Impact of non-neoplastic vs intratumoural hepatitis B viral DNA and replication on hepatocellular carcinoma recurrence

Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can Be a Predictor of Sustained Response to Therapy

This study was designed to evaluate the prognostic value of three systemic inflammation markers, neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and prognostic nutritional index (PNI), for hepatocellular carcinoma (HCC) associated with hepatitis B (HBV). This analysis included 234 HBV-HCC patients who underwent primary surgical resection at the Mount Sinai Medical Center between 1988 and 2013. Serum albumin and circulating neutrophil, lymphocyte, and platelet counts immediately before surgery were obtained to calculate NLR, PLR, and PNI. Patients with larger tumor size (>3 cm) had higher NLR, higher PLR, and lower PNI. Stratified analysis showed that the impact of three markers on outcome depends on the severity of liver fibrosis. High NLR, high PLR, or low PNI was associated with poor outcome only in patients without end-stage fibrosis (Ishak stage 0-5) and not in those with cirrhosis (Ishak stage 6). Multivariate analysis in Ishak stage 0-5 patients showed that only high NLR was associated with poor outcome independent of tumor size. Of the three markers, only NLR correlated with PD-L1 expression in center of tumor, but not in nonneoplastic liver. The prognostic value of these three markers following surgery was only significant for HBV-HCC patients without end-stage fibrosis, and among the three markers, only NLR remained a significant prognostic indicator independent of tumor size. The correlation of NLR with intratumoral PD-L1 expression raises a hypothesis for shared pathways leading to PD-L1-mediated local tolerance within tumor and systemic inflammatory responses represented by elevated NLR in HBV-HCC.

We read with great interest the study by Heo et al. which revealed that the tumour recurrence associated with the baseline viral replication activity in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients who underwent hepatectomy varied according to cirrhosis status [1]. Notably, patients who underwent liver transplantation were not included in this study; thus, the association between the HCC recurrence and the baseline viral replication activity for these individuals remains unclear. Liver transplantation stands as the optimal curative treatment for HCC patients [2]. However, tumour recurrence significantly compromises the efficacy of the operation [3, 4]. Our previous studies have identified HBV reactivation following liver transplantation as a critical risk factor for post-transplant tumour recurrence of HCC patients [5-7]. Here, we conducted a retrospective analysis of our cohort (NCT06114251), which included 462 patients with HBV-related HCC. Our aim was to explore the association between baseline viral replication activity and both HCC recurrence and HBV reactivation in these individuals with cirrhotic status. Among the 462 HBV-related HCC patients, 211 (45.7%) had detectable HBV-DNA prior to transplantation, while 431 (93.3%) had cirrhosis. The cumulative HCC recurrence rates at 1, 2, and 5 years were 23.6%, 32.3%, and 36.0%, respectively. The cumulative HBV reactivation rates at 1, 2, and 5 years were 12.3%, 15.5%, and 17.8%, respectively. Antiviral therapy with nucleotide analogues was routinely administered after the diagnosis of HBV infection prior to surgery. However, some patients were unaware of their HBV infection until HCC was diagnosed and thus may not have received adequate antiviral treatment before transplantation. Other baseline characteristics of these patients can be found in our previous research [5]. In cirrhotic patients, HCC recurrence risk showed a non-linear association with baseline HBV DNA, peaking at 2–4 log10 IU/mL. Compared to the non-detectable (ND) HBV DNA group, the adjusted HR was 1.72 (95% CI, 1.07–2.77; p = 0.025) for the 2–3 log10 IU/mL group and 1.73 (95% CI, 1.03–2.95; p = 0.038) for the 3–4 log10 IU/mL group. Risks at levels ≥ 4 log10 IU/mL were non-significant (Figure 1A). A similar pattern was observed for HBV reactivation risk, which was elevated at 2–3 log10 IU/mL (adjusted HR, 2.30; 95% CI, 1.23–4.31; p = 0.009) but lower at ≥ 4 log10 IU/mL (Figure 1B). Our analysis confirms a non-linear association between baseline HBV DNA levels and the risks of HCC recurrence or HBV reactivation after transplantation in cirrhotic patients. This result is inconsistent with that reported by Heo et al. The discrepancy may be attributed to some factors. First, the cohort in Heo et al. exclusively comprised BCLC 0-A patients, whereas such patients constituted only 9.3% of our cohort. Second, the distinct context of liver transplantation-involving complete native liver removal, graft-recipient interactions, long-term immunosuppression, and so on-fundamentally differs from that of hepatectomy. Unfortunately, the sample size of this study was not large enough. There were only 31 non-cirrhotic patients in our cohort, preventing a corresponding analysis. We look forward to future validation of our conclusions in larger, multicenter cohorts and to more comprehensive analyses. Huigang Li: writing and original draft, conceptualization, methodology, software, formal analysis, investigation, validation, data curation. Ruijie Zhao: conceptualization, methodology, software, formal analysis, investigation, validation, data curation. Jinyan Chen: conceptualization, methodology, software, formal analysis, investigation, validation, data curation. Shusen Zheng: conceptualization, writing and review editing, resources, project administration, supervision. Di Lu: writing and original draft, writing and review editing, conceptualization, methodology, software, data curation, supervision, visualization, validation. Xiao Xu: conceptualization, methodology, supervision, funding acquisition, resources, writing and review editing, writing and original draft, project administration. This work was supported by the Key Research & Development Plan of Zhejiang Province (2024C03051), the Major Research Plan of the National Natural Science Foundation of China (92159202), the Innovation Team, Hangzhou Medical College (CXLJ202401), and Ningbo Top Medical and Health Research Program (2024020818). Ethical approval for the HBV-related HCC cohort was obtained from the Ethics Committee of two centers (Shulan (Hangzhou) Hospital and the First Affiliated Hospital, Zhejiang University School of Medicine), consistent with prior research (approval IDs: No. 2020–510 and No. KY2021014). The authors declare no conflicts of interest. This article is linked to Heo et al. paper. To view this article, visit https://doi.org/10.1111/apt.70085. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Eliminating cccDNA to cure hepatitis B virus infection

