Diagnostic accuracy of AGILE 3+ score for advanced fibrosis in patients with NAFLD: A systematic review and meta-analysis.

BACKGROUND AND AIMS: A simple noninvasive score, the Agile 3+ score, combining liver stiffness measurement, aspartate aminotransferase/alanine aminotransferase ratio, platelet count, diabetes status, sex, and age, has been proposed for the identification of advanced fibrosis in patients with suspected NAFLD. We performed a systematic review and meta-analysis of observational studies to evaluate the diagnostic accuracy of the Agile 3+ score in identifying patients with NAFLD and advanced fibrosis. Recently, an International consensus changed the nomenclature of NAFLD into metabolic-associated steatotic liver disease, so currently, the two terms are interchangeable. APPROACH AND RESULTS: We systematically searched MEDLINE, Ovid Embase, Scopus, and Cochrane Library electronic databases for full-text published articles in any language from the inception to the April 24, 2023. We included original articles reporting data on the sensitivity and specificity of the Agile 3+ score, according to previously described rule-out (≤ 0.451) and rule-in (≥ 0.679) cutoffs. We included 6 observational studies (total of 6955 participants) with biopsy-proven NAFLD [mean age 53 (SE 4) years, mean body mass index 30.9 (SE 2.3) kg/m 2 , 54.0% men, prevalence of diabetes 59.6%]. The pooled prevalence of advanced fibrosis (≥ F3) was 42.1%. By the rule-out cutoff, the overall sensitivity and specificity were 88% (95% CI: 81-93%; I2 = 89.2%) and 65% (95% CI: 54-75%; I2 = 97.6%), respectively. By the rule-in cutoff, the overall sensitivity and specificity were 68% (95% CI: 57-78%; I2 =91.1%) and 87% (95% CI: 80%-92%; I2 =96.7%), respectively. Meta-regression analyses reported that the diagnostic accuracy was partly mediated by age ( p < 0.01), body mass index ( p < 0.01), and, although not statistically significant, sex ( p = 0.06). CONCLUSIONS: Our systematic review and meta-analysis suggests that Agile 3+ accurately diagnoses NAFLD with advanced fibrosis and can identify patients eligible for biopsy and emerging pharmacotherapies.

Risk of liver-related events in metabolic dysfunction-associated steatohepatitis (MASH) patients with fibrosis: A comparative analysis of various risk stratification criteria.

BACKGROUND AND AIMS: International regulatory agencies recommend testing drug therapy for patients with noncirrhotic high-risk metabolic dysfunction-associated steatohepatitis (MASH) because they are at risk of liver-related events (LRE). We aimed to compare the risk of LRE in patients with MASLD stratified for F2-F4 fibrosis and MASH. APPROACH AND RESULTS: Overall, 1938 consecutive patients with biopsy-proven MASLD were enrolled. High-risk MASH was defined as MASH with F2-F4 fibrosis. LSM was measured by transient elastography. LRE were recorded during follow-up. Cox multivariate models were used to assess the association between high-risk MASH or F2-F4 fibrosis without MASH, of LSM (≥8 or ≥10 Kpa), and of AGILE 3+ with LRE. The diagnostic performance for the prediction of LRE was assessed using the area under the receiver operating characteristic curves. The observed 5-year actuarial rate of LRE was 0.4%, 0.2%, 5.1%, and 6.6% in patients with F0-F1 fibrosis without MASH, F0-F1 fibrosis with MASH, F2-F4 fibrosis without MASH, and high-risk MASH, respectively. At multivariate Cox regression analysis using F0-F1 fibrosis without MASH as a reference, both F2-F4 fibrosis without MASH [adjusted HR (aHR) 9.96] and high-risk MASH (aHR 10.14) were associated with LRE. In the 1074 patients with available LSM, LSM ≥ 10 kPa (aHR 6.31) or AGILE 3+ > 0.67 (aHR 27.45) independently predicted the development of LRE and had similarly acceptable 5-year area under the receiver operating characteristic to high-risk MASH and F2-F4 fibrosis (0.772, 0.818, 0.739, and 0.780, respectively). CONCLUSIONS: The risk of LRE is similar in patients with high-risk MASH and with F2-F4 fibrosis without MASH. The use of LSM ≥ 10 kPa or AGILE 3+ > 0.67 could be an accurate option to identify patients with MASLD worthy to be included in clinical trials.

ANGPTL3 Downregulation Increases Intracellular Lipids by Reducing Energy Utilization.

