The overarching aim of LITMUS is to develop, robustly validate and advance towards regulatory qualification biomarkers that diagnose, risk stratify and/or monitor NAFLD/NASH progression and fibrosis stage. This will be achieved through a goal-oriented, tri-partite collaboration delivering a definitive and impartial evaluation platform for biomarkers, bringing together: (i) End-users of biomarker technologies (clinicians with expertise in NAFLD and the pharmaceutical industry); (ii) Independent academics with expertise in the evaluation of medical test/biomarker performance; and (iii) Biomarker researchers and developers (academic or commercial).
For deliverable D3.2, we aimed to validate a 7-tier histological staging system for NAFLD-associated fibrosis using digital slides. The EPOS staging system is a 7-tier system for semi-quantitating the extent of fibrosis that has been created and tested by the EPOS Histopathology Group, members of which are included in the LITMUS Histopathology Group – LHG (Bedossa, J Hepatol 2018).
The performance of biomarkers in LITMUS has to be evaluated against well-defined criteria for acceptability. For this purpose, the context of use/minimally acceptable performance criteria document is prepared by LITMUS partners. These criteria include a definition of the context of use, the appropriate target condition(s), acceptable reference standard, required clinical evidence, and minimum levels of accuracy measurements. The whole document could be accessed here and is meant to be a “living” resource, further modified, if needed, in the course of the LITMUS project and will be periodically updated with additional terms and clarifying information.
Poster presentation by Whalley, D. et al at the EASL Digital International Liver Congress, 27th – 29th August 2020
“Patient-Reported Symptoms and Impact in Nonalcoholic Steatohepatitis: An Evaluation of the NASH-CHECK, a Novel PROM in NASH”
Poster presentation by Breckons, M. et al at the EASL Digital International Liver Congress, 27th – 29th August 2020
“Evaluating the patient-perceived impact of non-alcoholic steatohepatitis with compensated cirrhosis”
Poster presentation by Younes, R. et al at the EASL Digital International Liver Congress, 27th – 29th August 2020
“Insulin secretion is an independent predictor of hepatic ballooning in non-diabetic subjects with non-alcoholic fatty liver disease”
Poster presentation by Rosso, C. et al at the EASL Digital International Liver Congress, 27th – 29th August 2020
“Interplay between metabolic derangement, biomarkers of collagen remodeling and macrophage activation in non-diabetic patients with non-alcoholic fatty liver disease”
Poster presentation by Lee, J. et al at the EASL Digital International Liver Congress, 27th – 29th August 2020
“Accuracy of CK-18 (M30 and M65) in detecting NASH and fibrosis: a systematic review and meta-analysis”
Poster presentation by Selvaraj, E. et al at the EASL Digital International Liver Congress, 27th – 29th August 2020
“Diagnostic accuracy of acoustic radiation force impulse elastography for the staging of hepatic fibrosis in non-alcoholic fatty liver disease: a systematic review and meta-analysis”
Poster presentation by Mozes, F. et al at the EASL Digital International Liver Congress, 27th – 29th August 2020
“Diagnostic accuracy of magnetic resonance elastography for the staging of fibrosis and diagnosis of steatohepatitis in patients with non-alcoholic fatty liver disease: a systematic review and meta-analysis”
Poster presentation by Hvid, H. et al at the 3rd Global NASH Congress, London, UK, 11th February 2020
“Liver stiffness assessed with Shear Wave Elastography correlates with biopsy data in Gottingen Minipigs with non-alcoholic steatohepatitis”
Poster presentation by Johnson, K. et al at the AASLD Liver Meeting, Boston, MA, USA, 8th – 12th November 2019
“High-throughput detection of novel circulating miRNA biomarkers of non-alcoholic fatty liver disease”
Poster presentation by Rosso, C. et al, at EASL International Liver Congress, Vienna, Austria, 10th – 14th April 2019
“Selenoprotein P levels discriminate the degree of hepatic steatosis and are related to the NAS score in patients with Non-Alcoholic Fatty Liver Disease”
Poster presentation by Carli, F. et al, at EASL International Liver Congress, Paris, France, 11th – 15th April 2018
“In non obese NAFLD increased plasma saturated fatty acids and insulin resistance are metabolic signatures of severity of liver disease”
Poster presentation by Svegliati-Baroni, G. et al, at EASL International Liver Congress, Paris, France, 11th – 15th April 2018
“Predisposition to diabetes is related to insulin resistance in NAFLD patients and to decreased insulin secretion in HCV patients”
Journal article: Hardy, T. et al “The European NAFLD Registry: A real-world longitudinal cohort study of nonalcoholic fatty liver disease”, Contemporary Clinical Trials 98, 1st November 2020.
