acFibroMASH Index for the Diagnosis of Fibrotic MASH and Prediction of Liver-related Events: An International Multicenter Study.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) and fibrotic MASH are significant health challenges. This multi-national study aimed to validate the acMASH index (including serum creatinine and aspartate aminotransferase concentrations) for MASH diagnosis and develop a new index (acFibroMASH) for non-invasively identifying fibrotic MASH and exploring its predictive value for liver-related events (LREs). METHODS: We analyzed data from 3004 individuals with biopsy-proven metabolic dysfunction-associated fatty liver disease (MAFLD) across 29 Chinese and 9 international cohorts to validate the acMASH index and develop the acFibroMASH index. Additionally, we utilized the independent external data from a multi-national cohort of 9034 patients with MAFLD to examine associations between the acFibroMASH index and the risk of LREs. RESULTS: In the pooled global cohort, the acMASH index identified MASH with an area under the receiver operating characteristic curve (AUROC) of 0.802 (95% confidence interval [CI], 0.786-0.818). The acFibroMASH index (including the acMASH index plus liver stiffness measurement) accurately identified fibrotic MASH with an AUROC of 0.808 in the derivation cohort and 0.800 in the validation cohort. Notably, the AUROC for the acFibroMASH index was 0.835 (95% CI, 0.786-0.882), superior to that of the FAST score at 0.750 (95% CI, 0.693-0.800; P < .01) in predicting the 5-year risk of LREs. Patients with acFibroMASH >0.39 had a higher risk of LREs than those with acFibroMASH <0.15 (adjusted hazard ratio, 11.23; 95% CI, 3.98-31.66). CONCLUSIONS: This multi-ethnic study validates the acMASH index as a reliable, noninvasive test for identifying MASH. The newly proposed acFibroMASH index is a reliable test for identifying fibrotic MASH and predicting the risk of LREs.

Glycated haemoglobin is a major predictor of disease severity in patients with NAFLD.

OBJECTIVES: Currently, non-invasive scoring systems to stage the severity of non-alcoholic fatty liver disease (NAFLD) do not consider markers of glucose control (glycated haemoglobin, HbA1c); this study aimed to define the relationship between HbA1c and NAFLD severity in patients with and without type 2 diabetes. RESEARCH DESIGN AND METHODS: Data were obtained from 857 patients with liver biopsy staged NAFLD. Generalized-linear models and binomial regression analysis were used to define the relationships between histological NAFLD severity, age, HbA1c, and BMI. Paired biopsies from interventional studies (n = 421) were used to assess the impact of change in weight, HbA1c and active vs. placebo treatment on improvements in steatosis, non-alcoholic steatohepatitis (NASH), and fibrosis. RESULTS: In the discovery cohort (n = 687), risk of severe steatosis, NASH and advanced fibrosis correlated positively with HbA1c, after adjustment for obesity and age. These data were endorsed in a separate validation cohort (n = 170). Predictive modelling using HbA1c and age was non-inferior to the established non-invasive biomarker, Fib-4, and allowed the generation of HbA1c, age, and BMI adjusted risk charts to predict NAFLD severity. Following intervention, reduction in HbA1c was associated with improvements in steatosis and NASH after adjustment for weight change and treatment, whilst fibrosis change was only associated with weight change and treatment. CONCLUSIONS: HbA1c is highly informative in predicting NAFLD severity and contributes more than BMI. Assessments of HbA1c must be a fundamental part of the holistic assessment of patients with NAFLD and, alongside age, can be used to identify patients with highest risk of advanced disease.

Machine learning approaches to enhance diagnosis and staging of patients with MASLD using routinely available clinical information.

