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Daniel Geh,1 Quentin M Anstee,2,3 Helen L Reeves2– 4 1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK; 2Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK; 3The Liver Unit, Newcastle Upon Tyne Hospitals NHS Foundation Trust, Freeman Hospital, Newcastle upon Tyne, UK; 4Hepatopancreatobiliary Multidisciplinary Team, Newcastle Upon Tyne Hospitals NHS Foundation Trust, Freeman Hospital, Newcastle upon Tyne, UKCorrespondence: Helen L ReevesTranslational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UKTel +44 191 208 4423Fax +44 191 208 4301Email h.l.reeves@ncl.ac.ukAbstract: Due to an increase in the obesity-associated metabolic syndrome of epidemic proportions, nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of hepatocellular carcinoma (HCC) in western countries. This presents added challenges, as NAFLD-associated HCC tends to present at an advanced stage in older patients with co-morbidities. Their prognosis is generally poor with the benefits of standard therapies less certain. The pathogenesis of NAFLD-associated HCC is multifactorial and not well understood, although the risk of HCC developing undoubtedly increases as NAFLD progresses to steatohepatitis and cirrhosis. Recent advances in our understanding of the drivers of NAFLD and HCC will hopefully lead to the development of clinically relevant biomarkers, tools and strategies to aid the identification of high-risk patients, inform preventive measures, and introduction of better tolerated targeted therapies. Lifestyle modification and chemoprevention with drugs such as anti-platelets, statins and anti-diabetics are being evaluated for HCC prevention. The landmark IMBrave150 study introducing the combination of atezolizumab and bevacizumab has recently transformed the landscape of systemic therapies in HCC, with follow-up analyses and real-world data for patients with NAFLD-associated HCC eagerly anticipated. While responses may vary in ways not yet appreciated, the rate of discovery and progress suggests imminent change and opportunities.Keywords: hepatocellular carcinoma, HCC, nonalcoholic fatty liver disease, NAFLD, liver cirrhosis, biomarkers, prevention, systemic therapy, immunotherapy

Munirah Alsaleh,1 Thomas A Barbera,1 Helen L Reeves,2 Matthew E Cramp,3 Stephen Ryder,4,5 Hani Gabra,1,6 Kathryn Nash,7 Yi-Liang Shen,1,8 Elaine Holmes,1 Roger Williams,9 Simon D Taylor-Robinson11Division of Surgery and Cancer, Imperial College London, London; 2Northern Institute for Cancer Research, Medical School, University of Newcastle, Newcastle upon Tyne, UK; 3Liver Unit, Derriford Hospital, Plymouth, Devon , UK; 4Nottingham Digestive Diseases Centre, University of Nottingham, Nottingham, UK; 5NIHR Biomedical Research Unit, Nottingham University Hospitals NHS Trust, Queen’s Medical Centre, Nottingham, UK; 6Early Clinical Development, IMED Biotech Unit, AstraZeneca, Cambridge, UK; 7Liver Unit, Southampton General Hospital, Southampton, Hampshire, UK; 8Department of Radiation Oncology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan; 9Institute of Hepatology, UKBackground: Outside South-East Asia, most cases of cholangiocarcinoma (CCA) have an obscure etiology. There is often diagnostic uncertainty. Metabolomics using ultraperformance liquid chromatography mass spectrometry (UPLC-MS) offers the portent to distinguish disease-specific metabolic signatures. We aimed to define such a urinary metabolic signature in a patient cohort with sporadic CCA and investigate whether there were characteristic differences from those in patients with hepatocellular carcinoma (HCC), metastatic secondary liver cancer, pancreatic cancer and ovarian cancer (OCA).Methods: Spot urine specimens were obtained from 211 subjects in seven participating centers across the UK. Samples were collected from healthy controls and from patients with benign hepatic disease (gallstone, biliary strictures, sphincter of Oddi dysfunction and viral hepatitis) and patients with malignant conditions (HCC, pancreatic cancer, OCA and metastatic cancer in the liver). The spectral metabolite profiles were generated using a UPLC-MS detector and data were analyzed using multivariate and univariate statistical analyses.Results: The greatest class differences were seen between the metabolic profiles of disease-free controls compared to individuals with CCA with altered acylcarnitine, bile acid and purine levels. Individuals with benign strictures showed comparable urine profiles to patients with malignant bile duct lesions. The metabolic signatures of patients with bile duct tumors were distinguishable from patients with hepatocellular and ovarian tumors, but no difference was observed between CCA cases and patients with pancreatic cancer or hepatic secondary metastases.Conclusion: CCA causes subtle but detectable changes in the urine metabolic profiles. The findings point toward potential applications of metabonomics in early tumor detection. However, it is key to utilize both global and targeted metabonomics in a larger cohort for in-depth characterization of the urine metabolome in hepato-pancreato-biliary disease.Keywords: cholangiocarcinoma, metabolomics, diagnostic biomarkers

Daniel Geh,1,* Fahd A Rana,2,* Helen L Reeves1,3 1Northern Institute for Cancer Research, Newcastle University Medical School, Newcastle upon Tyne, UK; 2The Liver Unit, Freeman Hospital, Newcastle upon Tyne, UK; 3The Hepatopancreatobiliary Multidisciplinary Team, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK *These authors contributed equally to this work Abstract: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and usually occurs in people with liver cirrhosis. Both the incidence and mortality of HCC are increasing worldwide, making it a growing public health issue. HCC diagnosed at an early stage has a far better prognosis than HCC diagnosed at a late stage, mainly because early stage HCC can be treated with potentially curative therapies such as resection and transplantation. This makes surveillance for HCC in patients with liver cirrhosis an important strategy in improving outcomes. Serial measurements of serum alpha fetoprotein (AFP) and abdominal ultrasound (US) are the established methods of surveillance. Surveillance using a combination of these techniques has reasonable sensitivity and specificity and reduces mortality from HCC by varying degrees, depending on the patient population. However, there are potential harms. The main harms result from false-positive and false-negative results. False-positive results commit patients to undergo further, potentially invasive and ultimately unnecessary diagnostic testing – which has both financial and emotional costs. False-negative results can have devastating consequences for patients who later present with more advanced HCC. Obesity is increasingly prevalent and reduces the sensitivity of US in detecting HCC. Obesity-associated non-alcoholic fatty liver disease (NAFLD) presents an additional challenge, where HCC can develop in the absence of cirrhosis. As surveillance with US and AFP is not cost-effective in NAFLD without cirrhosis, it is not advocated. These aspects will be reviewed. Keywords: hepatocellular carcinoma, liver cirrhosis, surveillance, NAFLD, screening

