Association of HBsAg levels with differential gene expression in NK, CD8 T, and memory B cells in treated patients with chronic HBV.

Background & Aims: HBsAg secretion may impact immune responses to chronic HBV infection. Thus, therapeutic approaches to suppress HBsAg production are being investigated. Our study aims to examine the immunomodulatory effects of high and low levels of circulating HBsAg and thereby improve our understanding of anti-HBV immunity. Methods: An optimized 10x Genomics single-cell RNA sequencing workflow was applied to blood samples and liver fine-needle aspirates from 18 patients undergoing tenofovir/entecavir (NUC) treatment for chronic HBV infection. They were categorized based on their HBsAg levels: high (920-12,447 IU/ml) or low (1-100 IU/ml). Cluster frequencies, differential gene expression, and phenotypes were analyzed. Results: In the blood of HBV-infected patients on NUC, the proportion of KLRC2+ "adaptive" natural killer (NK) cells was significantly lower in the HBsAg-high group and, remarkably, both KLRC2+ NK and KLRG1+ CD8 T cells display enrichment of lymphocyte activation-associated gene sets in the HBsAg-low group. High levels of HBsAg were associated with mild immune activation in the liver. However, no suppression of liver-resident CXCR6+ NCAM1+ NK or CXCR6+ CD69+ CD8 T cells was detected, while memory B cells showed signs of activation in both the blood and liver. Conclusions: Among NUC-treated patients, we observed a minimal impact of HBsAg on leukocyte populations in the blood and liver. However, for the first time, we found that HBsAg has distinct effects, restricted to NK-, CD8 T-, and memory B-cell subsets, in the blood and liver. Our findings are highly relevant for current clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Impact and implications: This study provides unique insight into the impact of HBsAg on gene expression levels of immune cell subsets in the blood and liver, particularly in the context of NUC-treated chronic HBV infection. It holds significant relevance for current and future clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Our findings raise questions about the effectiveness of such treatment strategies and challenge the previously hypothesized immunomodulatory effects of HBsAg on immune responses against HBV.

Cryptogenic non-cirrhotic HCC: Clinical, prognostic and immunologic aspects of an emerging HCC etiology.

The incidence of hepatocellular carcinoma (HCC) in non-cirrhotic livers is rising significantly, but clear risk factors for screening remain elusive. This study sought to characterize non-cirrhotic HCC etiologies. HCC cases from 2009 to 2020 in a Dutch referral center were examined, revealing 371 out of 1654 cases (22%) as non-cirrhotic. Notably, the incidence of non-cirrhotic HCC increased by 61% in the time frame between 2009 and 2020. Interestingly 39% of non-cirrhotic HCC cases had cryptogenic origins. Cryptogenic non-cirrhotic HCC exhibited similarities with non-cirrhotic NAFLD HCC, but displayed advanced tumor stages, lower surgical rates, and a more frequent presence of symptoms, which substantiated in poor survival rates. Advanced cryptogenic non-cirrhotic HCC stages exhibited elevated serum interleukin-6 levels compared to non-cirrhotic HCC with defined etiologies. Comparative analysis encompassing cryptogenic and NAFLD non-cirrhotic HCC cohorts and controls unveiled comparable circulating immune biomarker profiles and PNPLA3 polymorphisms. To conclude, the primary etiology of non-cirrhotic HCC in our cohort has not defined risk factors. This cryptogenic variant exhibits distinct traits, such as advanced tumors and increased symptoms, and most resemble burned-out NAFLD. Understanding this HCC variant is crucial for improving screening and management strategies.

Addition of PEG-interferon to long-term nucleos(t)ide analogue therapy enhances HBsAg decline and clearance in HBeAg-negative chronic hepatitis B: Multicentre Randomized Trial (PAS Study).