BACKGROUND: ANGPTL3 (angiopoietin-like protein 3) is a circulating protein with a key role in maintaining lipoprotein homeostasis. A monoclonal antibody against ANGPTL3 is an approved and well-tolerated treatment to reduce lipoproteins in familial hypercholesterolemia homozygotes. However, the reduction of hepatic ANGPTL3 synthesis using an antisense oligonucleotide unexpectedly resulted in a dose-dependent increase in liver lipid content and circulating transaminases, resulting in the termination of the clinical trial. Meanwhile, the use of silencing RNAs remains an area of active investigation. Our study sought to investigate whether intracellular downregulation of ANGPTL3 may lead to a primary increase in neutral lipids within the hepatocyte. METHODS: We downregulated ANGPTL3 by silencing RNA in primary human hepatocytes 3-dimensional spheroids, HepG2/LX-2 3-dimensional spheroids, and in HepG2, Hep3B2, and Huh7 cultured in 2 dimensions. RESULTS: ANGPTL3 downregulation increased neutral lipids in all models investigated. Interestingly, ANGPTL3 induced lower intracellular deiodinase type 1 protein levels resulting in a reduction in beta-oxidation and causing an increase in triglycerides stored in lipid droplets. CONCLUSIONS: In conclusion, intracellular ANGPTL3 downregulation by silencing RNA led to an increase in triglycerides content due to a reduction in energy substrate utilization resembling a primary intracellular hepatocyte hypothyroidism.

Serum ferritin levels can predict long-term outcomes in patients with metabolic dysfunction-associated steatotic liver disease.

OBJECTIVE: Hyperferritinaemia is associated with liver fibrosis severity in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), but the longitudinal implications have not been thoroughly investigated. We assessed the role of serum ferritin in predicting long-term outcomes or death. DESIGN: We evaluated the relationship between baseline serum ferritin and longitudinal events in a multicentre cohort of 1342 patients. Four survival models considering ferritin with confounders or non-invasive scoring systems were applied with repeated five-fold cross-validation schema. Prediction performance was evaluated in terms of Harrell's C-index and its improvement by including ferritin as a covariate. RESULTS: Median follow-up time was 96 months. Liver-related events occurred in 7.7%, hepatocellular carcinoma in 1.9%, cardiovascular events in 10.9%, extrahepatic cancers in 8.3% and all-cause mortality in 5.8%. Hyperferritinaemia was associated with a 50% increased risk of liver-related events and 27% of all-cause mortality. A stepwise increase in baseline ferritin thresholds was associated with a statistical increase in C-index, ranging between 0.02 (lasso-penalised Cox regression) and 0.03 (ridge-penalised Cox regression); the risk of developing liver-related events mainly increased from threshold 215.5 µg/L (median HR=1.71 and C-index=0.71) and the risk of overall mortality from threshold 272 µg/L (median HR=1.49 and C-index=0.70). The inclusion of serum ferritin thresholds (215.5 µg/L and 272 µg/L) in predictive models increased the performance of Fibrosis-4 and Non-Alcoholic Fatty Liver Disease Fibrosis Score in the longitudinal risk assessment of liver-related events (C-indices>0.71) and overall mortality (C-indices>0.65). CONCLUSIONS: This study supports the potential use of serum ferritin values for predicting the long-term prognosis of patients with MASLD.

Long-term liver-related outcomes and liver stiffness progression of statin usage in steatotic liver disease.

BACKGROUND: Statins have multiple benefits in patients with metabolic-associated steatotic liver disease (MASLD). AIM: To explore the effects of statins on the long-term risk of all-cause mortality, liver-related clinical events (LREs) and liver stiffness progression in patients with MASLD. METHODS: This cohort study collected data on patients with MASLD undergoing at least two vibration-controlled transient elastography examinations at 16 tertiary referral centres. Cox regression analysis was performed to examine the association between statin usage and long-term risk of all-cause mortality and LREs stratified by compensated advanced chronic liver disease (cACLD): baseline liver stiffness measurement (LSM) of ≥10 kPa. Liver stiffness progression was defined as an LSM increase of ≥20% for cACLD and from <10 kPa to ≥10 or LSM for non-cACLD. Liver stiffness regression was defined as LSM reduction from ≥10 kPa to <10 or LSM decrease of ≥20% for cACLD. RESULTS: We followed up 7988 patients with baseline LSM 5.9 kPa (IQR 4.6-8.2) for a median of 4.6 years. At baseline, 40.5% of patients used statins, and cACLD was present in 17%. Statin usage was significantly associated with a lower risk of all-cause mortality (adjusted HR=0.233; 95% CI 0.127 to 0.426) and LREs (adjusted HR=0.380; 95% CI 0.268 to 0.539). Statin usage was also associated with lower liver stiffness progression rates in cACLD (HR=0.542; 95% CI 0.389 to 0.755) and non-cACLD (adjusted HR=0.450; 95% CI 0.342 to 0.592), but not with liver stiffness regression (adjusted HR=0.914; 95% CI 0.778 to 1.074). CONCLUSIONS: Statin usage was associated with a relatively lower long-term risk of all-cause mortality, LREs and liver stiffness progression in patients with MASLD.