Objectives: The aim of this study was to examine the methylation status of intrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and to elucidate the possible relationship between the cccDNA methylation and viral replicative activity in patients with HBV-related liver cirrhosis (HBV-LC). Methods: The methylation status of HBV cccDNA was investigated by bisulfite sequencing in nonneoplastic tissues from 12 patients with HBV-LC who underwent surgical resection for combined hepatocellular carcinoma. Clinical, biochemical and virologic factors were evaluated with respect to the degrees of cccDNA methylation. We also examined the effect of methylation of cccDNA on viral transcription by an in vitro transcription assay. Results: Variable degrees of CpG methylation were present in the HBV cccDNA from patients with HBV-LC. Old age, low serum HBV DNA levels and low virion productivity were significantly associated with elevated cccDNA methylation. Virion productivity of cccDNA was also lower in HepAD38 cells with a higher degree of cccDNA methylation. In vitro transcription assays showed that the transcriptional activity of HBV cccDNA was suppressed by increased methylation of cccDNA. Conclusions: Increased CpG methylation of cccDNA is associated with old age, low serum HBV DNA levels and suppressed replicative activity in HBV-LC.

Baseline viral replication activity influences the risk of hepatocellular carcinoma (HCC) development in patients with chronic hepatitis B virus (HBV) infection. To evaluate the impact of baseline viral replication activity on recurrence in HBV-related HCC after curative resection. A multinational retrospective cohort of 2384 patients with very early or early-stage HBV-related HCC who consecutively underwent curative resection and received antiviral therapy (AVT) between 2010 and 2018 was analysed. Patients were categorised into ongoing-AVT (previously on AVT with viral suppression) and initiation-AVT (initiated AVT at the time of resection) groups. HCC recurrence was compared between these two groups based on baseline viral replication activity. During a median follow-up of 4.9 years, 1188 (49.8%) patients developed recurrence. Multivariable analysis showed similar recurrence risk between the ongoing-AVT and initiation-AVT groups (HR, 1.09; 95% CI, 0.96-1.24). However, in cirrhotic patients, the initiation-AVT group had a higher recurrence risk than the ongoing-AVT group (HR, 1.22; 95% CI, 1.02-1.45) but not in non-cirrhotic patients (HR, 0.90; 95% CI, 0.73-1.09). Intriguingly, in the non-cirrhotic initiation-AVT group, a parabolic association was observed between baseline HBV DNA levels and the risk of recurrence, with those having 5-6 log10 IU/mL HBV DNA levels showing significantly higher recurrence risk compared to the ongoing-AVT group (HR, 1.78; 95% CI, 1.32-2.42). The association between HBV replication activity and the risk of HCC recurrence varied depending on cirrhosis, providing important insights for optimising the timing of AVT and post-operative surveillance strategies.

