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.

Prognostic performance of non-invasive tests for portal hypertension is comparable to that of hepatic venous pressure gradient.

BACKGROUND & AIMS: Non-invasive tests to assess the probability of clinically significant portal hypertension (CSPH) - including the ANTICIPATE±NASH models based on liver stiffness measurement and platelet count±BMI, and the von Willebrand factor antigen to platelet count ratio (VITRO) - have fundamentally changed the management of compensated advanced chronic liver disease (cACLD). However, their prognostic utility has not been compared head-to-head to the gold standard for prognostication in cACLD, i.e. the hepatic venous pressure gradient (HVPG). METHODS: Patients with cACLD (liver stiffness measurement ≥10 kPa) who underwent advanced characterization via same-day HVPG/non-invasive test assessment from 2007-2022 were retrospectively included. Long-term follow-up data on hepatic decompensation was recorded. RESULTS: Four hundred and twenty patients with cACLD of varying etiologies, with a CSPH prevalence of 67.6%, were included. The cumulative incidence of hepatic decompensation at 1 and 2 years was 4.7% and 8.0%, respectively. HVPG, VITRO, and ANTICIPATE±NASH-CSPH-probability showed similar time-dependent prognostic value (AUROCs 0.683-0.811 at 1 year and 0.699-0.801 at 2 years). In competing risk analyses adjusted for MELD score and albumin, HVPG (adjusted subdistribution hazard ratio [aSHR] 1.099 [95% CI 1.054-1.150] per mmHg; p <0.001), or VITRO (aSHR 1.134 [95% CI 1.062-1.211] per unit; p <0.001), or ANTICIPATE±NASH-CSPH-probability (aSHR 1.232 [95% CI 1.094-1.387] per 10%; p <0.001) all predicted first decompensation during follow-up. Previously proposed cut-offs (HVPG ≥10 mmHg vs. <10 mmHg, VITRO ≥2.5 vs. <2.5, and ANTICIPATE-CSPH probability ≥60% vs. <60%) all accurately discriminated between patients at negligible risk and those at substantial risk of hepatic decompensation. CONCLUSIONS: The prognostic performance of ANTICIPATE±NASH-CSPH-probability and VITRO is comparable to that of HVPG, supporting their utility for identifying patients who may benefit from medical therapies to prevent first hepatic decompensation. IMPACT AND IMPLICATIONS: Non-invasive tests have revolutionized the diagnosis and management of clinically significant portal hypertension in patients with compensated advanced chronic liver disease (cACLD). However, limited data exists regarding the prognostic utility of non-invasive tests in direct comparison to the gold standard for prognostication in cACLD, i.e. the hepatic venous pressure gradient. In our study including 420 patients with cACLD, the ANTICIPATE±NASH model and VITRO yielded similar AUROCs to hepatic venous pressure gradient for hepatic decompensation within 1 to 2 years. Thus, non-invasive tests should be applied and updated in yearly intervals in clinical routine to identify patients at short-term risk, thereby identifying patients who may benefit from treatment aimed at preventing hepatic decompensation.

Characteristics and outcomes of immunotherapy-related liver injury in patients with hepatocellular carcinoma versus other advanced solid tumours.

BACKGROUND & AIMS: Immune-related liver injury (irLI) is commonly observed in patients with cancer treated with immune checkpoint inhibitors (ICIs). We aimed to compare the incidence, clinical characteristics, and outcomes of irLI between patients receiving ICIs for hepatocellular carcinoma (HCC) vs. other solid tumours. METHODS: Two separate cohorts were included: 375 patients with advanced/unresectable HCC, Child-Pugh A class treated with first-line atezolizumab+bevacizumab from the AB-real study, and a non-HCC cohort including 459 patients treated with first-line ICI therapy from the INVIDIa-2 multicentre study. IrLI was defined as a treatment-related increase of aminotransferase levels after exclusion of alternative aetiologies of liver injury. The incidence of irLI was adjusted for the duration of treatment exposure. RESULTS: In patients with HCC, the incidence of any grade irLI was 11.4% over a median treatment exposure of 4.4 months (95% CI 3.7-5.2) vs. 2.6% in the INVIDIa-2 cohort over a median treatment exposure of 12.4 months (95% CI 11.1-14.0). Exposure-adjusted-incidence of any grade irLI was 22.1 per 100-patient-years in patients with HCC and 2.1 per 100-patient-years in patients with other solid tumours (p <0.001), with median time-to-irLI of 1.4 and 4.7 months, respectively. Among patients who developed irLI, systemic corticosteroids were administered in 16.3% of patients with HCC and 75.0% of those without HCC (p <0.001), and irLI resolution was observed in 72.1% and 58.3%, respectively (p = 0.362). In patients with HCC, rates of hepatic decompensation and treatment discontinuation due to irLI were 7%. Grade 1-2 irLI was associated with improved overall survival only in patients with HCC (hazard ratio 0.53, 95% CI 0.29-0.96). CONCLUSIONS: Despite higher incidence and earlier onset, irLI in patients with HCC is characterised by higher rates of remission and lower requirement for corticosteroid therapy (vs. irLI in other solid tumours), low risk of hepatic decompensation and treatment discontinuation, not negatively affecting oncological outcomes. IMPACT AND IMPLICATIONS: Immune-related liver injury (irLI) is common in patients with cancer receiving immune checkpoint inhibitors (ICIs), but whether irLI is more frequent or it is associated with a worse clinical course in patients with hepatocellular carcinoma (HCC), compared to other tumours, is not known. Herein, we compared characteristics and outcomes of irLI in two prospective cohorts including patients treated with ICIs for HCC or for other oncological indications. irLI is significantly more common and it occurs earlier in patients with HCC, also after adjustment for duration of treatment exposure. However, outcomes of patients with HCC who developed irLI are not negatively affected in terms of requirement for corticosteroid therapy, hepatic decompensation, treatment discontinuation and overall survival.

