HBV integrations reshaping genomic structures promote hepatocellular carcinoma.

OBJECTIVE: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further. DESIGN: Here, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice. RESULTS: These HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials. CONCLUSION: HBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient's life.

Ocean acidification alters shellfish-algae nutritional value and delivery.

The ecological effects of climate change and ocean acidification (OA) have been extensively studied. Various microalgae are ecologically important in the overall pelagic food web as key contributors to oceanic primary productivity. Additionally, no organism exists in isolation in a complex environment, and shifts in food quality may lead to indirect OA effects on consumers. This study aims to investigate the potential effects of OA on algal trophic composition and subsequent bivalve growth. Here, the growth and nutrient fractions of Chlorella sp., Phaeodactylum tricornutum and Chaetocetos muelleri were used to synthesize and assess the impact of OA on primary productivity. Total protein content, total phenolic compounds, and amino acid (AA) and fatty acid (FA) content were evaluated as nutritional indicators. The results demonstrated that the three microalgae responded positively to OA in the future environment, significantly enhancing growth performance and nutritional value as a food source. Additionally, certain macromolecular fractions found in consumers are closely linked to their dietary sources, such as phenylalanine, C14:0, C16:0, C16:1, C20:1n9, C18:0, and C18:3n. Our findings illustrate that OA affects a wide range of crucial primary producers in the oceans, which can disrupt nutrient delivery and have profound impacts on the entire marine ecosystem and human food health.

Cancer screening in China: a steep road from evidence to implementation.

Cancer screening has the potential to decrease mortality from several common cancer types. The first cancer screening programme in China was initiated in 1958 and the Cancer High Incidence Fields established in the 1970s have provided an extensive source of information for national cancer screening programmes. From 2012 onwards, four ongoing national cancer screening programmes have targeted eight cancer types: cervical, breast, colorectal, lung, oesophageal, stomach, liver, and nasopharyngeal cancers. By synthesising evidence from pilot screening programmes and population-based studies for various screening tests, China has developed a series of cancer screening guidelines. Nevertheless, challenges remain for the implementation of a fully successful population-based programme. The aim of this Review is to highlight the key milestones and the current status of cancer screening in China, describe what has been achieved to date, and identify the barriers in transitioning from evidence to implementation. We also make a set of implementation recommendations on the basis of the Chinese experience, which might be useful in the establishment of cancer screening programmes in other countries.

The clinical application value of gonadotropin-releasing hormone antagonist combined with low-dose HCG regimen in patients with ovarian hyper-stimulation based on clinical characteristics and laboratory indicators.

OBJECTIVE: To explore the clinical application value of gonadotropin-releasing hormone antagonist (GnRH-A) combined with low-dose HCG regimen in patients with high ovarian response based on clinical characteristics and laboratory indicators. METHODS: The clinical data of 305 patients who received IVF/ICSI in the Hechi People's Hospital Reproductive Medicine Center from March 2018 to December 2021 were retrospectively included, and all patients were treated with GnRH-A combined with low-dose HCG regimen protocol. The patients were separated into an ovarian hyper-response group and a normal ovarian reaction group according to their ovarian reactivity. Risk factors for ovarian hyper-response in IVF/ICSI patients were screened by univariate and multivariate logistic analysis. The ROC curve area was used to evaluate the prediction effect. RESULTS: Of the 305 patients, 6 (1.97%) had poor ovarian reaction, 123 (40.33%) had ovarian hyper response, and 176 (57.70%) had normal ovarian reaction. The proportion of ovarian hyper response and normal ovarian reaction was 98.03% (299/305); the basic serum FSH level, AMH level, E2 on HCG level on HCG injection day and the incidence of moderate to severe OHSS in the Ovarian hyper-response group were compared with those in the normal ovarian reaction group (P < 0.05). Logistic reversion analysis showed that AMH (OR = 1.246, 95% CI = 1.107-1.402), E2 level on HCG injection day (OR = 1.050, 95% CI = 1.028-1.072) and P level on HCG injection day (OR = 5.831, 95% CI = 1.231-27.616) were factors for ovarian hyper response. Basal serum FSH (OR = 0.781, 95% CI = 0.647-0.94) and LH level on HCG injection day (OR = 0.594, 95% CI = 0.405-0.871) were negatively correlated with the occurrence of high response (P < 0.05). ROC curve analysis showed that AMH (AUC = 0.779), E2 level on HCG injection day (AUC = 0.802), P level on HCG injection day (AUC = 0.636), combined detection (AUC = 0.843), AUC > 0.5. Among them, the prediction effect of joint detection is better. CONCLUSION: GnRH-A combined with low-dose HCG regimen is feasible for patients with ovarian hyper-response during IVF-ET/ICSI, and does not affect the implantation rate, clinical pregnancy rate, live birth rate, and early abortion rate of such patients. Combined detection of basal serum FSH, AMH, LH, E2 and P levels on HCG injection day can effectively predict the occurrence of ovarian hyper-response.