To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. HBV DNA was isolated from patients' liver biopsies and sera. A sensitive real-time PCR method, which is capable of differentiation of HBV viral genomic DNA and cccDNA, was used to quantify the total HBV cccDNA. The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech, LTD, Shenzhen, China) described previously. For the first time, we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera. In the liver biopsies, cccDNA was detected from all the biopsy samples. The copy number of cccDNA ranged from from 0.03 to 173.1 per cell, the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell. The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406. In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples. The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%. To further investigate the reason why cccDNA could only be detected in some patients' sera, we performed longitudinal studies. The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation. The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. HBV cccDNA is actively transcribed and replicated in some patients' hepatocytes, which is reflected by a high ratio of HBV total DNA vs cccDNA. Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy. The occurrence of cccDNA in the sera is an early signal of liver damage, which may be another important clinical parameter.

Objectives: Taiwan has a high prevalence of hepatitis B viral (HBV) infection with rising alcoholic liver disease. We investigated the histological assessment of viral hepatitis B activity in patients with concomitant HBV infection and alcoholism. Methods: 229 patients (33 with concomitant heavy alcoholism and HBV infection, 114 with HBV infection alone, and 82 with heavy alcoholism alone) were enrolled between 2009 and 2012 at Cathy General hospital and E-Da hospital. Results: Patients with concomitant alcoholism and HBV infection are male predominant and younger. 97.4% and 91.4% patients have detectable HBV DNA in patients with HBV infection without or with alcoholism, respectively. Patients with concomitant HBV infection and alcoholism have much piecemeal necrosis, confluent necrosis, focal necrosis, portal inflammation, necroinflammatory grading, and cirrhosis with Ishak stage 5-6 fibrosis. Moreover, patients with concomitant HBV infection and alcoholism also have much pericelluar fibrosis, sclerosing hyaline necrosis, non-alcoholic fatty liver disease (NAFLD) ballooning, NAFLD activity score (NAS) and NAFLD Stage 4 fibrosis (P<0.001). However, patients with alcoholism alone have much more steatosis than those with HBV infection with and without alcoholism. Conclusions: Patients having concomitant alcoholism and HBV infection develop the histological features of both alcoholic liver disease and viral hepatitis B. The assessment of hepatitis B viral activity in alcoholic liver disease depends on detectable viral load and histological features of viral hepatitis B in patients with concomitant HBV infection and alcoholism.

Collagen proportionate area (CPA) has a better correlation with hepatic venous pressure gradient (HVPG) than with Ishak stage. Liver stiffness measurement (LSM) is proposed as non invasive marker of portal hypertension/disease progression. Our aim was to compare LSM and CPA with Ishak staging in chronic viral hepatitis, and HVPG in HCV hepatitis after transplantation. One hundred and sixty-nine consecutive patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections pre/post liver transplantation (LT), had a liver biopsy combined with LSM (transient elastography), CPA (biopsies stained with Sirius Red and evaluated by digital image analysis and expressed as CPA) and HVPG (measured contemporaneously with transjugular biopsies in LT HCV patients). LSM was dependent on CPA in HBV (r (2)=0.61, p<0.0001), HCV (r (2)=0.59, p<0.0001) and LT groups (r (2)=0.64, p<0.0001). In all three groups, CPA and Ishak were predictors of LSM, but multivariately CPA was better related to LSM (HBV: r (2)=0.61, p<0.0001; HCV: r (2)=0.59, p<0.0001; post-LT: r (2)=0.68, p<0.0001) than Ishak stage. In the LT group, multiple regression analysis including HVPG, LSM, aspartate aminotransferase to platelet ratio index (APRI) and Ishak stage/grade, showed that only CPA was related to HVPG (r (2)=0.41, p=0.01), both for HVPG ≥6mmHg (OR 1.34, 95% CI 1.14-1.58; p<0.0001) or ≥10mmHg (OR 1.25, 95% CI 1.06-1.47; p=0.007). CPA was related to LSM in HBV or HCV hepatitis pre/post-LT. CPA was better related to LSM than Ishak stage. In the LT HCV group, CPA was better related to HVPG than Ishak stage/grade, LSM or APRI. CPA may represent a better comparative histological index for LSM, rather than histological stages.

