Molecularly generated rat hepatitis E virus strains from human and rat show efficient replication in a human hepatoma cell line.

The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Whereas HEV genotypes 1-4 of species Paslahepevirus balayani are commonly found in humans, infections with ratHEV (species Rocahepevirus ratti) were previously considered to be restricted to rats. However, several cases of human ratHEV infections have been described recently. To investigate the zoonotic potential of this virus, a genomic clone was constructed here based on sequence data of ratHEV strain pt2, originally identified in a human patient with acute hepatitis from Hongkong. For comparison, genomic clones of ratHEV strain R63 from a rat and of HEV genotype 3 strain 47832mc from a human patient were used. After transfection of in vitro-transcribed RNA from the genomic clones into the human hepatoma cell line HuH-7-Lunet BLR, virus replication was shown for all strains by increasing genome copy numbers in cell culture supernatants. These cells developed persistent virus infections, and virus particles in the culture supernatant as well as viral antigen within the cells were demonstrated. All three generated virus strains successfully infected fresh HuH-7-Lunet BLR cells. In contrast, the human hepatoma cell lines HuH-7 and PLC/PRF/5 could only be infected with the genotype 3 strain and to a lesser extent with ratHEV strain R63. Infection of the rat-derived hepatoma cell lines clone 9, MH1C1 and H-4-II-E did not result in efficient virus replication for either strain. The results indicate that ratHEV strains from rats and humans can infect human hepatoma cells. The replication efficiency is strongly dependent on the cell line and virus strain. The investigated rat hepatoma cell lines could not be infected and other rat-derived cells should be tested in future to identify permissive cell lines from rats. The developed genomic clone can represent a useful tool for future research investigating pathogenicity and zoonotic potential of ratHEV.

[Overexpression of tuftelin and KLF-5 and its clinicopathological features in hepatitis B virus-related hepatocellular carcinoma].

Objective: To analyze and evaluate the expressions and clinical value of tuftelin (TUFT1) and Krüppel-like factor 5 (KLF5) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) tissues. Method: KLF5 mRNA and TUFT1 mRNA transcriptional status in cancer and non-cancer groups were compared according to the Cancer Genome Atlas (TCGA) database. The differences and prognostic value between the groups were analyzed. Postoperative liver cancer and its paired pericancerous tissues, with the approval of the ethics committee, were collected to build tissue chips. The expression of KLF5 and TUFT1 and their intracellular localization were verified by immunohistochemistry. Tissue expression and clinicopathological characteristics were analyzed by immunoblotting. SPSS software was used to analyze the relationship between SPSS and patient prognosis. Results: The transcription level of TUFT1 or KLF5 mRNA was significantly higher in the HCC group than the non-cancer group (P < 0.001), according to TCGA data. Immunohistochemistry and Western blotting examination confirmed the overexpression of TUFT1 and KLF5 in human HCC tissues, which were mainly localized in the cytoplasm and cell membrane. The positivity rates of TUFT1 and KLF5 were 87.1% ( χ(2) = 18.563, P < 0.001) and 95.2% ( χ(2) = 96.435, P < 0.001) in HCC tissues, and both were significantly higher than those in the adjacent group. The expression intensity was higher in stage III-IV than stage I-II of the International Union Against Cancer standard (P < 0.01). The clinicopathological features showed that the abnormalities of the two were significantly related to HBV infection, tumor size, extrahepatic metastasis, TNM stage, and ascites. Univariate analysis was related to tumor size, HBV infection, and survival. Multivariate analysis was an independent prognostic factor for patients with HCC. Conclusion: TUFT1 and KLF5 may both be novel markers possessing clinical value in the diagnosis and prognosis of HBV-related HCC.

Post-transplant hepatitis B virus reactivation impacts the prognosis of patients with hepatitis B-related hepatocellular carcinoma: a dual-centre retrospective cohort study in China.