We studied whether 48 weeks of PEG-IFN alfa-2a add-on increases HBsAg-decline and clearance in HBeAg-negative patients on long-term nucleo(s)tide analogue (NA) therapy. In this investigator-initiated, randomized, controlled trial conducted in Europe and Canada, HBeAg-negative patients treated with NA > 12 months, with HBVDNA < 200 IU/mL, were enrolled. Patients were randomized 2:1 to 48 weeks of PEG-IFN alfa-2a add-on (180 µg per week) or continued NA-monotherapy with subsequent follow-up to Week 72. Endpoints were HBsAg decline (≥1 log10 IU/mL) and HBsAg clearance at Week 48. Of the 86 patients in the modified-intention-to-treat analysis, 58 patients received PEG-IFN add-on, and 28 continued NA monotherapy. At Week 48, 16(28%) patients achieved HBsAg decline ≥1 log10 in the add-on arm versus none on NA-monotherapy (p < .001), and HBsAg clearance was observed in 6 (10%) PEG-IFN add-on patients versus 0% NA-monotherapy (p = .01). HBVRNA was only detected in 2% after PEG-IFN treatment versus 19% in NA-monotherapy (p = .002) at Week 48. PEG-IFN add-on therapy was well tolerated in majority of patients. Low baseline HBsAg levels (<10 IU/mL) identified patients most likely to achieve HBsAg loss with PEG-IFN add-on, whereas an HBsAg level > 200 IU/mL at on-treatment Week 12 was highly predictive of non-response (NPV = 100%). Addition of PEG-IFN to long-term NA enhanced HBsAg decline and increased the chance of HBsAg clearance in HBeAg-negative patients on long-term NA. On-treatment HBsAg levels >200 IU/mL identify patients unlikely to benefit from PEG-IFN add-on and could be used as a potential stopping-rule for PEG-IFN therapy. Our findings support further exploration of immune modulation add-on to antiviral therapy, preferably using response-guided strategies, to increase functional cure rates in patients with CHB.

Assessment of TLL1 variant and risk of hepatocellular carcinoma in Latin Americans and Europeans.

INTRODUCTION AND OBJECTIVES: Tolloid like protein 1 (TLL1) rs17047200 has been reported to be associated with HCC development and liver fibrosis. However, to our knowledge, no studies have been performed on Latin Americans and comparative differences between TLL1 rs17047200 in HCC patients from Latin America and Europe are undefined. MATERIALS AND METHODS: Cross-sectional analysis was performed on Latin American and European individuals. We analyzed TLL1 rs17047200 on DNA from 1194 individuals, including 420 patients with HCC (86.0 % cirrhotics) and 774 without HCC (65.9 % cirrhotics). RESULTS: TLL1 rs17047200 genotype AT/TT was not associated with HCC development in Latin Americans (OR: 0.699, 95 %CI 0.456-1.072, p = 0.101) or Europeans (OR: 0.736, 95 %CI 0.447-1.211, p = 0.228). TLL1 AT/TT was not correlated with fibrosis stages among metabolic dysfunction-associated steatotic liver disease (MASLD) patients from Latin America (OR: 0.975, 95 %CI 0.496-1.918, p = 0.941). Among Europeans, alcohol-related HCC had lower TLL1 AT/TT frequencies than cirrhosis (18.3 % versus 42.3 %, OR: 0.273, 95 %CI 0.096-0.773, p = 0.015). CONCLUSIONS: We found no evidence that the TLL1 rs17047200 AT/TT genotype is a risk factor for HCC development in Latin Americans or Europeans. A larger study integrating ethnic and etiology backgrounds is needed to determine the importance of the TLL1 SNP in HCC development.

HBV DNA and HBsAg Levels at 24 Weeks Off-Treatment Predict Clinical Relapse and HBsAg Loss in HBeAg-Negative Patients Who Discontinued Antiviral Therapy.