Performance and effectiveness of hepatocellular carcinoma screening in individuals with HBsAg seropositivity in China: a multicenter prospective study.

Current guidelines recommend hepatocellular carcinoma (HCC) surveillance for at-risk individuals, including individuals with hepatitis B virus infection. However, the performance and survival benefits of annual screening have not been evaluated through multicenter prospective studies in a Chinese population. Between 2017 and 2021, we included 14,426 participants with hepatitis B surface antigen seropositivity in an annual HCC screening study in China using a multicenter prospective design with ultrasonography and serum alpha-fetoprotein. After four rounds of screening and follow-up, the adjusted hazard ratios of death after correction for lead-time and length-time biases for screen-detected cancers at the prevalent and incident rounds were 0.74 (95% confidence interval = 0.60-0.91) and 0.52 (95% confidence interval = 0.40-0.68), respectively. A meta-analysis demonstrated that HCC screening was associated with improved survival after adjusting for lead-time bias. Our findings highlight the 'real-world' feasibility and effectiveness of annual HCC screening in community settings for the early detection of HCC and to improve survival.

Abnormal generation of IL-17A represses tumor infiltration of stem-like exhausted CD8+ T cells to demote the antitumor immunity.

BACKGROUND: Variated anti-cancer therapies are combined with immune checkpoint blockades (ICBs) for improving ICB therapeutic efficacy. Occurrence of tissue damage is common that triggers multiple inflammatory cytokine generation. Gastrointestinal organs are the commonly affected. We investigated the impact of acute colitis on tumor infiltration of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) for controlling tumor growth and responding to antibody against PD-1 (anti-PD-1). METHODS: Several tumor cell lines were inoculated into syngeneic mice subcutaneously or intra-hepatically. When tumor mass formed, activated CTLs were intravenously transferred into the tumor-bearing mice, that were given the drinking water containing 2% dextran sulfate sodium (DSS) for acute colitis induction. Tumor growth, infiltration of two exhausted CTL subsets, and the CTL interaction with tumor vascular endothelium were examined. RESULTS: Acute colitis dampened CTL-mediated antitumor effects, correlating with IL-17A elevation in the inflamed intestine. In the tumor bed, stem-like exhausted CTLs, which were defined as PD-1+Slamf6+Tim3-, expressed higher IL-17A receptor heterodimers and lower leukocyte function-associated antigen-1 (LFA-1) than terminally exhausted CTLs did, that were defined as PD-1+Slamf6-Tim3+. IL-17A stimulation reduced LFA-1 surface expression on stem-like exhausted CTLs and the counterpart ICAM-1 (intracellular adhesion molecule-1) on tumor vascular endothelium. IL-17A stimulation suppressed the extravasation across tumor vascular endothelium and self-renewal of stem-like, not the terminally exhausted CTLs. Administration of anti-IL-17A neutralizing antibody to the colitis mice restored the CTL tumor infiltration and enhanced anti-PD-1 treatment efficacy against tumors. In 33 hepatocellular carcinoma patients being treated with anti-PD-1 plus antibody against vascular endothelial growth factor, disease progression of 15 patients, that exhibited serum IL-17A increase 24 h post-therapy as compared to pre-therapy level, was poorer than that of 18 patients that exhibited serum IL-17A no-increase. CONCLUSIONS: Abnormal generation of IL-17A mainly repressed tumor infiltration of stem-like exhausted CTLs. ICB-based immunotherapeutic efficacy could be upgraded with administration of anti-IL-17A, when treatment-related IL-17A elevation occurred due to tissue damage, such as acute colitis.