BACKGROUND: Highly active hepatitis B virus (HBV) is known to be associated with poor outcomes in patients with hepatocellular carcinoma (HCC). This study aims to investigate the relationship between HBV status and HCC recurrence after liver transplantation. METHODS: The study retrospectively analyzed HCC patients undergoing liver transplantation in two centres between January 2015 and December 2020. The authors reviewed post-transplant HBV status and its association with outcomes. RESULTS: The prognosis of recipients with hepatitis B surface antigen (HBsAg) reappearance ( n =58) was poorer than those with HBsAg persistent negative ( n =351) and positive ( n =53). In HBsAg persistent positive group, recipients with HBV DNA reappearance or greater than 10-fold increase above baseline had worse outcomes than those without ( P <0.01). HBV reactivation was defined as (a) HBsAg reappearance or (b) HBV DNA reappearance or greater than 10-fold increase above baseline. After propensity score matching, the 5-year overall survival rate and recurrence-free survival rate after liver transplantation in recipients with HBV reactivation were significantly lower than those without (32.0% vs. 62.3%; P <0.01, and 16.4% vs. 63.1%; P <0.01, respectively). Moreover, HBV reactivation was significantly related to post-transplant HCC recurrence, especially lung metastasis. Cox regression analysis revealed that beyond Milan criteria, microvascular invasion and HBsAg-positive graft were independent risk factors for post-transplant HBV reactivation, and a novel nomogram was established accordingly with a good predictive efficacy (area under the time-dependent receiver operating characteristic curve=0.78, C-index =0.73). CONCLUSIONS: Recipients with HBV reactivation had worse outcomes and higher tumour recurrence rates than those without. The nomogram could be used to evaluate the risk of post-transplant HBV reactivation effectively.

Deep whole-genome analysis of 494 hepatocellular carcinomas.

Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.

Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

NAMPT promotes the malignant progression of HBV-associated hepatocellular carcinoma through activation of SREBP1-mediated lipogenesis.

Metabolic reprogramming is a hallmark of cancer. The nicotinamide phosphoribosyltransferase (NAMPT)-mediated salvage pathway maintains sufficient cellular NAD levels and is required for tumorigenesis and development. However, the molecular mechanism by which NAMPT contributes to HBV-associated hepatocellular carcinoma (HCC) remains not fully understood. In the present study, our results showed that NAMPT protein was obviously upregulated in HBV-positive HCC tissues compared with HBV-negative HCC tissues. NAMPT was positively associated with aggressive HCC phenotypes and poor prognosis in HBV-positive HCC patients. NAMPT overexpression strengthened the proliferative, migratory, and invasive capacities of HBV-associated HCC cells, while NAMPT-insufficient HCC cells exhibited decreased growth and mobility. Mechanistically, we demonstrated that NAMPT activated SREBP1 (sterol regulatory element-binding protein 1) by increasing the expression and nuclear translocation of SREBP1, leading to the transcription of SREBP1 downstream lipogenesis-related genes and the production of intracellular lipids and cholesterol. Altogether, our data uncovered an important molecular mechanism by which NAMPT promoted HBV-induced HCC progression through the activation of SREBP1-triggered lipid metabolism reprogramming and suggested NAMPT as a promising prognostic biomarker and therapeutic target for HBV-associated HCC patients.

HLA-DR genetic polymorphisms and hepatitis B virus mutations affect the risk of hepatocellular carcinoma in Han Chinese population.