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.
Journal article: Lee, J. et al, “Prognostic accuracy of FIB‐4, NAFLD fibrosis score, and APRI for NAFLD‐related events: a systematic review”, Liver International, 18th September 2020.
Background & Aims:
Fibrosis is the strongest predictor for long‐term clinical outcomes among patients with non‐alcoholic fatty liver disease (NAFLD). There is growing interest in employing non‐invasive methods for risk stratification based on prognosis. FIB‐4, NFS and APRI are models commonly used for detecting fibrosis among NAFLD patients. We aimed to synthesize existing literature on the ability of these models in prognosticating NAFLD‐related events.
A sensitive search was conducted in two medical databases to retrieve studies evaluating the prognostic accuracy of FIB‐4, NFS and APRI among NAFLD patients. Target events were change in fibrosis, liver‐related event, and mortality. Two reviewers independently performed reference screening, data extraction and quality assessment (QUAPAS tool).
A total of 13 studies (FIB‐4: 12, NFS: 11, APRI: 10), published between 2013 and 2019, were retrieved. All studies were conducted in a secondary or tertiary care setting, with follow‐up ranging from one to 20 years. All three markers showed consistently good prognostication of liver‐related events (AUC from 0.69 to 0.92). For mortality, FIB‐4 (AUC of 0.67 to 0.82) and NFS (AUC of 0.70 to 0.83) outperformed APRI (AUC of 0.52 to 0.73) in all studies. All markers had inconsistent performance for predicting change in fibrosis stage.
FIB‐4, NFS and APRI have demonstrated ability to risk stratify patients for liver‐related morbidity and mortality, with comparable performance to a liver biopsy, although more head‐to‐head studies are needed to validate this. More refined models to prognosticate NAFLD‐events may further enhance performance and clinical utility of non‐invasive markers.
Journal article: Lee, J. et al, “Accuracy of cytokeratin 18 (M30 and M65) in detecting non-alcoholic steatohepatitis and fibrosis: A systematic review and meta-analysis”, Plos One, 11th September 2020.
Association between elevated cytokeratin 18 (CK-18) levels and hepatocyte death has made circulating CK-18 a candidate biomarker to differentiate non-alcoholic fatty liver from non-alcoholic steatohepatitis (NASH). Yet studies produced variable diagnostic performance. We aimed to provide summary estimates with increased precision for the accuracy of CK-18 (M30, M65) in detecting NASH and fibrosis among non-alcoholic fatty liver disease (NAFLD) adults.
We searched five databases to retrieve studies evaluating CK-18 against a liver biopsy in NAFLD adults. Reference screening, data extraction and quality assessment (QUADAS-2) were independently conducted by two authors. Meta-analyses were performed for five groups based on the CK-18 antigens and target conditions, using one of two methods: linear mixed-effects multiple thresholds model or bivariate logit-normal random-effects model.