AIMS: Metabolic dysfunction Associated Steatotic Liver Disease (MASLD) outcomes such as MASH (metabolic dysfunction associated steatohepatitis), fibrosis and cirrhosis are ordinarily determined by resource-intensive and invasive biopsies. We aim to show that routine clinical tests offer sufficient information to predict these endpoints. METHODS: Using the LITMUS Metacohort derived from the European NAFLD Registry, the largest MASLD dataset in Europe, we create three combinations of features which vary in degree of procurement including a 19-variable feature set that are attained through a routine clinical appointment or blood test. This data was used to train predictive models using supervised machine learning (ML) algorithm XGBoost, alongside missing imputation technique MICE and class balancing algorithm SMOTE. Shapley Additive exPlanations (SHAP) were added to determine relative importance for each clinical variable. RESULTS: Analysing nine biopsy-derived MASLD outcomes of cohort size ranging between 5385 and 6673 subjects, we were able to predict individuals at training set AUCs ranging from 0.719-0.994, including classifying individuals who are At-Risk MASH at an AUC = 0.899. Using two further feature combinations of 26-variables and 35-variables, which included composite scores known to be good indicators for MASLD endpoints and advanced specialist tests, we found predictive performance did not sufficiently improve. We are also able to present local and global explanations for each ML model, offering clinicians interpretability without the expense of worsening predictive performance. CONCLUSIONS: This study developed a series of ML models of accuracy ranging from 71.9-99.4% using only easily extractable and readily available information in predicting MASLD outcomes which are usually determined through highly invasive means.

Translating the potential of the urine steroid metabolome to stage NAFLD (TrUSt-NAFLD): study protocol for a multicentre, prospective validation study.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) affects approximately one in four individuals and its prevalence continues to rise. The advanced stages of NAFLD with significant liver fibrosis are associated with adverse morbidity and mortality outcomes. Currently, liver biopsy remains the 'gold-standard' approach to stage NAFLD severity. Although generally well tolerated, liver biopsies are associated with significant complications, are resource intensive, costly, and sample only a very small area of the liver as well as requiring day case admission to a secondary care setting. As a result, there is a significant unmet need to develop non-invasive biomarkers that can accurately stage NAFLD and limit the need for liver biopsy. The aim of this study is to validate the use of the urine steroid metabolome as a strategy to stage NAFLD severity and to compare its performance against other non-invasive NAFLD biomarkers. METHODS AND ANALYSIS: The TrUSt-NAFLD study is a multicentre prospective test validation study aiming to recruit 310 patients with biopsy-proven and staged NAFLD across eight centres within the UK. 150 appropriately matched control patients without liver disease will be recruited through the Oxford Biobank. Blood and urine samples, alongside clinical data, will be collected from all participants. Urine samples will be analysed by liquid chromatography-tandem mass spectroscopy to quantify a panel of predefined steroid metabolites. A machine learning-based classifier, for example, Generalized Matrix Relevance Learning Vector Quantization that was trained on retrospective samples, will be applied to the prospective steroid metabolite data to determine its ability to identify those patients with advanced, as opposed to mild-moderate, liver fibrosis as a consequence of NAFLD. ETHICS AND DISSEMINATION: Research ethical approval was granted by West Midlands, Black Country Research Ethics Committee (REC reference: 21/WM/0177). A substantial amendment (TrUSt-NAFLD-SA1) was approved on 26 November 2021. TRIAL REGISTRATION NUMBER: ISRCTN19370855.

Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS): Assessment & validation of imaging modality performance across the NAFLD spectrum in a prospectively recruited cohort study (the LITMUS imaging study): Study protocol.

Non-alcoholic fatty liver disease (NAFLD) is the liver manifestation of the metabolic syndrome with global prevalence reaching epidemic levels. Despite the high disease burden in the population only a small proportion of those with NAFLD will develop progressive liver disease, for which there is currently no approved pharmacotherapy. Identifying those who are at risk of progressive NAFLD currently requires a liver biopsy which is problematic. Firstly, liver biopsy is invasive and therefore not appropriate for use in a condition like NAFLD that affects a large proportion of the population. Secondly, biopsy is limited by sampling and observer dependent variability which can lead to misclassification of disease severity. Non-invasive biomarkers are therefore needed to replace liver biopsy in the assessment of NAFLD. Our study addresses this unmet need. The LITMUS Imaging Study is a prospectively recruited multi-centre cohort study evaluating magnetic resonance imaging and elastography, and ultrasound elastography against liver histology as the reference standard. Imaging biomarkers and biopsy are acquired within a 100-day window. The study employs standardised processes for imaging data collection and analysis as well as a real time central monitoring and quality control process for all the data submitted for analysis. It is anticipated that the high-quality data generated from this study will underpin changes in clinical practice for the benefit of people with NAFLD. Study Registration: clinicaltrials.gov: NCT05479721.

Health-related Quality of Life in Patients With Nonalcoholic Fatty Liver Disease: A Prospective Multi-center UK Study.