Drug-induced liver injury (DILI) is a significant cause of acute liver failure (ALF) and liver transplantation in the Western world. Acetaminophen (APAP) overdose is a main contributor of DILI, leading to hepatocyte cell death through necrosis. Here, we identified that neddylation, an essential post-translational modification involved in the mitochondria function, was upregulated in liver biopsies from patients with APAP-induced liver injury (AILI) and in mice treated with an APAP overdose. MLN4924, an inhibitor of the neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8)-activating enzyme (NAE-1), ameliorated necrosis and boosted liver regeneration in AILI. To understand how neddylation interferes in AILI, whole-body biotinylated NEDD8 ( bio NEDD8) and ubiquitin ( bio UB) transgenic mice were investigated under APAP overdose with and without MLN4924. The cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41, responsible for producing cardiolipin essential for mitochondrial activity, was found modulated under AILI and restored its levels by inhibiting neddylation. Understanding this ubiquitin-like crosstalk in AILI is essential for developing promising targeted inhibitors for DILI treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogen bonding is a crucial feature of biomolecules, but its characterization in glycans dissolved in aqueous solutions is challenging due to rapid hydrogen exchange between hydroxyl groups and H 2 O. In principle, the scalar (J) coupling constant can reveal the relative orientation of the atoms in the molecule. In contrast to J-coupling through H-bonds reported in proteins and nucleic acids, research on J-coupling through H-bonds in glycans dissolved in water is lacking. Here, we use sucrose as a model system for H-bonding studies; its structure, which consists of glucose (Glc) and fructose (Frc), is well-studied, and it is readily available. We apply the in-phase, antiphase-HSQC-TOCSY and quantify previously unreported through H-bond J-values for Frc-OH1-Glc-OH2 in H 2 O. While earlier reports of Brown and Levy indicate this H-bond as having only a single direction, our reported findings indicate the potential presence of two involving these same atoms, namely, G2OH ➔ F1O and F1OH ➔ G2O (where F and G stand for Frc and Glc, respectively). The calculated density functional theory J-values for the G2OH ➔ F1O agree with the experimental values. Additionally, we detected four other possible H-bonds in sucrose, which require different phi, psi (ϕ, ψ) torsion angles. The ϕ, ψ values are consistent with previous predictions of du Penhoat et al. and Venable et al. Our results will provide new insights into the molecular structure of sucrose and its interactions with proteins., (© 2024 The Author(s). Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Objectives: The study aimed to assess the feasibility, acceptability and safety of delivering a home-based telehealth exercise intervention to older patients with hepatocellular carcinoma (HCC)., Design: Non-randomised feasibility study., Setting: Patients were recruited from UK outpatient liver cancer clinics., Participants: Patients were aged ≥60 years with HCC, with post-treatment imaging reporting a complete response, partial response or stable disease., Intervention and Data Collection: Patients were invited to attend synchronous online exercise sessions, twice weekly for 10 weeks. Physical function and patient-reported outcomes were assessed pre-intervention and post-intervention. Qualitative data were collected via semistructured interviews after intervention completion., Primary Outcome Measures: Recruitment, retention, exercise adherence and safety., Results: 40 patients were invited to participate and 19 (mean age 74 years) provided consent (recruitment rate 48%). Patients completed 76% of planned exercise sessions and 79% returned to the clinic for follow-up. Hand grip strength (95% CI 1.0 to 5.6), Liver Frailty Index (95% CI -0.46 to -0.23) and time taken to perform five sit-to-stands (95% CI -3.2 to -1.2) improved from pre-intervention to post-intervention. Patients reported that concerns they had relating to their cancer had improved following the intervention (95% CI 0.30 to 5.85). No adverse events occurred during exercise sessions.Qualitative data highlighted the importance of an instructor in real time to ensure that the sessions were achievable, tailored and well balanced, which helped to foster motivation and commitment within the group. Patients reported enjoying the exercise intervention, including the benefits of peer support and highlighted perceived benefits to both their physical and mental health. Patients felt that the online sessions overcame some of the barriers to exercise participation and preferred attending virtual sessions over face-to-face classes., Conclusions: It is feasible, acceptable and safe to deliver supervised group exercise via videoconferencing to patients with HCC in their own homes. These findings will inform the design of a future, adequately powered randomised controlled trial to evaluate the efficacy of the intervention., Trial Registration Number: ISRCTN14411809., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global epidemic and is the most rapidly rising cause of HCC. Clonal hematopoiesis of indeterminate potential (CHIP) contributes to neoplastic and cardiometabolic disorders and is considered a harbinger of tissue inflammation. CHIP was recently associated with increased risk of liver disease. The aim of this study was to examine whether CHIP is associated with HCC development in patients with SLD., Approach and Results: We considered individuals with MASLD-HCC (n=208) and controls with (n =414) and without (n =259) advanced fibrosis who underwent whole exome sequencing. CHIP was diagnosed when ≥2 variant callers identified a known myeloid mutation with variant allele frequency ≥2%. CHIP was observed in 116 participants (13.1%), most frequently in DNMT3A, TET2, TP53 , and ASXL1 , and correlated with age ( p <0.0001) and advanced liver fibrosis (p=0.001). Higher aspartate aminotransferase levels predicted non- DNMT3A -CHIP, in particular with variant allele frequency ≥10% (OR: 1.14, 1.03 -1.28 and OR: 1.30, 1.12 -1.49, respectively, p <0.05). After adjustment for sex, diabetes, and a polygenic risk, a score of inherited MASLD predisposition CHIP was associated with cirrhosis (2.00, 1.30 -3.15, p =0.02), and with HCC even after further adjustment for cirrhosis (OR: 1.81, 1.11 -2.00, 1.30 -3.15, p =0.002). Despite the strong collinearity among aging and development of CHIP and HCC, non- DNTM3A -CHIP, and TET2 lesions remained associated with HCC after full correction for clinical/genetics covariates and age (OR: 2.45, 1.35 -4.53; OR: 4.8, 1.60 -17.0, p =0.02)., Conclusions: We observed an independent association between CHIP, particularly related to non- DNTM3A and TET2 genetic lesions and MASLD-HCC., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background and Aims: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC., Approach and Results: Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone., Conclusion: Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Introduction: The COVID-19 pandemic has caused severe disruption of healthcare services worldwide and interrupted patients' access to essential services. During the first lockdown, many healthcare services were shut to all but emergencies. In this study, we aimed to determine the immediate and long-term indirect impact of COVID-19 health services utilisation on hepatocellular cancer (HCC) outcomes., Methods: A prospective cohort study was conducted from 1 March 2020 until 30 June 2020, correlating to the first wave of the COVID-19 pandemic. Patients were enrolled from tertiary hospitals in the UK and Germany with dedicated HCC management services. All patients with current or past HCC who were discussed at a multidisciplinary meeting (MDM) were identified. Any delay to treatment (DTT) and the effect on survival at one year were reported., Results: The median time to receipt of therapy following MDM discussion was 49 days. Patients with Barcelona Clinic Liver Cancer (BCLC) stages-A/B disease were more likely to experience DTT. Significant delays across all treatments for HCC were observed, but delay was most marked for those undergoing curative therapies. Even though severe delays were observed in curative HCC treatments, this did not translate into reduced survival in patients., Conclusion: Interruption of routine healthcare services because of the COVID-19 pandemic caused severe delays in HCC treatment. However, DTT did not translate to reduced survival. Longer follow is important given the delay in therapy in those receiving curative therapy.