Von Willebrand Factor Antigen Improves Risk Stratification for Patients with a Diagnosis of Resectable Hepatocellular Carcinoma.

BACKGROUND: Posthepatectomy liver failure (PHLF), complications of portal hypertension, and disease recurrence determine the outcome for hepatocellular carcinoma (HCC) patients undergoing liver resection. This study aimed to evaluate the von Willebrand factor antigen (vWF-Ag) as a non-invasive test for clinically significant portal hypertension (CSPH) and a predictive biomarker for time to recurrence (TTR) and overall survival (OS). METHODS: The study recruited 72 HCC patients with detailed preoperative workup from a prospective trial (NCT02118545) and followed for complications, TTR, and OS. Additionally, 163 compensated patients with resectable HCC were recruited to evaluate vWF-Ag cutoffs for ruling out or ruling in CSPH. Finally, vWF-Ag cutoffs were prospectively evaluated in an external validation cohort of 34 HCC patients undergoing liver resection. RESULTS: In receiver operating characteristic (ROC) analyses, vWF-Ag (area under the curve [AUC], 0.828) was similarly predictive of PHLF as indocyanine green clearance (disappearance rate: AUC, 0.880; retention rate: AUC, 0.894), whereas computation of future liver remnant was inferior (AUC, 0.756). Cox-regression showed an association of vWF-Ag with TTR (per 10%: hazard ratio [HR], 1.056; 95% confidence interval [CI] 1.017-1.097) and OS (per 10%: HR, 1.067; 95% CI 1.022-1.113). In the analyses, VWF-Ag yielded an AUC of 0.824 for diagnosing CSPH, with a vWF-Ag of 182% or lower ruling out and higher than 291% ruling in CSPH. Therefore, a highest-risk group (> 291%, 9.7% of patients) with a 57.1% incidence of PHLF was identified, whereas no patient with a vWF-Ag of 182% or lower (52.7%) experienced PHLF. The predictive value of vWF-Ag for PHLF and OS was externally validated. CONCLUSION: For patients with resectable HCC, VWF-Ag allows for simplified preoperative risk stratification. Patients with vWF-Ag levels higher than 291% might be considered for alternative treatments, whereas vWF-Ag levels of 182% or lower identify patients best suited for surgery.

Artificial intelligence-based pathology as a biomarker of sensitivity to atezolizumab-bevacizumab in patients with hepatocellular carcinoma: a multicentre retrospective study.

BACKGROUND: Clinical benefits of atezolizumab plus bevacizumab (atezolizumab-bevacizumab) are observed only in a subset of patients with hepatocellular carcinoma and the development of biomarkers is needed to improve therapeutic strategies. The atezolizumab-bevacizumab response signature (ABRS), assessed by molecular biology profiling techniques, has been shown to be associated with progression-free survival after treatment initiation. The primary objective of our study was to develop an artificial intelligence (AI) model able to estimate ABRS expression directly from histological slides, and to evaluate if model predictions were associated with progression-free survival. METHODS: In this multicentre retrospective study, we developed a model (ABRS-prediction; ABRS-P), which was derived from the previously published clustering-constrained attention multiple instance learning (or CLAM) pipeline. We trained the model fit for regression analysis using a multicentre dataset from The Cancer Genome Atlas (patients treated by surgical resection, n=336). The ABRS-P model was externally validated on two independent series of samples from patients with hepatocellular carcinoma (a surgical resection series, n=225; and a biopsy series, n=157). The predictive value of the model was further tested in a series of biopsy samples from a multicentre cohort of patients with hepatocellular carcinoma treated with atezolizumab-bevacizumab (n=122). All samples in the study were from adults (aged ≥18 years). The validation sets were sampled between Jan 1, 2008, to Jan 1, 2023. For the multicentre validation set, the primary objective was to assess the association of high versus low ABRS-P values, defined relative to cross-validation median split thresholds in the first biopsy series, with progression-free survival after treatment initiation. Finally, we performed spatial transcriptomics and matched prediction heatmaps with in situ expression profiles. FINDINGS: Of the 840 patients sampled, 641 (76%) were male and 199 (24%) were female. Across the development and validation datasets, hepatocellular carcinoma risk factors included alcohol intake, hepatitis B and C virus infections, and non-alcoholic steatohepatitis. Using cross-validation in the development series, the mean Pearson's correlation between ABRS-P values and ABRS score (mean expression of ABRS genes) was r=0·62 (SD 0·09; mean p<0·0001, SD<0·0001). The ABRS-P generalised well on the external validation series (surgical resection series, r=0·60 [95% CI 0·51-0·68], p<0·0001; biopsy series, r=0·53 [0·40-0·63], p<0·0001). In the 122 patients treated with atezolizumab-bevacizumab, those with ABRS-P-high tumours (n=74) showed significantly longer median progression-free survival than those with ABRS-P-low tumours (n=48) after treatment initiation (12 months [95% CI 7-not reached] vs 7 months [4-9]; p=0·014). Spatial transcriptomics showed significantly higher ABRS score, along with upregulation of various other immune effectors, in tumour areas with high ABRS-P values versus areas with low ABRS-P values. INTERPRETATION: Our study indicates that AI applied on hepatocellular carcinoma digital slides is able to serve as a biomarker for progression-free survival in patients treated with atezolizumab-bevacizumab. This approach could be used in the development of inexpensive and fast biomarkers for targeted therapies. The combination of AI heatmaps with spatial transcriptomics provides insight on the molecular features associated with predictions. This methodology could be applied to other cancers or diseases and improve understanding of the biological mechanisms that drive responses to treatments. FUNDING: Institut National du Cancer, Fondation ARC, China Scholarship Council, Ligue Contre le Cancer du Val de Marne, Fondation de l'Avenir, Ipsen, and Fondation Bristol Myers Squibb Pour la Recherche en Immuno-Oncologie.