SIRT5 rs12216101 T>G variant is associated with liver damage and mitochondrial dysfunction in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Sirtuin 5, encoded by the SIRT5 gene, is a NAD+-dependent deacylase that modulates mitochondrial metabolic processes through post-translational modifications. In this study, we aimed to examine the impact of the SIRT5 rs12216101 T>G non-coding single nucleotide polymorphism on disease severity in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: The rs12216101 variant was genotyped in 2,606 consecutive European patients with biopsy-proven NAFLD. Transcriptomic analysis, expression of mitochondrial complexes and oxidative stress levels were measured in liver samples from a subset of bariatric patients. Effects of SIRT5 pharmacological inhibition were evaluated in HepG2 cells exposed to excess free fatty acids. Mitochondrial energetics in vitro were investigated by high-performance liquid chromatography. RESULTS: In the whole cohort, the frequency distribution of SIRT5 rs12216101 TT, TG and GG genotypes was 47.0%, 42.3% and 10.7%, respectively. At multivariate logistic regression analysis adjusted for sex, age >50 years, diabetes, and PNPLA3 rs738409 status, the SIRT5 rs12216101 T>G variant was associated with the presence of non-alcoholic steatohepatitis (odds ratio 1.20, 95% CI 1.03-1.40) and F2-F4 fibrosis (odds ratio 1.18; 95% CI 1.00-1.37). Transcriptomic analysis showed that the SIRT5 rs12216101 T>G variant was associated with upregulation of transcripts involved in mitochondrial metabolic pathways, including the oxidative phosphorylation system. In patients carrying the G allele, western blot analysis confirmed an upregulation of oxidative phosphorylation complexes III, IV, V and consistently higher levels of reactive oxygen species, reactive nitrogen species and malondialdehyde, and lower ATP levels. Administration of a pharmacological SIRT5 inhibitor preserved mitochondrial energetic homeostasis in HepG2 cells, as evidenced by restored ATP/ADP, NAD+/NADH, NADP+/NADPH ratios and glutathione levels. CONCLUSIONS: The SIRT5 rs12216101 T>G variant, heightening SIRT5 activity, is associated with liver damage, mitochondrial dysfunction, and oxidative stress in patients with NAFLD. IMPACT AND IMPLICATIONS: In this study we discovered that the SIRT5 rs12216101 T>G variant is associated with higher disease severity in patients with non-alcoholic fatty liver disease (NAFLD). This risk variant leads to a SIRT5 gain-of-function, enhancing mitochondrial oxidative phosphorylation and thus leading to oxidative stress. SIRT5 may represent a novel disease modulator in NAFLD.

Prevalence and Determinants of Liver Disease in Relatives of Italian Patients With Advanced MASLD.

BACKGROUND & AIMS: Metabolic dysfunction associated steatotic liver disease (MASLD) has a strong genetic component. The aim of this study was to examine noninvasively the prevalence of MASLD and of advanced fibrosis in relatives of patients with advanced MASLD and the risk factors for liver involvement, with a focus on the contribution of common genetic risk variants. METHODS: We prospectively enrolled 98 consecutive probands with advanced fibrosis and/or hepatocellular carcinoma caused by MASLD and 160 nontwin first-degree relatives noninvasively screened for MASLD and advanced fibrosis at 4 Italian centers. We evaluated common genetic determinants and polygenic risk scores of liver disease. RESULTS: Among relatives, prevalence of MASLD was 56.8% overall, whereas advanced fibrosis was observed in 14.4%. At multivariable analysis in relatives, MASLD was associated with body mass index (odds ratio [OR], 1.31 [1.18-1.46]) and tended to be associated with diabetes (OR, 5.21 [0.97-28.10]), alcohol intake (OR, 1.32 [0.98-1.78]), and with female sex (OR, 0.54 [0.23-1.15]), whereas advanced fibrosis was associated with diabetes (OR, 3.13 [1.16-8.45]) and nearly with body mass index (OR, 1.09 [1.00-1.19]). Despite that the PNPLA3 risk variant was enriched in probands (P = .003) and overtransmitted to relatives with MASLD (P = .045), evaluation of genetic risk variants and polygenic risk scores was not useful to guide noninvasive screening of advanced fibrosis in relatives. CONCLUSIONS: We confirmed that about 1 in 7 relatives of patients with advanced MASLD has advanced fibrosis, supporting clinical recommendations to perform family screening in this setting. Genetic risk variants contributed to liver disease within families but did not meaningfully improve fibrosis risk stratification.

