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

BackgroundHistologically 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.MethodsThis 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: 2·67; NFS: 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.FindingsOf 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.InterpretationSimple 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

An unbiased ranking of murine dietary models based on their proximity to human Metabolic Dysfunction–associated Steatotic Liver Disease (MASLD)

Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), encompasses steatosis and steatohepatitis (NASH/MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASH research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male), and ranked them using an unbiased MASLD “Human Proximity Score” (MHPS) to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western Diets align closely with human MASH; high cholesterol content, extended study duration, and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with selection of appropriate in vivo models to accelerate preclinical research.This study has been conducted as part of the Preclinical work package of the LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis) project. The LITMUS study is a large multi-centre study aiming to evaluate Non-Alcoholic Fatty Liver Disease biomarkers. The Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under Grant Agreement 777377, funded the LITMUS study. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation program and EFPIA. EMBL-EBI Core funding supported EP and IK through funding and computing resources from EMBL-EBI. Funding from the MRC (Medical Research Council) supported IK. M.V. is supported by the University of Bari (Horizon Europe Seed cod. id. S06-miRNASH), the Foundation for Liver Research (Intramural Funding), Associazione Italiana Ricerca sul Cancro (IG2022 Grant n. 27521) and Ministry of University and Research on Next Generation EU Funds [COD: P202222FCC, CUP: H53D23009960001, D.D. MUR 1366 (01-09-2023), Title: “System Biology” approaches in HCV Patients with Residual Hepatic Steatosis after Viral Eradication; Cod PE00000003, CUP: H93C22000630001, DD MUR 1550, Title: “ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security – Working ON Foods”; Cod: CN00000041, CUP: H93C22000430007, Title PNRR “National Center for Gene Therapy and Drugs based on RNA Technology”, M4C2-Investment 1.4; Code: CN00000013, CUP: H93C22000450007, Title PNNR: “National Centre for HPC, Big Data and Quantum Computing”). A.V-P. is funded by MRC MDU, MRC Metabolic Diseases Unit (MC_UU_00014/5): Disease Model Core, Biochemistry Assay Lab, Histology Core and British Heart Foundation. F.O. is funded by UK Medical Research Council Program Grants MR/K0019494/1 and MR/R023026/1. C.M.P.R. is supported by Fundação para a Ciência e Tecnologia (PTDC/MED-FAR/3492/2021) and La Caixa Foundation (LCF/PR/HR21/52410028). Q.M.A. is supported by the Newcastle NIHR Biomedical Research Centre. S.L.F. and W.S. are supported by the NIH (NIH R01 DK128289; NCI 5P30CA196521-08 to S.L.F.; NIH R01 DK136016 to W.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript

Choline-deficient high-fat diet-induced MASH in Göttingen Minipigs: Characterisation and effects of a chow reversal period

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) is increasing, and translational animal models are needed to develop novel treatments for this disease. The physiology and metabolism of pigs have a relatively high resemblance to humans, and the present study aimed to characterise choline-deficient, and high-fat diet (CDAHFD) fed Göttingen Minipigs as a novel animal model of MASLD/MASH. Göttingen Minipigs were fed CDAHFD for up to 5 months, and the phenotype was investigated by analysis of plasma parameters and repeated collection of liver biopsies. Furthermore, changes in hepatic gene expression during the experiment were explored by RNA sequencing. For a subset of the minipigs, the diet was changed from CDAHFD back to chow to investigate if the liver pathology was reversible. Göttingen Minipigs on CDAHFD gained bodyweight, and plasma levels of cholesterol, AST, ALT, ALP and GGT were increased. CDAHFD-fed minipigs developed hepatic steatosis, inflammation, and fibrosis, which in 5 of 16 animals progressed to cirrhosis. During an 11-week chow reversal period, steatosis regressed while fibrosis persisted. Regarding inflammation, the findings were less clear, depending on the type of readout. MASH Human Proximity Scoring (combined evaluation of transcriptional, phenotypic and histopathological parameters) showed that CDAHFD-fed Göttingen Minipigs resemble human MASLD/MASH better than most rodent models. In conclusion, CDAHFD-fed minipigs develop a MASH-like phenotype which in several aspects resemble the changes observed in human patients with MASLD/MASH. Furthermore, repeated collection of liver biopsies allow detailed characterisation of histopathological changes over time in individual animals.European Federation of Pharmaceutical Industries and Associations 10.13039/100013322EC | Horizon 2020 Framework Programme 10.13039/10001066

An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.This study has been conducted as part of the Preclinical work package of the LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis) project. The LITMUS study is a large multi-centre study aiming to evaluate Non-Alcoholic Fatty Liver Disease biomarkers. The Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under Grant Agreement 777377, funded the LITMUS study. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation program and EFPIA. EMBL-EBI Core funding supported EP and IK through funding and computing resources from EMBL-EBI. Funding from the MRC (Medical Research Council) supported IK. M.V. is supported by the University of Bari (Horizon Europe Seed cod. id. S06-miRNASH), the Foundation for Liver Research (Intramural Funding), Associazione Italiana Ricerca sul Cancro (IG2022 Grant n. 27521) and Ministry of University and Research on Next Generation EU Funds [COD: P202222FCC, CUP: H53D23009960001, D.D. MUR 1366 (01-09-2023), Title: “System Biology” approaches in HCV Patients with Residual Hepatic Steatosis after Viral Eradication; Cod PE00000003, CUP: H93C22000630001, DD MUR 1550, Title: “ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security – Working ON Foods”; Cod: CN00000041, CUP: H93C22000430007, Title PNRR “National Center for Gene Therapy and Drugs based on RNA Technology”, M4C2-Investment 1.4; Code: CN00000013, CUP: H93C22000450007, Title PNNR: “National Centre for HPC, Big Data and Quantum Computing”). A.V-P. is funded by MRC MDU, MRC Metabolic Diseases Unit (MC_UU_00014/5): Disease Model Core, Biochemistry Assay Lab, Histology Core and British Heart Foundation. F.O. is funded by UK Medical Research Council Program Grants MR/K0019494/1 and MR/R023026/1. C.M.P.R. is supported by Fundação para a Ciência e Tecnologia (PTDC/MED-FAR/3492/2021) and La Caixa Foundation (LCF/PR/HR21/52410028). Q.M.A. is supported by the Newcastle NIHR Biomedical Research Centre. S.L.F. and W.S. are supported by the NIH (NIH R01 DK128289; NCI 5P30CA196521-08 to S.L.F.; NIH R01 DK136016 to W.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript

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

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

Abstract: 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\u20134), 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 6515). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0\u20132 vs F3 vs F4; LSM: 2\ub767; NFS: 0\ub7676), 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\ub77%] were female, median age was 54 years [IQR 44\u201363), and 1161 [46\ub71%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33\u201391], the composite endpoint was observed in 145 (5\ub78%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0\ub70001 for all comparisons). The tAUC at 5 years were 0\ub772 (95% CI 0\ub762\u20130\ub781) for histology, 0\ub776 (0\ub770\u20130\ub783) for LSM-VCTE, 0\ub774 (0\ub764\u20130\ub782) for FIB-4, and 0\ub770 (0\ub763\u20130\ub780) 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