Carvedilol Achieves Higher Hemodynamic Response and Lower Rebleeding Rates Than Propranolol in Secondary Prophylaxis.

BACKGROUND & AIMS: Carvedilol induces stronger decreases in hepatic venous pressure gradient (HVPG) than conventional nonselective ß-blockers (ie, propranolol). Limited data exist on the efficacy of carvedilol in secondary prophylaxis of variceal bleeding. METHODS: Patients undergoing paired HVPG measurements for guiding secondary prophylaxis with either carvedilol or propranolol were included in this retrospective analysis. All patients also underwent band ligation. Changes in HVPG and systemic hemodynamics were compared between the 2 groups. Long-term follow-up data on rebleeding, acute kidney injury, nonbleeding decompensation, and liver-related death were analyzed applying competing risk regression. RESULTS: Eighty-seven patients (carvedilol/propranolol, n = 45/42) were included in our study. The median baseline HVPG was 21 mm Hg (interquartile range, 18-24 mm Hg), and 39.1%/48.3%/12.6% had Child-Turcotte-Pugh A/B/C cirrhosis, respectively. Upon nonselective ß-blocker initiation, HVPG decreased more strongly in carvedilol users (median relative decrease, -20% [interquartile range: -29% to -10%] vs -11% [-22% to -5%] for propranolol; P = .027), who also achieved chronic HVPG response more often (53.3% vs 28.6%; P = .034). Cumulative incidences for rebleeding (Gray test, P = .027) and liver-related death (P = .036) were significantly lower in patients taking carvedilol compared with propranolol. Notably, ascites development/worsening also was observed less commonly in carvedilol patients (P = .012). Meanwhile, acute kidney injury rates did not differ between the 2 groups (P = .255). Stratifying patients by HVPG response status yielded similar results. The prognostic value of carvedilol intake was confirmed in competing risk regression models. CONCLUSIONS: Carvedilol induces more marked reductions in HVPG than propranolol in secondary prophylaxis of variceal bleeding, and thus is associated with lower rates of rebleeding, liver-related death, and further nonbleeding decompensation.

The Sequential Application of Baveno VII Criteria and VITRO Score Improves Diagnosis of Clinically Significant Portal Hypertension.

BACKGROUND & AIMS: Baveno VII proposed liver stiffness measurement (LSM)/platelet count (PLT)-based criteria ('ruled out,' LSM ≤15 kPa plus PLT ≥150 G/L; 'ruled in': LSM ≥25 kPa) for clinically significant portal hypertension (CSPH) in compensated advanced chronic liver disease (cACLD). However, a substantial proportion of patients remains 'unclassified.' METHODS: Patients with evidence of cACLD (LSM ≥10 kPa) undergoing hepatic venous pressure gradient (HVPG) measurement at the Vienna General Hospital 2004 to 2021 (derivation [2004-2016], n = 221; validation [2017-2021], n = 81) were included. The performance of noninvasive tests (NITs) including von Willebrand factor antigen to PLT ratio (VITRO) for the detection of CSPH (HVPG ≥10 mmHg) were evaluated. RESULTS: Overall, viral hepatitis was the predominant (50.7%) etiology, followed by alcoholic liver disease (15.2%) and nonalcoholic steatohepatitis (13.2%); CSPH prevalence was 62.3%. In the derivation cohort, 45.7% were 'unclassified' according to Baveno VII criteria; in this group, VITRO showed an excellent diagnostic performance for the detection of CSPH (area under the receiver operating curve, 0.909; 95% confidence interval, 0.823-0.965). VITRO ≤1.5 and ≥2.5 ruled out (sensitivity, 97.7%; negative predictive value, 97.5%) and ruled in (specificity, 94.7%; positive predictive value, 91.2%), respectively, CSPH in patients who were 'unclassifiable' by Baveno VII criteria. The application of a sequential Baveno VII-VITRO algorithm reallocated 73% and 70% of 'unclassified' patients to the 'ruled in' and 'ruled out' group, respectively, while maintaining high sensitivity and negative predictive value and specificity and positive predictive value in the derivation and validation cohort, respectively. No patient allocated to the 'CSPH ruled out' group by the Baveno VII-VITRO algorithm developed decompensation within 5 years, whereas 5-year decompensation rates were negligible (4%) and substantial (23.9%) among 'unclassified' and 'CSPH ruled in' patients, respectively. CONCLUSIONS: The sequential application of VITRO in patients with cACLD who were 'unclassifiable' with regard to CSPH by Baveno VII criteria substantially decreased the number of 'unclassifiable' patients to <15% and refined prognostication.