Noninvasive assessment of liver disease severity in patients with nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes.

BACKGROUND AND AIMS: We evaluated the diagnostic accuracy of simple, noninvasive tests (NITs) in NAFLD patients with type 2 diabetes (T2D). METHODS AND RESULTS: This was an individual patient data meta-analysis of 1780 patients with biopsy-proven NAFLD and T2D. The index tests of interest were FIB-4, NAFLD Fibrosis Score (NFS), aspartate aminotransferase-to-platelet ratio index, liver stiffness measurement (LSM) by vibration-controlled transient elastography, and AGILE 3+. The target conditions were advanced fibrosis, NASH, and fibrotic NASH(NASH plus F2-F4 fibrosis). The diagnostic performance of noninvasive tests. individually or in sequential combination, was assessed by area under the receiver operating characteristic curve and by decision curve analysis. Comparison with 2278 NAFLD patients without T2D was also made. In NAFLD with T2D LSM and AGILE 3+ outperformed, both NFS and FIB-4 for advanced fibrosis (area under the receiver operating characteristic curve:LSM 0.82, AGILE 3+ 0.82, NFS 0.72, FIB-4 0.75, aspartate aminotransferase-to-platelet ratio index 0.68; p < 0.001 of LSM-based versus simple serum tests), with an uncertainty area of 12%-20%. The combination of serum-based with LSM-based tests for advanced fibrosis led to a reduction of 40%-60% in necessary LSM tests. Decision curve analysis showed that all scores had a modest net benefit for ruling out advanced fibrosis at the risk threshold of 5%-10% of missing advanced fibrosis. LSM and AGILE 3+ outperformed both NFS and FIB-4 for fibrotic NASH (area under the receiver operating characteristic curve:LSM 0.79, AGILE 3+ 0.77, NFS 0.71, FIB-4 0.71; p < 0.001 of LSM-based versus simple serum tests). All noninvasive scores were suboptimal for diagnosing NASH. CONCLUSIONS: LSM and AGILE 3+ individually or in low availability settings in sequential combination after FIB-4 or NFS have a similar good diagnostic accuracy for advanced fibrosis and an acceptable diagnostic accuracy for fibrotic NASH in NAFLD patients with T2D.

Clinical outcomes of MAFLD versus NAFLD: A meta-analysis of observational studies.

IMPORTANCE: The recent change in terminology from nonalcoholic fatty liver disease (NAFLD) to metabolic dysfunction-associated fatty liver disease (MAFLD) and metabolic dysfunction-associated steatotic liver disease (MASLD) highlights the link between hepatic steatosis and metabolic dysfunction, taking out the stigmata of alcohol. OBJECTIVE: We compared the effects of NAFLD and MAFLD definitions on the risk of overall and cardiovascular (CV) mortality, liver-related events (LRE), nonfatal CV events (CVE), chronic kidney disease (CKD), and extra-hepatic cancers (EHC). DATA SOURCES AND STUDY SELECTION: We systematically searched four large electronic databases for cohort studies (published through August 2023) that simultaneously used NAFLD and MAFLD definitions for examining the risk of mortality and adverse CV, renal, or oncological outcomes associated with both definitions. In total, 21 eligible cohort studies were identified. Meta-analysis was performed using random-effects modelling. RESULTS: Compared with those with NAFLD, individuals with MAFLD had significantly higher rates of overall mortality (random-effect OR 1.12, 95% CI 1.04-1.21, p = .004) and CV mortality (random-effect OR 1.15, 95% CI 1.04-1.26, p = .004), and a marginal trend towards higher rates of developing CKD (random-effect OR 1.06, 95% CI 1.00-1.12, p = .058) and EHC events (random-effect OR 1.11, 95% CI 1.00-1.23, p = .052). We found no significant differences in the risk LREs and nonfatal CVE between MAFLD and NAFLD. Meta-regression analyses identified male sex and metabolic comorbidities as the strongest risk factors related to the risk of adverse clinical outcomes in MAFLD compared to NAFLD. CONCLUSIONS AND RELEVANCE: Individuals with MAFLD have higher rates of overall and CV mortality and higher rates of developing CKD and EHC events than those with NAFLD, possibly due to the dysmetabolic risk profile related to MAFLD.

Mitochondrial amidoxime-reducing component 1 p.Ala165Thr increases protein degradation mediated by the proteasome.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization. METHODS: We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization. RESULTS: MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization. CONCLUSIONS: This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.