BACKGROUND & AIMS: Patients who discontinue nucleo(s)tide analogue therapy are at risk of viral rebound and severe hepatitis flares, necessitating intensive off-treatment follow-up. METHODS: We studied the association between hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels at off-treatment follow-up week 24 (FU W24), with subsequent clinical relapse, and HBsAg loss in a multicenter cohort of hepatitis B e antigen (HBeAg)-negative patients with chronic hepatitis B who discontinued nucleo(s)tide analogue therapy. RESULTS: We studied 475 patients, 82% Asian, and 55% treated with entecavir. Patients with higher HBV DNA levels at FU W24 had a higher risk of clinical relapse (hazard ratio [HR], 1.576; P < .001) and a lower chance of HBsAg loss (HR, 0.454; P < .001). Similarly, patients with higher HBsAg levels at FU W24 had a higher risk of clinical relapse (HR, 1.579; P < .001) and a lower chance of HBsAg loss (HR, 0.263; P < .001). A combination of both HBsAg <100 IU/mL and HBV DNA <100 IU/mL at FU W24 identified patients with excellent outcomes (9.9% clinical relapse and 58% HBsAg loss at 216 weeks of follow-up). Conversely, relapse rates were high and HBsAg loss rates negligible among patients with both HBsAg >100 IU/mL and HBV DNA >100 IU/mL (P < .001). CONCLUSIONS: Among HBeAg-negative patients with chronic hepatitis B who discontinued antiviral therapy and who did not experience clinical relapse before FU W24, serum levels of HBV DNA and HBsAg at FU W24 can be used to predict subsequent clinical relapse and HBsAg clearance. A combination of HBsAg <100 IU/mL with HBV DNA <100 IU/mL identifies patients with a low risk of relapse and excellent chances of HBsAg loss and could potentially be used as an early surrogate end point for studies aiming at finite therapy in HBV.

Sustained response and HBsAg loss after nucleo(s)tide analogue discontinuation in chronic hepatitis B patients: the prospective SNAP study.

BACKGROUND & AIM(S): Current guidelines suggest that nucleos(t)ide analogues (NA) can be discontinued before HBsAg loss in a selected group of chronic hepatitis B (CHB) patients. We aimed to study the safety and off-treatment response after NA cessation. METHODS: This is a prospective, multicentre, cohort study in which eligible patients discontinued NA therapy. Adult patients, with a CHB mono-infection, HBeAg-negative, without a (history of) liver cirrhosis, who had achieved long-term viral suppression were eligible. Follow-up visits were planned at week 2-4-8-12-24-36-48-72-96. Re-treatment criteria included severe hepatitis (ALT >10x ULN), signs of imminent liver failure (bilirubin >1.5x ULN or INR >1.5), or at the physician's own discretion. RESULTS: In total, 33 patients were enrolled. Patients were predominantly Caucasian (45.5%) and had genotype A/B/C/D/unknown in 3/4/6/10/10 (9.1/12.1/18.2/30.3/30.3%). At week 48, 15 patients (45.5%) achieved a sustained response (HBV DNA <2,000 IU/mL). At week 96, 13 patients (39.4%) achieved a sustained response, 4 (12.1%) achieved HBsAg loss, and 12 (36.4%) were re-treated. Severe hepatitis was the main reason for re-treatment (n=7, 21.2%). One patient with severe hepatitis developed jaundice, without signs of hepatic decompensation. Re-treatment was successful in all patients. CONCLUSION: NA therapy can be ceased in a highly selected group of CHB patients if close follow-up can be guaranteed. Treatment cessation may increase the chance of HBsAg loss in selected patients, which is counterbalanced by a significant risk of severe hepatitis.

Hypermethylation of DNA Methylation Markers in Non-Cirrhotic Hepatocellular Carcinoma.

Aberrant DNA methylation changes have been reported to be associated with carcinogenesis in cirrhotic HCC, but DNA methylation patterns for these non-cirrhotic HCC cases were not examined. Therefore, we sought to investigate DNA methylation changes on non-cirrhotic HCC using reported promising DNA methylation markers (DMMs), including HOXA1, CLEC11A, AK055957, and TSPYL5, on 146 liver tissues using quantitative methylation-specific PCR and methylated DNA sequencing. We observed a high frequency of aberrant methylation changes in the four DMMs through both techniques in non-cirrhotic HCC compared to cirrhosis, hepatitis, and benign lesions (p < 0.05), suggesting that hypermethylation of these DMMs is specific to non-cirrhotic HCC development. Also, the combination of the four DMMs exhibited 78% sensitivity at 80% specificity with an AUC of 0.85 in discriminating non-cirrhotic HCC from hepatitis and benign lesions. In addition, HOXA1 showed a higher aberrant methylation percentage in non-cirrhotic HCC compared to cirrhotic HCC (43.3% versus 13.3%, p = 0.039), which was confirmed using multivariate linear regression (p < 0.05). In summary, we identified aberrant hypermethylation changes in HOXA1, CLEC11A, AK055957, and TSPYL5 in non-cirrhotic HCC tissues compared to cirrhosis, hepatitis, and benign lesions, providing information that could be used as potentially detectable biomarkers for these unusual HCC cases in clinical practice.

