Background & aims

Non-invasive tests (NITs) offer a practical solution for advanced fibrosis identification in non-alcoholic fatty liver disease (NAFLD). Despite increasing implementation, their use is not standardised, which can lead to inconsistent interpretation and risk stratification. We aimed to assess the types of NITs and the corresponding cut-offs used in a range of healthcare settings.

Methods

A survey was distributed to a convenience sample of liver health experts who participated in a global NAFLD consensus statement. Respondents provided information on the NITs used in their clinic with the corresponding cut-offs and those used in established care pathways in their areas.

Results

There were 35 respondents from 24 countries, 89% of whom practised in tertiary level settings. A total of 14 different NITs were used, and each respondent reported using at least one (median = 3). Of the respondents, 80% reported using FIB-4 and liver stiffness by vibration-controlled transient elastography (Fibroscan®), followed by the NAFLD fibrosis score (49%). For FIB-4, 71% of respondents used a low cut-off of <1.3 (range <1.0 to <1.45) and 21% reported using age-specific cut-offs. For Fibroscan®, 21% of respondents used a single liver stiffness cut-off: 8 kPa in 50%, while the rest used 7.2 kPa, 7.8 kPa and 8.7 kPa. Among the 63% of respondents who used lower and upper liver stiffness cut-offs, there were variations in both values (<5 to <10 kPa and >7.5 to >20 kPa, respectively).

Conclusions

The cut-offs used for the same NITs for NAFLD risk stratification vary between clinicians. As cut-offs impact test performance, these findings underscore the heterogeneity in risk-assessment and support the importance of establishing consistent guidelines on the standardised use of NITs in NAFLD management.

Lay summary

Owing to the high prevalence of non-alcoholic fatty liver disease (NAFLD) in the general population it is important to identify those who have more advanced stages of liver fibrosis, so that they can be properly treated. Non-invasive tests (NITs) provide a practical way to assess fibrosis risk in patients. However, we found that the cut-offs used for the same NITs vary between clinicians. As cut-offs impact test performance, these findings highlight the importance of establishing consistent guidelines on the standardised use of NITs to optimise clinical management of NAFLD.

Objective

In retrospective studies, liver stiffness (LS) by vibration-controlled transient elastography (VCTE) is associated with the risk of liver decompensation in patients with non-alcoholic steatohepatitis (NASH), but prospective data in biopsy-confirmed cohorts with advanced fibrosis are limited. We aimed to establish thresholds for LS by VCTE that predict progression to cirrhosis among patients with bridging fibrosis and hepatic decompensation among patients with cirrhosis due to NASH.

Design

We used data from four randomised placebo-controlled trials of selonsertib and simtuzumab in participants with advanced fibrosis (F3-F4). The trials were discontinued due to lack of efficacy. Liver fibrosis was staged centrally at baseline and week 48 (selonsertib study) or week 96 (simtuzumab study). Associations between LS by VCTE with disease progression were determined using Cox proportional hazards regression analysis.

Results

Progression to cirrhosis occurred in 16% (103/664) of participants with bridging fibrosis and adjudicated liver-related events occurred in 4% (27/734) of participants with baseline cirrhosis. The optimal baseline LS thresholds were ≥16.6 kPa for predicting progression to cirrhosis, and ≥30.7 kPa for predicting liver-related events. Baseline LS ≥16.6 kPa (adjusted HR 3.99; 95% CI 2.66 to 5.98, p<0.0001) and a ≥5 kPa (and ≥20%) increase (adjusted HR 1.98; 95% CI 1.20 to 3.26, p=0.008) were independent predictors of progression to cirrhosis in participants with bridging fibrosis, while baseline LS ≥30.7 kPa (adjusted HR 10.13, 95% CI 4.38 to 23.41, p<0.0001) predicted liver-related events in participants with cirrhosis.

Conclusion

The LS thresholds identified in this study may be useful for risk stratification of NASH patients with advanced fibrosis.

Background & aims

We conducted an individual patient data meta-analysis to establish stiffness cut-off values for magnetic resonance elastography (MRE) in staging liver fibrosis and to assess potential confounding factors.

Methods

A systematic review of the literature identified studies reporting MRE data in patients with NAFLD. Data were obtained from the corresponding authors. The pooled diagnostic cut-off value for the various fibrosis stages was determined in a two-stage meta-analysis. Multilevel modelling methods were used to analyse potential confounding factors influencing the diagnostic accuracy of MRE in staging liver fibrosis.