BACKGROUND: Human leucocyte antigen (HLA)-DR plays a crucial role in the immune response against hepatitis B virus (HBV). We aimed to investigate the associations of HLA-DR single nucleotide polymorphisms (SNPs) with the generation of hepatocellular carcinoma (HCC)-related HBV mutations. The effects of HLA-DR SNPs and their interactions with HBV mutations on HCC risks were also determined. METHODS: Five HLA-DR SNPs (rs3135363, rs9268644, rs35445101, rs24755213, and rs984778) were genotyped in 792 healthy controls, 586 chronic hepatitis B (CHB) patients, 536 liver cirrhosis (LC) patients, and 1500 HCC patients using quantitative PCR. Sanger sequencing was used to identify the HBV mutations. Logistic regression model was performed to evaluate the association of HLA-DR SNPs with HCC risk and the frequencies of HCC-related HBV mutations. RESULTS: The variant genotypes at rs3135363, rs9268644, rs35445101, rs24755213, and rs984778 were associated with decreased HCC risks. In genotype C HBV-infected subjects, variant genotypes of these SNPs were associated with decreased frequencies of HCC-related HBV mutations such as C1653T, T1674C/G, G1719T, T1753A/C, A1762T/G1764A, A1846T, G1896A, G1899A, and preS deletion. AG genotype at rs3135363, CA genotype at rs9268644, and AG genotype at rs24755213 reduced the generation of T1753A/C and G1896A in genotype B HBV-infected subjects, respectively. In addition, the interactions of rs3135363, rs9268644, rs24755213 with C1653T, T1753A/C, A1846T, and G1896A decreased the risks of HCC. CONCLUSIONS: HLA-DR genetic polymorphisms might predispose the host to immunoselection of HCC-related HBV mutations and affect the HCC risks possibly through interacting with HBV mutations.

Hepatitis B doubly spliced protein (HBDSP) promotes hepatocellular carcinoma cell apoptosis via ETS1/GATA2/YY1-mediated p53 transcription.

IMPORTANCE: Hepatitis B virus (HBV) spliced variants are associated with viral persistence or pathogenicity. Hepatitis B doubly spliced protein (HBDSP), which has been previously reported as a pleiotropic transactivator protein, can potentially serve as an HBV virulence factor. However, the underlying mechanisms of HBDSP in HBV-associated liver diseases remain to be elucidated. In this study, we revealed that HBDSP promotes cellular apoptosis and induces wt-p53-dependent apoptotic signaling pathway in wt-p53 hepatocellular cells by transactivating p53 transcription, and increases the release of HBV progeny. Therefore, HBDSP may promote the HBV particles release through wt-p53-dependent hepatocellular apoptosis. Our findings suggest that blocking HBDSP-induced wt-p53-dependent apoptosis might have therapeutic values for chronic hepatitis B.

Nuclear translocation of YAP drives BMI-associated hepatocarcinogenesis in hepatitis B virus infection.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) development and progression. The aim of this study was to mechanistically investigate the involvement of Hippo signalling in HBV surface antigen (HBsAg)-dependent neoplastic transformation. METHODS: Liver tissue and hepatocytes from HBsAg-transgenic mice were examined for the Hippo cascade and proliferative events. Functional experiments in mouse hepatoma cells included knockdown, overexpression, luciferase reporter assays and chromatin immunoprecipitation. Results were validated in HBV-related HCC biopsies. RESULTS: Hepatic expression signatures in HBsAg-transgenic mice correlated with YAP responses, cell cycle control, DNA damage and spindle events. Polyploidy and aneuploidy occurred in HBsAg-transgenic hepatocytes. Suppression and inactivation of MST1/2 led to the loss of YAP phosphorylation and the induction of BMI1 expression in vivo and in vitro. Increased BMI1 directly mediated cell proliferation associated with decreased level of p16INK4a , p19ARF , p53 and Caspase 3 as well as increased Cyclin D1 and γ-H2AX expression. Chromatin immunoprecipitation and the analysis of mutated binding sites in dual-luciferase reporter assays confirmed that the YAP/TEAD4 transcription factor complex bound and activated the Bmi1 promoter. In chronic hepatitis B patients, paired liver biopsies of non-tumour and tumour tissue indicated a correlation between YAP expression and the abundance of BMI1. In a proof-of-concept, treatment of HBsAg-transgenic mice with YAP inhibitor verteporfin directly suppressed the BMI1-related cell cycle. CONCLUSION: HBV-associated proliferative HCC might be related to the HBsAg-YAP-BMI1 axis and offer a potential target for the development of new therapeutic approaches.

