antiviral therapy in these patients. METHODS: Seven hundred and forty-nine patients were analyzed. Significant variables were weighted to develop a scoring system for HCC prediction. The area under receiver operating curves (AUROC) were estimated and validated by REVEAL-HBV cohort (n = 3527). RESULTS: Older age (p < 0.001), male sex (p = 0.036), family history of HCC (p = 0.002) and HBV DNA ≥ 2000 IU/ml (p = 0.045) were independently associated with HCC. A 14-point risk score system predicts 3 and 5-years HCC risk to be 0.866 and 0.868 of AUROC, respectively in the derivation cohort; 0.821 and 0.820, in the REVEAL-HBV cohort. The cumulative HCC incidence was higher in the high risk (score ≥ 8) group both in derivation and validation cohorts (p < 0.001). Patients with antiviral therapy had lower HCC incidence compared to those without (p = 0.016). Of note, antiviral therapy significantly decreased HCC in the high risk group (p = 0.005), but not in the low risk group (p = 0.705). CONCLUSIONS: A risk scoring system is established and validated. Of CHB patients in gray zone of APASL guidelines, those with risk scores ≥ 8 had higher risk of HCC, but the risk could be significantly reduced by antiviral therapy.

Detection of Hepatitis B Virus-Host Junction Sequences in Urine of Infected Patients.

Integrated hepatitis B virus (HBV) DNA, found in more than 85% of HBV-associated hepatocellular carcinomas (HBV-HCCs), can play a significant role in HBV-related liver disease progression. HBV-host junction sequences (HBV-JSs), created through integration events, have been used to determine HBV-HCC clonality. Here, we investigate the feasibility of analyzing HBV integration in a noninvasive urine liquid biopsy. Using an HBV-targeted next-generation sequencing (NGS) assay, we first identified HBV-JSs in eight HBV-HCC tissues and designed short-amplicon junction-specific polymerase chain reaction assays to detect HBV-JSs in matched urine. We detected and validated tissue-derived junctions in five of eight matched urine samples. Next, we screened 32 urine samples collected from 25 patients infected with HBV (5 with hepatitis, 10 with cirrhosis, 4 with HCC, and 6 post-HCC). Encouragingly, all 32 urine samples contained HBV-JSs detectable by HBV-targeted NGS. Of the 712 total HBV-JSs detected in urine, 351 were in gene-coding regions, 11 of which, including TERT (telomerase reverse transcriptase), had previously been reported as recurrent integration sites in HCC tissue and were found only in the urine patients with cirrhosis or HCC. The integration breakpoints of HBV DNA detected in urine were found predominantly (~70%) at a previously identified integration hotspot, HBV DR1-2 (down-regulator of transcription 1-2). Conclusion: HBV viral-host junction DNA can be detected in urine of patients infected with HBV. This study demonstrates the potential for a noninvasive urine liquid biopsy of integrated HBV DNA to monitor patients infected with HBV for HBV-associated liver diseases and the efficacy of antiviral therapy.

Detection of CTNNB1 Hotspot Mutations in Cell-Free DNA from the Urine of Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. The beta-catenin gene, CTNNB1, is among the most frequently mutated in HCC tissues. However, mutational analysis of HCC tumors is hampered by the difficulty of obtaining tissue samples using traditional biopsy. Here, we explored the feasibility of detecting tumor-derived CTNNB1 mutations in cell-free DNA (cfDNA) extracted from the urine of HCC patients. Using a short amplicon qPCR assay targeting HCC mutational hotspot CTNNB1 codons 32-37 (exon 3), we detected CTNNB1 mutations in 25% (18/73) of HCC tissues and 24% (15/62) of pre-operative HCC urine samples in two independent cohorts. Among the CTNNB1-mutation-positive patients with available matched pre- and post-operative urine (n = 13), nine showed apparent elimination (n = 7) or severalfold reduction (n = 2) of the mutation in urine following tumor resection. Four of the seven patients with no detectable mutations in postoperative urine remained recurrence-free within five years after surgery. In contrast, all six patients with mutation-positive in post-operative urine recurred, including the two with reduced mutation levels. This is the first report of association between the presence of CTNNB1 mutations in pre- and post-operative urine cfDNA and HCC recurrence with implications for minimum residual disease detection.

Herbal and dietary supplement-induced liver injury in Taiwan: comparison with conventional drug-induced liver injury.