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.

Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection.

Cell-free DNA (cfDNA)-based liquid biopsy is a promising approach for the early detection of cancer. A major hurdle is the limited yield of cfDNA from one blood draw, limiting the use of most samples to one test of either mutation or methylation. Here, we develop a technology, Mutation Capsule Plus (MCP), which enables multiplex profiling of one cfDNA sample, including simultaneous detection of genetic and epigenetic alterations and genome-wide discovery of methylation markers. With this technology, we performed de novo screening of methylation markers on cfDNA samples from 30 hepatocellular carcinoma (HCC) cases and 30 non-HCC controls. The methylation markers enriched in HCC cfDNA were further profiled in parallel with a panel of mutations on a training cohort of 60 HCC and 60 non-HCC cases, resulting in an HCC detection model. We validated the model in an independent retrospective cohort with 58 HCC and 198 non-HCC cases and got 90% sensitivity with 94% specificity. Furthermore, we applied the model to a prospective cohort of 311 asymptomatic hepatitis B virus carriers with normal liver ultrasonography and serum AFP concentration. The model detected four of the five HCC cases in the cohort, showing 80% sensitivity and 94% specificity. These findings demonstrate that the MCP technology has potential for the discovery and validation of multiomics biomarkers for the noninvasive detection of cancer. This study also provides a comprehensive database of genetic and epigenetic alterations in the cfDNA of a large cohort of HCC cases and high-risk non-HCC individuals.

Occult infection with hepatitis B virus PreS variants synergistically promotes hepatocellular carcinoma development in a high-fat diet context by generating abnormal ceramides.

BACKGROUND: Some occult hepatitis B virus (HBV) infections are resulted from PreS mutations that reduce secretion of envelope protein (HBsAg). We investigated the ceramide amounts and species in hepatocytes infected with PreS variants that were isolated from HBsAg-seronegative patients with hepatocellular carcinoma (HCC) and the ceramide effects on autochthonous HCC development in murine models. METHODS: HBV PreS/S regions from 35 HBsAg-seronegative HCC patients were sequenced. Hepatocyte cell lines and male C57BL/6J mouse livers were transfected with two PreS variant representatives. The ceramides with variated lengths of fatty acyl chains were quantified. Tumour development was examined in the HBV-transfected mice fed different diet types. RESULTS: In HBsAg-seronegative HCC patients, nonneoplastic liver tissues harboured HBsAg and replication-competent HBV. The most frequently detected PreS/S variants carried mutations of altered amino acid properties in HBsAg compared with an isolate from one HBsAg-seronegative HCC patient. Hepatocyte infection with PreS variants caused HBsAg retention within the endoplasmic reticulum and generated more amounts of ceramides with C16:0 ceramide elevated the highest. Saturated fatty acids aggravated the PreS variant-infected hepatocytes to generate abnormal amounts and species of ceramides, which with HBV proteins synergistically activated NLRP3 inflammasome in liver inflammatory macrophages. Liver tumours were only detected in HBV-transfected mice fed high-fat diet, with higher tumour loads in the PreS variant-transfected, associated with abnormal ceramide generation. CONCLUSIONS: HBV PreS mutations which altered amino acid properties of envelope proteins inhibited HBsAg secretion. Hepatocyte infection with PreS variants generated abnormal ceramides which with HBV proteins coactivated NLRP3 inflammasome in liver macrophages to promote autochthonous HCC development.

Epidemiological Characteristics of Primary Liver Cancer in Mainland China From 2003 to 2020: A Representative Multicenter Study.