Preliminary evidence of safety and tolerability of atezolizumab plus bevacizumab in patients with hepatocellular carcinoma and Child-Pugh A and B cirrhosis: A real-world study.

BACKGROUND AND AIMS: Atezolizumab plus bevacizumab (AtezoBev) is the standard of care for first-line treatment of unresectable HCC. No evidence exists as to its use in routine clinical practice in patients with impaired liver function. APPROACH AND RESULTS: In 216 patients with HCC who were consecutively treated with AtezoBev across 11 tertiary centers, we retrospectively evaluated treatment-related adverse events (trAEs) graded (G) according to Common Terminology Criteria for Adverse Events v5.0, including in the analysis all patients treated according to label (n = 202, 94%). We also assessed overall survival (OS), progression-free survival (PFS), overall response (ORR), and disease control rates (DCR) defined by Response Evaluation Criteria in Solid Tumors v1.1. Disease was mostly secondary to viral hepatitis, namely hepatitis C (n = 72; 36%) and hepatitis B infection (n = 35, 17%). Liver function was graded as Child-Pugh (CP)-A in 154 patients (76%) and CP-B in 48 (24%). Any grade trAEs were reported by 143 patients (71%), of which 53 (26%) were G3 and 3 (2%) G4. Compared with CP-A, patients with CP-B showed comparable rates of trAEs. Presence and grade of varices at pretreatment esophagogastroduodenoscopy did not correlate with bleeding events. After a median follow-up of 9.0 months (95% CI, 7.8-10.1), median OS was 14.9 months (95% CI, 13.6-16.3), whereas median PFS was 6.8 months (95% CI, 5.2-8.5). ORR and DCR were respectively 25% and 73%, with no difference across CP classes. CONCLUSIONS: This study confirms reproducible safety and efficacy of AtezoBev in routine practice. Patients with CP-B reported similar tolerability compared with CP-A, warranting prospective evaluation of AtezoBev in this treatment-deprived population.

Progression patterns and therapeutic sequencing following immune checkpoint inhibition for hepatocellular carcinoma: An international observational study.

BACKGROUND AND AIMS: Different approaches are available after the progression of disease (PD) to immune checkpoint inhibitors (ICIs) for hepatocellular carcinoma (HCC), including the continuation of ICI, treatment switching to tyrosine kinase inhibitors (TKIs) and cessation of anticancer therapy. We sought to characterise the relationship between radiological patterns of progression and survival post-ICI, also appraising treatment strategies. METHODS: We screened 604 HCC patients treated with ICIs, including only those who experienced PD by data cut-off. We evaluated post-progression survival (PPS) according to the treatment strategy at PD and verified its relationship with radiological patterns of progression: intrahepatic growth (IHG), new intrahepatic lesion (NIH), extrahepatic growth (EHG), new extrahepatic lesion (NEH) and new vascular invasion (nVI). RESULTS: Of 604 patients, 364 (60.3%) experienced PD during observation. Median PPS was 5.3 months (95% CI: 4.4-6.9; 271 events). At the data cut-off, 165 patients (45%) received no post-progression anticancer therapy; 64 patients (17.6%) continued ICI beyond PD. IHG (HR 1.64 [95% CI: 1.21-2.22]; p = .0013) and nVI (HR 2.15 [95% CI: 1.38-3.35]; p = .0007) were associated with shorter PPS. Multivariate models adjusted for progression patterns, treatment line and albumin-bilirubin grade and Eastern Cooperative Oncology Group performance status at PD confirmed receipt of ICI beyond PD with (HR 0.17, 95% CI: 0.09-0.32; p < .0001) or without subsequent TKI (HR 0.39, 95% CI: 0.26-0.58; p < .0001) as predictors of prolonged PPS versus no anticancer therapy. CONCLUSIONS: ICI-TKI sequencing is a consolidated option in advanced HCC. nVI and IHG predict a poorer prognosis. Despite lack of recommendation, the continuation of ICI beyond progression in HCC is adopted clinically: future efforts should appraise which patients benefit from this approach.

A bilateral tumor model identifies transcriptional programs associated with patient response to immune checkpoint blockade.

Immune checkpoint blockade (ICB) is efficacious in many diverse cancer types, but not all patients respond. It is important to understand the mechanisms driving resistance to these treatments and to identify predictive biomarkers of response to provide best treatment options for all patients. Here we introduce a resection and response-assessment approach for studying the tumor microenvironment before or shortly after treatment initiation to identify predictive biomarkers differentiating responders from nonresponders. Our approach builds on a bilateral tumor implantation technique in a murine metastatic breast cancer model (E0771) coupled with anti-PD-1 therapy. Using our model, we show that tumors from mice responding to ICB therapy had significantly higher CD8+ T cells and fewer Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) at early time points following therapy initiation. RNA sequencing on the intratumoral CD8+ T cells identified the presence of T cell exhaustion pathways in nonresponding tumors and T cell activation in responding tumors. Strikingly, we showed that our derived response and resistance signatures significantly segregate patients by survival and associate with patient response to ICB. Furthermore, we identified decreased expression of CXCR3 in nonresponding mice and showed that tumors grown in Cxcr3-/- mice had an elevated resistance rate to anti-PD-1 treatment. Our findings suggest that the resection and response tumor model can be used to identify response and resistance biomarkers to ICB therapy and guide the use of combination therapy to further boost the antitumor efficacy of ICB.