MBOAT7 rs641738 Variant Is Not Associated with an Increased Risk of Hepatocellular Carcinoma in a Latin American Cohort.

BACKGROUND: The rs641738 C > T single-nucleotide polymorphism of MBOAT7 has been associated with hepatocellular carcinoma (HCC) and nonalcoholic fatty liver disease (NAFLD). Latin Americans have high rates of HCC and NAFLD, but no assessment between MBOAT7 and HCC has been performed in this population. AIMS: We provide the first assessment of the impact of MBOAT7 on HCC risk in Latin Americans. METHODS: Patients were prospectively recruited into the ESCALON network, designed to collect samples from Latin American patients with HCC in 6 South American countries (Argentina, Ecuador, Brazil, Chile, Peru, and Colombia). A European cohort and the general Hispanic population of gnomAD database were included for comparison. Associations between HCC and MBOAT7 were evaluated using logistic regression. RESULTS: In total, 310 cases of HCC and 493 cases of cirrhosis without HCC were assessed. The MBOAT7 TT genotype was not predictive of HCC in Latin Americans (TT vs CC OR adjusted = 1.15, 95% CI 0.66-2.01, p = 0.610) or Europeans (TT vs CC OR adjusted = 1.20, 95% CI 0.59-2.43, p = 0.621). No significant association was noted on subgroup analysis for NAFLD, viral hepatitis, or alcohol-related liver disease. The TT genotype was increased in the NAFLD-cirrhosis cohort of Latin Americans compared to a non-cirrhotic NAFLD cohort (TT vs CC + CT OR = 2.75, 95% CI 1.10-6.87, p = 0.031). CONCLUSION: The rs631738 C > T allele of MBOAT7 was not associated with increased risk of HCC in Latin Americans or Europeans. An increase in the risk of cirrhosis was noted with the TT genotype in Latin Americans with NAFLD.

Validation and optimization of AFP-based biomarker panels for early HCC detection in Latin America and Europe.

BACKGROUND: HCC is a major cause of cancer death worldwide. Serum biomarkers such as alpha-fetoprotein (AFP), protein induced by vitamin K absence-II, and the Gender, Age, AFP-L3, AFP, Des-gamma-carboxy prothrombin (GALAD) score have been recommended for HCC surveillance. However, inconsistent recommendations in international guidelines limit their clinical utility. METHODS: In this multicenter study, over 2000 patient samples were collected in 6 Latin American and 2 European countries. The performance of the GALAD score was validated in cirrhotic cases, and optimized versions were tested for early-stage HCC and prediagnostic HCC detection. RESULTS: The GALAD score could distinguish between HCC and cirrhosis in Latin American patients with an AUC of 0.76, sensitivity of 70%, and specificity of 83% at the conventional cutoff value of -0.63. In a European cohort, GALAD had an AUC of 0.69, sensitivity of 66%, and specificity of 72%. Optimizing the score in the 2 large multicenter cohorts revealed that AFP-L3 contributed minimally to early-stage HCC detection. Thus, we developed a modified GALAD score without AFP-L3, the ASAP (age, sex, AFP, and protein induced by vitamin K absence-II), which showed promise for early-stage HCC detection upon validation. The ASAP score also identified patients with cirrhosis at high risk for advanced-stage HCC up to 15 months before diagnosis (p < 0.0001) and differentiated HCC from hemangiomas, with a specificity of 100% at 71% sensitivity. CONCLUSION: Our comprehensive analysis of large sample cohorts validates the GALAD score's utility in Latin American, Spanish, and Dutch patients for early-stage HCC detection. The optimized GALAD without AFP-L3, the ASAP score, is a good alternative and shows greater promise for HCC prediction.