Long-term Therapy With Adefovir Dipivoxil for HBeAg-Negative Chronic Hepatitis B for up to 5 Years

Major risk factors for hepatocellular carcinoma (HCC) vary by region and population. Chronic infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV), heavy alcohol intake, aflatoxin exposure, haemochromatosis, nonalcoholic fatty liver disease, obesity, diabetes mellitus and tobacco smoking have been suggested to be risk factors of HCC. However, chronic infection with HBV remains the major cause of HCC in the world. More than 50% of HCC cases worldwide and 80% of HCC cases in most high-risk areas, such as the Asia‐Pacific region and Africa, are attributable to HBV infection. The relative risk of HCC among individuals infected with HBV ranges from 5 to 49 in case‐control studies and from 7 to 98 in cohort studies [1]. In HBV endemic regions, HBV infection is usually acquired perinatally or in childhood, whereas in HBV nonendemic areas, it is mostly transmitted during adolescence or adulthood. Newborns become chronic HBV carriers at a very high rate (about 90%), while immune-competent adults are generally described as developing chronic infection at a rate of 5‐10%. HBV subgenotype C2 is more prone to causing chronic infection than HBV subgenotype B2 following acute hepatitis B [2]. Up to 40% of patients with chronic HBV infection will develop the life-threatening complications of HCC or decompensated liver cirrhosis (LC). Chronic hepatitis B (CHB), LC and HCC are progressive liver diseases and consecutive stages of HBVassociated hepatocarcinogenesis, although CHB may precede the asymptomatic hepatitis B surface antigen (HBsAg) carrier (ASC) state and HCC does not have to pass through the CHB or LC stage. Hepatitis B e antigen (HBeAg) expression indicates active viral replication. HBeAg expression, high viral load and alanine aminotransferase (ALT) levels are associated with an increased risk of HCC. HBV subgenotypes B2 and C2 are endemic in most parts of Asia, while subgenotype B1 is endemic in Japan. Chronic infection with HBV C2 is frequently associated with an increased risk of LC and HCC in patients older than 50 years, whereas chronic infection with HBV B2 is associated with HCC or HCC recurrence in young, mostly non-cirrhotic, patients. In comparison with infection with HBV B2 or C2, infection with HBV B1 is associated with fulminant hepatitis B, a lower incidence of HCC and the development of HCC when older. Different HBV genotypes display their own distinct mutation pattern in the preS region and in the enhancer II (EnhII)/basic core promoter (BCP)/precore region of the HBV genome. Takahashi and colleagues identified HCC-associated HBV mutations through the comparative analysis of full-length HBV isolates (95% genotype C) from sera of 40 Japanese patients with HCC. They found that deletions and missense mutations in the preS2 region, A1762T and G1764A (often appearing simultaneously, termed A1762T/G1764A) and T1753C/A mutations in the BCP region and G1613A and C1653T mutations in the EnhII region were more frequent in HCC patients [3]. This pioneering work has aroused great interest in the field. Many studies pertaining to the association of HBV mutations with the risk of HCC have been published during the past decade. It has been found that the HBV preS deletion, A1762T/G1764A, T1753V, C1653T and T31C are each associated with a significantly increased risk of HCC [4]. Of the two major HBV

The effects of hepatocyte steatosis on hepatitis B virus (HBV) DNA replication and HBV-related antigen secretion are incompletely understood. The aims of this study are to explore the effects and mechanism of hepatocyte steatosis on HBV replication and secretion. Stearic acid (SA) and oleic acid (OA) were used to induce HepG2.2.15 cell steatosis in this study. The expressions of glucose-regulated protein 78 (GRP78), phosphorylation of protein kinase R-like endoplasmic reticulum (ER) kinase (p-PERK), and eukaryotic translation initiation factor 2α (p-eIF2α) were detected by Western blotting (WB). HBV DNA, HBsAg, and HBeAg in the supernatant were determined by real-time fluorescent polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. Intracellular HBV DNA, HBsAg level, and HBV RNA were measured by real-time fluorescent PCR, WB, and real-time quantitative reverse transcriptase-PCR, respectively. The results showed that SA and OA significantly increased intracellular lipid droplets and triglyceride levels. SA and OA significantly induced GRP78, p-PERK, and p-eIF2α expressions from 24 to 72 h. 4-phenylbutyric acid (PBA) alleviated ER stress induced by SA. SA promoted intracellular HBsAg and HBV DNA accumulation; however, it inhibited the transcript of HBV 3.5 kb mRNA and S mRNA. The secretion of HBsAg and HBV DNA inhibited by SA or OA could be partially restored by pretreatment with PBA but not by inhibiting GRP78 expression with siRNA. Hepatocyte steatosis inhibits HBsAg and HBV DNA secretion via induction of ER stress in hepatocytes, but not via induction of GRP78.