We included 41 studies, with data on 5,815 participants. A wide range of disease prevalence was observed. No study reported a pre-defined cut-off. Thirty of 41 studies provided sufficient data for inclusion in any of the meta-analyses. Summary AUC [95% CI] were: 0.75 [0.69–0.82] (M30) and 0.82 [0.69–0.91] (M65) for NASH; 0.73 [0.57–0.85] (M30) for fibrotic NASH; 0.68 (M30) for significant (F2-4) fibrosis; and 0.75 (M30) for advanced (F3-4) fibrosis. Thirteen studies used CK-18 as a component of a multimarker model.
For M30 we found lower diagnostic accuracy to detect NASH compared to previous meta-analyses, indicating a limited ability to act as a stand-alone test, with better performance for M65. Additional external validation studies are needed to obtain credible estimates of the diagnostic accuracy of multimarker models.
Journal article: Vali, Y. et al, “Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: A systematic review and meta-analysis”, Journal of Hepatology 73(2), August 2020.
Background & Aims:
The enhanced liver fibrosis (ELF) test has been proposed for the non-invasive assessment of advanced fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). We performed a systematic review to estimate the accuracy of this test against biopsy.
In this systematic review, we searched MEDLINE, Embase, Web of Science and the Cochrane Library for studies that included patients with NAFLD and that used both liver biopsy (as the reference standard) and the ELF test. Two authors independently screened the references, extracted the data and assessed the quality of included studies. Due to the variation in reported thresholds, we used a multiple thresholds random effects model for meta-analysis (diagmeta R-package).
The meta-analysis of 11 studies reporting advanced fibrosis and 5 studies reporting significant fibrosis showed that the ELF test had a sensitivity of >0.90 for excluding fibrosis at a threshold of 7.7. However, as a diagnostic test at high thresholds, the test only achieved specificity and positive predictive value >0.80 in very high prevalence settings (>50%). To achieve a specificity of 0.90 for advanced and significant fibrosis, thresholds of 10.18 (sensitivity: 0.57) and 9.86 (sensitivity: 0.55) were required, respectively.
The ELF test showed high sensitivity but limited specificity to exclude advanced and significant fibrosis at low cut-offs. The diagnostic performance of the test at higher thresholds was found to be more limited in low-prevalence settings. We conclude that clinicians should carefully consider the likely disease prevalence in their practice setting and adopt suitable test thresholds to achieve the desired performance.
Journal article: McSweeney, L. et al, “Health-related quality of life and patient-reported outcome measures in NASH-related cirrhosis”, JHEP Reports 2(3), June 2020.
Background & Aims:
Non-alcoholic steatohepatitis (NASH) is known to have a negative impact on patients’ health-related quality of life (HRQoL), even before progression to cirrhosis has occurred. The burden of NASH-related cirrhosis from the patient perspective remains poorly understood. Herein, we aimed to identify the burden of disease and HRQoL impairment among patients with NASH-related compensated cirrhosis.
This targeted literature review sought first to identify the humanistic burden of disease from the perspective of patients with diagnosed NASH-cirrhosis and, secondly, to identify generic or disease-specific patient-reported outcome measures (PROMs) used to assess the impact of NASH-cirrhosis. Searches were conducted in bibliographical databases, grey or unpublished literature, liver disease websites, support group websites and online blogs. A quality assessment of specific PROMs was conducted.
Patients with NASH-cirrhosis are reported to suffer from lower HRQoL than patients with non-cirrhotic NASH and the general population with respect to physical health/functioning, emotional health and worry, and mental health. Thirteen PROMs were identified, of which 4 were liver-disease specific: CLDQ, CLDQ-NAFLD, LDQoL and LDSI. The most commonly used measures do not comply with current industry or regulatory standards for PROMs and/or are not validated for use in a cirrhotic NASH population.
Patients with NASH-cirrhosis have lower HRQoL and poorer physical health than patients with non-cirrhotic NASH. However, the literature lacked detail of the everyday impact on patients’ lives. Currently, a number of PROMs are available to measure the impact of the disease in patients with chronic liver conditions. The lack of studies that include qualitative insights in this population mandates further exploration and research.