Assessment of STAT4 Variants and Risk of Hepatocellular Carcinoma in Latin Americans and Europeans.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. The STAT4 rs7574865 genetic variant has been associated with an increased risk of developing HCC in Asian populations. However, this association has not been studied in Latin America and is poorly assessed in European populations. This case-control study investigated the association between STAT4 rs7574865 and HCC risk in these populations. We evaluated DNA samples from seven medical institutions across six Latin American countries and one Dutch institution in 1060 individuals (344 HCC and 716 controls). STAT4 rs7574865 SNP was genotyped using TaqMan-genotyping assay and analyzed using logistic regression. We found no significant association between the homozygous risk allele (G) of STAT4 and HCC development in either population, with odds ratios (OR) for GG versus TT of 0.85 (CI: 0.48-1.52, p = 0.58) and 0.81 (CI: 0.34-1.93, p = 0.67) for Latin Americans and Europeans respectively. No correlation was found between the risk allele and HCC based on underlying liver disease. However, we found that Latin Americans of European ancestry were more likely to carry the risk allele. Our results suggest that the STAT4 SNP rs7574865 does not influence the risk of developing HCC in Latin American or European populations, highlighting the importance of evaluating genetic risk factors in various ethnic groups and understanding the possible influence of ancestry on the genetic basis of disease.

Lower pretreatment HBV DNA levels are associated with better off-treatment outcomes after nucleo(s)tide analogue withdrawal in patients with HBeAg-negative chronic hepatitis B: A multicentre cohort study.

Background & Aims: Pretreatment predictors of finite nucleo(s)tide analogue (NUC) therapy remain elusive. We studied the association between pretreatment HBV DNA levels and outcomes after therapy cessation. Methods: Patients with chronic hepatitis B who were HBeAg negative at the start of NUC treatment were enrolled from sites in Asia and Europe. We studied the association between pretreatment HBV DNA levels and (1) clinical relapse (defined as HBV DNA >2,000 IU/ml + alanine aminotransferase >2 × the upper limit of normal or retreatment) and (2) HBsAg loss after NUC withdrawal. Results: We enrolled 757 patients, 88% Asian, 57% treated with entecavir, with a median duration of treatment of 159 (IQR 156-262) weeks. Mean pretreatment HBV DNA levels were 5.70 (SD 1.5) log IU/ml and were low (<20,000 IU/ml) in 150 (20%) and high (>20,000 IU/ml) in 607 (80%). The cumulative risk of clinical relapse at 144 weeks after therapy cessation was 22% among patients with pretreatment HBV DNA levels <20,000 IU/ml vs. 60% among patients with pretreatment HBV DNA levels >20,000 IU/ml, whereas the cumulative probabilities of HBsAg loss were 17.5% vs. 5% (p <0.001). In multivariable analysis, pretreatment HBV DNA levels <20,000 IU/ml were independently associated with a reduced likelihood of clinical relapse (adjusted hazard ratio 0.379, p <0.001) and with an increased chance of HBsAg loss (adjusted hazard ratio 2.872, p <0.001). Conclusions: Lower pretreatment HBV DNA levels are associated with a lower risk of clinical relapse and a higher chance of HBsAg loss after cessation of NUC therapy, independent of end-of-treatment viral antigen levels. Further studies are needed to confirm these findings in non-Asian populations. Impact and Implications: A subgroup of patients with chronic hepatitis B may not require retreatment after stopping antiviral therapy. In this study, comprising 757 patients with chronic hepatitis B from Europe and Asia, we found that higher viral load before initiation of treatment was a risk factor for relapse after stopping treatment. Patients with a low HBV DNA level before starting antiviral therapy had the lowest risk of relapse, and a high chance of HBsAg loss, after stopping treatment. These findings can help select patients for treatment withdrawal and guide intensity of off-treatment monitoring.