Performance of ultrasound-guided attenuation parameter and 2D shear wave elastography in patients with metabolic dysfunction-associated steatotic liver disease.

PURPOSE: To assess the performance and the reproducibility of ultrasound-guided attenuation parameter (UGAP) and two-dimensional shear wave elastography (2D-SWE) in patients with biopsy-proven metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: This study included consecutive adult patients with MASLD who underwent ultrasound with UGAP, 2D-SWE and percutaneous liver biopsy. The median values of 12 consecutive UGAP measurements were acquired by two independent radiologists (R1 and R2). Hepatic steatosis was graded by liver biopsy as: (0) < 5%; (1) 5-33%; (2) > 33-66%; (3) > 66%. Areas under the curve (AUCs) were calculated to determine the diagnostic performance. Inter- and intra-observer reliability was assessed with intraclass correlation coefficient (ICC). RESULTS: A hundred patients (median age 55.0 years old) with MASLD were prospectively enrolled. At histopathology, 70 and 42 patients had grade ≥ 2 and 3 steatosis, respectively. Median UGAP was 0.78 dB/cm/MHz (IQR/Med: 5.55%). For the diagnosis of grade ≥ 2 steatosis, the AUCs of UGAP were 0.828 (95% CI: 0.739, 0.896) for R1 and 0.779 (95% CI: 0.685, 0.856) for R2. The inter- and intra-operator reliability of UGAP were excellent, with an ICC of 0.92 (95% CI: 0.87-0.95) and 0.95 (95% CI: 0.92-0.96), respectively. The median liver stiffness was 6.76 kPa (IQR/Med: 16.30%). For the diagnosis of advanced fibrosis, 2D-SWE had an AUC of 0.862 (95% CI: 0.757, 0.934), and the optimal cutoff value was > 6.75 kPa with a sensitivity of 80.6% and a specificity of 75.7%. CONCLUSION: UGAP and 2D-SWE provide a good performance for the staging of steatosis and fibrosis in patients with MASLD with an excellent intra-operator reliability of UGAP. KEY POINTS: Question How well do ultrasound-guided attenuation parameter (UGAP) and two-dimensional shear wave elastography (2D-SWE) perform for quantifying hepatic steatosis and fibrosis? Findings UGAP had a maximum AUC of 0.828 for the diagnosis of grade ≥ 2 steatosis, and 2D-SWE had an AUC of 0.862 for diagnosing advanced fibrosis. Clinical relevance UGAP and 2D-SWE allow rapid, reproducible, and accurate quantification of hepatic steatosis and fibrosis that can be used for the noninvasive assessment of patients with metabolic dysfunction-associated steatotic liver disease.

Vibration-Controlled Transient Elastography Scores to Predict Liver-Related Events in Steatotic Liver Disease.

Importance: Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the most common chronic liver disease worldwide. It is important to develop noninvasive tests to assess the disease severity and prognosis. Objective: To study the prognostic implications of baseline levels and dynamic changes of the vibration-controlled transient elastography (VCTE)-based scores developed for the diagnosis of advanced fibrosis (Agile 3+) and cirrhosis (Agile 4) in patients with MASLD. Design, Setting, and Participants: This cohort study included data from a natural history cohort of patients with MASLD who underwent VCTE examination at 16 tertiary referral centers in the US, Europe, and Asia from February 2004 to January 2023, of which the data were collected prospectively at 14 centers. Eligible patients were adults aged at least 18 years with hepatic steatosis diagnosed by histologic methods (steatosis in ≥5% of hepatocytes) or imaging studies (ultrasonography, computed tomography or magnetic resonance imaging, or controlled attenuation parameter ≥248 dB/m by VCTE). Main Outcomes and Measures: The primary outcome was liver-related events (LREs), defined as hepatocellular carcinoma or hepatic decompensation (ascites, variceal hemorrhage, hepatic encephalopathy, or hepatorenal syndrome), liver transplant, and liver-related deaths. The Agile scores were compared with histologic and 8 other noninvasive tests. Results: A total of 16 603 patients underwent VCTE examination at baseline (mean [SD] age, 52.5 [13.7] years; 9600 [57.8%] were male). At a median follow-up of 51.7 (IQR, 25.2-85.2) months, 316 patients (1.9%) developed LREs. Both Agile 3+ and Agile 4 scores classified fewer patients between the low and high cutoffs than most fibrosis scores and achieved the highest discriminatory power in predicting LREs (integrated area under the time-dependent receiver-operating characteristic curve, 0.89). A total of 10 920 patients (65.8%) had repeated VCTE examination at a median interval of 15 (IQR, 11.3-27.7) months and were included in the serial analysis. A total of 81.9% of patients (7208 of 8810) had stable Agile 3+ scores and 92.6% of patients (8163 of 8810) had stable Agile 4 scores (same risk categories at both assessments). The incidence of LREs was 0.6 per 1000 person-years in patients with persistently low Agile 3+ scores and 30.1 per 1000 person-years in patients with persistently high Agile 3+ scores. In patients with high Agile 3+ score at baseline, a decrease in the score by more than 20% was associated with substantial reduction in the risk of LREs. A similar trend was observed for the Agile 4 score, although it missed more LREs in the low-risk group. Conclusions and Relevance: Findings of this study suggest that single or serial Agile scores are highly accurate in predicting LREs in patients with MASLD, making them suitable alternatives to liver biopsy in routine clinical practice and in phase 2b and 3 clinical trials for steatohepatitis.