Primary non-response to antiviral therapy affects the prognosis of hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND AND AIM: Although antiviral treatments have been shown to affect the recurrence and long-term survival of patients with hepatocellular carcinoma (HCC) who have high viral loads, the effect of different responses to antiviral therapy on the clinical outcomes remains unclear. This study aimed to assess the effect of primary non-response (no-PR) to antiviral therapy on the survival or prognosis of patients with HCC with a high load of hepatitis B virus (HBV) DNA. METHODS: A total of 493 HBV-HCC patients hospitalized at Beijing Ditan Hospital of Capital Medical University were admitted to this retrospective study. Patients were divided into two groups based on viral response (no-PR and primary response). Kaplan-Meier (KM) curves were used to compare the overall survival of the two cohorts. Serum viral load comparison and subgroup analysis were performed. Additionally, risk factors were screened and the risk score chart was created. RESULTS: This study consisted of 101 patients with no-PR and 392 patients with primary response. In the different categories based on hepatitis B e antigen and HBV DNA, no-PR group had a poor 1-year overall survival (OS). In addition, in the alanine aminotransferase < 50 IU/L and cirrhosis groups, primary nonresponse was related to poor overall survival and progression-free survival. Based on multivariate risk analysis, primary non-response (hazard ratio (HR) = 1.883, 95% CI 1.289-2.751, P = 0.001), tumor multiplicity (HR = 1.488, 95% CI 1.036-2.136, P = 0.031), portal vein tumor thrombus (HR = 2.732, 95% CI 1.859-4.015, P < 0.001), hemoglobin < 120 g/L (HR = 2.211, 95% CI 1.548-3.158, P < 0.001) and tumor size ≥ 5 cm (HR = 2.202, 95% CI 1.533-3.163, P < 0.001) were independent risk factors for 1-year OS. According to the scoring chart, patients were divided into three risk groups (high-, medium-, and low-risk groups) with mortality rates of 61.7%, 30.5%, and 14.1%, respectively. CONCLUSIONS: The level of viral decline at 3 months post-antiviral treatment may predict the OS of patients with HBV-related HCC, and primary non-response may shorten the median survival time of patients with high HBV-DNA levels.

HLA-G susceptibility to hepatitis B infection and related hepatocellular carcinoma in the Japanese population.

AIMS: Human leukocyte antigen (HLA)-G plays a role in various physiological immunomodulatory functions. Aberrant HLA-G expression is observed in various disease states, including tumors, autoimmune disorders, and viral infections. The present study investigated the association between HLA-G functional gene polymorphisms (rs1736933 [-486 C > A], rs1049033 [+2018 C > T], 14 bp Insertion [Ins]/Deletion [Del] [+2961 Del > Ins], and rs1063320 [+3142 C > G]) and disease susceptibility, hepatocellular carcinoma (HCC) development, and hepatitis B surface antigen (HBsAg) clearance. METHODS: Allele discrimination of the 3 SNPs (-486 C > A, +2018 C > T, +3142 C > G) was determined by a TaqMan 5' exonuclease assay, while the 14 bp Ins/Del polymorphism was typed by fragment analysis using Genetic Analyzer and GeneMapper software. The above polymorphisms were analyzed for 325 Japanese hepatitis B virus (HBV) patients, 355 Japanese healthy subjects (Controls) as healthy controls, and 799 Japanese hepatitis C virus (HCV) patients as disease controls, respectively. RESULTS: The 14 bp Insertion allele was significantly more frequent in HBV patients than Controls (27.1 % vs 20.6 %, odds ratio [OR] 1.43, P = 0.005) but did not differ between HCV patients and Controls. Similar results were found for the rs1063320 G allele (38.9 % vs 26.3 %, OR 1.78, P < 0.001) and the rs1736933 T allele (32.2 % vs 26.9 %, OR 1.29, P = 0.034) between HBV and Controls. The rs1049033 T allele showed a weak but significant association with HCC development in the dominant model (OR 1.95, P = 0.04). Regarding HBsAg clearance, the A allele at rs1736933 was significantly correlated in the recessive model (OR 3.23, P = 0.003). CONCLUSIONS: This study revealed significant associations of HLA-G gene polymorphisms with disease susceptibility, HCC development, and HBsAg clearance in HBV patients.