BACKGROUND & AIMS: It is unclear whether health-related quality of life (HRQoL) is impaired in patients with nonalcoholic fatty liver disease (NAFLD) without advanced fibrosis and how this compares with the general population. We aimed to assess HRQoL in patients with NAFLD in comparison to the general population and any associations of fibrosis severity and metabolic comorbidities with impairments in HRQoL. METHODS: We prospectively enrolled 513 consecutive patients with NAFLD who completed the EuroQol 5-dimensional questionnaire (EQ-5D) and Chronic Liver Disease Questionnaires (CLDQ). Demographic and clinical information, liver biopsy results, and/or liver stiffness (LS) by transient elastography were recorded. A general population sub-cohort of the Health Survey for England 2018 was used as a comparator (n = 5483), and a 1:1 propensity-score (PS) matching was performed, according to age, sex, body mass index, and type 2 diabetes mellitus (T2DM). RESULTS: EQ-5D-5L utility was significantly lower in 466 PS-matched patients with NAFLD compared with PS-matched controls (0.77 ± 0.27 vs 0.84 ± 0.19; P < .001), even in those without advanced fibrosis (F ≤2 or LS <8kPa) (0.80 ± 0.24 vs 0.84 ± 0.19; P = .024). HRQoL measures (EQ-5D-5L, EQ-VAS, CLDQ) did not differ between patients with NAFLD with and without advanced fibrosis. LS was independently associated with lower EQ-5D-5L in all patients with NAFLD but not in those without advanced fibrosis. In the latter, lower EQ-5D-5L was associated with female sex, T2DM, and depression. CONCLUSIONS: Patients with NAFLD, even those without advanced fibrosis, have worse HRQoL compared with the general population. In patients with NAFLD without advanced fibrosis, HRQoL is independently associated with non-liver comorbidities but not LS. Multi-disciplinary management is therefore required in NAFLD, irrespective of fibrosis severity.

Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis.

BACKGROUND: Histologically assessed liver fibrosis stage has prognostic significance in patients with non-alcoholic fatty liver disease (NAFLD) and is accepted as a surrogate endpoint in clinical trials for non-cirrhotic NAFLD. Our aim was to compare the prognostic performance of non-invasive tests with liver histology in patients with NAFLD. METHODS: This was an individual participant data meta-analysis of the prognostic performance of histologically assessed fibrosis stage (F0-4), liver stiffness measured by vibration-controlled transient elastography (LSM-VCTE), fibrosis-4 index (FIB-4), and NAFLD fibrosis score (NFS) in patients with NAFLD. The literature was searched for a previously published systematic review on the diagnostic accuracy of imaging and simple non-invasive tests and updated to Jan 12, 2022 for this study. Studies were identified through PubMed/MEDLINE, EMBASE, and CENTRAL, and authors were contacted for individual participant data, including outcome data, with a minimum of 12 months of follow-up. The primary outcome was a composite endpoint of all-cause mortality, hepatocellular carcinoma, liver transplantation, or cirrhosis complications (ie, ascites, variceal bleeding, hepatic encephalopathy, or progression to a MELD score ≥15). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0-2 vs F3 vs F4; LSM: <10 vs 10 to <20 vs ≥20 kPa; FIB-4: <1·3 vs 1·3 to ≤2·67 vs >2·67; NFS: <-1·455 vs -1·455 to ≤0·676 vs >0·676), calculated areas under the time-dependent receiver operating characteristic curves (tAUC), and performed Cox proportional-hazards regression to adjust for confounding. This study was registered with PROSPERO, CRD42022312226. FINDINGS: Of 65 eligible studies, we included data on 2518 patients with biopsy-proven NAFLD from 25 studies (1126 [44·7%] were female, median age was 54 years [IQR 44-63), and 1161 [46·1%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33-91], the composite endpoint was observed in 145 (5·8%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0·0001 for all comparisons). The tAUC at 5 years were 0·72 (95% CI 0·62-0·81) for histology, 0·76 (0·70-0·83) for LSM-VCTE, 0·74 (0·64-0·82) for FIB-4, and 0·70 (0·63-0·80) for NFS. All index tests were significant predictors of the primary outcome after adjustment for confounders in the Cox regression. INTERPRETATION: Simple non-invasive tests performed as well as histologically assessed fibrosis in predicting clinical outcomes in patients with NAFLD and could be considered as alternatives to liver biopsy in some cases. FUNDING: Innovative Medicines Initiative 2.