Results

Eight independent cohorts comprising 798 patients were included in the meta-analysis. The area under the receiver operating characteristic curve (AUROC) for MRE in detecting significant fibrosis was 0.92 (sensitivity, 79%; specificity, 89%). For advanced fibrosis, the AUROC was 0.92 (sensitivity, 87%; specificity, 88%). For cirrhosis, the AUROC was 0.94 (sensitivity, 88%, specificity, 89%). Cut-offs were defined to explore concordance between MRE and histopathology: ≥F2, 3.14 kPa (pretest probability, 39.4%); ≥F3, 3.53 kPa (pretest probability, 24.1%); and F4, 4.45 kPa (pretest probability, 8.7%). In generalized linear mixed model analysis, histological steatohepatitis with higher inflammatory activity (odds ratio 2.448, 95% CI 1.180-5.079, p <0.05) and high gamma-glutamyl transferase (GGT) concentration (>120U/L) (odds ratio 3.388, 95% CI 1.577-7.278, p <0.01] were significantly associated with elevated liver stiffness, and thus affecting accuracy in staging early fibrosis (F0-F1). Steatosis, as measured by magnetic resonance imaging proton density fat fraction, and body mass index(BMI) were not confounders.

Conclusions

MRE has excellent diagnostic performance for significant, advanced fibrosis and cirrhosis in patients with NAFLD. Elevated inflammatory activity and GGT level may lead to overestimation of early liver fibrosis, but anthropometric measures such as BMI or the degree of steatosis do not.

Impact and implications

This individual patient data meta-analysis of eight international cohorts, including 798 patients, demonstrated that MRE achieves excellent diagnostic accuracy for significant, advanced fibrosis and cirrhosis in patients with NAFLD. Cut-off values (significant fibrosis, 3.14 kPa; advanced fibrosis, 3.53 kPa; and cirrhosis, 4.45 kPa) were established. Elevated inflammatory activity and gamma-glutamyltransferase level may affect the diagnostic accuracy of MRE, leading to overestimation of liver fibrosis in early stages. We observed no impact of diabetes, obesity, or any other metabolic disorder on the diagnostic accuracy of MRE.

Background & aims

Nonalcoholic fatty liver disease (NAFLD) is a consequence of defects in diverse metabolic pathways that involve hepatic accumulation of triglycerides. Features of these aberrations might determine whether NAFLD progresses to nonalcoholic steatohepatitis (NASH). We investigated whether the diverse defects observed in patients with NAFLD are caused by different NAFLD subtypes with specific serum metabolomic profiles, and whether these can distinguish patients with NASH from patients with simple steatosis.

Methods

We collected liver and serum from methionine adenosyltransferase 1a knockout (MAT1A-KO) mice, which have chronically low levels of hepatic S-adenosylmethionine (SAMe) and spontaneously develop steatohepatitis, as well as C57Bl/6 mice (controls); the metabolomes of all samples were determined. We also analyzed serum metabolomes of 535 patients with biopsy-proven NAFLD (353 with simple steatosis and 182 with NASH) and compared them with serum metabolomes of mice. MAT1A-KO mice were also given SAMe (30 mg/kg/day for 8 weeks); liver samples were collected and analyzed histologically for steatohepatitis.

Results

Livers of MAT1A-KO mice were characterized by high levels of triglycerides, diglycerides, fatty acids, ceramides, and oxidized fatty acids, as well as low levels of SAMe and downstream metabolites. There was a correlation between liver and serum metabolomes. We identified a serum metabolomic signature associated with MAT1A-KO mice that also was present in 49% of the patients; based on this signature, we identified 2 NAFLD subtypes. We identified specific panels of markers that could distinguish patients with NASH from patients with simple steatosis for each subtype of NAFLD. Administration of SAMe reduced features of steatohepatitis in MAT1A-KO mice.

Conclusions

In an analysis of serum metabolomes of patients with NAFLD and MAT1A-KO mice with steatohepatitis, we identified 2 major subtypes of NAFLD and markers that differentiate steatosis from NASH in each subtype. These might be used to monitor disease progression and identify therapeutic targets for patients.