Efficacy and safety of PD-1/PD-L1 inhibitors combined with anti-angiogenic therapy for the unresectable hepatocellular carcinoma and the benefit for hepatitis B virus etiology subgroup: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death, worldwide. The predominant causative factor for HCC is hepatitis B virus (HBV) infection. We conducted a meta-analysis to estimate the efficacy and safety of PD-1/PD-L1 inhibitors combined with anti-angiogenic therapy for the first-line treatment of the unresectable HCC and to evaluate the benefits of different geographic regions and etiology stratifications. METHODS: Randomized clinical trials published up to 12th November 2022 were searched by online databases. Moreover, effects of hazard ratio (HR) for overall survival (OS) and progression-free survival (PFS) were extracted from included studies. Pooled odds ratio (OR) and 95% CI for objective response rate (ORR), disease control rate (DCR), and treatment-related adverse events (TRAEs) were calculated. RESULTS: A total of 3057 patients from five phase III randomized clinical trials were collected and reviewed for this meta-analysis. The pooled HR of OS (HR = 0.71; 95% CI: 0.60-0.85) and PFS (HR = 0.64; 95% CI: 0.53-0.77) demonstrated significantly better benefit in PD-1/PD-L1 inhibitors combination group than targeted monotherapy to treat unresectable HCC. In addition, combination therapy showed better ORR and DCR, with ORs of 3.29 (95% CI: 1.92-5.62) and 1.88 (95% CI: 1.35-2.61), respectively. The subgroup analysis indicated that PD-1/PD-L1 inhibitors combination therapy was significantly superior to anti-angiogenic monotherapy for HBV-related HCC in terms of OS (HR = 0.64; 95% CI: 0.55-0.74) and PFS (HR = 0.53; 95% CI:0.47-0.59), while there was no significant difference in patients with HCV (OS, HR = 0.81, p = 0.1) or non-viral (OS, HR = 0.91, p = 0.37; PFS, HR = 0.77, p = 0.05). CONCLUSIONS: Meta-analysis revealed for the first-time that PD-1/PD-L1 inhibitors combination therapy for unresectable HCC was associated with better clinical outcomes than anti-angiogenic monotherapy, especially for HBV infection and Asian population.

Prediction Model with HLA-A*33:03 Reveals Number of Days to Develop Liver Cancer from Blood Test.

The development of liver cancer in patients with hepatitis B is a major problem, and several models have been reported to predict the development of liver cancer. However, no predictive model involving human genetic factors has been reported to date. For the items incorporated in the prediction model reported so far, we selected items that were significant in predicting liver carcinogenesis in Japanese patients with hepatitis B and constructed a prediction model of liver carcinogenesis by the Cox proportional hazard model with the addition of Human Leukocyte Antigen (HLA) genotypes. The model, which included four items-sex, age at the time of examination, alpha-fetoprotein level (log10AFP) and presence or absence of HLA-A*33:03-revealed an area under the receiver operating characteristic curve (AUROC) of 0.862 for HCC prediction within 1 year and an AUROC of 0.863 within 3 years. A 1000 repeated validation test resulted in a C-index of 0.75 or higher, or sensitivity of 0.70 or higher, indicating that this predictive model can distinguish those at high risk of developing liver cancer within a few years with high accuracy. The prediction model constructed in this study, which can distinguish between chronic hepatitis B patients who develop hepatocellular carcinoma (HCC) early and those who develop HCC late or not, is clinically meaningful.

Elucidating the role of missense SNP of protein kinase C epsilon in HCV-induced hepatocellular carcinoma.