Background: The contribution of hepatitis B virus (HBV) and hepatitis C virus (HCV) to primary liver cancer (PLC) and their association with cancer aggressiveness remains uncertain in China, a country with half of global PLC. We aimed to characterize this using data from four representative medical centers. Methods: In total, 15,801 PLC patients were enrolled from the centers distributed in Easter5n, Southern, Northern, and Western China from 2003 to 2020. Of those, 7585 with curative surgery were involved in survival analysis. A nomogram was constructed using preoperative parameters to predict postoperative survival. Results: Hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, and combined hepatocellular cholangiocarcinoma accounted for 93.0%, 4.3%, and 1.6% in PLC, respectively. The seropositivities of HBV and HCV were 84.4% and 3.2% in HCC, respectively. The seropositivity of anti-HCV antibody was significantly higher in HBV-negative than in HBV-positive HCC patients (13.2% vs. 1.1%). Compared to HCV-positive HCC (HCV-HCC), HBV-positive HCC (HBV-HCC) was associated with 12-year earlier onset, higher proportions of males, high α-fetoprotein, large tumor size, advanced Barcelona Clinic Liver Cancer (BCLC) stage, and vascular tumor thrombus. The proportions of HCC and HBV seropositivity increased, whereas that of anti-HCV decreased, from 2003 to 2020. Postoperative five-year survival rate was 73.5%, 64.1%, 34.9%, and 19.7% in HCC at BCLC stage 0, A, B, and C, respectively. The multivariate Cox regression analysis showed that HBV seropositivity, incomplete tumor capsule, vascular tumor thrombus, tumor diameter (≥3 cm), advanced BCLC stage (B+C), α-fetoprotein (≥20ng/ml), and direct bilirubin (>8µmol/L) contributed independently to shorter overall survival (OS); whereas post-operative radiofrequency ablation and second resection independently improved OS in HCC. HCV-HCC had a more favorable prognosis than did HBV-HCC (Log-rank test, P<0.001). A nomogram composed of age, gender, and the preoperative independent risk factors was accurate in predicting postoperative survival in HCC (C-index: 0.735; 95% confidence interval: 0.727-0.743). Conclusion: HBV contributes to 84.4% of HCC in China, and actively promotes hepatocarcinogenesis and HCC progression. A favorable postoperative survival obtained in patients at the early BCLC stage highlights the importance of screening for early HCC in high-risk populations. Our preoperative prognosis prediction model is important in clinical decision-making.

The population-level economic burden of liver cancer in China, 2019-2030: prevalence-based estimations from a societal perspective.

BACKGROUND: Benchmark data on the population-level economic burden are critical to inform policymakers about liver cancer control. However, comprehensive data in China are currently limited. METHODS: A prevalence-based approach from a societal perspective was used to quantify the annual economic burden of liver cancer in China from 2019 to 2030. Detailed per-case data on medical/non-medical expenditure and work-loss days were extracted from a multicenter survey. The numbers/rates of new/prevalent cases and deaths, survival, and population-related parameters were extracted from the Global Burden of Disease 2019 and the literature. All expenditure data were reported in both 2019 Chinese Yuan (CNY) and United States dollar (US$, for main estimations). RESULT: The overall economic burden of liver cancer was estimated at CNY76.7/US$11.1 billion in China in 2019 (0.047% of the local GDP). The direct expenditure was CNY21.6/US$3.1 billion, including CNY19.7/US$2.9 billion for medical expenditure and CNY1.9/US$0.3 billion for non-medical expenditure. The indirect cost was CNY55.1/US$8.0 billion (71.8% of the overall burden), including CNY3.0/US$0.4 billion due to disability and CNY52.0/US$7.5 billion due to premature death. The total burden would increase to CNY84.2/US$12.2 billion, CNY141.7/US$20.5 billion, and CNY234.3/US$34.0 billion in 2020, 2025, and 2030, accounting for 0.102%, 0.138%, and 0.192% of China's GDP, respectively. However, if China achieves the goals of Healthy China 2030 or the United Nations' Sustainable Development Goals for non-communicable diseases, the burden in 2030 would be < CNY144.4/US$20.9 billion. CONCLUSIONS: The population-level economic burden of liver cancer in China is currently substantial and will consistently increase in the future. Sustainable efforts in primary and secondary interventions for liver cancer need to be further strengthened.