Targeting DNA repair to enhance the efficacy of sorafenib in hepatocellular carcinoma.

The multityrosine kinase inhibitor sorafenib remains an important systemic treatment option for hepatocellular carcinoma (HCC). Signaling pathways, which are targeted by sorafenib, are involved in checkpoint and DNA repair response, RAD51 being a candidate protein. Here, we aim to evaluate the effect of the human RAD51 inhibitor B02 in combination with sorafenib in human HCC cells. Impact of RAD51 expression on HCC patient survival was evaluated by an in silico approach using Human Protein Atlas dataset. Cell viability of HUH7, AKH12, AKH13, and 3P was assessed by neutral red assay. To measure the cytotoxicity, we quantified loss of membrane integrity by lactate dehydrogenase release. We also employed colony formation assay and hanging drop method to assess clonogenic and invasive ability of HCC cell lines upon sorafenib and B02 treatment. Cell cycle distribution and characterization of apoptosis was evaluated by flow cytometry. In silico approach revealed that HCC patients with higher expression of RAD51 messenger RNA had a significantly shorter overall survival. The RAD51 inhibitor B02 alone and in combination with sorafenib significantly reduced viability, colony formation ability, and invasion capacity of HCC cells. Cell cycle analysis revealed that the combination of both agents reduces the proportion of cells in the G2/M phase while leading to an accumulating in the subG1 phase. The RAD51 inhibitor B02 seems to be a promising agent for HCC treatment and enhances the antitumor effects of sorafenib in vitro.

Factor VIII/protein C ratio independently predicts liver-related events but does not indicate a hypercoagulable state in ACLD.

BACKGROUND & AIMS: It has been suggested that the ratio of procoagulant factor VIII to anticoagulant protein C (FVIII/PC) reflects the hemostatic equilibrium. Moreover, FVIII/PC predicted decompensation/death in a small study not accounting for portal hypertension severity. We investigated (i) the prognostic value of FVIII/PC (outcome-cohort) and (ii) whether FVIII/PC reflects the hypercoagulable state (assessed by thrombomodulin-modified thrombin generation assay [TM-TGA]) or the risk of bleeding/thrombotic events in patients undergoing hepatic venous pressure gradient (HVPG) measurement during follow-up. METHODS: (i) The outcome-cohort comprised 576 patients with evidence of advanced chronic liver disease (liver stiffness measurement ≥10 kPa and/or HVPG ≥6 mmHg). (ii) TM-TGA-cohort patients (n = 142) were recruited from the prospective VIenna CIrrhosis Study (VICIS: NCT03267615). RESULTS: (i) FVIII/PC significantly increased across clinical stages (p <0.001) as well as HVPG (p <0.001) and MELD score (p <0.001) strata and remained independently associated with decompensation/liver-related death (adjusted hazard ratio 1.06; 95% CI 1.01-1.11; p = 0.013), even after multivariable adjustment. It was also associated with acute-on-chronic liver failure (ACLF) development (adjusted hazard ratio 1.10; 95% CI 1.02-1.19; p = 0.015) in patients with decompensated cirrhosis. (ii) FVIII/PC showed a weak positive correlation with endogenous thrombin potential (Spearman's ρ = 0.255; p = 0.002), but this association disappeared after adjusting for the severity of liver disease. FVIII/PC was not associated with the development of bleeding (p = 0.272) or thrombotic events (p = 0.269). However, FVIII/PC correlated with biomarkers of different pathophysiological mechanisms that promote liver disease progression. CONCLUSION: FVIII/PC provides prognostic information regarding hepatic decompensation/death and ACLF, independently of established prognostic indicators. However, this is not evidence that hypercoagulability drives disease progression, as the correlation between FVIII/PC and thrombin generation is confounded by liver disease severity and FVIII/PC was not associated with thrombosis. Therefore, FVIII/PC does not reflect coagulation and results from previous studies on FVIII/PC require re-interpretation. CLINICAL TRIAL NUMBER: NCT03267615 (in part). LAY SUMMARY: A balanced coagulation system is essential for preventing bleeding episodes and blood clot formation (thrombosis). Blood of patients with advanced liver disease may have increased coagulation potential, possibly promoting the worsening of liver disease via thrombosis in the blood vessels of the liver. The ratio between the results of 2 blood tests (procoagulant factor VIII to anticoagulant protein C) has been suggested to reflect these increases in coagulation potential. Our study demonstrates, on the one hand, that this ratio is a versatile predictor of the development of complications of cirrhosis, yet on the other hand, that it is unrelated to coagulation.