BACKGROUND: The protein kinase C (PKC) family of serine/threonine kinases contains more than ten isozymes that are involved in multiple signaling pathways, including cell cycle regulation and carcinogenesis. The PKCε isozyme is an oncogene known to be upregulated in various signaling pathways involved in hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). However, there is no known association of missense SNPs in PKCε with this disease, which can be a potential biomarker for early diagnosis and treatment. This research reveals a novel missense SNP in PKCε that is associated with HCV-induced HCC in the Pakistani population. METHODS: The PKCε SNP with amino acid substitution of E14K was chosen for wet lab analysis. Tetra ARMS-PCR was employed for the identification of high-risk SNP in PKCε of HCV-induced HCC patients. Liver function testing was also performed for comparison between the liver condition of the HCC patient and control group, and the viral load of HCC patient samples was evaluated to determine any alteration in the viral infectivity between different genotypes of the selected high-risk PKCε variant SNP. RESULTS: Frequency distribution of the homozygous GG genotype was found to be highest among HCV-induced HCC patients and was also found to be significantly associated with disease development and progression. The p values of comparative data obtained for the other two genotypes, heterozygous AG and homozygous AA, of the SNP also showed the significance of the data for these alleles. Still, their odds ratio and relative risk analysis did not indicate their association with HCV-induced HCC. CONCLUSION: The distribution of a genotype GG of PKCε has been found in HCV- induced HCC patients. Therefore, these PKCε SNP have the potential to be biomarkers for HCV-induced HCC. Further investigation using a larger sample size would provide additional insight into these initial data and open a new avenue for a better prognosis of this disease.

Low Level of Hepatitis B Viremia Compared With Undetectable Viremia Increases the Risk of Hepatocellular Carcinoma in Patients With Untreated Compensated Cirrhosis.

INTRODUCTION: The initiation of antiviral treatment in patients with chronic hepatitis B with compensated cirrhosis and low-level viremia (LLV; HBV DNA 15-2,000 IU/mL) remains controversial. We sought to compare the long-term outcomes of these untreated patients according to their viremic status. METHODS: Six hundred twenty-seven untreated patients with chronic hepatitis B with compensated cirrhosis were analyzed retrospectively. The risk of hepatocellular carcinoma (HCC) and liver-related clinical events, including hepatic decompensation, were compared between patients with LLV and undetectable HBV DNA. Patients who received antiviral treatment were censored during treatment initiation. RESULTS: The mean age of the patients was 54.7 years, 64.4% of whom were male. During the study period, 59 patients developed HCC (20 and 39 in the undetectable and LLV groups, respectively) with an annual incidence of 2.44/100 person-years. Multivariable analysis revealed that the LLV group was associated with a significantly higher risk of HCC (adjusted hazard ratio: 2.36, P = 0.002) than the undetectable group. In the 204 propensity score-matched cohort, the LLV group had a 2.16-fold greater risk of HCC than the undetectable group ( P = 0.014). Liver-related clinical events occurred in 121 patients with an annual incidence of 5.25/100 person-years. Despite not reaching statistical significance, the LLV group tended to have a higher risk of liver-related events in the propensity score-matched cohort (hazard ratio: 1.14, P = 0.50). DISCUSSION: Compared with patients with undetectable HBV DNA, those with compensated cirrhosis and LLV had a significantly higher risk of HCC. Antiviral treatment should be advised for these patients.

Therapies against chronic hepatitis B infections: The times they are a-changin', but the changing is slow!

PREAMBULAR NOTA BENE: As a tribute to Dr Mike Bray, the following review of literature willbe mainly based on published data andconcepts, but will also contain my personal views, and in this respect could be more considered as a bioassay. Even though a cost-effective and excellent prophylactic vaccine exists since many years to protect against hepatitis B virus (HBV) infection, academic-researcher/drug-developers/stakeholders are still busy with the R&D of novel therapies that could eventually have an impact on its worldwide incidence. The Taiwanese experience have univocally demonstrated the effectiveness of constrained national HBV prophylactic vaccination programs to prevent the most dramatic HBV-induced end-stage liver disease, which is hepatocellular carcinoma; but yet the number of individuals chronically infected with the virus, for whom the existing prophylactic vaccine is no longer useful, remains high, with around 300 million individuals around the globe. In this review/bioassay, recent findings and novel concepts on prospective therapies against HBV infections will be discussed; yet it does not have the pretention to be exhaustive, as "pure immunotherapeutic concepts" will be mainly let aside (or referred to other reviews) due to a lack of expertise of this writer, but also due to the lack of, or incremental, positive results in clinical trials as-off today with these approaches.