Objectives: The Glucokinase Regulatory Protein GKRP, encoded by GCKR, enables acute regulation of liver glucokinase to support metabolic demand. The common human GCKR rs1260326:Pro446 > Leu variant within a large linkage disequilibrium region associates with pleiotropic traits including lower Type 2 diabetes risk and raised blood triglycerides and cholesterol. Whether the GCKR-P446 > L substitution is causal to the raised lipids is unknown. We determined whether mouse GKRP phenocopies the human GKRP:P446 > L substitution and studied a GKRP:P446L knockin mouse to identify physiological consequences to P446 > L., Methods: GKRP-deficient hepatocytes were transfected with adenoviral vectors for human or mouse GKRP:446 P or 446 L for cellular comprehensive analysis including transcriptomics consequent to P446 > L. Physiological traits in the diet-challenged P446L mouse were compared with pleiotropic associations at the human rs1260326 locus. Transcriptomics was compared in P446L mouse liver with hepatocytes overexpressing glucokinase or GKRP:446 P/L., Results: 1. P446 > L substitution in mouse or human GKRP similarly compromises protein expressivity of GKRP:446 L, nuclear sequestration of glucokinase and counter-regulation of gene expression. 2. The P446L knockin mouse has lower liver glucokinase and GKRP protein similar to human liver homozygous for rs1260326-446 L. 3. The diet-challenged P446L mouse has lower blood glucose, raised blood cholesterol and altered hepatic cholesterol homeostasis consistent with relative glucokinase-to-GKRP excess, but not raised blood triglycerides., Conclusions: Mouse GKRP phenocopies the human GKRP:P446 > L substitution despite the higher affinity for glucokinase of human GKRP. The diet-challenged P446L mouse replicates several traits found in association with the rs1260326 locus on chromosome 2 including raised blood cholesterol, lower blood glucose and lower liver glucokinase and GKRP protein but not raised blood triglycerides., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Hepatocellular carcinoma (HCC), the most common type of liver cancer, has very poor outcomes. Current therapies often have low efficacy and significant toxicities. Thus, there is a critical need for the development of novel therapeutic approaches for HCC. We have developed a novel bioinformatics pipeline, which integrates genome-wide DNA methylation and gene expression data, to identify genes required for the survival of specific molecular cancer subgroups but not normal cells. Targeting these genes may induce cancer-specific "synthetic lethality". Initially, five potential HCC molecular subgroups were identified based on global DNA methylation patterns. Subgroup-2 exhibited the most unique methylation profile and two candidate subtype-specific vulnerability or SL-like genes were identified for this subgroup, including TIAM1, a guanine nucleotide exchange factor encoding gene known to activate Rac1 signalling. siRNA targeting TIAM1 inhibited cell proliferation in TIAM1-positive (subgroup-2) HCC cell lines but had no effect on the normal hepatocyte HHL5 cell line. Furthermore, TIAM1 -positive/subgroup-2 cell lines were significantly more sensitive to the TIAM1/RAC1 inhibitor NSC23766 compared with TIAM1 -negative HCC lines or the normal HHL5 cell line. The results are consistent with a synthetic lethal role for TIAM1 in a methylation-defined HCC subgroup and suggest it may be a viable therapeutic target in this subset of HCC patients.

Dihedral angles in organic molecules and biomolecules are vital structural parameters that can be indirectly probed by nuclear magnetic resonance (NMR) measurements of vicinal J-couplings. The empirical relations that map the measured couplings to dihedral angles are typically determined by fitting using static structural models, but this neglects the effects of thermal fluctuations at the finite temperature conditions under which NMR measurements are often taken. In this study, we calculate ensemble-averaged J-couplings for several structurally rigid carbohydrate derivatives using first-principles molecular dynamics simulations to sample the thermally accessible conformations around the minimum energy structure. Our results show that including thermal fluctuation effects significantly shifts the predicted couplings relative to single-point calculations at the energy minima, leading to improved agreement with experiments. This provides evidence that accounting for conformational sampling in first-principles calculations can improve the accuracy of NMR-based structure determination for structurally complex carbohydrates., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury., (© 2022. The Author(s).)

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disorders and has a strong heritable component. The aim of this study was to identify new loci that contribute to severe NAFLD by examining rare variants., Methods: We performed whole-exome sequencing in individuals with NAFLD and advanced fibrosis or hepatocellular carcinoma (n = 301) and examined the enrichment of likely pathogenic rare variants vs. the general population. This was followed by validation at the gene level., Results: In patients with severe NAFLD, we observed an enrichment of the p.P426L variant (rs143545741 C>T; odds ratio [OR] 5.26, 95% CI 2.1-12.6; p = 0.003) of autophagy-related 7 (ATG7), which we characterized as a loss-of-function, vs. the general population, and an enrichment in rare variants affecting the catalytic domain (OR 13.9; 95% CI 1.9-612; p = 0.002). In the UK Biobank cohort, loss-of-function ATG7 variants increased the risk of cirrhosis and hepatocellular carcinoma (OR 3.30; 95% CI 1.1-7.5 and OR 12.30, 95% CI 2.6-36, respectively; p <0.001 for both). The low-frequency loss-of-function p.V471A variant (rs36117895 T>C) was also associated with severe NAFLD in the clinical cohort (OR 1.7; 95% CI 1.2-2.5; p = 0.003), predisposed to hepatocellular ballooning (p = 0.007) evolving to fibrosis in the Liver biopsy cohort (n = 2,268), and was associated with liver injury in the UK Biobank (aspartate aminotransferase levels, p <0.001), with a larger effect in severely obese individuals in whom it was linked to hepatocellular carcinoma (p = 0.009). ATG7 protein localized to periportal hepatocytes, particularly in the presence of ballooning. In the Liver Transcriptomic cohort (n = 125), ATG7 expression correlated with suppression of the TNFα pathway, which was conversely upregulated in p.V471A carriers., Conclusions: We identified rare and low-frequency ATG7 loss-of-function variants that promote NAFLD progression by impairing autophagy and facilitating ballooning and inflammation., Lay Summary: We found that rare mutations in a gene called autophagy-related 7 (ATG7) increase the risk of developing severe liver disease in individuals with dysmetabolism. These mutations cause an alteration in protein function and impairment of self-renewal of cellular content, leading to liver damage and inflammation., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest relevant to the present study. LV has received speaking fees from MSD, Gilead, AlfaSigma and AbbVie, served as a consultant for Gilead, Pfizer, AstraZeneca, Novo Nordisk, Intercept, Diatech Pharmacogenetics and Ionis Pharmaceuticals, and received research grants from Gilead. SR has served as a consultant for AstraZeneca, Celgene, Sanofi, Amgen, Akcea Therapeutics, Camp4, AMbys, Medacorp and Pfizer in the past 5 years, and received research grants from AstraZeneca, Sanofi and Amgen. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Information about the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with liver cancer is lacking. This study characterizes the outcomes and mortality risk in this population., Methods: Multicentre retrospective, cross-sectional, international study of liver cancer patients with SARS-CoV-2 infection registered between February and December 2020. Clinical data at SARS-CoV-2 diagnosis and outcomes were registered., Results: Two hundred fifty patients from 38 centres were included, 218 with hepatocellular carcinoma (HCC) and 32 with intrahepatic cholangiocarcinoma (iCCA). The median age was 66.5 and 64.5 years, and 84.9% and 21.9% had cirrhosis in the HCC and iCCA cohorts respectively. Patients had advanced cancer stage at SARS-CoV-2 diagnosis in 39.0% of the HCC and 71.9% of the iCCA patients. After a median follow-up of 7.20 (IQR: 1.84-11.24) months, 100 (40%) patients have died, 48% of the deaths were SARS-CoV-2-related. Forty (18.4%) HCC patients died within 30-days. The death rate increase was significantly different according to the BCLC stage (6.10% [95% CI 2.24-12.74], 11.76% [95% CI 4.73-22.30], 20.69% [95% CI 11.35-31.96] and 34.52% [95% CI 17.03-52.78] for BCLC 0/A, B, C and D, respectively; p = .0017). The hazard ratio was 1.45 (95% CI 0.49-4.31; p = .5032) in BCLC-B versus 0/A, and 3.13 (95% CI 1.29-7.62; p = .0118) in BCLC-C versus 0/A in the competing risk Cox regression model. Nineteen out of 32 iCCA (59.4%) died, and 12 deaths were related to SARS-CoV-2 infection., Conclusions: This is the largest cohort of liver cancer patients infected with SARS-CoV-2. It characterizes the 30-day mortality risk of SARS-CoV-2 infected patients with HCC during this period., (© 2022 The Authors. Liver International published by John Wiley & Sons Ltd.)

Introduction: Heparin sulphate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signalling, modulated by sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME., Methods: In liver biopsies from 60 patients with HCC, expression and localization of SULF2 were analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids, and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, β-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68, and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cell phenotype using fluorescent activated cell sorting., Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type 2 diabetes, SULF2 was more commonly expressed in cancer-associated fibroblasts (CAFs) (52%) and independently associated with shorter survival (7.2 vs. 29.2 months, p = 0.003). Stromal SULF2 modulated glypican-3/β-catenin signalling in vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFβ1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFβ1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRβ/STAT3 signalling was evident in stromal cells, with the release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In human PBMCs, SULF2 preferentially induced the migration of macrophage precursors (monocytes), inducing a phenotypic change consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2-induced paracrine activation of the IKKβ/NF-κB pathway, tumour cell proliferation, invasion, and sorafenib resistance., Conclusion: SULF2 derived from CAFs modulates glypican-3/β-catenin signalling but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKβ/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC., Competing Interests: F.O. is a director and shareholder in Fibrofind Ltd. J.L. is a shareholder in Fibrofind Ltd, all other authors declare no competing interests., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

Introduction: The burden of metabolic (dysfunction) associated fatty liver disease (MAFLD) is rising mirrored by an increase in hepatocellular cancer (HCC). MAFLD and its sequelae are characterized by perturbations in lipid handling, inflammation, and mitochondrial damage. The profile of circulating lipid and small molecule metabolites with the development of HCC is poorly characterized in MAFLD and could be used in future studies as a biomarker for HCC., Methods: We assessed the profile of 273 lipid and small molecule metabolites by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry in serum from patients with MAFLD ( n = 113) and MAFLD-associated HCC ( n = 144) from six different centers. Regression models were used to identify a predictive model of HCC., Results: Twenty lipid species and one metabolite, reflecting changes in mitochondrial function and sphingolipid metabolism, were associated with the presence of cancer on a background of MAFLD with high accuracy (AUC 0.789, 95% CI: 0.721-0.858), which was enhanced with the addition of cirrhosis to the model (AUC 0.855, 95% CI: 0.793-0.917). In particular, the presence of these metabolites was associated with cirrhosis in the MAFLD subgroup ( p < 0.001). When considering the HCC cohort alone, the metabolic signature was an independent predictor of overall survival (HR 1.42, 95% CI: 1.09-1.83, p < 0.01)., Conclusion: These exploratory findings reveal a metabolic signature in serum which is capable of accurately detecting the presence of HCC on a background of MAFLD. This unique serum signature will be taken forward for further investigation of diagnostic performance as biomarker of early stage HCC in patients with MAFLD in the future., Competing Interests: Augusto Villanueva is an Editorial Board Member of Liver Cancer. Rohini Sharma received lecture fees from Bayer Healthcare and Shingoi; consulting fees from EISAI, Roche, SIRTEX; and research funding (to institution) from Incyte, Boston Scientific, AAA, and Astex Pharmaceuticals. Haonan Lu, Jacob George, Mohammed Eslam, Luigi Bolondi, Helen L. Reeves, Misti McCain, Edward Chambers, Caroline Ward, Dewi Sartika, Caroline Sands, Lynn Maslen, Matthew R. Lewis, and Ramya Ramaswami have no conflicts of interest to disclose., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

Objective: Hepatocellular carcinoma (HCC) deaths are rising alarmingly. Many patients are unsuitable for available therapies. Poor response rates further hamper outcomes for those that are. Selective internal radiation therapy (SIRT) offers hope, although which patients benefit over standard approaches remains unclear., Design/method: As a quality/service improvement, we audited consecutive patients treated with SIRT (2015-2020) by the Newcastle upon Tyne Hospitals National Health Service Foundation Trust HCC multidisciplinary team. Indications, Barcelona clinic liver cancer (BCLC) stage, treatment response, subsequent therapies and survival at 30 September 2021 were assessed., Results: Fifty-one patients received SIRT. Thirty-day mortality was zero. Three months partial response, stable disease and progressive disease on imaging were 50%, 22% and 28%, respectively. Overall median survival was 21 months. There were four subgroups: (1) BCLC-B: HCC>7 cm too large for transarterial chemoembolisation (TACE) alone (n=21); (2) BCLC-B: HCC progressed post TACE (n=7); (3) BCLC-C: HCC with any combination of large tumour burden, branch portal vein thrombosis, non-hepatitis C virus aetiology (n=16); (4) BCLC-C: sorafenib inappropriate (n=7). In group 1, 5/21 (23.8%) of patients were downstaged to resection, 33% received subsequent medical therapies and median survival was >40 months. In BCLC-B patients treated second line (group 2), median survival was 14.2 months. In BCLC-C, median survival was 20.2 months for group 3 and 4.2 months for group 4., Conclusion: SIRT outcomes for advanced HCC, often bridging patients with adverse predictive factors to subsequent surgery or medical therapies, were encouraging. A role after TACE or for BCLC-C patients requires further assessment., Competing Interests: Competing interests: HLR is a member of the European Association for Study of Liver Disease (EASL) education committee, the HCC-UK Committee, the NCRI hepatobiliary subgroup and the Patient Advocacy group for the International Association for Liver cancer (ILCA). She is an expert advisor to the LiverNorth patient Charity and the British Liver Trust and has acted as an expert advisor to NICE Committee Multiple Technology Appraisal (MTA) assessing selective internal radiation therapies (SIRT) for treating hepatocellular carcinoma (ID1276). On behalf of Newcastle University, she has acted as an advisor to Boston Scientific UK and SIRTEX. JR is a member of the British Society of Interventional Radiology Education Committee. GP is a member of the British Society of Interventional Radiology Scientific Program Committee. DMM is the medical director for NHS British Transplant, council member for British Transplant Society, secretary for Cholangiocarcinoma-UK, member of the Transplant Oncology Group and trustee for British Association for Cancer Surgery (BASO). He has been on advisory boards for Boston Scientific UK and SIRTEX. PL is the secretary for Interventional Oncology UK (IOUK) and the Chair for the British Society of Interventional Radiology Scientific Programme Committee. He has received honoraria for lectures from Boston Scientific UK, Sirtex and Johnson and Johnson. He has participated in advisory boards for Boston Scientific UK, Guerbet and Johnson and Johnson., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Background: The number of incident cases and deaths from primary liver cancer, predominantly hepatocellular carcinoma (HCC), has increased markedly in the last two decades. HCC is generally diagnosed at an advanced stage, and most new cases are in people aged over 70 years with age-related comorbidities. Treatment options are often limited, with most patients receiving palliative treatment or supportive care only. As a consequence, maintaining quality of life (QoL) through symptom management is critically important and is a core objective of clinical care. Strong evidence supports the efficacy of supervised exercise training for addressing certain cancer-related symptoms, including QoL, physical function, and fatigue. However, there are many barriers to implementing supervised exercise programmes within cancer care pathways, including economic pressures on healthcare systems and personal barriers for patients. Recent advances in technology allow patients to exercise at home under the 'virtual' supervision of an exercise professional through videoconferencing software (termed 'telehealth exercise'). Despite its potential, there are uncertainties relating to the feasibility, acceptability, and safety of telehealth exercise in people living with HCC., Methods: This is a protocol for a prospective, single-centre, single-arm, pretest-posttest feasibility trial. We aim to recruit 20 patients aged 60 years or older who have received treatment for HCC and are undergoing routine clinical monitoring. Patients will be invited to take part in two online, home-based, group exercise sessions per week for 10 consecutive weeks. The 'virtual' exercise sessions will be delivered in real time by an exercise professional through videoconferencing software. Each session will comprise 30 min of aerobic and resistance exercise performed at a moderate intensity, as guided by the 10-point Borg rating of perceived exertion scale. Feasibility outcomes include recruitment, retention, adherence, intervention fidelity, and safety. Acceptability of the intervention will be assessed using a mixed-methods approach via monthly online surveys and an exit telephone interview. Physical function, accelerometry-measured physical activity, mid-upper arm circumference, and patient-reported outcome measures (PROMS) will be assessed before and after the intervention to determine the feasibility of assessing outcome measures. Physical function outcomes include the short physical performance battery and Liver Frailty Index. PROMS include the Functional Assessment of Cancer Therapy-Hepatobiliary questionnaire, Functional Assessment of Chronic Illness Therapy-Fatigue questionnaire, Activities-specific Balance Confidence scale, Hospital Anxiety and Depression Scale, and the Godin Leisure-Time Exercise Questionnaire., Discussion: This mixed-methods study will address uncertainties relating to the feasibility and acceptability of delivering live, online, home-based, group exercise sessions to patients with HCC. The findings will inform whether any modifications are required to refine and optimise the intervention, and the assessment of outcome measures will provide information on the likely size and variability of intervention effects. Collectively, the data generated will inform the design of a subsequent, adequately powered, randomised controlled trial to evaluate the efficacy of the telehealth exercise intervention., Trial Registration: ISRCTN14411809., (© 2022. The Author(s).)

Objective: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy., Design: Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry., Results: Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1 + dendritic cell activation and CD8 + T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8 + T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8 + T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs., Conclusion: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC., Competing Interests: Competing interests: DM is a director of Fibrofind. JL and DM are shareholders in Fibrofind limited. SB owns shares in AstraZeneca. OJS receives funding from AstraZeneca and Novartis. TGB receives research funding support from AstraZeneca. JML receives research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, Boehringer-Ingelheim and Ipsen, and consulting fees from Eli Lilly, Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Genentech, Roche, Glycotest, Nucleix, Sirtex, Mina Alpha and AstraZeneca., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Background: Northern England has been experiencing a persistent rise in the number of primary liver cancers, largely driven by an increasing incidence of hepatocellular carcinoma (HCC) secondary to alcohol-related liver disease and non-alcoholic fatty liver disease. Here we review the effect of the COVID-19 pandemic on primary liver cancer services and patients in our region., Objective: To assess the impact of the COVID-19 pandemic on patients with newly diagnosed liver cancer in our region., Design: We prospectively audited our service for the first year of the pandemic (March 2020-February 2021), comparing mode of presentation, disease stage, treatments and outcomes to a retrospective observational consecutive cohort immediately prepandemic (March 2019-February 2020)., Results: We observed a marked decrease in HCC referrals compared with previous years, falling from 190 confirmed new cases to 120 (37%). Symptomatic became the the most common mode of presentation, with fewer tumours detected by surveillance or incidentally (% surveillance/incidental/symptomatic; 34/42/24 prepandemic vs 27/33/40 in the pandemic, p=0.013). HCC tumour size was larger in the pandemic year (60±4.6 mm vs 48±2.6 mm, p=0.017), with a higher incidence of spontaneous tumour haemorrhage. The number of new cases of intrahepatic cholangiocarcinoma (ICC) fell only slightly, with symptomatic presentation typical. Patients received treatment appropriate for their cancer stage, with waiting times shorter for patients with HCC and unchanged for patients with ICC. Survival was associated with stage both before and during the pandemic. 9% acquired COVID-19 infection., Conclusion: The pandemic-associated reduction in referred patients in our region was attributed to the disruption of routine healthcare. For those referred, treatments and survival were appropriate for their stage at presentation. Non-referred or missing patients are expected to present with more advanced disease, with poorer outcomes. While protective measures are necessary during the pandemic, we recommend routine healthcare services continue, with patients encouraged to engage., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Hepatocellular carcinoma (HCC) remains one of the most prevalent and deadliest cancers. The poor outcome associated with HCC is dramatically changing due to the advent of effective systemic therapies. Here we discuss the molecular pathogenesis of HCC, molecular classes and determinants of heterogeneity. In addition, effective single-agent and combination systemic therapies involving immunotherapies as standard of care are analyzed. Finally, we propose a flowchart of sequential therapies, explore mechanisms of resistance and address the need for predictive biomarkers., (© 2022. Springer Nature America, Inc.)

The success of atezolizumab plus bevacizumab treatment contributed to a shift in systemic therapies for hepatocellular carcinoma (HCC) towards combinations that include cancer immunotherapeutic agents. Thus far, the principal focus of cancer immunotherapy has been on interrupting immune checkpoints that suppress antitumour lymphocytes. As well as lymphocytes, the HCC environment includes numerous other immune cell types, among which neutrophils are emerging as an important contributor to the pathogenesis of HCC. A growing body of evidence supports neutrophils as key mediators of the immunosuppressive environment in which some cancers develop, as well as drivers of tumour progression. If neutrophils have a similar role in HCC, approaches that target or manipulate neutrophils might have therapeutic benefits, potentially including sensitization of tumours to conventional immunotherapy. Several neutrophil-directed therapies for patients with HCC (and other cancers) are now entering clinical trials. This Review outlines the evidence in support of neutrophils as drivers of HCC and details their mechanistic roles in development, progression and metastasis, highlighting the reasons that neutrophils are well worth investigating despite the challenges associated with studying them. Neutrophil-modulating anticancer therapies entering clinical trials are also summarized., (© 2022. Springer Nature Limited.)

Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1 f/f ) and hepatocyte-specific knockout Xbp1 mice (Xbp1 ∆hepa ) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1-2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1 ∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1 ∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury., (© 2022. The Author(s).)

Background and Aims: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies., Methods: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays., Results: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-β proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved., Conclusions: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature., Lay Summary: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC., Competing Interests: Conflict of interest J.M.L. receives research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, Boehringer-Ingelheim and Ipsen, and consulting fees from Eli Lilly, Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Genentech, Roche, Glycotest, Leerink Swann LLC, Fortress Biotech, Nucleix, Can-Fite Biopharma, Sirtex, Mina Alpha Ltd and AstraZeneca. S.L.F consults for the following companies: 89 Bio, Amgen, Axcella Health, Blade Therapeutics, Bristol Myers Squibb, Can-Fite Biopharma, ChemomAb, Escient Pharmaceuticals, Forbion, Foresite laboratories, Galmed, Gordian Biotechnology, Glycotest, Glympse Bio, Hepgene, In sitro, Morphic Therapeutics, North Sea Therapeutics, Novartis, Ono Pharmaceuticals, Pfizer Pharmaceuticals, Scholar Rock Surrozen. He has stock options in the following companies: Blade Therapeutics, Escient, Galectin, Galmed, Genfit, Glympse, Hepgene, Lifemax, Metacrine, Morphic Therapeutics, Nimbus, North Sea Therapeutics, Scholar Rock, Surrozen. He receives research support from Morphic Therapeutics, Novo Nordisk, and Galmed, and has an SBIR grant with Abalone Bio. A.L. is a consultant for Neuwave and Histosonics. C.P.M.S.O. consults for Bayer, Novartis, Novonordisk, Allergan, Pfizer, Roche and Zambon. P.S. is receiving research grants from BMS, Roche, Incyte, Chugai and consulting fees from BMS, MSD, Incyte, Janssen, Roche, AstraZeneca and Amgen. H.W., P.T., and A.V.U. are or were salaried employees of Sema4 at the time of the study. H.W., P.T., and A.V.U. hold Sema4 stock options. B.M. received consultancy fees from Bayer-Shering Pharma, and speaker fees from Eisai and MSD. The rest of authors have nothing to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) is rising, even in the absence of cirrhosis. We aimed to develop a murine model that would facilitate further understanding of NAFLD-HCC pathogenesis. A total of 144 C3H/He mice were fed either control or American lifestyle (ALIOS) diet, with or without interventions, for up to 48 weeks of age. Gross, liver histology, immunohistochemistry (IHC) and RNA-sequencing data were interpreted alongside human datasets. The ALIOS diet promoted obesity, elevated liver weight, impaired glucose tolerance, non-alcoholic fatty liver disease (NAFLD) and spontaneous HCC. Liver weight, fasting blood glucose, steatosis, lobular inflammation and lipogranulomas were associated with development of HCC, as were markers of hepatocyte proliferation and DNA damage. An antioxidant diminished cellular injury, fibrosis and DNA damage, but not lobular inflammation, lipogranulomas, proliferation and HCC development. An acquired CD44 phenotype in macrophages was associated with type 2 diabetes and NAFLD-HCC. In this diet induced NASH and HCC (DINAH) model, key features of obesity associated NAFLD-HCC have been reproduced, highlighting roles for hepatic steatosis and proliferation, with the acquisition of lobular inflammation and CD44 positive macrophages in the development of HCC-even in the absence of progressive injury and fibrosis., (© 2021. The Author(s).)

Background & Aims: The coronavirus disease 2019 (COVID-19) pandemic has posed unprecedented challenges to healthcare systems and it may have heavily impacted patients with liver cancer (LC). Herein, we evaluated whether the schedule of LC screening or procedures has been interrupted or delayed because of the COVID-19 pandemic., Methods: An international survey evaluated the impact of the COVID-19 pandemic on clinical practice and clinical trials from March 2020 to June 2020, as the first phase of a multicentre, international, and observational project. The focus was on patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma, cared for around the world during the first COVID-19 pandemic wave., Results: Ninety-one centres expressed interest to participate and 76 were included in the analysis, from Europe, South America, North America, Asia, and Africa (73.7%, 17.1%, 5.3%, 2.6%, and 1.3% per continent, respectively). Eighty-seven percent of the centres modified their clinical practice: 40.8% the diagnostic procedures, 80.9% the screening programme, 50% cancelled curative and/or palliative treatments for LC, and 41.7% modified the liver transplantation programme. Forty-five out of 69 (65.2%) centres in which clinical trials were running modified their treatments in that setting, but 58.1% were able to recruit new patients. The phone call service was modified in 51.4% of centres which had this service before the COVID-19 pandemic (n = 19/37)., Conclusions: The first wave of the COVID-19 pandemic had a tremendous impact on the routine care of patients with liver cancer. Modifications in screening, diagnostic, and treatment algorithms may have significantly impaired the outcome of patients. Ongoing data collection and future analyses will report the benefits and disadvantages of the strategies implemented, aiding future decision-making., Lay Summary: The coronavirus disease 2019 (COVID-19) pandemic has posed unprecedented challenges to healthcare systems globally. Herein, we assessed the impact of the first wave pandemic on patients with liver cancer and found that routine care for these patients has been majorly disrupted, which could have a significant impact on outcomes., Competing Interests: SM.-M.: Speaker fees from Bayer and travel funding from 10.13039/100004326Bayer and 10.13039/501100014382Eisai. V.S.: Travel grants from 10.13039/100004326Bayer. A.F.: Lecture fees from Bayer, Gilead and MSD; consultancy fees from Bayer, AstraZeneca, Roche and Guerbert. J-C.N.: Received research grant from 10.13039/100004326Bayer for Inserm UMR1138. L.R.: Reports receiving consulting fees from Amgen, ArQule, AstraZeneca, Basilea, Bayer, Celgene, Eisai, Exelixis, Hengrui, Incyte, Ipsen, Lilly, MSD, Nerviano Medical Sciences, Roche, Sanofi; lectures fees from AbbVie, Amgen, Eisai, Gilead, Incyte, Ipsen, Lilly, Roche, Sanofi; travel fees from Ipsen; and institutional research funding from Agios, ARMO BioSciences, 10.13039/100004325AstraZeneca, BeiGene, 10.13039/501100003769Eisai, 10.13039/100010544Exelixis, 10.13039/100006591Fibrogen, Incyte, 10.13039/501100014382Ipsen, 10.13039/100004312Lilly, 10.13039/100007054MSD, 10.13039/100004337Roche. B.S.: Reports consultancy fees from Adaptimmune, AstraZeneca, Bayer, BMS, BTG, Eli Lilly, Ipsen, Novartis, Merck, Roche, Sirtex Medical, Terumo; and research grants from 10.13039/100002491BMS and Sirtex Medical. J. Bruix: Consultancy: AbbVie, ArQule, Astra, Basilea, Bayer, BMS, Daiichi Sankyo, GlaxoSmithKline, Gilead, Kowa, Lilly, Medimune, Novartis, Onxeo, Polaris, Quirem, Roche, Sanofi-Aventis, Sirtex, Terumo/Grants: 10.13039/100004326Bayer and 10.13039/501100014382Ipsen. M.S.Z.: Received speaker fees and travel grants from 10.13039/100004326Bayer and 10.13039/100014869BTG, 10.13039/100007054MSD. M.B.: Consultant and Advisory Board for: Bayer Pharma, Ipsen, BMS, Eisai, Roche, AstraZeneca, Sirtex Medical. D.J.P.: Received lecture fees from ViiV Healthcare and Bayer Healthcare and travel expenses from BMS and Bayer Healthcare; consulting fees for Mina Therapeutics, EISAI, Roche, and AstraZeneca; received research funding (to institution) from MSD and BMS. T.M.: Consultancy: Eisai, Roche, BTG, Ipsen, Bayer, Adaptimmune. Research funding: Bayer, BTG. H.W.: Served as speaker for Bayer, Eisai, and Ipsen, and as a consultant for Bayer, Eisai, Lilly, BMS, Roche, and Ipsen. B.M.: Consultancy: Bayer-Shering Pharma /Speaker fees: Eisai, MSDG. C. Consultancy fees from Bayer, Ipsen. P.R.G.: Bayer, BMS, MSD, AstraZeneca, Adaptimmune, Sirtex, Lilly Ipsen, Roche, Eisai. M.R.A.S.: Has received Research grants, advisory board or speaker fees for 10.13039/100006483AbbVie, 10.13039/100004326Bayer, Biolab, Intercept, 10.13039/501100014382Ipsen, 10.13039/100008799Gilead, 10.13039/100009947MSD, 10.13039/100004336Novartis, and 10.13039/100004337Roche. J.T.: Has received research grants from 10.13039/100004337Roche and 10.13039/501100014382Ipsen. He has received speaker and consulting honoraria from AstraZeneca, Amgen, Bayer Healthcare, Bristol Myers-Squibb, Eisai, Ipsen, Merck Serono, Merck Sharp & Dome, Lilly Imclone, and Roche. J. Bridgewater: Consultancy Bayer, BMS, Incyte, Taiho, Roche, MSD and Merck Serono. Research funding from Incyte. G.C.: Consultancy fees from Bayer, Ipsen. A.L.: Consultancy CAScination, Advisory Board Neuwave and Histosonics. H.T.: Speaker fees from AbbVie, Gilead, MSD, and Bayer. R.V.: Research grant from 10.13039/100006483Abbvie. A.M.M.P.: Speaker honorarium from Bayer, BMS, Boston Scientific and EISAI. Consulting honorarium from Bayer, AstraZeneca and EISAI. Advisory honorarium from Bayer, AstraZeneca and EISAI. Grants from 10.13039/100004326Bayer and 10.13039/100008497Boston Scientific. M.D.: Has received consulting and training fees from Bayer and Eisai. B.B.: Reports Consultancy for GenMab (paid to Institution); Advisory Boards for Roche (paid to Institution), Eisai Europe Limited (paid to Institution), research grant from 10.13039/100006436Celgene Ltd (paid to Institution), Speakers Bureau for Eisai Europe Limited (paid to Institution), Travel and registration for Congress from Bayer. L.d.F.: Lectures fees from BMS, Roche and Bayer. B.M.M.: Advisory/Speaker: Amgen, AstraZeneca, Bayer, BMS, Eisai, Ipsen, Merck, Roche, Sanoffi Genzyme, Taiho. Expert Testimony: Eisai, Roche. Travel: Eisai, Merck. Research: Sillajen (Individual); AstraZeneca, H3/Eisai, Galera, GSK, Exelixis (Institution). M.S.: Travel/ accommodation/meeting expenses: Bayer. Eisai. Speaker fees: Bayer. C.R.L.: Travel grants from 10.13039/100004326Bayer. M.R-G.: Reports grants from Intercept, grants from 10.13039/100005564Gilead-Sciences, personal fees from Shionogi, personal fees from Alfa-Wasserman, personal fees from Prosciento, personal fees from Kaleido, personal fees from Novonrdisk, personal fees from MSD, personal fees from BMS, personal fees from Allergan, personal fees from Boehriger-Ingelheim, personal fees from Zydus, personal fees from Intercept Pharma, personal fees from Gilead-Sciences, outside the submitted work. F.P.: Disclosures: Received speaker honoraria from Bayer, Roche, LKM-Biotoscana, RAFFO. Research Grants from INC Argentinean 10.13039/100013137National Institute of Corrections, 10.13039/100004337Roche. V.M.: Lectures sponsored by Bayer. G.B.: Advisory board Eli-Lilly and Incyte. M. Vergara: Travel grants from 10.13039/100004326Bayer, 10.13039/100008799Gilead, 10.13039/100009947MSD and 10.13039/100006483Abbvie. Lectures sponsored by Gilead, Abbvie, Intercept, and MSD. M.L.: Lectures and educational presentations: Abbvie. Travel/accommodation, meeting expenses covered by Bayer, Gilead, Abbvie. M.I.: Received speaker honoraria from Bayer, Gilead Sciences, BMS, Janssen, Ipsen, MSD, BTG-Boston Scientific, AbbVie, EISAI, and was consultant for BTG-Boston Scientific, Bayer, and Guerbet. M.R.: Consultancy: Bayer-Schering Pharma, BMS, Roche, Ipsen, AstraZeneca, Lilly, BTG/Paid conferences: Bayer-Schering Pharma, BMS, Gilead, Lilly/Research Grants: 10.13039/100004326Bayer-Schering Pharma, 10.13039/501100014382Ipsen. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

Dysregulation of miRNAs is a hallmark of cancer, modulating oncogenes, tumor suppressors, and drug responsiveness. The multi-kinase inhibitor sorafenib is one of the first-line drugs for advanced hepatocellular carcinoma (HCC), although the outcome for treated patients is heterogeneous. The identification of predictive biomarkers and targets of sorafenib efficacy are sorely needed. Thus, selected top upregulated miRNAs from the C19MC cluster were analyzed in different hepatoma cell lines compared to immortalized liver human cells, THLE-2 as control. MiR-518d-5p showed the most consistent upregulation among them. Thus, miR-518d-5p was measured in liver tumor/non-tumor samples of two distinct cohorts of HCC patients (n = 16 and n = 20, respectively). Circulating miR-518d-5p was measured in an independent cohort of HCC patients receiving sorafenib treatment (n = 100), where miR-518d-5p was analyzed in relation to treatment duration and patient's overall survival. In vitro and in vivo studies were performed in human hepatoma BCLC3 and Huh7 cells to analyze the effect of miR-518d-5p inhibition/overexpression during the response to sorafenib. Compared with healthy individuals, miR-518d-5p levels were higher in hepatic and serum samples from HCC patients (n = 16) and in an additional cohort of tumor/non-tumor paired samples (n = 20). MiR-518d-5p, through the inhibition of c-Jun and its mitochondrial target PUMA, desensitized human hepatoma cells and mouse xenograft to sorafenib-induced apoptosis. Finally, serum miR-518d-5p was assessed in 100 patients with HCC of different etiologies and BCLC-stage treated with sorafenib. In BCLC-C patients, higher serum miR-518d-5p at diagnosis was associated with shorter sorafenib treatment duration and survival. Hence, hepatic miR-518d-5p modulates sorafenib resistance in HCC through inhibition of c-Jun/PUMA-induced apoptosis. Circulating miR-518d-5p emerges as a potential lack of response biomarker to sorafenib in BCLC-C HCC patients.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Obesity and non-alcoholic fatty liver disease (NAFLD) are contributing to the global rise in deaths from hepatocellular carcinoma (HCC). The pathogenesis of NAFLD-HCC is not well understood. The severity of hepatic steatosis, steatohepatitis and fibrosis are key pathogenic mechanisms, but animal studies suggest altered immune responses are also involved. Genetic studies have so far highlighted a major role of gene variants promoting fat deposition in the liver ( PNPLA3 rs738409; TM6SF2 rs58542926). Here, we have considered single-nucleotide polymorphisms (SNPs) in candidate immunoregulatory genes ( MICA rs2596542; CD44 rs187115; PDCD1 rs7421861 and rs10204525), in 594 patients with NAFLD and 391 with NAFLD-HCC, from three European centres. Associations between age, body mass index, diabetes, cirrhosis and SNPs with HCC development were explored. PNPLA3 and TM6SF2 SNPs were associated with both progression to cirrhosis and NAFLD-HCC development, while PDCD1 SNPs were specifically associated with NAFLD-HCC risk, regardless of cirrhosis. PDCD1 rs7421861 was independently associated with NAFLD-HCC development, while PDCD1 rs10204525 acquired significance after adjusting for other risks, being most notable in the smaller numbers of women with NAFLD-HCC. The study highlights the potential impact of inter individual variation in immune tolerance induction in patients with NAFLD, both in the presence and absence of cirrhosis.

Importance: Non-alcoholic fatty liver disease (NAFLD) is a rapidly growing cause of chronic liver disease and is becoming a leading cause of hepatocellular carcinoma (HCC) in many developed countries. This presents major challenges for the surveillance, diagnosis and treatment of HCC., Objective: To discuss the clinical challenges faced by clinicians in managing the rising number of NAFLD-HCC cases., Evidence Review: MEDLINE, PubMed and Embase databases were searched using the keywords; NAFLD, HCC, surveillance, hepatectomy, liver transplantation, percutaneous ablation, transarterial chemoembolization (TACE), selective internal radiotherapy treatment (SIRT) and sorafenib. Relevant clinical studies were included., Findings: Current HCC surveillance programmes are inadequate because they only screen for HCC in patients with cirrhosis, whereas in NAFLD a significant proportion of HCC develops in the absence of cirrhosis. Consequently NAFLD patients often present with a more advanced stage of HCC, with a poorer prognosis. NAFLD-HCC patients also tend to be older and to have more co-morbidities compared to HCC of other etiologies. This limits the use of curative treatments such as liver resection and orthotopic liver transplantation (OLT). Evidence suggests that although NAFLD-HCC patients who undergo liver resection or OLT have worse perioperative and short-term outcomes, overall long-term survival is comparable to HCC of other etiologies. This highlights the importance of careful patient selection, pre-habilitation and perioperative planning for NAFLD-HCC patients being considered for surgical treatment. Careful consideration is also important for non-surgical treatments, although the evidence supporting treatment selection is frequently lacking, as these patients tend to be poorly represented in clinical trials. Locoregional therapies such as percutaneous ablation and TACE may be less well tolerated and less effective in NAFLD patients with obesity or diabetes. The tyrosine kinase inhibitor sorafenib may also be less effective., Conclusions and Relevance: This review highlights how international guidelines, for which NAFLD traditionally has made up a small part of the evidence base, may not be appropriate for all NAFLD-HCC patients. Future guidelines need to reflect the changing landscape of HCC, by making specific recommendations for the management of NAFLD-HCC., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/hbsn.2019.08.08). The authors have no conflicts of interest to declare., (2021 Hepatobiliary Surgery and Nutrition. All rights reserved.)

Background: The aim of current study was to (1) construct and validate a novel hepatocellular carcinoma (HCC)-specific inflammatory index; (2) compare the performances of the Integrated Liver Inflammatory Score (ILIS) to existing 4 inflammatory indices in HCC; (3) explore the association between the inflammatory indices and systemic/intratumoral inflammatory markers., Methods: Two cohorts from Hong Kong (HK; n = 1,315) and Newcastle ( n = 574) were studied. A novel index was constructed from the HK training set ( n = 627). The index was constructed from the training set by combing independent prognostic circulating parameters, followed by validating in the validation set of HK cohort ( n = 688) and the Newcastle cohort. Its prognostic performance was compared to 4 inflammatory indices, namely, the neutrophil to lymphocyte ratio, platelet-to-lymphocyte ratio, prognostic nutrition index, and systemic immune-inflammation index, were compared in the HK cohort. Circulating cytokines and intratumoral gene expression were analyzed in a subset of patients with available samples and correlated with the inflammatory indices., Results: In the training set of the HK cohort, the ILIS, was generated: -0.057 × albumin (g/L) + 0.978 × log (Bilirubin, µmol/L) + 1.341 × log (alkaline phosphatase, IU/L) + 0.086 × Neutrophil (10 9 /L) + 0.301 × log (alpha-fetoprotein, µg/L). With cutoff of 2.60 and 3.87, the ILIS could categorize patients into 3 risk groups in the both validation cohorts. ILIS outperforms other inflammatory indices and remains an independent prognosticator for overall survival after adjustment with Barcelona Clinic Liver Cancer (hazard ratio 31.90, p < 0.001). The ILIS had the best prognostic performances as compared to other inflammatory indices. In exploratory analyses, the ILIS correlated with circulating inflammatory cytokines (e.g., IL-8) but not with any intratumoral inflammatory gene expression., Conclusions: ILIS is an HCC-specific prognostic index built on 5 readily available blood parameters. Its versatility is validated both Eastern and Western population of HCC. The score is correlated with levels of circulating cytokines., Competing Interests: The authors declare that they have no conflicts of interest to disclose., (Copyright © 2020 by S. Karger AG, Basel.)

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

Background: A distinct serum metabonomic pattern has been previously revealed to be associated with various forms of liver disease. Here, we aimed to apply mass spectrometry to obtain serum metabolomic profiles from individuals with cholangiocarcinoma and benign hepatobiliary diseases to gain an insight into pathogenesis and search for potential early-disease biomarkers., Methods: Serum samples were profiled using a hydrophilic interaction liquid chromatography platform, coupled to a mass spectrometer. A total of 47 serum specimens from 8 cholangiocarcinoma cases, 20 healthy controls, 8 benign disease controls (bile duct strictures) and 11 patients with hepatocellular carcinoma (as malignant disease controls) were included. Data analysis was performed using univariate and multivariate statistics., Results: The serum metabolome disparities between the metabolite profiles from healthy controls and patients with hepatobiliary disease were predominantly related to changes in lipid and lipid-derived compounds (phospholipids, bile acids and steroids) and amino acid metabolites (phenylalanine). A metabolic pattern indicative of inflammatory response due to cirrhosis and cholestasis was associated with the disease groups. The abundance of phospholipid metabolites was altered in individuals with liver disease, particularly cholangiocarcinoma, but no significant difference was seen between profiles from patients with benign biliary strictures and cholangiocarcinoma., Conclusion: The serum metabolome in cholangiocarcinoma exhibited changes in metabolites related to inflammation, altered energy production and phospholipid metabolism. This study serves to highlight future avenues for biomarker research in large-scale studies., (© 2019 Indian National Association for Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Aims: To evaluate early outcomes of patients with hepatocellular carcinoma (HCC) treated with a novel radiopaque bead, the 75-150 μm DC Bead LUMI™ (Biocompatibles UK Ltd)., Materials and Methods: This was a retrospective review of the first 40 consecutive patients at a UK tertiary hepato-biliary centre, treated for HCC with TACE using radiopaque beads, between May 2017 and March 2019. Information regarding complications, mortality, lesion response and subsequent ablation procedures was collected from electronic records and case notes. Intra- and post-operative imaging was reviewed for visibility of the embolised territory., Results: Fifty-five TACE procedures were performed in 40 patients, with a median age of 70 years (range 28-88) and median lesion size of 3.8 cm (range 1.5-7.8). The median follow-up period was 30 weeks (range 6-101). Mean post-procedure hospital stay was 1.2 days. Complications of CIRSE Grade II or above occurred after 4/55 procedures (7.3%). Mortality at 30 days was zero. Objective response rates (mRECIST) at 1, 3 and 6 months were 32/35 (91.4%), 21/24 (87.5%) and 12/15 (80%), respectively. Complete response rates at 1, 3 and 6 months were 16/35 (45.7%), 12/24 (50%) and 9/15 (60%). The embolised territory was visible on intra-operative and follow-up CT imaging in all patients. The radiopaque beads were used as a fiducial marker to guide ablation in 5/40 patients (12.5%)., Conclusion: TACE with radiopaque beads shows promising tolerability and efficacy. The radiopaque beads ensure visualisation of the embolised lesion on intra- and post-operative imaging and, in selected cases, can act as a marker for CT-guided ablation.

Caloric excess and sedentary lifestyle have led to a global epidemic of obesity and metabolic syndrome. The hepatic consequence of metabolic syndrome and obesity, nonalcoholic fatty liver disease (NAFLD), is estimated to affect up to one-third of the adult population in many developed and developing countries. This spectrum of liver disease ranges from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Owing to the high prevalence of NAFLD, especially in industrialized countries but also worldwide, and the consequent burden of progressive liver disease, there is mounting epidemiological evidence that NAFLD has rapidly become a leading aetiology underlying many cases of hepatocellular carcinoma (HCC). In this Review, we discuss NAFLD-associated HCC, including its epidemiology, the key features of the hepatic NAFLD microenvironment (for instance, adaptive and innate immune responses) that promote hepatocarcinogenesis and the management of HCC in patients with obesity and associated metabolic comorbidities. The challenges and future directions of research will also be discussed, including clinically relevant biomarkers for early detection, treatment stratification and monitoring as well as approaches to therapies for both prevention and treatment in those at risk or presenting with NAFLD-associated HCC.

Nonalcoholic fatty liver disease (NAFLD) is a rising cause of hepatocellular carcinoma (HCC). We examined whether inherited pathogenic variants in candidate genes (n = 181) were enriched in patients with NAFLD-HCC. To this end, we resequenced peripheral blood DNA of 142 NAFLD-HCC, 59 NAFLD with advanced fibrosis, and 50 controls, and considered 404 healthy individuals from 1000 G. Pathogenic variants were defined according to ClinVar, likely pathogenic as rare variants predicted to alter protein activity. In NAFLD-HCC patients, we detected an enrichment in pathogenic (p = 0.024), and likely pathogenic variants (p = 1.9*10 -6 ), particularly in APOB (p = 0.047). APOB variants were associated with lower circulating triglycerides and higher HDL cholesterol (p < 0.01). A genetic risk score predicted NAFLD-HCC (OR 4.96, 3.29-7.55; p = 5.1*10 -16 ), outperforming the diagnostic accuracy of common genetic risk variants, and of clinical risk factors (p < 0.05). In conclusion, rare pathogenic variants in genes involved in liver disease and cancer predisposition are associated with NAFLD-HCC development.

With the growing number of novel therapeutic approaches for liver diseases, significant research efforts have been devoted to the development of liquid biopsy tools for precision medicine. This can be defined as non-invasive reliable biomarkers that can supplement and eventually replace the invasive liver biopsy for diagnosis, disease stratification and monitoring of response to therapeutic interventions. Similarly, detection of liver cancer at an earlier stage of the disease, potentially susceptible to curative resection, can be critical to improve patient survival. Circulating extracellular vesicles, nucleic acids (DNA and RNA) and tumour cells have emerged as attractive liquid biopsy candidates because they fulfil many of the key characteristics of an ideal biomarker. In this review, we summarise the currently available information regarding these promising and potential transformative tools, as well as the issues still needed to be addressed for adopting various liquid biopsy approaches into clinical practice. These studies may pave the way to the development of a new generation of reliable, mechanism-based disease biomarkers., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. No commercial re-use. See rights and permissions. Published by BMJ.)