Machine learning algorithm improves the detection of NASH (NAS-based) and at-risk NASH: A development and validation study.

BACKGROUND AND AIMS: Detecting NASH remains challenging, while at-risk NASH (steatohepatitis and F≥ 2) tends to progress and is of interest for drug development and clinical application. We developed prediction models by supervised machine learning techniques, with clinical data and biomarkers to stage and grade patients with NAFLD. APPROACH AND RESULTS: Learning data were collected in the Liver Investigation: Testing Marker Utility in Steatohepatitis metacohort (966 biopsy-proven NAFLD adults), staged and graded according to NASH CRN. Conditions of interest were the clinical trial definition of NASH (NAS ≥ 4;53%), at-risk NASH (NASH with F ≥ 2;35%), significant (F ≥ 2;47%), and advanced fibrosis (F ≥ 3;28%). Thirty-five predictors were included. Missing data were handled by multiple imputations. Data were randomly split into training/validation (75/25) sets. A gradient boosting machine was applied to develop 2 models for each condition: clinical versus extended (clinical and biomarkers). Two variants of the NASH and at-risk NASH models were constructed: direct and composite models.Clinical gradient boosting machine models for steatosis/inflammation/ballooning had AUCs of 0.94/0.79/0.72. There were no improvements when biomarkers were included. The direct NASH model produced AUCs (clinical/extended) of 0.61/0.65. The composite NASH model performed significantly better (0.71) for both variants. The composite at-risk NASH model had an AUC of 0.83 (clinical and extended), an improvement over the direct model. Significant fibrosis models had AUCs (clinical/extended) of 0.76/0.78. The extended advanced fibrosis model (0.86) performed significantly better than the clinical version (0.82). CONCLUSIONS: Detection of NASH and at-risk NASH can be improved by constructing independent machine learning models for each component, using only clinical predictors. Adding biomarkers only improved the accuracy of fibrosis.

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.

Biomarkers for staging fibrosis and non-alcoholic steatohepatitis in non-alcoholic fatty liver disease (the LITMUS project): a comparative diagnostic accuracy study.

BACKGROUND: The reference standard for detecting non-alcoholic steatohepatitis (NASH) and staging fibrosis-liver biopsy-is invasive and resource intensive. Non-invasive biomarkers are urgently needed, but few studies have compared these biomarkers in a single cohort. As part of the Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) project, we aimed to evaluate the diagnostic accuracy of 17 biomarkers and multimarker scores in detecting NASH and clinically significant fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) and identify their optimal cutoffs as screening tests in clinical trial recruitment. METHODS: This was a comparative diagnostic accuracy study in people with biopsy-confirmed NAFLD from 13 countries across Europe, recruited between Jan 6, 2010, and Dec 29, 2017, from the LITMUS metacohort of the prospective European NAFLD Registry. Adults (aged ≥18 years) with paired liver biopsy and serum samples were eligible; those with excessive alcohol consumption or evidence of other chronic liver diseases were excluded. The diagnostic accuracy of the biomarkers was expressed as the area under the receiver operating characteristic curve (AUC) with liver histology as the reference standard and compared with the Fibrosis-4 index for liver fibrosis (FIB-4) in the same subgroup. Target conditions were the presence of NASH with clinically significant fibrosis (ie, at-risk NASH; NAFLD Activity Score ≥4 and F≥2) or the presence of advanced fibrosis (F≥3), analysed in all participants with complete data. We identified thres holds for each biomarker for reducing the number of biopsy-based screen failures when recruiting people with both NASH and clinically significant fibrosis for future trials. FINDINGS: Of 1430 participants with NAFLD in the LITMUS metacohort with serum samples, 966 (403 women and 563 men) were included after all exclusion criteria had been applied. 335 (35%) of 966 participants had biopsy-confirmed NASH and clinically significant fibrosis and 271 (28%) had advanced fibrosis. For people with NASH and clinically significant fibrosis, no single biomarker or multimarker score significantly reached the predefined AUC 0·80 acceptability threshold (AUCs ranging from 0·61 [95% CI 0·54-0·67] for FibroScan controlled attenuation parameter to 0·81 [0·75-0·86] for SomaSignal), with accuracy mostly similar to FIB-4. Regarding detection of advanced fibrosis, SomaSignal (AUC 0·90 [95% CI 0·86-0·94]), ADAPT (0·85 [0·81-0·89]), and FibroScan liver stiffness measurement (0·83 [0·80-0·86]) reached acceptable accuracy. With 11 of 17 markers, histological screen failure rates could be reduced to 33% in trials if only people who were marker positive had a biopsy for evaluating eligibility. The best screening performance for NASH and clinically significant fibrosis was observed for SomaSignal (number needed to test [NNT] to find one true positive was four [95% CI 4-5]), then ADAPT (six [5-7]), MACK-3 (seven [6-8]), and PRO-C3 (nine [7-11]). INTERPRETATION: None of the single markers or multimarker scores achieved the predefined acceptable AUC for replacing biopsy in detecting people with both NASH and clinically significant fibrosis. However, several biomarkers could be applied in a prescreening strategy in clinical trial recruitment. The performance of promising markers will be further evaluated in the ongoing prospective LITMUS study cohort. FUNDING: The Innovative Medicines Initiative 2 Joint Undertaking.