Journal article: Aron-Wisnewsky, J. et al, “Nonalcoholic Fatty Liver Disease: Modulating Gut Microbiota to Improve Severity?”, Gastroenterology 158(7), May 2020.
Gut microbiota plays a role in the pathophysiology of metabolic diseases, which include nonalcoholic fatty liver diseases, through the gut–liver axis. To date, clinical guidelines recommend a weight loss goal of 7%–10% to improve features of nonalcoholic fatty liver diseases. Because this target is not easily achieved by all patients, alternative therapeutic options are currently being evaluated. This review focuses on therapeutics that aim to modulate the gut microbiota and the gut–liver axis. We discuss how probiotics, prebiotics, synbiotic, fecal microbiota transfer, polyphenols, specific diets, and exercise interventions have been found to modify gut microbiota signatures; improve nonalcoholic fatty liver disease outcomes; and detail, when available, the different mechanisms by which these beneficial outcomes might occur. Apart from probiotics that have already been tested in human randomized controlled trials, most of these potential therapeutics have been studied in animals. Their efficacy still warrants confirmation in humans using appropriate design.
Journal article: Luukkonen, P.K. et al, “Hydroxysteroid 17-β dehydrogenase 13 variant increases phospholipids and protects against fibrosis in nonalcoholic fatty liver disease”, JCI insight 5(5), March 2020.
Carriers of the hydroxysteroid 17-β dehydrogenase 13 (HSD17B13) gene variant (rs72613567:TA) have a reduced risk of NASH and cirrhosis but not steatosis. We determined its effect on liver histology, lipidome, and transcriptome using ultra performance liquid chromatography-mass spectrometry and RNA-seq. In carriers and noncarriers of the gene variant, we also measured pathways of hepatic fatty acids (de novo lipogenesis [DNL] and adipose tissue lipolysis [ATL] using 2H2O and 2H-glycerol) and insulin sensitivity using 3H-glucose and euglycemic-hyperinsulinemic clamp) and plasma cytokines. Carriers and noncarriers had similar age, sex and BMI. Fibrosis was significantly less frequent while phospholipids, but not other lipids, were enriched in the liver in carriers compared with noncarriers. Expression of 274 genes was altered in carriers compared with noncarriers, consisting predominantly of downregulated inflammation-related gene sets. Plasma IL-6 concentrations were lower, but DNL, ATL and hepatic insulin sensitivity were similar between the groups. In conclusion, carriers of the HSD17B13 variant have decreased fibrosis and expression of inflammation-related genes but increased phospholipids in the liver. These changes are not secondary to steatosis, DNL, ATL, or hepatic insulin sensitivity. The increase in phospholipids and decrease in fibrosis are opposite to features of choline-deficient models of liver disease and suggest HSD17B13 as an attractive therapeutic target.
Journal article: Aron-Wisnewsky, J. et al, “Gut microbiota and human NAFLD: disentangling microbial signatures from metabolic disorders”, Nature Reviews Gasteroenterology & Hepatology 17, March 2020.
Gut microbiota dysbiosis has been repeatedly observed in obesity and type 2 diabetes mellitus, two metabolic diseases strongly intertwined with non-alcoholic fatty liver disease (NAFLD). Animal studies have demonstrated a potential causal role of gut microbiota in NAFLD. Human studies have started to describe microbiota alterations in NAFLD and have found a few consistent microbiome signatures discriminating healthy individuals from those with NAFLD, non-alcoholic steatohepatitis or cirrhosis. However, patients with NAFLD often present with obesity and/or insulin resistance and type 2 diabetes mellitus, and these metabolic confounding factors for dysbiosis have not always been considered. Patients with different NAFLD severity stages often present with heterogeneous lesions and variable demographic characteristics (including age, sex and ethnicity), which are known to affect the gut microbiome and have been overlooked in most studies. Finally, multiple gut microbiome sequencing tools and NAFLD diagnostic methods have been used across studies that could account for discrepant microbiome signatures. This Review provides a broad insight into microbiome signatures for human NAFLD and explores issues with disentangling these signatures from underlying metabolic disorders. More advanced metagenomics and multi-omics studies using system biology approaches are needed to improve microbiome biomarkers.