Outcomes following liver resection and clinical pathologic characteristics of hepatocellular carcinoma occurring in patients with chronic hepatitis B and minimally fibrotic liver

To study the relationship between metastasis or recurrence of hepatocellular carcinoma (HCC) and hepatitis B virus (HBV) DNA load or the presence of double mutation at 1762/1764 in the basic core promoter (BCP). One-hundred-and-fifty-seven patients with HCC were included in the study. Events of tumor metastasis or recurrence were recorded during 120 weeks of clinical follow-up after treatment by surgery or transarterial chemoembolization (TACE). The 1-year follow-up included monthly alpha fetoprotein (AFP) measurement and abdominal ultrasonography (US), as well as helical computed tomographic (CT) scan performed every 3 months. Follow-up beyond 1-year (surveillance) included AFP measurement and abdominal US every 2 months and helical CT scan every 6 months. Suspected metastasis or recurrence was investigated by hepatic angiography and confirmed according to the combined imaging findings. Serum HBV DNA level was measured by real-time PCR. HBV genotypes were determined by PCR-restriction fragment length polymorphism analysis. Of the 157 HCC cases 110 experienced tumor metastasis or recurrence; the cumulative probability of post-treatment HCC metastasis or recurrence was 4 (2.55%) at week 12, 14 (8.92%) at week 24, 28 (17.83%) at week 48, 64 (40.76%) at week 72, 92 (58.60%) at week 96, and 110 (70.06%) at week 120. Multivariate analysis indicated that both the BCP 1762/1764 double mutations and HBV DNA levels were risk factors for HCC recurrence or metastasis. In particular, the incidence of HCC recurrence or metastasis increased with baseline serum HBV DNA levels in a dose-response manner, ranging from 8/19 (42.1%) for less than 3 log10 copies/ml HBV DNA to 35/61 (57.3%) for 3-5 log10 copies/ml and 67/77 (87.0%) for more than 5 log10 copies/ml. After adjusting for potential confounders, serum HBV DNA level remained independently associated with HCC metastasis or recurrence. HCC recurrence or metastasis occurred in 22/43 (51.2%) of patients without BCP 1762/1764 mutations and 88/114 (77.2%) of patients with BCP 1762/1764 mutations. The adjusted odds ratio for patients infected with BCP 1762/1764 double mutation HBV, compared with those infected with non-BCP 1762/1764 mutation HBV, was 5.264 (95% CI: 1.436-12.574, P less than 0.05). Infection with HBV carrying the BCP 1762/1764 double mutation and presence of high HBV DNA load are independent risk factors for developing HCC metastasis or recurrence after surgery or TACE.

The objective of this study was to explore the effects of dendritic cells (DCs) from hepatitis B virus (HBV) transgenic mice-stimulated autologous lymphocytes on in vitro HBV replication. DCs from HBV transgenic mice were induced to maturity by lipopolysaccharide, followed by incubation with hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in vitro. Mature DCs and autologous lymphocytes were co-stimulated to form specific sensitized immune effector cells (IEC), which were then co-cultured with the human hepatoma cell line HepG2.2.15. Changes in morphology and activity of hepatocytes were then observed, as well as analysis of changes in liver enzyme, and HBV DNA and inflammatory cytokine levels in the culture supernatant. Intracellular HBV DNA and covalently closed circular DNA (cccDNA) concentration were measured by real-time polymerase chain reaction. Co-stimulation by mature DCs and IEC showed no impact on the morphology and liver enzyme expression level of HepG2.2.15 cells, but the supernatant HBV DNA and intracellular HBV DNA and cccDNA levels decreased significantly compared with those cells co-cultured with immature DCs. Secretion of inflammatory cytokines in the supernatant showed that when HBV DNA was highly expressed, the concentration of IFN-γ and IL-2 decreased, while IL-10 increased. Contrastingly, when HBV DNA had low expression, the concentration of IFN-γ and IL-2 increased and IL-10 decreased. Co-stimulation of HBV-related antigen-induced mature DCs and autologous lymphocytes showed inhibitory effects on ex vivo HBV replication, and cytokines were suggested to mediate this effect.