Association of changes in histologic severity of nonalcoholic steatohepatitis and changes in patient-reported quality of life.

Nonalcoholic steatohepatitis (NASH) is a prevalent chronic disease that is associated with a spectrum of liver fibrosis and can lead to cirrhosis. Patients with NASH report lower health-related quality of life (HRQoL) than the general population. It remains uncertain how changes in histologic severity are associated with changes in HRQoL. This is a secondary analysis of the Farnesoid X Receptor Ligand Obeticholic Acid in NASH Treatment (FLINT) and Pioglitazone, Vitamin E, or Placebo for Nonalcoholic Steatohepatitis (PIVENS) randomized controlled trials in patients with biopsy-proven NASH. HRQoL was assessed using short form-36 at baseline and at follow-up biopsy (at 72 and 96 weeks, respectively). Adjusted linear regression models were used to examine the association between changes in liver fibrosis (primary analysis), nonalcoholic fatty liver disease (NAFLD) activity score (secondary analysis), and changes in HRQoL scores. Compared with stable fibrosis, improvement of fibrosis by at least one stage was significantly associated with improvements only in the physical function component by 1.8 points (95% confidence interval, 0.1, 3.5). Worsening of fibrosis by at least one stage was not associated with statistically significant changes in any HRQoL domain compared with stable fibrosis. Associations between HRQoL and NAFLD disease activity score in the secondary analysis were of similar magnitude. Weight loss was associated with small improvements in physical function, general health, and energy levels. Conclusion: Improvements in fibrosis stage were associated with improvements in the physical component of HRQoL, but the clinical impact was modest. As improving fibrosis may not meaningfully improve well-being, treatment for NASH will be cost effective only if it prevents long-term hepatic and cardiovascular disease.

Infection with the hepatitis C virus causes viral genotype-specific differences in cholesterol metabolism and hepatic steatosis.

Lipids play essential roles in the hepatitis C virus (HCV) life cycle and patients with chronic HCV infection display disordered lipid metabolism which resolves following successful anti-viral therapy. It has been proposed that HCV genotype 3 (HCV-G3) infection is an independent risk factor for hepatocellular carcinoma and evidence suggests lipogenic proteins are involved in hepatocarcinogenesis. We aimed to characterise variation in host lipid metabolism between participants chronically infected with HCV genotype 1 (HCV-G1) and HCV-G3 to identify likely genotype-specific differences in lipid metabolism. We combined several lipidomic approaches: analysis was performed between participants infected with HCV-G1 and HCV-G3, both in the fasting and non-fasting states, and after sustained virological response (SVR) to treatment. Sera were obtained from 112 fasting patients (25% with cirrhosis). Serum lipids were measured using standard enzymatic methods. Lathosterol and desmosterol were measured by gas-chromatography mass spectrometry (MS). For further metabolic insight on lipid metabolism, ultra-performance liquid chromatography MS was performed on all samples. A subgroup of 13 participants had whole body fat distribution determined using in vivo magnetic resonance imaging and spectroscopy. A second cohort of (non-fasting) sera were obtained from HCV Research UK for comparative analyses: 150 treatment naïve patients and 100 non-viraemic patients post-SVR. HCV-G3 patients had significantly decreased serum apoB, non-HDL cholesterol concentrations, and more hepatic steatosis than those with HCV-G1. HCV-G3 patients also had significantly decreased serum levels of lathosterol, without significant reductions in desmosterol. Lipidomic analysis showed lipid species associated with reverse cholesterol transport pathway in HCV-G3. We demonstrated that compared to HCV-G1, HCV-G3 infection is characterised by low LDL cholesterol levels, with preferential suppression of cholesterol synthesis via lathosterol, associated with increasing hepatic steatosis. The genotype-specific lipid disturbances may shed light on genotypic variations in liver disease progression and promotion of hepatocellular cancer in HCV-G3.