HCC risk stratification after cure of hepatitis C in patients with compensated advanced chronic liver disease.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a major cause of morbidity and mortality in patients with advanced chronic liver disease (ACLD) caused by chronic hepatitis C who have achieved sustained virologic response (SVR). We developed risk stratification algorithms for de novo HCC development after SVR and validated them in an independent cohort. METHODS: We evaluated the occurrence of de novo HCC in a derivation cohort of 527 patients with pre-treatment ACLD and SVR to interferon-free therapy, in whom alpha-fetoprotein (AFP) and non-invasive surrogates of portal hypertension including liver stiffness measurement (LSM) were assessed pre-/post-treatment. We validated our results in 1,500 patients with compensated ACLD (cACLD) from other European centers. RESULTS: During a median follow-up (FU) of 41 months, 22/475 patients with cACLD (4.6%, 1.45/100 patient-years) vs. 12/52 decompensated patients (23.1%, 7.00/100 patient-years, p <0.001) developed de novo HCC. Since decompensated patients were at substantial HCC risk, we focused on cACLD for all further analyses. In cACLD, post-treatment-values showed a higher discriminative ability for patients with/without de novo HCC development during FU than pre-treatment values or absolute/relative changes. Models based on post-treatment AFP, alcohol consumption (optional), age, LSM, and albumin, accurately predicted de novo HCC development (bootstrapped Harrel's C with/without considering alcohol: 0.893/0.836). Importantly, these parameters also provided independent prognostic information in competing risk analysis and accurately stratified patients into low- (~2/3 of patients) and high-risk (~1/3 of patients) groups in the derivation (algorithm with alcohol consumption; 4-year HCC-risk: 0% vs. 16.5%) and validation (3.3% vs. 17.5%) cohorts. An alternative approach based on alcohol consumption (optional), age, LSM, and albumin (i.e., without AFP) also showed a robust performance. CONCLUSIONS: Simple algorithms based on post-treatment age/albumin/LSM, and optionally, AFP and alcohol consumption, accurately stratified patients with cACLD based on their risk of de novo HCC after SVR. Approximately two-thirds were identified as having an HCC risk <1%/year in both the derivation and validation cohort, thereby clearly falling below the cost-effectiveness threshold for HCC surveillance. LAY SUMMARY: Simple algorithms based on age, alcohol consumption, results of blood tests (albumin and α-fetoprotein), as well as liver stiffness measurement after the end of hepatitis C treatment identify a large proportion (approximately two-thirds) of patients with advanced but still asymptomatic liver disease who are at very low risk (<1%/year) of liver cancer development, and thus, might not need to undergo 6-monthly liver ultrasound.

Prognosis of patients with hepatocellular carcinoma treated with immunotherapy - development and validation of the CRAFITY score.

BACKGROUND & AIMS: Immunotherapy with atezolizumab plus bevacizumab represents the new standard of care in systemic front-line treatment of hepatocellular carcinoma (HCC). However, biomarkers that predict treatment success and survival remain an unmet need. METHODS: Patients with HCC put on PD-(L)1-based immunotherapy were included in a training set (n = 190; 6 European centers) and a validation set (n = 102; 8 European centers). We investigated the prognostic value of baseline variables on overall survival using a Cox model in the training set and developed the easily applicable CRAFITY (CRP and AFP in ImmunoTherapY) score. The score was validated in the independent, external cohort, and evaluated in a cohort of patients treated with sorafenib (n = 204). RESULTS: Baseline serum alpha-fetoprotein ≥100 ng/ml (hazard ratio [HR] 1.7; p = 0.007) and C-reactive protein ≥1 mg/dl (HR, 1.7; p = 0.007) were identified as independent prognostic factors in multivariable analysis and were used to develop the CRAFITY score. Patients who fulfilled no criterion (0 points; CRAFITY-low) had the longest median overall survival (27.6 (95% CI 19.5-35.8) months), followed by those fulfilling 1 criterion (1 point; CRAFITY-intermediate; 11.3 (95% CI 8.0-14.6) months), and patients meeting both criteria (2 points; CRAFITY-high; 6.4 (95% CI 4.8-8.1) months; p <0.001). Additionally, best radiological response (complete response/partial response/stable disease/progressive disease) was significantly better in patients with lower CRAFITY score (CRAFITY-low: 9%/20%/52%/20% vs. CRAFITY-intermediate: 3%/25%/36%/36% vs. CRAFITY-high: 2%/15%/22%/61%; p = 0.003). These results were confirmed in the independent validation set and in different subgroups, including Child-Pugh A and B, performance status 0 and ≥1, and first-line and later lines. In the sorafenib cohort, CRAFITY was associated with survival, but not radiological response. CONCLUSIONS: The CRAFITY score is associated with survival and radiological response in patients receiving PD-(L)1 immunotherapy. The score may help with patient counseling but requires prospective validation. LAY SUMMARY: The immunotherapy-based regimen of atezolizumab plus bevacizumab represents the new standard of care in systemic first-line therapy of hepatocellular carcinoma (HCC). Biomarkers to predict treatment outcome are an unmet need in patients undergoing immunotherapy for HCC. We developed and externally validated a score that predicts outcome in patients with HCC undergoing immunotherapy with immune checkpoint blockers.

Decreasing von Willebrand Factor Levels Upon Nonselective Beta Blocker Therapy Indicate a Decreased Risk of Further Decompensation, Acute-on-chronic Liver Failure, and Death.