A Novel Diagnostic Model for Early Hepatitis B Virus-Related Hepatocellular Carcinoma Diagnosis.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is closely related to the diagnostic stage. Due to the difficulty diagnosing early-stage HCC, most patients with HCC are diagnosed at the advanced stage. In this study, we used protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein (AFP) combined with aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-Bil) to establish a novel diagnostic model for early-stage hepatitis B virus (HBV)-related HCC. METHODS: The serum levels of PIVKA-II, AFP, AST, ALT, and T-Bil were measured in 148 patients with early-stage HBV-related HCC and 940 patients with chronic hepatitis B. The receiver operating characteristic (ROC) curves were used to verify the diagnostic efficacy of the novel diagnostic model for early-stage HBV-related HCC. RESULTS: The mathematical model of [1.5 x PIVKA-II/(AST x T-Bil) + AFP/(ALT x T-Bil)] was selected as the novel diagnostic model. The areas under ROC curves (AUROCs) of the novel diagnostic model for detecting early-stage HBV-related HCC were significantly higher than those of PIVKA-II, AFP, and PIVKA-II combined with AFP (HCC ≤ 5 cm: 0.925 vs. 0.826, 0.666, and 0.821; HCC < 3 cm: 0.896 vs. 0.741, 0.651, and 0.765, respectively) (all p < 0.001). Using serum levels of AFP ≥ 20 ng/mL, the diagnostic model had the highest AUROC values of 0.960 and 0.933 for HCC ≤ 5 cm (89 cases) and HCC < 3 cm (40 cases), respectively, with a sensitivity of 83.15%, and 77.50% and specificity of 95.34% and 90.69%, respectively. CONCLUSIONS: The novel diagnostic model is superior to PIVKA-II and AFP for diagnosing early-stage HBV-related HCC, especially in patients with abnormal serum AFP levels.

Association of hepatitis B virus DNA levels with overall survival for advanced hepatitis B virus-related hepatocellular carcinoma under immune checkpoint inhibitor therapy.

BACKGROUND: High hepatitis B virus (HBV) DNA level is an independent risk factor for postoperative HBV-associated liver cancer recurrence. We sought to examine whether HBV DNA level and antiviral therapy are associated with survival outcomes in patients with advanced hepatocellular carcinoma (HCC) treated with anti-programmed cell death protein 1 (PD-1)based immunotherapy. METHODS: This single-institution retrospective analysis included 217 patients with advanced HBV-related HCC treated from 1 June 2018, through 30 December 2020. Baseline information was compared between patients with low and high HBV DNA levels. Overall survival (OS) and progression-free survival (PFS) were compared, and univariate and multivariate analyses were applied to identify potential risk factors for oncologic outcomes. RESULTS: The 217 patients included in the analysis had a median survival time of 20.6 months. Of these HBV-associated HCC patients, 165 had known baseline HBV DNA levels. Baseline HBV DNA level was not significantly associated with OS (P = 0.59) or PFS (P = 0.098). Compared to patients who did not receive antiviral therapy, patients who received antiviral therapy had significantly better OS (20.6 vs 11.1 months, P = 0.020), regardless of HBV DNA levels. Moreover, antiviral status (adjusted HR = 0.24, 95% CI 0.094-0.63, P = 0.004) was an independent protective factor for OS in a multivariate analysis of patients with HBV-related HCC. CONCLUSIONS: HBV viral load does not compromise the clinical outcome of patients with HBV-related HCC treated with anti-PD-1-based immunotherapy. The use of antiviral therapy significantly improves survival time of HBV-related HCC patients.