Phytotoxicity of environmental norfloxacin concentrations on the aquatic plant Spirodela polyrrhiza: Evaluation of growth parameters, photosynthetic toxicity and biochemical traits.

As an emerging pollutant, the increasing use of antibiotics in wastewater posed a serious threat to non-target organisms in the environment. Duckweed (Spirodela polyrrhiza) is a common higher aquatic plant broadly used in phytotoxicity tests for xenobiotic substances. The aim of this study was to evaluate the chronic toxicity of norfloxacin (NOR) on Spirodela polyrrhiza during 18 days of exposure. Our study investigated the addition of NOR into the medium with environment-related concentrations (0, 0.1, 10, and 1000 µg L-1). Subsequently, biomarkers of toxicity such as growth, pigment, chlorophyll fluorescence parameters, indicators of oxidative stress, and osmotic regulatory substances content were analyzed in duckweed. In response to NOR exposure, obvious chlorosis, declines in growth and photosynthetic pigment, and photosystem II inhibition were noted in a concentration dependent manner. Reactive oxygen species (ROS) and antioxidant activity content increased in the treated fronds, which indicated that oxidative stress was specifically affected by NOR exposure. A slight increase in osmotic regulatory substances in NOR treated setups than in the control represented the increasing stress resistance. These results suggest NOR exerts its toxic effects on the aquatic plant Spirodela polyrrhiza.

Bi-specific T1 positive-contrast-enhanced magnetic resonance imaging molecular probe for hepatocellular carcinoma in an orthotopic mouse model.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. HCC-targeted magnetic resonance imaging (MRI) is an effective noninvasive diagnostic method that involves targeting clinically-related HCC biomarkers, such as alpha-fetoprotein (AFP) or glypican-3 (GPC3), with iron oxide nanoparticles. However, in vivo studies of HCC-targeted MRI utilize single-target iron oxide nanoprobes as negative (T2) contrast agents, which might weaken their future clinical applications due to tumor heterogeneity and negative MRI contrast. Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles (approximately 5 nm) are potential optimal positive (T1) contrast agents. We previously verified the efficiency of AFP/GPC3-double-antibody-labeled iron oxide MR molecular probe in vitro. AIM: To validate the effectiveness of a bi-specific probe in vivo for enhancing T1-weighted positive contrast to diagnose the early-stage HCC. METHODS: The single- and double-antibody-conjugated 5-nm USPIO probes, including anti-AFP-USPIO (UA), anti-GPC3-USPIO (UG), and anti-AFP-USPIO-anti-GPC3 (UAG), were synthesized. T1- and T2-weighted MRI were performed on day 10 after establishment of the orthotopic HCC mouse model. Following intravenous injection of U, UA, UG, and UAG probes, T1- and T2-weighted images were obtained at 12, 12, and 32 h post-injection. At the end of scanning, mice were euthanized, and a histologic analysis was performed on tumor samples. RESULTS: T1- and T2-weighted MRI showed that absolute tumor-to-background ratios in UAG-treated HCC mice peaked at 24 h post-injection, with the T1- and T2-weighted signals increasing by 46.7% and decreasing by 11.1%, respectively, relative to pre-injection levels. Additionally, T1-weighted contrast in the UAG-treated group at 24 h post-injection was enhanced 1.52-, 2.64-, and 4.38-fold compared to those observed for single-targeted anti-GPC3-USPIO, anti-AFP-USPIO, and non-targeted USPIO probes, respectively. Comparison of U-, UA-, UG-, and UAG-treated tumor sections revealed that UAG-treated mice exhibited increased stained regions compared to those observed in UG- or UA-treated mice. CONCLUSION: The bi-specific T1-positive contrast-enhanced MRI probe (UAG) for HCC demonstrated increased specificity and sensitivity to diagnose early-stage HCC irrespective of tumor size and/or heterogeneity.