Journal article: Meroni, M. et al, “MBOAT7 down-regulation by hyper-insulinemia induces fat accumulation in hepatocytes”, EBioMedicine 52, February 2020.
Naturally occurring variation in Membrane-bound O-acyltransferase domain-containing 7 (MBOAT7), encoding for an enzyme involved in phosphatidylinositol acyl-chain remodelling, has been associated with fatty liver and hepatic disorders. Here, we examined the relationship between hepatic MBOAT7 down-regulation and fat accumulation.
Hepatic MBOAT7 expression was surveyed in 119 obese individuals and in experimental models. MBOAT7 was acutely silenced by antisense oligonucleotides in C57Bl/6 mice, and by CRISPR/Cas9 in HepG2 hepatocytes.
In obese individuals, hepatic MBOAT7 mRNA decreased from normal liver to steatohepatitis, independently of diabetes, inflammation and MBOAT7 genotype. Hepatic MBOAT7 levels were reduced in murine models of fatty liver, and by hyper-insulinemia. In wild-type mice, MBOAT7 was down-regulated by refeeding and insulin, concomitantly with insulin signalling activation. Acute hepatic MBOAT7 silencing promoted hepatic steatosis I and enhanced expression of fatty acid transporter Fatp1. MBOAT7 deletion in hepatocytes reduced the incorporation of arachidonic acid into phosphatidylinositol, consistently with decreased enzymatic activity, determining the accumulation of saturated triglycerides, enhanced lipogenesis and FATP1 expression, while FATP1 deletion rescued the phenotype.
MBOAT7 down-regulation by hyper-insulinemia contributes to hepatic fat accumulation, impairing phosphatidylinositol remodelling and up-regulating FATP1.
Journal article: Majo, J. et al, “Pathology and natural history of organ fibrosis”, Current Opinion in Pharmacology 49, December 2019.
Histopathological assessment of fibrosis focusing on morphological patterns provides important information for the management of patients with chronic diseases of the kidney, liver and the lung. This review summarizes key histopathological features of pulmonary, renal and hepatic fibrosis and discusses advances in the understanding of the pathogenesis of pulmonary fibrosis and pathogenetic insights with translational implications for renal fibrosis. The review also tackles new staging approaches based on liver fibrosis dynamics and evaluation of fibrosis regression, digital pathology and second harmonic generation microscopy methods for hepatic fibrosis assessment and critical appraisal of non-invasive tests for liver and renal fibrosis evaluation.
Journal article: Luukkonen, P.K. et al, “Human PNPLA3-I148M variant increases hepatic retention of polyunsaturated fatty acids”, JCI insight 4(16), August 2019.
The common patatin-like phospholipase domain–containing protein 3 (PNPLA3) variant I148M predisposes to nonalcoholic liver disease but not its metabolic sequelae. We compared the handling of labeled polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFA) in vivo in humans and in cells harboring different PNPLA3 genotypes. In 148M homozygous individuals, triglycerides (TGs) in very low–density lipoproteins (VLDL) were depleted of PUFAs both under fasting and postprandial conditions compared with 148I homozygotes, and the PUFA/SFA ratio in VLDL-TGs was lower relative to the chylomicron precursor pool. In human PNPLA3-148M and PNPLA3-KO cells, PUFA but not SFA incorporation into TGs was increased at the expense of phosphatidylcholines, and under lipolytic conditions, PUFA-containing diacylglycerols (DAGs) accumulated compared with PNPLA3-148I cells. Polyunsaturated TGs were increased, while phosphatidylcholines (PCs) were decreased in the human liver in 148M homozygous individuals as compared with 148I homozygotes. We conclude that human PNPLA3-I148M is a loss-of-function allele that remodels liver TGs in a polyunsaturated direction by impairing hydrolysis/transacylation of PUFAs from DAGs to feed phosphatidylcholine synthesis.