BACKGROUND & AIMS: Nonselective beta blockers (NSBBs) exert beneficial effects beyond lowering hepatic venous pressure gradient (HVPG), which may be particularly relevant in patients with decompensated cirrhosis (DC), in whom bacterial translocation and bacterial-induced systemic inflammation drive the development of complications such as acute-on-chronic liver failure (ACLF). We evaluated whether NSBB-related changes in von Willebrand factor (VWF) may serve as a biomarker for these effects. METHODS: In this retrospective analysis, 159 prospectively characterized patients with clinically stable DC (ie, without acute decompensation) who underwent paired HVPG/VWF assessments before/on NSBB therapy were classified as 'VWF-responders' (as defined by a ≥5% decrease in VWF) versus 'VWF-non-responders.' RESULTS: There were no major differences in baseline characteristics between VWF-responders (61%) and VWF-non-responders. VWF-responders showed more pronounced decreases in inflammation (procalcitonin), whereas rates of HVPG-response were similar. In line, NSBB-related changes in VWF correlated with the dynamics of bacterial translocation/inflammation (lipopolysaccharide-binding protein, C-reactive protein, and procalcitonin), rather than those of HVPG. Interestingly, VWF-responders also showed less pronounced NSBB-related decreases in mean arterial pressure, suggesting an amelioration of systemic vasodilatation. Finally, VWF-response was associated with decreased risks of further decompensation (adjusted hazard ratio [aHR], 0.555; 95% confidence interval [CI], 0.337-0.912; P = .020), ACLF (aHR, 0.302; 95% CI, 0.126-0.721; P = .007), and liver-related death (aHR, 0.332; 95% CI, 0.179-0.616; P < .001) in Cox regression models adjusted for prognostic factors including changes in HVPG. CONCLUSIONS: Decreases in VWF upon NSBB therapy reflect their anti-inflammatory activity, are accompanied by less pronounced adverse effects on systemic hemodynamics, and are independently associated with a decreased risk of further decompensation, ACLF, and death. VWF-response may discriminate between decompensated patients who benefit from NSBB treatment and have a favorable prognosis versus patients with poor outcomes.

Clinical Course of Porto-Sinusoidal Vascular Disease Is Distinct From Idiopathic Noncirrhotic Portal Hypertension.

BACKGROUND & AIMS: Porto-sinusoidal vascular disease (PSVD) was recently proposed as novel clinical entity characterized by typical histological changes with or without portal hypertension (PH) in the absence of cirrhosis. Thus, we aimed to describe clinical characteristics and the outcome of PSVD patients and to compare these to patients meeting traditional idiopathic non-cirrhotic portal hypertension (INCPH) criteria. METHODS: Patients undergoing liver biopsy (baseline) ±hepatic venous pressure gradient (HVPG) measurement at the Vienna General Hospital between 2000-2019 were screened for PSVD and INCPH criteria. RESULTS: 91 patients were diagnosed with PSVD of which 28 (30.8%) also fulfilled INCPH criteria (INCPH+/PSVD+). Specific histological and specific clinical PH signs were found in 72 (79.1%) and 54 (59.3%) patients, respectively. INCPH+/PSVD+ showed higher Child-Pugh-scores (7±2 vs 6±1 points; P = .002) and a higher prevalence of decompensation (57.1% vs 28.6%; P = .009) than INCPH-/PSVD+ patients. Importantly, hepatic decompensation after three years (3Y) occurred in 11.2% of PSVD patients with specific clinical signs of PH, while no decompensation occurred in patients with only specific histological or with unspecific clinical/histological signs (P = .002). When categorizing by INCPH definition, 3Y decompensation was 13.4% in INCPH+/PSVD+ and 3.8% in INCPH-/PSVD+ (P = .120). While overall mortality was similar in INCPH+/PSVD+ (n = 6; 21.4%) and INCPH-/PSVD+ (n = 10; 15.9%) patients (P = .558), liver-related mortality tended to be higher in INCPH+/PSVD+ (6.9%) than in INCPH-/PSVD+ (0%; P = .078). CONCLUSION: Novel PSVD criteria facilitate diagnosis. Compared to INCPH, clinical course of PSVD patients is more favorable. Importantly, specific signs of PH including varices and collaterals are associated with hepatic decompensation and mortality.

Noninvasive Risk Stratification After HCV Eradication in Patients With Advanced Chronic Liver Disease.

