Publications

Below please find relevant publications, including reports, posters, and journal articles.

Public deliverables

LITMUS Detailed plan for submission of biomarkers to the regulatory authorities (EMA and FDA) for qualification

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).

LITMUS-Report-Histological-staging-system-NAFLD-associated-fibrosisitled

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.

Posters

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 articles

Journal article: Luukkonen, P.K. et al, “Human PNPLA3-I148M variant increases hepatic retention of polyunsaturated fatty acids”, JCI insight 4(16), August 2019.

Abstract
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.

Abstract
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.

Abstract
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.

Copyright © 2020 LITMUS Project

This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No. 777377. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. Visit IMI.

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