Long term outcome of prevention of liver cancer by hepatitis B vaccine: Results from an RCT with 37 years.

We aimed to evaluate the long-term efficacy of the hepatitis B vaccine in China. In an initial efficacy study, participants were collected from a cluster-randomized clinical trial conducted in 1983-90 in Qidong. All the participants in the vaccination group were vaccinated at birth, 1 and 6 months of age, and no intervention was implemented to the control group. In this 37-year extended follow-up study, the Poisson regression method was employed to derive rates per 105 person-years. The frailty Cox proportional hazard regression models obtained the hazard ratio (HR). Cumulative incidence/mortality rates were calculated and compared with log-rank tests. 41,136 in the vaccination and 41,730 in the control group were recorded. The incidence rate of liver cancer was significantly lower in the vaccinated group than in the control group [HR, 0.28; 95% confidence interval (CI) 0.11-0.70, P = 0.007]. The vaccine offers 72% (95% CI, 30-89) protection to prevent the occurrence of liver cancer. There is 70% (95% CI, 23-88) protective efficacy against liver cancer deaths and 64% (95% CI, 27-82) benefits in the prevention of deaths associated with liver diseases. Hepatitis B vaccine given at birth shows excellent protective effects in preventing the development of liver cancer and reducing mortality from liver cancer and liver diseases.

Apoptosis-induced translocation of centromere protein F in its corresponding autoantibody production in hepatocellular carcinoma.

Serum autoantibodies against tumor-associated antigen have important value in the early diagnosis of hepatocellular carcinoma (HCC), but the mechanism of autoantibody production is poorly understood. We previously showed that autoantibodies against the centromere protein F (CENPF) may be useful as an early diagnostic marker for HCC. Here we explored the mechanism of cell apoptosis-based CENPF autoantibody production and verified the correlation of CENPF autoantibody level with HCC development. We demonstrated that CENPF was overexpressed and aberrantly localized throughout the nuclei and cytoplasm in human HCC cells compared with hepatic cells. CENPF overexpression promoted the production of CENPF autoantibodies in a manner that correlated with tumor growth of mouse HCC model. During apoptosis of HCC cells, CENPF protein translocated to apoptotic vesicles and relocalized at the cell surface. Through isolating apoptotic components, we found apoptotic body and blebs with lower CD31 and CD47 expression more effectively induced DC phagocytosis and maturation compared with apoptotic intact cells in vitro, and this DC response was independent of CENPF expression. Moreover, injection of mice with apoptotic bodies and blebs effectively induced an immune response and the production of CENPF-specific antibodies. Our findings provide a first elucidation of mechanisms underlying the CENPF autoantibody production via cell apoptosis-induced CENPF translocation, and demonstrate a direct correlation between CENPF autoantibody levels and HCC progression, suggesting the potential of CENPF autoantibody as an HCC diagnostic marker.

Booster immunization improves the generation of T follicular helper (Tfh) cells specific to hepatitis B surface antigen (HBsAg) after prenatal HBsAg exposure.

Breakthrough infections of hepatitis B virus (HBV) after neonatal vaccination occurred in some adolescents and young adults who were born to mothers with hepatitis B surface antigen (HBsAg). We aimed to determine the impacts of prenatal HBsAg exposure on the generation of T follicular helper (Tfh) cells and antibodies (anti-HBs) specific to HBsAg. To mimic human prenatal HBsAg exposure, we mated female Alb1-HBV (HBV-M) mice with male C57BL/6J mice. Of their first filial generation (F1), HBV-M/F1+ expressed HBsAg in liver tissues and blood, and HBV-M/F1- mice exposed HBsAg in amniotic fluid. At their four weeks old, each HBV-M/F1 mouse was immunized with hepatitis B vaccine containing 5 µg HBsAg subcutaneously. Both HBV-M/F1- and HBV-M/F1+ mice had reduced generation of HBsAg-specific CD4+CXCR5+PD1+ Tfh cells and CD138+IgD- plasma cells in comparison with C57BL/6J mice. Results of coculturing the Tfh cells with B cells that were isolated from different strains of mice indicated that CD4+ T cell activation in response to HBsAg was critical for anti-HBs generation after prenatal HBsAg exposure. When interleukin (IL) 21 was supplemented, the generation of HBsAg-specific Tfh and plasma cells in HBV-M/F1- mice was improved, while supplementation showed little effect in HBV-M/F1+ mice. In HBV-M/F1- mice, HBV vaccine booster improved the generation of Tfh cells and plasma cells, and enhanced anti-HBs production. CONCLUSION: Impaired generation of HBsAg-specific Tfh cells and plasma cells after prenatal HBsAg exposure can be improved by HBV vaccine booster, most likely increasing IL-21 production.