BACKGROUND AND AIMS: Risk stratification after cure from hepatitis C virus (HCV) infection remains a clinical challenge. We investigated the predictive value of noninvasive surrogates of portal hypertension (liver stiffness measurement [LSM] by vibration-controlled transient elastography and von Willebrand factor/platelet count ratio [VITRO]) for development of hepatic decompensation and hepatocellular carcinoma in patients with pretreatment advanced chronic liver disease (ACLD) who achieved HCV cure. APPROACH AND RESULTS: A total of 276 patients with pretreatment ACLD and information on pretreatment and posttreatment follow-up (FU)-LSM and FU-VITRO were followed for a median of 36.6 months after the end of interferon-free therapy. FU-LSM (area under the receiver operating characteristic curve [AUROC]: 0.875 [95% confidence interval [CI]: 0.796-0.954]) and FU-VITRO (AUROC: 0.925 [95% CI: 0.874-0.977]) showed an excellent predictive performance for hepatic decompensation. Both parameters provided incremental information and were significantly associated with hepatic decompensation in adjusted models. A previously proposed combined approach (FU-LSM < 12.4 kPa and/or FU-VITRO < 0.95) to rule out clinically significant portal hypertension (CSPH, hepatic venous pressure gradient ≥10 mm Hg) at FU assigned most (57.3%) of the patients to the low-risk group; none of these patients developed hepatic decompensation. In contrast, in patients in whom FU-CSPH was ruled in (FU-LSM > 25.3 kPa and/or FU-VITRO > 3.3; 25.0% of patients), the risk of hepatic decompensation at 3 years following treatment was high (17.4%). Patients within the diagnostic gray-zone for FU-CSPH (17.8% of patients) had a very low risk of hepatic decompensation during FU (2.6%). The prognostic value of this algorithm was validated in an internal (n = 86) and external (n = 162) cohort. CONCLUSION: FU-LSM/FU-VITRO are strongly and independently predictive of posttreatment hepatic decompensation in HCV-induced ACLD. An algorithm combining these noninvasive markers not only rules in or rules out FU-CSPH, but also identifies populations at negligible versus high risk for hepatic decompensation. FU-LSM/FU-VITRO are readily accessible and enable risk stratification after sustained virological response, and thus facilitate personalized management.

Systemic inflammation is linked to liver fibrogenesis in patients with advanced chronic liver disease.

BACKGROUND & AIMS: Experimental evidence indicates that systemic inflammation (SI) promotes liver fibrogenesis. This study investigated the potential link between SI and fibrogenesis in patients with advanced chronic liver disease (ACLD). METHODS: Serum biomarkers of SI (CRP, IL-6, procalcitonin [PCT]) and extracellular matrix (ECM) turnover (i.e., fibrogenesis/fibrolysis) were analysed in 215 prospectively recruited patients with ACLD (hepatic venous pressure gradient [HVPG] ≥6 mm Hg) undergoing hepatic vein catheterization. Patients with non-elective hospitalization or bacterial infection were excluded. Histological alpha-smooth muscle actin (α-SMA) area was quantified on full biopsy scans by automated morphometric quantification in a subset of 34 patients who underwent concomitant transjugular liver biopsy. RESULTS: Histological α-SMA proportionate area correlated with enhanced liver fibrosis (ELF) score (Spearman's ρ = 0.660, p < .001), markers of collagen formation (PRO-C3, ρ = 0.717, p < .001; PRO-C6, ρ = 0.526, p = .002) and tissue inhibitor of metalloproteinases-1 (TIMP1; ρ = 0.547, p < .001), indicating that these blood biomarkers are capable of reflecting the dynamic process of ECM turnover. CRP, IL-6 and PCT levels correlated with ELF, biomarkers of collagen synthesis/degradation and TIMP1, both in compensated and decompensated patients. Multivariate linear regression models (adjusted for HVPG) confirmed that CRP, IL-6 and PCT were independently linked to markers of liver fibrogenesis and ECM turnover. CONCLUSION: Systemic inflammation is linked to both liver fibrogenesis and ECM turnover in ACLD and this association is not confounded by the severity of liver disease, as evaluated by HVPG. Our study confirms experimental data on the detrimental impact of SI on ECM deposition and fibrosis progression in a thoroughly characterized cohort of patients with ACLD.

Impact of older age in patients receiving atezolizumab and bevacizumab for hepatocellular carcinoma.

BACKGROUND AND AIMS: Combination atezolizumab/bevacizumab is the gold standard for first-line treatment of unresectable hepatocellular carcinoma (HCC). Our study investigated the efficacy and safety of combination therapy in older patients with HCC. METHODS: 191 consecutive patients from eight centres receiving atezolizumab and bevacizumab were included. Overall survival (OS), progression-free survival (PFS), overall response rate (ORR) and disease control rate (DCR) defined by RECIST v1.1 were measured in older (age ≥ 65 years) and younger (age < 65 years) age patients. Treatment-related adverse events (trAEs) were evaluated. RESULTS: The elderly (n = 116) had higher rates of non-alcoholic fatty liver disease (19.8% vs. 2.7%; p < .001), presenting with smaller tumours (6.2 cm vs 7.9 cm, p = .02) with less portal vein thrombosis (31.9 vs. 54.7%, p = .002), with fewer patients presenting with BCLC-C stage disease (50.9 vs. 74.3%, p = .002). There was no significant difference in OS (median 14.9 vs. 15.1 months; HR 1.15, 95% CI 0.65-2.02 p = .63) and PFS (median 7.1 vs. 5.5 months; HR 1.11, 95% CI 0.54-1.92; p = .72) between older age and younger age. Older patients had similar ORR (27.6% vs. 20.0%; p = .27) and DCR (77.5% vs. 66.1%; p = .11) compared to younger patients. Atezolizumab-related (40.5% vs. 48.0%; p = .31) and bevacizumab-related (44.8% vs. 41.3%; p = .63) trAEs were comparable between groups. Rates of grade ≥3 trAEs and toxicity-related treatment discontinuation were similar between older and younger age patients. Patients 75 years and older had similar survival and safety outcomes compared to younger patients. CONCLUSIONS: Atezolizumab and bevacizumab therapy is associated with comparable efficacy and tolerability in older age patients with unresectable HCC.

PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis.

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi-protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome-induced p53-activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro-tumorigenic effect of PIDDosome-mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence-free survival in HCC patients.