Sex Differences in Genomic Features of Hepatitis B-Associated Hepatocellular Carcinoma With Distinct Antitumor Immunity.

BACKGROUND & AIMS: Aflatoxin exposure increases the risk for hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected individuals, particularly males. We investigated sex-based differences in the HCC genome and antitumor immunity. METHODS: Whole-genome, whole-exome, and RNA sequencing were performed on 101 HCC patient samples (47 males, 54 females) that resulted from HBV infection and aflatoxin exposure from Qidong. Androgen on the expression of aflatoxin metabolism-related genes and nonhomologous DNA end joining (NHEJ) factors were examined in HBV-positive HCC cell lines, and further tested in tumor-bearing syngeneic mice. RESULTS: Qidong HCC differed between males and females in genomic landscape and transcriptional dysfunction pathways. Compared with females, males expressed higher levels of aflatoxin metabolism-related genes, such as AHR and CYP1A1, and lower levels of NHEJ factors, such as XRCC4, LIG4, and MRE11, showed a signature of up-regulated type I interferon signaling/response and repressed antitumor immunity. Treatment with AFB1 in HBV-positive cells, the addition of 2 nmol/L testosterone to cultures significantly increased the expression of aflatoxin metabolism-related genes, but reduced NHEJ factors, resulting in more nuclear DNA leakage into cytosol to activate cGAS-STING. In syngeneic tumor-bearing mice that were administrated tamoxifen daily via oral gavage, favorable androgen signaling repressed NHEJ factor expression and activated cGAS-STING in tumors, increasing T-cell infiltration and improving anti-programmed cell death protein 1 treatment effect. CONCLUSIONS: Androgen signaling in the context of genotoxic stress repressed DNA damage repair. The alteration caused more nuclear DNA leakage into cytosol to activate the cGAS-STING pathway, which increased T-cell infiltration into tumor mass and improved anti-programmed cell death protein 1 immunotherapy in HCCs.

Comparison of hepatitis B virus reactivation in hepatocellular carcinoma patients who received tyrosine kinase inhibitor alone or together with programmed cell death protein-1 inhibitors.

BACKGROUND AND AIMS: Programmed cell death protein-1 (PD-1) inhibitors plus tyrosine kinase inhibitor (TKI) have dramatically improved survival of patients with advanced hepatocellular carcinoma (HCC). However, the risk of hepatitis B virus (HBV) reactivation from these antitumor medications remains unclear. METHODS: Patients receiving TKI monotherapy (TKI group) or TKI combined with PD-1 inhibitors (combination group) were included. The primary endpoint was HBV reactivation as defined by an increase in HBV DNA titer by at least 1 log (tenfold) from baseline. The secondary endpoints included tumor progression and overall survival. RESULTS: Four hundred and ninety-nine patients met the inclusion criteria, including 296 patients in the TKI group and 203 patients in the combination group. The 3-, 6- and 12-month cumulative incidence rates of HBV reactivation in the TKI group vs. combination group were 7.8%, 12.8% and 21.3% vs. 9.9%, 19.2% and 30.0%, respectively (p = 0.02). The Cox proportional hazard model indicated that combination therapy (HR 1.41, 95% CI 1.00-1.99, p = 0.05), ALT > 40 U/ml (HR 1.50, 95% CI 1.05-2.16, p = 0.03), and tumor size > 5 cm (HR 1.58, 95% CI 1.10-2.28, p = 0.01) were independent risk factors for HBV reactivation. Compared with the HBV reactivation group, the progression-free survival and overall survival of patients in the HBV non-reactivation group were significantly prolonged (p < 0.001 and p = 0.001). CONCLUSIONS: Patients who received TKI combined with PD-1 inhibitors had a greater risk for HBV reactivation, and those with HBV reactivation had a higher rate of tumor progression and shorter survival time, than those receiving TKI alone.