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: Diagnostic and mechanistic relevance.

BACKGROUND & AIMS: Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. METHODS: We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. RESULTS: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2-4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5-8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2-4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p. CONCLUSIONS: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD. LAY SUMMARY: MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy.

Association of Weight Changes With Changes in Histological Features and Blood Markers in Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Weight loss is recommended for patients with non-alcoholic steatohepatitis (NASH) but the impact of weight change on disease activity remains unclear. We examined the association between weight change (gain/loss) and changes in biochemical and histological features of NASH. METHODS: This was an analysis of the PIVENS and FLINT trials in adults with NASH who had liver biopsies at baseline and at either 1.5 years or 2 years. Multivariable regression models examined how weight change was associated with changes in (a) blood liver markers, (b) NASH resolution with no fibrosis worsening, (c) fibrosis improving with no NASH worsening, and (d) individual histological features. RESULTS: The BMI of the 421 participants was 34.3 kg/m2 (SD:6.5) and their mean weight change was +0.5 kg (SD:6.5). Weight change was independently and positively associated with changes in liver enzymes and the Fibrosis-4 score (all P < .001). Each kg of weight loss was associated with 7% (95% CI, 3%-10%; P < .001) increase in odds of achieving NASH resolution with no fibrosis worsening and with 5% (95% CI, 1%-8%; P = .01) increase in odds of achieving fibrosis improvement with no NASH worsening. Weight gain was associated with worsening of disease activity. For every kg of weight lost, the odds of fibrosis improving were 5% (95% CI, 2%-8%; P = .001). There was no evidence that the association between weight change and outcome depended upon pharmacological treatment, trial, body mass index, and baseline fibrosis. CONCLUSIONS: Weight change was independently and monotonically associated with changes in biochemical and histological features of NASH. Guidelines for NASH management should incorporate recommendations for both avoidance of weight gain and support to lose weight.

Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.

OBJECTIVE: Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM-VCTE), Fibrosis-4 Index (FIB-4) and NAFLD (non-alcoholic fatty liver disease) Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies. DESIGN: Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individually and in sequential combinations. RESULTS: Data were included from 37 primary studies (n=5735; 45% women; median age: 54 years; median body mass index: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs (<1.3; ≥2.67) followed by LSM-VCTE cut-offs (<8.0; ≥10.0 kPa) to rule-in or rule-out advanced fibrosis had sensitivity and specificity (95% CI) of 66% (63-68) and 86% (84-87) with 33% needing a biopsy to establish a final diagnosis. FIB-4 cut-offs (<1.3; ≥3.48) followed by LSM cut-offs (<8.0; ≥20.0 kPa) to rule out advanced fibrosis or rule in cirrhosis had a sensitivity of 38% (37-39) and specificity of 90% (89-91) with 19% needing biopsy. CONCLUSION: Sequential combinations of markers with a lower cut-off to rule-out advanced fibrosis and a higher cut-off to rule-in cirrhosis can reduce the need for liver biopsies.

Assessment of hepatic steatosis by controlled attenuation parameter using the M and XL probes: an individual patient data meta-analysis.