The PD-1/PD-L1 Axis in the Biology of MASLD.

Metabolic Dysfunction-Associated Steatotic Liver (MASL), previously named nonalcoholic fatty liver (NAFL), is a multifactorial disease in which metabolic, genetic, and environmental risk factors play a predominant role. Obesity and type 2 diabetes act as triggers of the inflammatory response, which contributes to the progression of MASL to Metabolic Dysfunction-Associated Steatohepatitis and the development of hepatocellular carcinoma. In the liver, several parenchymal, nonparenchymal, and immune cells maintain immunological homeostasis, and different regulatory pathways balance the activation of the innate and adaptative immune system. PD-1/PD-L1 signaling acts, in the maintenance of the balance between the immune responses and the tissue immune homeostasis, promoting self-tolerance through the modulation of activated T cells. Recently, PD-1 has received much attention for its roles in inducing an exhausted T cells phenotype, promoting the tumor escape from immune responses. Indeed, in MASLD, the excessive fat accumulation dysregulates the immune system, increasing cytotoxic lymphocytes and decreasing their cytolytic activity. In this context, T cells exacerbate liver damage and promote tumor progression. The aim of this review is to illustrate the main pathogenetic mechanisms by which the immune system promotes the progression of MASLD and the transition to HCC, as well as to discuss the possible therapeutic applications of PD-1/PD-L1 target therapy to activate T cells and reinvigorate immune surveillance against cancer.

AGILE 3+ Score for the Diagnosis of Advanced Fibrosis and for Predicting Liver-related Events in NAFLD.

BACKGROUND & AIMS: We aimed to assess the diagnostic accuracy of AGILE 3+, a recently developed score based on the combination of aspartate aminotransferase/alanine aminotransferase ratio, platelet count, diabetes status, sex, age, and liver stiffness measurement (LSM) by transient elastography, when compared with Fibrosis-4 (FIB-4) and LSM, for the diagnosis of advanced fibrosis and for the prediction of liver-related events (LREs) occurrence in patients with NAFLD. METHODS: A total of 614 consecutive patients with biopsy-proven NAFLD or clinical diagnosis of NAFLD-related compensated cirrhosis were enrolled. LREs were recorded during follow-up. FIB-4, LSM by transient elastography (FibroScan device), and AGILE 3+ were measured. The diagnostic performance of noninvasive criteria for advanced fibrosis and for the prediction of LREs was assessed using the area under the receiver operating characteristic curve (AUROC) and decision curve analysis. RESULTS: In patients with biopsy-proven NAFLD (n = 520), LSM and AGILE 3+ had higher AUROC than FIB-4 (0.88 for LSM and AGILE 3+ vs 0.78 for FIB-4; P < .001) for advanced fibrosis, and AGILE 3+ exhibited a smaller indeterminate area in the test (25.2% for FIB-4 vs 13.1% for LSM vs 8.3% for AGILE 3+). Within the entire cohort of patients, AGILE 3+ had significantly higher AUROC for predicting LREs with respect to LSM (AUROC 36 months 0.95 vs 0.93; P =.008; 60 months 0.95 vs 0.92; P = .006; 96 months 0.97 vs 0.95; P = .001). Decision curve analysis showed that all scores had modest net benefit for ruling-out advanced fibrosis at the risk threshold of 5% to 10% where advanced fibrosis was absent. At the risk threshold of 5% of false negatives or false positives in LRE at 36, 60, 96, and 120 months, AGILE 3+ outperformed both FIB-4 and LSM for ruling out LRE. CONCLUSIONS: Depending on resource availability, clinical setting, and the risk scenarios, AGILE 3+ is an accurate and valid alternative to FIB-4 and LSM for the noninvasive assessment of disease severity and prognosis in patients with NAFLD.