DNA methylation markers in the detection of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and has a poor prognosis. Epigenetic modification has been shown to be deregulated during HCC development by dramatically impacting the differentiation, proliferation, and function of cells. One important epigenetic modification is DNA methylation during which methyl groups are added to cytosines without changing the DNA sequence itself. Studies found that methylated DNA markers can be specific for detection of HCC. On the basis of these findings, the utility of methylated DNA markers as novel biomarkers for early-stage HCC has been measured in blood, and indeed superior sensitivity and specificity have been found in several studies when compared to current surveillance methods. However, a variety of factors currently limit the immediate application of these exciting biomarkers. In this review, we provide a detailed rationalisation of the approach and basis for the use of methylation biomarkers for HCC detection and summarise recent studies on methylated DNA markers in HCC focusing on the importance of the aetiological cause of liver disease in the mechanisms leading to cancer.

Recent Insights into the Role of B Cells in Chronic Hepatitis B and C Infections.

Chronic viral hepatitis infections, caused by the hepatitis B or C virus, are a major global health problem causing an estimated one million deaths each year. Immunological studies have classically focused on T cells, while B cells have largely been neglected. Emerging evidence, however, highlights a role for B cells in the immunopathogenesis of chronic hepatitis B and C infections. B cell responses appear to be altered across different clinical phases of chronic HBV infection and across stages of disease in chronic HCV infection. These B cell responses show signs of a more activated state with a simultaneous enrichment of phenotypically exhausted atypical memory B cells. Despite the fact that studies show an activating B cell signature in chronic viral hepatitis infection, antibody responses to HBsAg remain impaired in chronic HBV infection, and glycoprotein E2-specific neutralizing antibody responses remain delayed in the acute phase of HCV infection. At the same time, studies have reported that a subset of HBV- and HCV-specific B cells exhibit an exhausted phenotype. This may, at least in part, explain why antibody responses in chronic HBV and HCV patients are suboptimal. Here, we summarize recent findings and discuss upcoming research questions while looking forward to how new single-cell technologies could provide novel insights into the role of B cells in chronic viral hepatitis infections.

Single-cell RNA sequencing of liver fine-needle aspirates captures immune diversity in the blood and liver in chronic hepatitis B patients.

BACKGROUND AND AIMS: HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. APPROACH AND RESULTS: We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S 3 picowell-based and the 10× Chromium reverse-emulsion droplet-based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S 3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. CONCLUSIONS: The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

End-of-treatment HBsAg, HBcrAg and HBV RNA predict the risk of off-treatment ALT flares in chronic hepatitis B patients.

BACKGROUND/PURPOSE(S): Since ALT flares after therapy withdrawal are associated with adverse outcomes, risk stratification is of major importance. We aimed to study whether off-treatment flares are related with virological outcomes, and if serum levels of novel biomarkers at end-of-treatment (EOT) can predict flares. METHODS: Chronic hepatitis B patients who participated in three global randomised trials of peginterferon-based therapy were studied (99-01, PARC, ARES). HBV RNA, HBsAg and HBcrAg were quantified at EOT. Associations between EOT biomarker levels and flares were assessed as continuous data and after categorisation. Flares were defined as ALT ≥5xULN during six months after therapy cessation. RESULTS: We included 344 patients; 230 HBeAg-positive and 114 HBeAg-negative. Patients were predominantly Caucasian (77.0%) and had genotype A/B/C/D in 23.3/7.3/13.4/52.3%. Flares were observed in 122 patients (35.5%). Flares were associated with lower rates of sustained response (3.5% vs 26.8% among patients with and without a flare; p < 0.001). Higher HBsAg (OR 1.586, 95%CI 1.231-2.043), HBV RNA (OR 1.695, 95%CI 1.371-2.094) and HBcrAg (OR 1.518, 95%CI 1.324-1.740) levels were associated with higher risk of flares (p < 0.001). Combinations of biomarkers further improved risk stratification, especially HBsAg + HBV RNA. Findings were consistent in multivariate analysis adjusted for potential predictors including HBeAg-status and EOT-response (HBV DNA <200 IU/mL). CONCLUSION: Off-treatment ALT flares were not associated with favourable virological outcomes. Higher EOT serum HBsAg, HBcrAg and HBV RNA were associated with a higher risk of flares after therapy withdrawal. These findings can be used to guide decision-making regarding therapy discontinuation and off-treatment follow-up. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00114361, NCT00146705, NCT00877760.