BACKGROUND: Diagnostic tools for liver disease can now include estimation of the grade of hepatic steatosis (S0 to S3). Controlled attenuation parameter (CAP) is a non-invasive method for assessing hepatic steatosis that has become available for patients who are obese (FibroScan XL probe), but a consensus has not yet been reached regarding cutoffs and its diagnostic performance. We aimed to assess diagnostic properties and identify relevant covariates with use of an individual patient data meta-analysis. METHODS: We did an individual patient data meta-analysis, in which we searched PubMed and Web of Science for studies published from database inception until April 30, 2019. Studies reporting original biopsy-controlled data of CAP for non-invasive grading of steatosis were eligible. Probe recommendation was based on automated selection, manual assessment of skin-to-liver-capsule distance, and a body-mass index (BMI) criterion. Receiver operating characteristic methods and mixed models were used to assess diagnostic properties and covariates. Patients with non-alcoholic fatty liver disease (NAFLD) were analysed separately because they are the predominant patient group when using the XL probe. This study is registered with PROSPERO, CRD42018099284. FINDINGS: 16 studies reported histology-controlled CAP including the XL probe, and individual data from 13 papers and 2346 patients were included. Patients with a mean age of 46·5 years (SD 14·5) were recruited from 20 centres in nine countries. 2283 patients had data for BMI; 673 (29%) were normal weight (BMI <25 kg/m2), 530 (23%) were overweight (BMI ≥25 to <30 kg/m2), and 1080 (47%) were obese (BMI ≥30 kg/m2). 1277 (54%) patients had NAFLD, 474 (20%) had viral hepatitis, 285 (12%) had alcohol-associated liver disease, and 310 (13%) had other liver disease aetiologies. The XL probe was recommended in 1050 patients, 930 (89%) of whom had NAFLD; among the patients with NAFLD, the areas under the curve were 0·819 (95% CI 0·769-0·869) for S0 versus S1 to S3 and 0·754 (0·720-0·787) for S0 to S1 versus S2 to S3. CAP values were independently affected by aetiology, diabetes, BMI, aspartate aminotransferase, and sex. Optimal cutoffs differed substantially across aetiologies. Risk of bias according to QUADAS-2 was low. INTERPRETATION: CAP cutoffs varied according to cause, and can effectively recognise significant steatosis in patients with viral hepatitis. CAP cannot grade steatosis in patients with NAFLD adequately, but its value in a NAFLD screening setting needs to be studied, ideally with methods beyond the traditional histological reference standard. FUNDING: The German Federal Ministry of Education and Research and Echosens.

The European NAFLD Registry: A real-world longitudinal cohort study of nonalcoholic fatty liver disease.

Non-Alcoholic Fatty Liver Disease (NAFLD), a progressive liver disease that is closely associated with obesity, type 2 diabetes, hypertension and dyslipidaemia, represents an increasing global public health challenge. There is significant variability in the disease course: the majority exhibit only fat accumulation in the liver but a significant minority develop a necroinflammatory form of the disease (non-alcoholic steatohepatitis, NASH) that may progress to cirrhosis and hepatocellular carcinoma. At present our understanding of pathogenesis, disease natural history and long-term outcomes remain incomplete. There is a need for large, well characterised patient cohorts that may be used to address these knowledge gaps and to support the development of better biomarkers and novel therapies. The European NAFLD Registry is an international, prospectively recruited observational cohort study that aims to establish a large, highly-phenotyped patient cohort and linked bioresource. Here we describe the infrastructure, data management and monitoring plans, and the standard operating procedures implemented to ensure the timely and systematic collection of high-quality data and samples. Already recruiting subjects at secondary/tertiary care centres across Europe, the Registry is supporting the European Union IMI2-funded LITMUS 'Liver Investigation: Testing Marker Utility in Steatohepatitis' consortium, which is a major international effort to robustly validate biomarkers that diagnose, risk stratify and/or monitor NAFLD progression and liver fibrosis stage. The European NAFLD Registry has the demonstrable capacity to support research and biomarker development at scale and pace.

Systemic iron reduction by venesection alters the gut microbiome in patients with haemochromatosis.

BACKGROUND & AIMS: Iron reduction by venesection has been the cornerstone of treatment for haemochromatosis for decades, and its reported health benefits are many. Repeated phlebotomy can lead to a compensatory increase in intestinal iron absorption, reducing intestinal iron availability. Given that most gut bacteria are highly dependent on iron for survival, we postulated that, by reducing gut iron levels, venesection could alter the gut microbiota. METHODS: Clinical parameters, faecal bacterial composition and metabolomes were assessed before and during treatment in a group of patients with haemochromatosis undergoing iron reduction therapy. RESULTS: Systemic iron reduction was associated with an alteration of the gut microbiome, with changes evident in those who experienced reduced faecal iron availability with venesection. For example, levels of Faecalibacterium prausnitzii, a bacterium associated with improved colonic health, were increased in response to faecal iron reduction. Similarly, metabolomic changes were seen in association with reduced faecal iron levels. CONCLUSION: These findings highlight a significant shift in the gut microbiome of patients who experience reduced colonic iron during venesection. Targeted depletion of faecal iron could represent a novel therapy for metabolic and inflammatory diseases, meriting further investigation. LAY SUMMARY: Iron depletion by repeated venesection is the mainstay of treatment for haemochromatosis, an iron-overload disorder. Venesection has been associated with several health benefits, including improvements in liver function tests, reversal of liver scarring, and reduced risk of liver cancer. During iron depletion, iron absorption from the gastrointestinal (GI) tract increases to compensate for iron lost with treatment. Iron availability is limited in the GI tract and is crucial to the growth and function of many gut bacteria. In this study we show that reduced iron availability in the colon following venesection treatment leads to a change in the composition of the gut bacteria, a finding that, to date, has not been studied in patients with haemochromatosis.