AHR mediates the aflatoxin B1 toxicity associated with hepatocellular carcinoma.

Aflatoxin exposure is a crucial factor in promoting the development of primary hepatocellular carcinoma (HCC) in individuals infected with the hepatitis virus. However, the molecular pathways leading to its bioactivation and subsequent toxicity in hepatocytes have not been well-defined. Here, we carried out a genome-wide CRISPR-Cas9 genetic screen to identify aflatoxin B1 (AFB1) targets. Among the most significant hits was the aryl hydrocarbon receptor (AHR), a ligand-binding transcription factor regulating cell metabolism, differentiation, and immunity. AHR-deficient cells tolerated high concentrations of AFB1, in which AFB1 adduct formation was significantly decreased. AFB1 triggered AHR nuclear translocation by directly binding to its N-terminus. Furthermore, AHR mediated the expression of P450 induced by AFB1. AHR expression was also elevated in primary tumor sections obtained from AFB1-HCC patients, which paralleled the upregulation of PD-L1, a clinically relevant immune regulator. Finally, anti-PD-L1 therapy exhibited greater efficacy in HCC xenografts derived from cells with ectopic expression of AHR. These results demonstrated that AHR was required for the AFB1 toxicity associated with HCC, and implicate the immunosuppressive regimen of anti-PD-L1 as a therapeutic option for the treatment of AFB1-associated HCCs.

A male-ABCD algorithm for hepatocellular carcinoma risk prediction in HBsAg carriers.

OBJECTIVE: Hepatocellular carcinoma (HCC) development among hepatitis B surface antigen (HBsAg) carriers shows gender disparity, influenced by underlying liver diseases that display variations in laboratory tests. We aimed to construct a risk-stratified HCC prediction model for HBsAg-positive male adults. METHODS: HBsAg-positive males of 35-69 years old (N=6,153) were included from a multi-center population-based liver cancer screening study. Randomly, three centers were set as training, the other three centers as validation. Within 2 years since initiation, we administrated at least two rounds of HCC screening using B-ultrasonography and α-fetoprotein (AFP). We used logistic regression models to determine potential risk factors, built and examined the operating characteristics of a point-based algorithm for HCC risk prediction. RESULTS: With 2 years of follow-up, 302 HCC cases were diagnosed. A male-ABCD algorithm was constructed including participant's age, blood levels of GGT (γ-glutamyl-transpeptidase), counts of platelets, white cells, concentration of DCP (des-γ-carboxy-prothrombin) and AFP, with scores ranging from 0 to 18.3. The area under receiver operating characteristic was 0.91 (0.90-0.93), larger than existing models. At 1.5 points of risk score, 26.10% of the participants in training cohort and 14.94% in validation cohort were recognized at low risk, with sensitivity of identifying HCC remained 100%. At 2.5 points, 46.51% of the participants in training cohort and 33.68% in validation cohort were recognized at low risk with 99.06% and 97.78% of sensitivity, respectively. At 4.5 points, only 20.86% of participants in training cohort and 23.73% in validation cohort were recognized at high risk, with positive prediction value of 22.85% and 12.35%, respectively. CONCLUSIONS: Male-ABCD algorithm identified individual's risk for HCC occurrence within short term for their HCC precision surveillance.