Journal article: Farrell, G. et al, “Mouse Models of Nonalcoholic Steatohepatitis: Toward Optimization of Their Relevance to Human Nonalcoholic Steatohepatitis”, Hepatology 69(5), May 2019.
Nonalcoholic steatohepatitis (NASH) arises from a variable interplay between environmental factors and genetic determinants that cannot be completely replicated in animals. Notwithstanding, preclinical models are needed to understand NASH pathophysiology and test mechanism-based therapies. Among several mouse models of NASH, some exhibit the key pathophysiologic as well as histopathologic criteria for human NASH, whereas others may be useful to address specific questions. Models based on overnutrition with adipose restriction/inflammation and metabolic complications, particularly insulin resistance, may be most useful to investigate critical etiopathogenic factors. In-depth pathologic description is required for all models. Some models demonstrate hepatocyte ballooning, which can be confused with microvesicular steatosis, whereas demonstration of an inflammatory infiltrate and pattern of liver fibrosis compatible with human NASH is desirable in models used for pharmacologic testing. When mice with specific genetic strains or mutations that cause overeating consume a diet enriched with fat, modest amounts of cholesterol, and/or simple sugars (“Western diet”), they readily develop obesity with liver disease similar to human NASH, including significant fibrosis. Purely dietary models, such as high-fat/high-cholesterol, Western diet, and choline-deficient, amino acid–defined, are similarly promising. We share concern about using models without weight gain, adipose pathology, or insulin resistance/hyperinsulinemia and with inadequate documentation of liver pathology. NASH-related fibrosis is a key endpoint in trials of possible therapies. When studied for this purpose, NASH models should be reproducible and show steatohepatitis (ideally with ballooning) and at least focal bridging fibrosis, while metabolic factors/disordered lipid partitioning should contribute to etiopathogenesis. Because murine models are increasingly used to explore pharmacologic therapies for NASH, we propose a minimum set of requirements that investigators, drug companies, and journals should consider to optimize their translational value.
Journal article: Younes, R. & Bugianesi, E., “NASH in lean individuals”, Seminars in Liver Disease 39(01), January 2019.
Nonalcoholic fatty liver disease (NAFLD) is generally associated with obesity and the related comorbidities but it can also develop in subjects with a body mass index (BMI) within the ethnic-specific cutoff of 25 kg/m2 BMI in Caucasian and 23 kg/m2 in Asian subjects, the so-called “lean” NAFLD. This sub-phenotype of NAFLD patients has been described across populations of different ethnicity, particularly in Asia, but it can be diagnosed in 10 to 20% of nonobese Americans and Caucasians. Pathophysiological mechanisms underpinning the “lean” phenotype are not completely understood, but they may include a more dysfunctional fat (visceral obesity, differences in adipocyte differentiation and altered lipid turnover), altered body composition (decreased muscle mass), a genetic background, not limited to patatin-like phospholipase domain-containing protein 3 (PNPLA3) C > G polymorphisms, epigenetic changes occurring early in life and a different pattern of gut microbiota. Lean subjects with NAFLD have milder features of the metabolic syndrome when compared with obese patients. Nonetheless they have a higher prevalence of metabolic alterations (e.g., dyslipidemia, arterial hypertension, insulin resistance, and diabetes) compared with healthy controls. Data on histological severity are controversial, but they can develop the full spectrum of liver disease associated with nonalcoholic steatohepatitis NASH. Since lean NAFLD usually present with less obesity-related comorbidities, it is commonly believed that this group would follow a relatively benign clinical course but recent data challenge this concept. Here, the authors describe the current knowledge about NAFLD in lean individuals and highlight the unanswered questions and gaps in the field.