Point-of-Care Noninvasive Prediction of Liver-Related Events in Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Individual risk prediction of liver-related events (LRE) is needed for clinical assessment of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) patients. We aimed to provide point-of-care validated liver stiffness measurement (LSM)-based risk prediction models for the development of LRE in patients with NAFLD, focusing on selecting patients for clinical trials at risk of clinical events. METHODS: Two large multicenter cohorts were evaluated, 2638 NAFLD patients covering all LSM values as the derivation cohort and 679 more advanced patients as the validation cohort. We used Cox regression to develop and validate risk prediction models based on LSM alone, and the ANTICIPATE and ANTICIPATE-NASH models for clinically significant portal hypertension. The main outcome of the study was the rate of LRE in the first 3 years after initial assessment. RESULTS: The 3 predictive models had similar performance in the derivation cohort with a very high discriminative value (c-statistic, 0.87-0.91). In the validation cohort, the LSM-LRE alone model had a significant inferior discrimination (c-statistic, 0.75) compared with the other 2 models, whereas the ANTICIPATE-NASH-LRE model (0.81) was significantly better than the ANTICIPATE-LRE model (0.79). In addition, the ANTICIPATE-NASH-LRE model presented very good calibration in the validation cohort (integrated calibration index, 0.016), and was better than the ANTICIPATE-LRE model. CONCLUSIONS: The ANTICIPATE-LRE models, and especially the ANTICIPATE-NASH-LRE model, could be valuable validated clinical tools to individually assess the risk of LRE at 3 years in patients with NAFLD/NASH.

Impact of PNPLA3 rs738409 Polymorphism on the Development of Liver-Related Events in Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is a complex disease, resulting from the interplay between environmental determinants and genetic variations. Single nucleotide polymorphism rs738409 C>G in the PNPLA3 gene is associated with hepatic fibrosis and with higher risk of developing hepatocellular carcinoma. Here, we analyzed a longitudinal cohort of biopsy-proven NAFLD subjects with the aim to identify individuals in whom genetics may have a stronger impact on disease progression. METHODS: We retrospectively analyzed 756 consecutive, prospectively enrolled biopsy-proven NAFLD subjects from Italy, United Kingdom, and Spain who were followed for a median of 84 months (interquartile range, 65-109 months). We stratified the study cohort according to sex, body mass index (BMI)

Programmed cell death 1 genetic variant and liver damage in nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Programmed cell death 1/programmed cell death-ligand 1 (PD-1/PDL-1) axis has been reported to modulate liver inflammation and progression to hepatocellular carcinoma (HCC) in patients with nonalcoholic fatty liver disease (NAFLD). Here, we examined whether the PDCD1 variation is associated with NAFLD severity in individuals with liver biopsy. METHODS: We examined the impact of PDCD1 gene variants on HCC, as robust severe liver disease phenotype in UK Biobank participants. The strongest genetic association with the rs13023138 G>C variation was subsequently tested for association with liver damage in 2889 individuals who underwent liver biopsy for suspected nonalcoholic steatohepatitis (NASH). Hepatic transcriptome was examined by RNA-Seq in a subset of NAFLD individuals (n = 121). Transcriptomic and deconvolution analyses were performed to identify biological pathways modulated by the risk allele. RESULTS: The rs13023138 C>G showed the most robust association with HCC in UK Biobank (p = 5.28E-4, OR = 1.32, 95% CI [1.1, 1.5]). In the liver biopsy cohort, rs13023138 G allele was independently associated with severe steatosis (OR 1.17, 95% CI 1.02-1.34; p = .01), NASH (OR 1.22, 95% CI 1.09-1.37; p < .001) and advanced fibrosis (OR 1.26, 95% CI 1.06-1.50; p = .007). At deconvolution analysis, rs13023138 G>C allele was linked to higher hepatic representation of M1 macrophages, paralleled by upregulation of pathways related to inflammation and higher expression of CXCR6. CONCLUSIONS: The PDCD1 rs13023138 G allele was associated with HCC development in the general population and with liver disease severity in patients at high risk of NASH.

Caucasian lean subjects with non-alcoholic fatty liver disease share long-term prognosis of non-lean: time for reappraisal of BMI-driven approach?