Prognostic value of multiparametric magnetic resonance imaging, transient elastography and blood-based fibrosis markers in patients with chronic liver disease.

BACKGROUND & AIMS: Liver cT1 , liver T1 , transient elastography (TE) and blood-based biomarkers have independently been shown to predict clinical outcomes but have not been directly compared in a single cohort of patients. Our aim was to compare these tests' prognostic value in a cohort of patients with compensated chronic liver disease. METHODS: Patients with unselected compensated liver disease aetiologies had baseline assessments and were followed up for development of clinical outcomes, blinded to the imaging results. The prognostic value of non-invasive liver tests at prespecified thresholds was assessed for a combined clinical endpoint comprising ascites, variceal bleeding, hepatic encephalopathy, hepatocellular carcinoma, liver transplantation and mortality. RESULTS: One hundred and ninety-seven patients (61% male) with median age of 54 years were followed up for 693 patient-years (median (IQR) 43 (26-58) months). The main diagnoses were NAFLD (41%), viral hepatitis (VH, 25%) and alcohol-related liver disease (ArLD; 14%). During follow-up 14 new clinical events, and 11 deaths occurred. Clinical outcomes were predicted by liver cT1  > 825ms with HR 9.9 (95% CI: 1.29-76.4, P = .007), TE > 8kPa with HR 7.8 (95% CI: 0.97-62.3, P = .02) and FIB-4 > 1.45 with HR 4.09 (95% CI: 0.90-18.4, P = .05). In analysis taking into account technical failure and unreliability, liver cT1  > 825 ms could predict clinical outcomes (P = .03), but TE > 8kPa could not (P = .4). CONCLUSIONS: We provide further evidence that liver cT1 , TE and serum-based biomarkers can predict clinical outcomes, but when taking into account technical failure/unreliability, TE cut-offs perform worse than those of cT1 and blood biomarkers.

Accurate non-invasive diagnosis and staging of non-alcoholic fatty liver disease using the urinary steroid metabolome.

BACKGROUND: The development of accurate, non-invasive markers to diagnose and stage non-alcoholic fatty liver disease (NAFLD) is critical to reduce the need for an invasive liver biopsy and to identify patients who are at the highest risk of hepatic and cardio-metabolic complications. Disruption of steroid hormone metabolic pathways has been described in patients with NAFLD. AIM(S): To assess the hypothesis that assessment of the urinary steroid metabolome may provide a novel, non-invasive biomarker strategy to stage NAFLD. METHODS: We analysed the urinary steroid metabolome in 275 subjects (121 with biopsy-proven NAFLD, 48 with alcohol-related cirrhosis and 106 controls), using gas chromatography-mass spectrometry (GC-MS) coupled with machine learning-based Generalised Matrix Learning Vector Quantisation (GMLVQ) analysis. RESULTS: Generalised Matrix Learning Vector Quantisation analysis achieved excellent separation of early (F0-F2) from advanced (F3-F4) fibrosis (AUC receiver operating characteristics [ROC]: 0.92 [0.91-0.94]). Furthermore, there was near perfect separation of controls from patients with advanced fibrotic NAFLD (AUC ROC = 0.99 [0.98-0.99]) and from those with NAFLD cirrhosis (AUC ROC = 1.0 [1.0-1.0]). This approach was also able to distinguish patients with NAFLD cirrhosis from those with alcohol-related cirrhosis (AUC ROC = 0.83 [0.81-0.85]). CONCLUSIONS: Unbiased GMLVQ analysis of the urinary steroid metabolome offers excellent potential as a non-invasive biomarker approach to stage NAFLD fibrosis as well as to screen for NAFLD. A highly sensitive and specific urinary biomarker is likely to have clinical utility both in secondary care and in the broader general population within primary care and could significantly decrease the need for liver biopsy.