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease worldwide and includes a broad spectrum of histologic phenotypes, ranging from simple hepatic steatosis or nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). While liver biopsy is the reference gold standard for NAFLD diagnosis and staging, it has limitations due to its sampling variability, invasive nature, and high cost. Thus, there is a need for noninvasive biomarkers that are robust, reliable, and cost effective. In this study, we measured 540 lipids and amino acids in serum samples from biopsy-proven subjects with normal liver (NL), NAFL, and NASH. Using logistic regression analysis, we identified two panels of triglycerides that could first discriminate between NAFLD and NL and second between NASH and NAFL. These noninvasive tests were compared to blinded histology as a reference standard. We performed these tests in an original cohort of 467 patients with NAFLD (90 NL, 246 NAFL, and 131 NASH) that was subsequently validated in a separate cohort of 192 patients (7 NL, 109 NAFL, 76 NASH). The diagnostic performances of the validated tests showed an area under the receiver operating characteristic curve, sensitivity, and specificity of 0.88 ± 0.05, 0.94, and 0.57, respectively, for the discrimination between NAFLD and NL and 0.79 ± 0.04, 0.70, and 0.81, respectively, for the discrimination between NASH and NAFL. When the analysis was performed excluding patients with glucose levels >136 mg/dL, the area under the receiver operating characteristic curve for the discrimination between NASH and NAFL increased to 0.81 ± 0.04 with sensitivity and specificity of 0.73 and 0.80, respectively. Conclusion: The assessed noninvasive lipidomic serum tests distinguish between NAFLD and NL and between NASH and NAFL with high accuracy. (Hepatology Communications 2018;2:807-820).

BACKGROUND AND AIMS: Manual histological assessment is currently the accepted standard for diagnosing and monitoring disease progression in NASH, but is limited by variability in interpretation and insensitivity to change. Thus, there is a critical need for improved tools to assess liver pathology in order to risk stratify NASH patients and monitor treatment response. APPROACH AND RESULTS: Here, we describe a machine learning (ML)-based approach to liver histology assessment, which accurately characterizes disease severity and heterogeneity, and sensitively quantifies treatment response in NASH. We use samples from three randomized controlled trials to build and then validate deep convolutional neural networks to measure key histological features in NASH, including steatosis, inflammation, hepatocellular ballooning, and fibrosis. The ML-based predictions showed strong correlations with expert pathologists and were prognostic of progression to cirrhosis and liver-related clinical events. We developed a heterogeneity-sensitive metric of fibrosis response, the Deep Learning Treatment Assessment Liver Fibrosis score, which measured antifibrotic treatment effects that went undetected by manual pathological staging and was concordant with histological disease progression. CONCLUSIONS: Our ML method has shown reproducibility and sensitivity and was prognostic for disease progression, demonstrating the power of ML to advance our understanding of disease heterogeneity in NASH, risk stratify affected patients, and facilitate the development of therapies.

Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from the phosphatidylethanolamine N-methyltransferase-mediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.

Background and aims

We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors.

Approach and results

We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL_5,6 , and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A.

Conclusions

Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.

The principal limitations of the terms NAFLD and NASH are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favor of a change in nomenclature and/or definition. A modified Delphi process was led by three large pan-national liver associations. The consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria. A total of 236 panelists from 56 countries participated in 4 online surveys and 2 hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83%, and 78%, respectively. Seventy-four percent of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms nonalcoholic and fatty were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease. There was consensus to change the definition to include the presence of at least 1 of 5 cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic steatotic liver disease. A new category, outside pure metabolic dysfunction-associated steatotic liver disease, termed metabolic and alcohol related/associated liver disease (MetALD), was selected to describe those with metabolic dysfunction-associated steatotic liver disease, who consume greater amounts of alcohol per week (140-350 g/wk and 210-420 g/wk for females and males, respectively). The new nomenclature and diagnostic criteria are widely supported and nonstigmatising, and can improve awareness and patient identification.

The principal limitations of the terms NAFLD and NASH are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favour of a change in nomenclature and/or definition. A modified Delphi process was led by three large pan-national liver associations. The consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria. A total of 236 panellists from 56 countries participated in 4 online surveys and 2 hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83%, and 78%, respectively. Seventy-four percent of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms "nonalcoholic" and "fatty" were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease (MASLD). There was consensus to change the definition to include the presence of at least 1 of 5 cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic steatotic liver disease. A new category, outside pure metabolic dysfunction-associated steatotic liver disease, termed metabolic and alcohol related/associated liver disease (MetALD), was selected to describe those with metabolic dysfunction-associated steatotic liver disease, who consume greater amounts of alcohol per week (140-350 g/wk and 210-420 g/wk for females and males, respectively). The new nomenclature and diagnostic criteria are widely supported and non-stigmatising, and can improve awareness and patient identification.