OBJECTIVE: The full phenotypic expression of non-alcoholic fatty liver disease (NAFLD) in lean subjects is incompletely characterised. We aimed to investigate prevalence, characteristics and long-term prognosis of Caucasian lean subjects with NAFLD. DESIGN: The study cohort comprises 1339 biopsy-proven NAFLD subjects from four countries (Italy, UK, Spain and Australia), stratified into lean and non-lean (body mass index (BMI) 10 483 person-years), 4.7% of lean vs 7.7% of non-lean patients reported liver-related events (p=0.37). No difference in survival was observed compared with non-lean NAFLD (p=0.069). CONCLUSIONS: Caucasian lean subjects with NAFLD may progress to advanced liver disease, develop metabolic comorbidities and experience cardiovascular disease (CVD) as well as liver-related mortality, independent of longitudinal progression to obesity and PNPLA3 genotype. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD where the disease manifests at lower overall BMI thresholds. LAY SUMMARY: NAFLD may affect and progress in both obese and lean individuals. Lean subjects are predominantly males, have a younger age at diagnosis and are more prevalent in some geographic areas. During the follow-up, lean subjects can develop hepatic and extrahepatic disease, including metabolic comorbidities, in the absence of weight gain. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD.

Exome-Wide Association Study on Alanine Aminotransferase Identifies Sequence Variants in the GPAM and APOE Associated With Fatty Liver Disease.

BACKGROUND & AIMS: Fatty liver disease (FLD) is a growing epidemic that is expected to be the leading cause of end-stage liver disease within the next decade. Both environmental and genetic factors contribute to the susceptibility of FLD. Several genetic variants contributing to FLD have been identified in exome-wide association studies. However, there is still a missing hereditability indicating that other genetic variants are yet to be discovered. METHODS: To find genes involved in FLD, we first examined the association of missense and nonsense variants with alanine aminotransferase at an exome-wide level in 425,671 participants from the UK Biobank. We then validated genetic variants with liver fat content in 8930 participants in whom liver fat measurement was available, and replicated 2 genetic variants in 3 independent cohorts comprising 2621 individuals with available liver biopsy. RESULTS: We identified 190 genetic variants independently associated with alanine aminotransferase after correcting for multiple testing with Bonferroni method. The majority of these variants were not previously associated with this trait. Among those associated, there was a striking enrichment of genetic variants influencing lipid metabolism. We identified the variants rs2792751 in GPAM/GPAT1, the gene encoding glycerol-3-phosphate acyltransferase, mitochondrial, and rs429358 in APOE, the gene encoding apolipoprotein E, as robustly associated with liver fat content and liver disease after adjusting for multiple testing. Both genes affect lipid metabolism in the liver. CONCLUSIONS: We identified 2 novel genetic variants in GPAM and APOE that are robustly associated with steatosis and liver damage. These findings may help to better elucidate the genetic susceptibility to FLD onset and progression.

RESIST-HCV Criteria to Monitor Progression of Low-Risk Esophageal Varices in Patients With Compensated Cirrhosis After HCV Eradication: The SIMPLE Study: SIMPLE: Scoring Index to Monitor Progression of Low-risk Esophageal varices.

INTRODUCTION: Noninvasive criteria to predict the progression of low-risk esophageal varices (EV) in patients with compensated hepatitis C virus (HCV) cirrhosis after sustained virological response (SVR) by direct-acting antivirals (DAAs) are lacking. Our aim was to assess the diagnostic performance of Rete Sicilia Selezione Terapia-HCV (RESIST-HCV) criteria for EV progression compared with elastography-based criteria (Baveno VI, Expanded Baveno VI, and Baveno VII-HCV criteria). METHODS: All consecutive patients observed at 3 referral centers with compensated HCV cirrhosis with or without F1 EV who achieved sustained virological response by DAAs were classified at last esophagogastroduodenoscopy (EGDS) as RESIST-HCV low risk (i.e., low probability of high-risk varices [HRV]) if platelets were >120 × 10 9 /L and serum albumin >3.6 g/dL or RESIST-HCV high risk (i.e., high probability of HRV) if platelets were <120 × 10 9 /L or serum albumin <3.6 g/dL. The primary outcome was the progression to HRV. The area under the receiver operating characteristic curve and decision curve analysis of noninvasive criteria were calculated. RESULTS: The cohort consisted of 353 patients in Child-Pugh class A (mean age 67.2 years, 53.8% males). During a mean follow-up of 44.2 months, 34 patients (9.6%, 95% CI 6.7%-13.5%) developed HRV. At the last EGDS, 178 patients (50.4%) were RESIST-low risk, and 175 (49.6%) were RESIST-high risk. RESIST-HCV criteria showed the highest area under the receiver operating characteristic curve (0.70, 95% confidence interval 0.65-0.75), correctly sparing the highest number of EGDS (54.3%), with the lowest false-positive rate (45.7%), compared with elastography-based criteria. Decision curve analysis showed that RESIST-HCV had higher clinical utility than elastography-based criteria. DISCUSSION: Biochemical-based RESIST-HCV criteria are useful to easily predict HRV development after HCV eradication by DAAs in patients with compensated cirrhosis and low-risk EV.