BACKGROUND AND AIMS: Whether herbal and dietary supplements (HDS) are safer than Western conventional drugs is controversial. The aim of this study was to explore the characteristics and risk factors for HDS-induced liver injury (HILI) in Taiwan. METHODS: This is a 9-year multi-center prospective study conducted in Taiwan from 2011 to 2019. Patients with HILI were compared to those with conventional drug-induced liver injury (CILI). RESULTS: A total of 1,297 patients were enrolled, of whom 285 (22.0%) had HILI and 1,012 (78.0%) had CILI. Compared to the CILI group, the HILI group had higher initial serum alanine aminotransferase, alkaline phosphatase (ALP), peak ALP and bilirubin levels, and higher rates of jaundice, ascites, encephalopathy, coagulopathy, sepsis and acute liver failure. In addition, the HILI group had a higher mortality rate than the CILI group (12.6 vs. 8.0%, p = 0.016). Hepatitis B carrier status, elevated baseline liver biochemical tests and the use of crude herbs (without processing) were associated with an increased risk of HILI-related mortality (adjusted hazard ratios [95% confidence intervals]: 2.90 [1.43-5.99], 2.40 [1.01-5.68] and 2.94 [1.45-5.97], respectively). CONCLUSIONS: HDS are popular and incriminated in more than one-fifth of drug-induced liver injuries in Taiwan. The patients with HILI were more severe than those with CILI in terms of liver biochemical tests, complications and mortality. Hepatitis B carriers, those with elevated baseline liver tests and crude herb users may have a higher risk of HILI-related mortality. The prudent use of HDS is suggested in these high-risk subjects.

Clinical Implications of Serum Hepatitis B Virus Pregenomic RNA Kinetics in Chronic Hepatitis B Patients Receiving Antiviral Treatment and Those Achieving HBsAg Loss.

Serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) is correlated with covalently closed circular DNA. We aimed to investigate the utility of serum HBV pgRNA in chronic hepatitis B patients receiving nucleos(t)ide analogue treatment and those achieving HBsAg loss. One hundred and eighty-five patients were enrolled for studying long-term HBV pgRNA kinetics during treatment. Twenty patients achieving HBsAg loss after treatment were enrolled for examining HBV pgRNA kinetics around HBsAg loss. HBV pgRNA significantly decreased in the high baseline HBV pgRNA (≥6 log copies/mL) group but significantly increased in the low baseline HBV pgRNA (<4 log copies/mL) group after 3-month entecavir treatment. Among the 20 patients achieving HBsAg loss, 13 (65%) patients had serum HBV pgRNA higher than the limit of detection (LOD, 1466 copies/mL) when they achieved HBsAg loss. Finally, all 20 patients had HBV pgRNA going below the LOD within 3 years after achieving HBsAg loss. In conclusion, baseline serum HBV pgRNA alone is insufficient for predicting the trajectory of HBV pgRNA. Most patients still had HBV pgRNA higher than the LOD when they achieved HBsAg loss. Further studies on HBV pgRNA kinetics around HBsAg loss would provide an enhanced basis for further applications of HBV pgRNA.

Recurrent HBV Integration Targets as Potential Drivers in Hepatocellular Carcinoma.

Chronic hepatitis B virus (HBV) infection is the major etiology of hepatocellular carcinoma (HCC), frequently with HBV integrating into the host genome. HBV integration, found in 85% of HBV-associated HCC (HBV-HCC) tissue samples, has been suggested to be oncogenic. Here, we investigated the potential of HBV-HCC driver identification via the characterization of recurrently targeted genes (RTGs). A total of 18,596 HBV integration sites from our in-house study and others were analyzed. RTGs were identified by applying three criteria: at least two HCC subjects, reported by at least two studies, and the number of reporting studies. A total of 396 RTGs were identified. Among the 28 most frequent RTGs, defined as affected in at least 10 HCC patients, 23 (82%) were associated with carcinogenesis and 5 (18%) had no known function. Available breakpoint positions from the three most frequent RTGs, TERT, MLL4/KMT2B, and PLEKHG4B, were analyzed. Mutual exclusivity of TERT promoter mutation and HBV integration into TERT was observed. We present an RTG consensus through comprehensive analysis to enable the potential identification and discovery of HCC drivers for drug development and disease management.