The Role of PIVKA-II as a Predictor of Early Hepatocellular Carcinoma Recurrence-Free Survival after Liver Transplantation in a Low Alpha-Fetoprotein Population.

INTRODUCTION: AFP and the RETREAT score are currently used to predict HCC recurrence after LT. However, superior discriminating models are needed for low AFP populations. The aim of this study is to investigate the predictive value of PIVKA-II on recurrence-free survival after LT in a low AFP population and microvascular invasion on explant. METHODS: A retrospective cohort study including all consecutive patients transplanted for HCC between 1989 and 2019 in the Erasmus MC University Medical Center in Rotterdam, the Netherlands, was used. AFP and PIVKA-II levels were determined in serum samples collected at the time of transplantation. Data on tumor load and microvascular invasion were retrieved from patients' records. RESULTS: The study cohort consisted of 121 patients, with HCC recurrence in 15 patients (12.4%). The median AFP was 7.7 ng/mL (4.4-20.2), and the median PIVKA-II was 72.0 mAU/mL (41.0-213.5). Patients with low AFP (≤8 ng/mL) and PIVKA-II (≤90 mAU/mL) had a 5-year recurrence-free survival of 100% compared to 85.7% in patients with low AFP and high PIVKA-II (p = 0.026). Regardless of the AFP level, patients within the Milan criteria (based on explant pathology) with a low PIVKA-II level had a 5-year recurrence-free survival of 100% compared to patients with a high PIVKA-II level of 81.1% (p = 0.002). In patients with microvascular invasion, the AUC for PIVKA-II was slightly better than the AUC for AFP (0.775 vs. 0.687). CONCLUSIONS: The dual model of PIVKA-II ≤ 90 mAU/mL with either AFP ≤ 8 ng/mL or with patients within the Milan criteria identifies patient groups which can be exempted from HCC surveillance after LT in a low AFP population. PIVKA-II may be a better predictor for explant microvascular invasion than AFP and could play a role in future models identifying LT candidates with the highest risk for HCC recurrence.

Circulating Cytokines Reflect the Etiology-Specific Immune Environment in Cirrhosis and HCC.

Background and Aims: Chronic liver disease­from any etiology­can progress to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The progression of liver cirrhosis to the end stages of disease is influenced by a variety of factors, including inflammatory cytokines. We pursued a study of cytokine-mediated inflammatory responses in hepatitis B (HBV), hepatitis C (HCV), alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) patients with liver cirrhosis. Methods: Immune profiles were determined through the serum multiplex profiling of >100 cytokines in a 188 cirrhotic patients, 35 healthy controls and 196 early-stage HCC patients. Results: Patients with liver cirrhosis exhibited a vast upregulation of proinflammatory cytokines (p < 0.0001), including those with pro-oncogenic features, when compared to healthy individuals. In contrast to prevailing assumptions, each etiological cause of cirrhosis exhibited a unique cytokine profile in blood. Regardless of antiviral therapy, HBV cirrhosis patients had the largest number of upregulated proinflammatory mediators, compared to HCV, ALD and NAFLD (p < 0.0001). To further evaluate the etiology-dependent modulation of cytokine response in relation to liver cancer, we studied cytokine profiles in early-stage HCC patients strictly stratified by underlying liver disease. We observed unique sets of differentially expressed cytokines in each cohort of early-stage HCC patients of different cirrhosis etiologies. Conclusions: Our findings, therefore, underscore the importance of stratification by the etiological cause of liver cirrhosis in immune-based studies.

Hepatitis B virus-specific CD4 T cell responses differentiate functional cure from chronic surface antigen+ infection.

BACKGROUND & AIMS: With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control. METHODS: We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining, and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly ex vivo. RESULTS: ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection. CONCLUSIONS: Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses. LAY SUMMARY: Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell).