NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Role of sex in liver tumor occurrence and clinical outcomes: A comprehensive review.

Clinical research on sex-based differences in the manifestations, pathophysiology, and prevalence of several diseases, including those affecting the liver, has expanded considerably in recent years. Increasing evidence suggests that liver diseases develop, progress, and respond to treatment differently depending on the sex. These observations support the concept that the liver is a sexually dimorphic organ in which estrogen and androgen receptors are present, which results in disparities between men and women in liver gene expression patterns, immune responses, and the progression of liver damage, including the propensity to develop liver malignancies. Sex hormones play protective or deleterious roles depending on the patient's sex, the severity of the underlying disease, and the nature of precipitating factors. Moreover, obesity, alcohol consumption, and active smoking, as well as social determinants of liver diseases leading to sex-related inequalities, may interact strongly with hormone-related mechanisms of liver damage. Drug-induced liver injury, viral hepatitis, and metabolic liver diseases are influenced by the status of sex hormones. Available data on the roles of sex hormones and gender differences in liver tumor occurrence and clinical outcomes are conflicting. Here, we critically review the main gender-based differences in the molecular mechanisms associated with liver carcinogenesis and the prevalence, prognosis, and treatment of primary and metastatic liver tumors.

Episodic overt hepatic encephalopathy after transjugular intrahepatic portosystemic shunt does not increase mortality in patients with cirrhosis.

BACKGROUND & AIMS: Overt hepatic encephalopathy (OHE) is a major complication of transjugular intrahepatic portosystemic shunt (TIPS) placement, given its high incidence and possibility of refractoriness to medical treatment. Nevertheless, the impact of post-TIPS OHE on mortality has not been investigated in a large population. METHODS: We designed a multicenter, non-inferiority, observational study to evaluate the mortality rate at 30 months in patients with and without OHE after TIPS. We analyzed a database of 614 patients who underwent TIPS in three Italian centers and estimated the cumulative incidence of OHE and mortality with competitive risk analyses, setting the non-inferiority limit at 0.12. RESULTS: During a median follow-up of 30 months (IQR 12-30), 293 patients developed at least one episode of OHE. Twenty-seven (9.2%) of them experienced recurrent/persistent OHE. Patients with OHE were older (64 [57-71] vs. 59 [50-67] years, p <0.001), had lower albumin (3.1 [2.8-3.5] vs. 3.25 [2.9-3.6] g/dl, p = 0.023), and had a higher prevalence of pre-TIPS OHE (15.4% vs. 9.0%, p = 0.023). Child-Pugh and MELD scores were similar. The 30-month difference in mortality between patients with and without post-TIPS OHE was 0.03 (95% CI -0.042 to 0.102). Multivariable analysis showed that age (subdistribution hazard ratio 1.04, 95% CI 1.02-1.05, p <0.001) and MELD score (subdistribution hazard ratio 1.09, 95% CI 1.05-1.13, p <0.001), but not post-TIPS OHE, were associated with a higher mortality rate. Similar results were obtained when patients undergoing TIPS for variceal re-bleeding prophylaxis (n = 356) or refractory ascites (n = 258) were analyzed separately. The proportion of patients with persistent OHE after TIPS was significantly higher in the group of patients who died. The robustness of these results was increased following propensity score matching. CONCLUSION: Episodic OHE after TIPS is not associated with mortality in patients undergoing TIPS, regardless of the indication. IMPACT AND IMPLICATIONS: Overt hepatic encephalopathy (OHE) is a common complication in patients with advanced liver disease and it is particularly frequent following transjugular intrahepatic portosystemic shunt (TIPS) placement. In patients with cirrhosis outside the setting of TIPS, the development of OHE negatively impacts survival, regardless of the severity of cirrhosis or the presence of acute-on-chronic liver failure. In this multicenter, non-inferiority, observational study we demonstrated that post-TIPS OHE does not increase the risk of mortality in patients undergoing TIPS, irrespective of the indication. This finding alleviates concerns regarding the weight of this complication after TIPS. Intensive research to improve patient selection and risk stratification remains crucial to enhance the quality of life of patients and caregivers and to avoid undermining the positive effects of TIPS on survival.

AGILE SCORES IN MASLD AND ALD: EXTERNAL VALIDATION AND THEIR UTILITY IN CLINICAL ALGORITHMS.

BACKGROUND: Agile scores, including liver stiffness measurements (LSM) and routine clinical/laboratory biomarkers, have been developed for advanced fibrosis (F≥3) and cirrhosis, respectively, in patients with metabolic-associated steatotic liver disease (MASLD). We independently validated the diagnostic accuracy of these scores in MASLD, alcohol-related liver disease (ALD) and chronic hepatitis B or C (CHB/C) and assessed them in clinical algorithms with FIB-4 and LSM. METHODS: We included 4,243 patients (MASLD:912, ALD:386, CHB:597, CHC:2348) with LSM, liver biopsy and laboratory tests within 6 months. FIB-4, Agile 3+ and Agile 4 scores were calculated. RESULTS: For F≥3, diagnostic accuracy of Agile 3+ and LSM were similar in MASLD (AUC: 0.86 vs 0.86, P=0.831) and ALD (0.92 vs 0.94, P=0.123). For cirrhosis, Agile 4 was similar to LSM in MASLD (0.89 vs 0.90, P=0.412) and ALD (0.94 vs 0.95, P=0.513). Agile 3+/4 performed worse than LSM in CHB/C. Using predefined dual thresholds of 90% Se/Sp, correct classification rates in MASLD were 66% vs 61% using Agile 3+ vs LS dual cut-offs and 71% vs 67% in ALD. When using Agile 3+ or LSM as a second step after FIB-4>1.3, correct classification rates were higher with Agile 3+ than LSM, both for MASLD (75% vs 71%) and for ALD patients (76% vs 72%) with fewer indeterminate results. Positive agreement of LSM and Agile 3+/4 significantly increased the specificity of a diagnosis of advanced fibrosis/cirrhosis. CONCLUSION: Agile 3+ and Agile 4 have equal diagnostic accuracy with LSM in both MASLD and ALD but result in fewer indeterminate results. Sequential use of FIB-4 and Agile 3+/4 or concurrent Agile 3+/4 and LSM can be used to further optimize F≥3 diagnosis.

Mortality after transjugular intrahepatic portosystemic shunt in older adult patients with cirrhosis: A validated prediction model.

BACKGROUND AND AIMS: Implantation of a transjugular intrahepatic portosystemic shunt (TIPS) improves survival in patients with cirrhosis with refractory ascites and portal hypertensive bleeding. However, the indication for TIPS in older adult patients (greater than or equal to 70 years) is debated, and a specific prediction model developed in this particular setting is lacking. The aim of this study was to develop and validate a multivariable model for an accurate prediction of mortality in older adults. APPROACH AND RESULTS: We prospectively enrolled 411 consecutive patients observed at four referral centers with de novo TIPS implantation for refractory ascites or secondary prophylaxis of variceal bleeding (derivation cohort) and an external cohort of 415 patients with similar indications for TIPS (validation cohort). Older adult patients in the two cohorts were 99 and 76, respectively. A cause-specific Cox competing risks model was used to predict liver-related mortality, with orthotopic liver transplant and death for extrahepatic causes as competing events. Age, alcoholic etiology, creatinine levels, and international normalized ratio in the overall cohort, and creatinine and sodium levels in older adults were independent risk factors for liver-related death by multivariable analysis. CONCLUSIONS: After TIPS implantation, mortality is increased by aging, but TIPS placement should not be precluded in patients older than 70 years. In older adults, creatinine and sodium levels are useful predictors for decision making. Further efforts to update the prediction model with larger sample size are warranted.

Impact of body mass index on the prognosis of unresectable HCC patients receiving first-line Lenvatinib or atezolizumab plus bevacizumab.

INTRODUCTION: Overweight is a negative prognostic factor in the general population in the long term. However, the role of body mass index (BMI) in the short-mid term in advanced tumours is unclear. The present analysis investigates the role of BMI weight classes in a large sample of patients affected by HCC and receiving atezolizumab plus bevacizumab or lenvatinib as first-line treatment. METHODS AND MATERIAL: The cohort included consecutive patients affected by BCLC-c and BCLC-B HCC patients from a multicenter international study group who received atezolizumab plus bevacizumab or lenvatinib as first-line therapy. Population was stratified according to the BMI in under-, over- and normal-weight according to the conventional thresholds. The primary objective of the study was to evaluate the prognostic and predictive impact of BMI in patients affected by advanced or intermediate HCC. Survival curves were estimated using the product-limit method of Kaplan-Meier. The role of stratification factors was analysed with log-rank tests. RESULTS: 1292 consecutive patients with HCC were analysed. 466 (36%) patients were treated with lenvatinib and 826 (64%) patients were treated with atezolizumab plus bevacizumab. In the atezolizumab plus bevacizumab arm, 510 (62%) patients were normal-weight, 52 (6%) underweight and 264 (32%) overweight. At the univariate analysis for OS, underweight patients had significantly shorter OS compared to normal-weight patients, whereas no differences were found between normal-weight versus overweight. Multivariate analysis confirmed that underweight patients had significantly shorter OS compared to normal-weight patients (HR: 1.7; 95% CI: 1.0-2.8; p = .0323). In the lenvatinib arm, 26 patients (5.6%) were categorized as underweight, 256 (54.9%) as normal-weight, and 184 (39.5%) as overweight. At the univariate analysis for OS, no significant differences were found between normal-weight versus underweight and between normal-weight versus overweight, which was confirmed at multivariate analysis. CONCLUSION: Our analysis highlighted a prognostic role of BMI in a cohort of patients with advanced HCC who received atezolizumab plus bevacizumab, while no prognostic role for low BMI was apparent in patients who received lenvatinib.

Epidemiological trends and trajectories of MAFLD-associated hepatocellular carcinoma 2002-2033: the ITA.LI.CA database.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) represents a new inclusive definition of the whole spectrum of liver diseases associated to metabolic disorders. The main objective of this study was to compare patients with MAFLD and non-MAFLD with hepatocellular carcinoma (HCC) included in a nationally representative cohort. METHODS: We analysed 6882 consecutive patients with HCC enrolled from 2002 to 2019 by 23 Italian Liver Cancer centres to compare epidemiological and future trends in three subgroups: pure, single aetiology MAFLD (S-MAFLD); mixed aetiology MAFLD (metabolic and others, M-MAFLD); and non-MAFLD HCC. RESULTS: MAFLD was diagnosed in the majority of patients with HCC (68.4%). The proportion of both total MAFLD and S-MAFLD HCC significantly increased over time (from 50.4% and 3.6% in 2002-2003, to 77.3% and 28.9% in 2018-2019, respectively, p<0.001). In Italy S-MAFLD HCC is expected to overcome M-MAFLD HCC in about 6 years. Patients with S-MAFLD HCC were older, more frequently men and less frequently cirrhotic with clinically relevant portal hypertension and a surveillance-related diagnosis. They had more frequently large tumours and extrahepatic metastases. After weighting, and compared with patients with non-MAFLD, S-MAFLD and M-MAFLD HCC showed a significantly lower overall (p=0.026, p=0.004) and HCC-related (p<0.001, for both) risk of death. Patients with S-MAFLD HCC showed a significantly higher risk of non-HCC-related death (p=0.006). CONCLUSIONS: The prevalence of MAFLD HCC in Italy is rapidly increasing to cover the majority of patients with HCC. Despite a less favourable cancer stage at diagnosis, patients with MAFLD HCC have a lower risk of HCC-related death, suggesting reduced cancer aggressiveness.

Baseline ALBI score and early variation of serum AFP predicts outcomes in patients with HCC treated by atezolizumab-bevacizumab.

BACKGROUND: The combination of atezolizumab and bevacizumab (AtezoBev) is the current first-line treatment for patients with hepatocellular carcinoma (HCC). Our aim was to evaluate the prognostic role of alpha-foetoprotein (AFP) early response and its combination with albumin-bilirubin (ALBI) in these patients. METHODS: Patients with HCC under AtezoBev with AFP > 20 ng/ml were included in three centres. The optimal threshold of AFP variation after 3 weeks of treatment was identified for overall survival (OS) and radiological response (RR) using RECIST 1.1 and mRECIST and its ability to predict progression-free survival (PFS) and OS was tested using univariate and multivariate analysis in derivation and validation cohorts. RESULTS: Seventy-five patients with AFP values >20 ng/ml were included. Fifty-eight patients were male with a median age of 63.5 years; 73% had cirrhosis and HCC stage was classified as BCLC B (18.7%) or C (81.3%). In the derivation cohort (n = 38), a decline in AFP ≥ 20% at 3 weeks (AFP early response) was associated with RR using mRECIST criteria (OR: 13.09 95% CI: 1.44-19.34 p = .02), PFS (HR: 0.42; 95% CI: 0.19-0.93, p = .03) and OS (HR: 0.35; 95% CI: 0.15-0.83, p = .01). AFP early response was confirmed as predictor of RR (p = .02 for mRECIST) and OS (p = .03) in the validation cohort (n= 37). In the whole cohort, the combination of ALBI and AFP early response was significantly associated with OS (p = .046) and PFS (p = .012) with a poor prognosis in patients belonging to the ALBI2-AFP non-responders class. CONCLUSION: AFP early response at 3 weeks predicts oncological outcomes in HCC patients treated with AtezoBev and combination with ALBI grade refines prognostic discrimination.

Oncostatin M is overexpressed in NASH-related hepatocellular carcinoma and promotes cancer cell invasiveness and angiogenesis.

Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Metabolic reprogramming in cholangiocarcinoma.

Metabolic reprogramming is a hallmark of cancer and allows tumour cells to meet the increased energy demands required for rapid proliferation, invasion, and metastasis. Indeed, many tumour cells acquire distinctive metabolic and bioenergetic features that enable them to survive in resource-limited conditions, mainly by harnessing alternative nutrients. Several recent studies have explored the metabolic plasticity of cancer cells with the aim of identifying new druggable targets, while therapeutic strategies to limit the access to nutrients have been successfully applied to the treatment of some tumours. Cholangiocarcinoma (CCA), a highly heterogeneous tumour, is the second most common form of primary liver cancer. It is characterised by resistance to chemotherapy and poor prognosis, with 5-year survival rates of below 20%. Deregulation of metabolic pathways have been described during the onset and progression of CCA. Increased aerobic glycolysis and glutamine anaplerosis provide CCA cells with the ability to generate biosynthetic intermediates. Other metabolic alterations involving carbohydrates, amino acids and lipids have been shown to sustain cancer cell growth and dissemination. In this review, we discuss the complex metabolic rewiring that occurs during CCA development and leads to unique nutrient addiction. The possible role of therapeutic interventions based on metabolic changes is also thoroughly discussed.

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA. APPROACH AND RESULTS: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA. CONCLUSIONS: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment.

Preneoplastic lesions in the liver: Molecular insights and relevance for clinical practice.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the most frequent primary liver cancers, accounting for approximately 80% and 15%, respectively. HCC carcinogenesis occurs mostly in cirrhosis and is a complex multi-step process, from precancerous lesions (low-grade and high-grade dysplastic nodules) to progressed HCC. During the different stages of liver carcinogenesis, there is an accumulation of pathological, genetic and epigenetic changes leading to initiation, malignant transformation and finally tumour progression. In contrast, a small subset of HCC occurs in normal liver from the transformation of hepatocellular adenoma (HCA), a benign hepatocellular tumour. The recent molecular classification enables to stratify HCAs according to their risk of complication, in particular malignant transformation, associated with mutations in exon 3 of the catenin beta 1 (CTNNB1) gene. Cholangiocarcinoma (CCA) derives from the multistep malignant transformation of preneoplastic lesions, like biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasm of the bile duct (IPNB), for which a pre-operative diagnosis remains difficult. Different genetic alterations are involved in BilIN and IPNB progression, leading to the development of tubular or intestinal adenocarcinoma. The aims of this review are to describe the main clinical and molecular features of preneoplastic lesions leading to the development of HCC and CCA, their implications in clinical practice and the perspectives for future research.

Portosystemic shunt is an effective treatment for complications of portal hypertension in hepatic myeloid metaplasia and improves nutritional status.

In patients affected by myelofibrosis with hepatic myeloid metaplasia (HMM), portal hypertension (PHT) complications may develop. In this case series, we analysed the efficacy and safety of transjugular portosystemic shunt (TIPS) in the treatment of PHT-related complications and its effects on the nutritional status. Six patients were evaluated and the average follow-up period after TIPS was 33 (IQR 5) months. None of the patients developed hepatic failure, nor any recurrence of variceal bleeding was recorded. No additional paracentesis or endoscopic prophylactic treatment for PHT-related complications were required. In all subjects, the average dose of diuretics was almost halved three months after TIPS. Three patients died during the follow-up, but none for liver-related causes. All patients showed an improvement in the global nutritional status. In conclusion, TIPS represent an effective and safe treatment option for patients affected by complications of PHT secondary to HMM and drives to an improvement of the nutritional status.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. METHODS: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+ ) cells in immune-deficient NOD-SCID/IL2Rgnull  (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. RESULTS: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (ß-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene ß-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. CONCLUSION: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Quality of life in liver transplant recipients during the Corona virus disease 19 pandemic: A multicentre study.

BACKGROUND: Liver transplant recipients require specific clinical and psychosocial attention given their frailty. Main aim of the study was to assess the quality of life after liver transplant during the current pandemic. METHODS: This multicentre study was conducted in clinically stable, liver transplanted patients. Enrollment opened in June and finished in September 2021. Patients completed a survey including lifestyle data, quality of life (Short Form health survey), sport, employment, diet. To examine the correlations, we calculated Pearson coefficients while to compare subgroups, independent samples t-tests and ANOVAs. To detect the predictors of impaired quality of life, we used multivariable logistic regression analysis. RESULTS: We analysed data from 511 patients observing significant associations between quality of life's physical score and both age and adherence to Mediterranean diet (p < .01). A significant negative correlation was observed between mental score and the sedentary activity (p < .05). Female patients scored significantly lower than males in physical and mental score. At multivariate analysis, females were 1.65 times more likely to report impaired physical score than males. Occupation and physical activity presented significant positive relation with quality of life. Adherence to Mediterranean diet was another relevant predictor. Regarding mental score, female patients were 1.78 times more likely to show impaired mental score in comparison with males. Sedentary activity and adherence to Mediterranean diet were further noteworthy predictors. CONCLUSIONS: Females and subjects with sedentary lifestyle or work inactive seem to show the worst quality of life and both physical activity and Mediterranean diet might be helpful to improve it.

Predictors of serious adverse events and non-response in cirrhotic patients with primary biliary cholangitis treated with obeticholic acid.

BACKGROUND & AIMS: Obeticholic acid (OCA) has recently been restricted in patients with primary biliary cholangitis (PBC) with "advanced cirrhosis" because of its narrow therapeutic index. We aimed to better define the predicting factors of hepatic serious adverse events (SAEs) and non-response in cirrhotic patients undergoing OCA therapy. METHODS: Safety and efficacy of treatment were evaluated in a cohort of consecutive PBC cirrhotic patients started with OCA. OCA response was evaluated according to the Poise criteria. Risk factors for hepatic SAEs and non-response were reported as risk ratios (RR) with 95% confidence intervals (CIs). RESULTS: One hundred PBC cirrhotics were included, 97 Child-Pugh class A and 3 class B. Thirty-one had oesophageal varices and 5 had a history of ascites. Thirty-three per cent and 32% of patients achieved a biochemical response at 6 and 12 months respectively. Male sex (adjusted-RR 1.75, 95%CI 1.42-2.12), INR (1.37, 1.00-1.87), Child-Pugh score (1.79, 1.28-2.50), MELD (1.17, 1.04-1.30) and bilirubin (1.83, 1.11-3.01) were independently associated with non-response to OCA. Twenty-two patients discontinued OCA within 12 months: 10 for pruritus, 9 for hepatic SAEs (5 for jaundice and/or ascitic decompensation; 4 for upper digestive bleeding). INR (adjusted-RR 1.91, 95%CI 1.10-3.36), lower albumin levels (0.18, 0.06-0.51), Child-Pugh score (2.43, 1.50-4.04), history of ascites (3.5, 1.85-6.5) and bilirubin (1.30, 1.05-1.56), were associated with hepatic SAEs. A total bilirubin≥1.4 mg/dl at baseline was the most accurate biochemical predictor of hepatic SAEs under OCA. CONCLUSIONS: An accurate baseline assessment is crucial to select cirrhotic patients who can benefit from OCA. Although OCA is effective in one third of cirrhotics, bilirubin level ≥1.4 mg/dl should discourage from its use.

Validation of the easy-to-use lenvatinib prognostic index to predict prognosis in advanced hepatocellular carcinoma patients treated with lenvatinib.

AIM: The identification of new prognostic factors able to stratify hepatocellular carcinoma patients candidate to first-line therapy is urgent. In the present work we validated the prognostic value of the lenvatinib prognostic index. METHODS: Data of Eastern and Western patients treated with lenvatinib as first-line for Barcelona Clinic Liver Cancer stage B or C hepatocellular carcinoma were recollected. The lenvatinib prognostic index was composed by three classes of risk according with our previous study. The "low risk" group includes patients with prognostic nutritional index (PNI) >43.3 and with previous transarterial chemoembolization. The "medium risk" group includes patients with PNI >43.3, but without previous transarterial chemoembolization and patients with PNI <43.3, albumin-bilirubin grade 1 and Barcelona Clinic Liver Cancer stage B. The "high risk" group includes patients with PNI <43.3, albumin-bilirubin grade 2, and patients with PNI <43.3, albumin-bilirubin grade 1 and Barcelona Clinic Liver Cancer stage C. RESULTS: A total of 717 patients were included. The median overall survival was 20.7 months (95% CI 16.1-51.6) in patients with low risk (n = 223), 16.7 months (95% CI 13.3-47.0) in patients with medium risk (n = 264), and 10.7 months (95% CI 9.3-12.2) in patients with high risk (n = 230; HR 1, 1.29, and 1.92, respectively; p < 0.0001). Median progression-free survival was 7.3 months (95% CI 6.3-46.5) in patients with low risk, 6.4 months (95% CI 5.3-8.0) in patients with medium risk ,and 4.9 months (95% CI 4.3-5.5) in patients with high risk (HR 1, 1.07, 1.47 respectively; p = 0.0009). CONCLUSION: The lenvatinib prognostic index confirms its prognostic value on an external cohort of hepatocellular carcinoma patients treated with Lenvatinib.

Predictors of solid extra-hepatic non-skin cancer in liver transplant recipients and analysis of survival: A long-term follow-up study.

INTRODUCTION AND OBJECTIVES: De novo malignancies represent an important cause of death for liver transplant recipients. Our aim was to analyze predictors of extra-hepatic non-skin cancer (ESNSC) and the impact of ESNSC on the long-term outcome. PATIENTS: We examined data from patients transplanted between 2000 and 2005 and followed-up in five Italian transplant clinics with a retrospective observational cohort study. Cox Regression was performed to identify predictors of ESNSC. A 1:2 cohort sub-study was developed to analyze the impact of ESNSC on 10-year survival. RESULTS: We analyzed data from 367 subjects (median follow-up: 15 years). Patients with ESNSC (n = 47) more often developed post-LT diabetes mellitus (DM) (57.4% versus 35,9%, p = 0.004). At multivariate analysis, post-LT DM independently predicted ESNSC (HR 1.929, CI 1.029-3.616, p = 0.040). Recipients with ESNSC showed a lower 10-year survival than matched controls (46,8% versus 68,1%, p = 0.023). CONCLUSIONS: Post-LT DM seems to be a relevant risk factor for post-LT ESNSC. ESNSC could have a noteworthy impact on the long-term survival of LT recipients.

Refining the Baveno VI elastography criteria for the definition of compensated advanced chronic liver disease.

BACKGROUND: The Baveno VI consensus proposed a dual liver stiffness (LS) by transient elastography threshold of <10 and >15 kPa for excluding and diagnosing compensated advanced chronic liver disease (cACLD) in the absence of other clinical signs. Herein, we aimed to validate these criteria in a real-world multicentre study. METHODS: We included 5,648 patients (mean age 51 ± 13 years, 53% males) from 10 European liver centres who had a liver biopsy and LS measurement within 6 months. We included patients with chronic hepatitis C (n = 2,913, 52%), non-alcoholic fatty liver disease (NAFLD, n = 1,073, 19%), alcohol-related liver disease (ALD, n = 946, 17%) or chronic hepatitis B (n = 716, 13%). cACLD was defined as fibrosis stage ≥F3. RESULTS: Overall, 3,606 (66%) and 987 (18%) patients had LS <10 and >15 kPa, respectively, while cACLD was histologically confirmed in 1,772 (31%) patients. The cut-offs of <10 and >15 kPa showed 75% sensitivity and 96% specificity to exclude and diagnose cACLD, respectively. Examining the ROC curve, a more optimal dual cut-off at <7 and >12 kPa, with 91% sensitivity and 92% specificity for excluding and diagnosing cACLD (AUC 0.87; 95% CI 0.86-0.88; p <0.001) was derived. Specifically, for ALD and NAFLD, a low cut-off of 8 kPa can be used (sensitivity=93%). For the unclassified patients, we derived a risk model based on common patient characteristics with better discrimination than LS alone (AUC 0.74 vs. 0.69; p <0.001). CONCLUSIONS: Instead of the Baveno VI proposed <10 and >15 kPa dual cut-offs, we found that the <8 kPa (or <7 kPa for viral hepatitis) and >12 kPa dual cut-offs have better diagnostic accuracy in cACLD. LAY SUMMARY: The term compensated advanced chronic liver disease (cACLD) was introduced in 2015 to describe the spectrum of advanced fibrosis and cirrhosis in asymptomatic patients. It was also suggested that cACLD could be diagnosed or ruled out based on specific liver stiffness values, which can be non-invasively measured by transient elastography. Herein, we assessed the suggested cut-off values and identified alternative values that offered better overall accuracy for diagnosing or ruling out cACLD.

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma.

BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.