Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis)

Teaser

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of disease occurring in the absence of excessive alcohol consumption that ranges from isolated hepatic triglyceride accumulation (steatosis, NAFL); through hepatic triglyceride accumulation plus inflammation and...

Summary

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of disease occurring in the absence of excessive alcohol consumption that ranges from isolated hepatic triglyceride accumulation (steatosis, NAFL); through hepatic triglyceride accumulation plus inflammation and hepatocyte injury (non-alcoholic steatohepatitis, NASH); and ultimately progresses to fibrosis/cirrhosis and potentially hepatocellular carcinoma (HCC). NAFLD is a common condition, strongly associated with the Metabolic Syndrome (obesity, type 2 diabetes mellitus and dyslipidaemia) and characterised by substantial inter-patient variability in severity and rate of progression. An important paradox exists: a significant proportion of the population have NAFLD but only a minority progress to advanced liver disease or morbidity/mortality.

The transition from NAFL to NASH and the stage of fibrosis are important discriminators between a relatively benign prognosis and an increased risk of morbidity/mortality. Liver biopsy remains the established but imperfect ‘gold standard’ investigation being invasive, resource intensive, prone to sampling error and carrying a small but significant risk of complications. Such invasive tests are not practical outside specialist practice, and are particularly unsuitable with such a large ‘at risk’ population. A lack of tractable non-invasive biomarkers has impeded the diagnosis, risk stratification and monitoring of patients. It has also hampered drug development and the conduct of clinical trials, which still depend on histological effect as an endpoint. 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. Success will help target care to those at greatest risk, facilitate drug development and ultimately give patients access to approved medicines.

The specific objectives of LITMUS are:
1. To leverage existing cross-sectional and longitudinal patient cohorts and bioresources into a single unified resource for biomarker validation. The infrastructure of the existing ‘European NAFLD Registry’ and additional large patient cohorts will be employed and expanded to fulfil this aim (Phase 1a). LITMUS has links with cohorts in North America and China to form the core of a nascent ‘Global NAFLD Registry’.
2. To expand our clinical network of European centres and accelerate the prospective recruitment of patients with histologically characterised NAFLD to further validate candidate biomarkers. Prospective recruitment and longitudinal follow-up collecting a predefined dataset and standardised sample set (Phase 1b).
3. To establish a robust technological and methodological platform and use it for the definitive validation of candidate biomarkers. LITMUS offers an impartial, technology-unbiased platform for biomarker discovery, assessment and validation across the NAFLD spectrum and all three FDA BEST biomarker domains (diagnostic, prognostic and monitoring), with a clear line of sight to regulatory qualification.
4. To identify and define the most accurate and tractable biomarkers relevant to NAFLD. LITMUS will provide validation data of the requisite standard to support regulatory qualification of biomarkers for trial use against both histological/biochemical indices and clinically relevant long-term outcome measures.
5. To develop consensus and qualify preclinical models of NAFLD/NASH and then back-translate biomarkers for validation in these models so that they may be used in pre-clinical drug development.

Work performed

WP2 developed the documents on contexts of use and minimally acceptable performance criteria. We also developed a protocol for the systematic review on the performance of diagnostic biomarkers and carried out this review.

WP3 highlights include: (i) Upgrading the European NAFLD Registry web portal and database to ensure high-quality data capture and audit capabilities; (ii) Collating data and biological samples into the Phase 1a (“Metacohort”), with biological samples held in the LITMUS biobank; (iii) Gaining ethical approval for prospective patient recruitment across the majority of territories across Europe; (iv) Writing the “LITMUS Investigator Handbook” in order to standardize the prospective sample collection and processing.

In WP4, A series of selected routine clinical laboratory assays as well as market-ready assays have been selected for implementation in the Central Laboratory. Here a complete list of 39 biomarkers, including a top-10 list, was agreed and frozen. DNA samples were collected for GWAS studies to identify genomic biomarkers. miRNA sequencing was performed on 284 serum samples with bioinfomatic analysis of these data now in progress. A comprehensive review of metabolomics/lipidomics biomarker literature in NAFLD was performed. SOPs were also developed for the key metabolomics protocols to be used in LITMUS. We have explored potential new metagenomics signatures in the Paris bariatric cohort and the EPoS NAFLD/NASH cohort (untargeted approach). Based on initial analyses, the group has uncovered some potential metagenomics signatures and their confirmation in the literature while also preparing a published review to increase NAFLD/NASH metagenomics biomarker visibility.

WP5 has prepared the Imaging Charter, which describes the roles and responsibilities of the participating partners and the Imaging Manual as a standardised means of data acquisition.

WP6 has collected phenotype and RNA Sequencing information on 586 animals to date. The team is now working to develop a coherent pipeline for data analysis to compare the different models to the progression of human disease. We have also started to generate a novel non-rodent pre-clinical model of NASH based on Göttingen Minipigs fed CDAHFD plus Fructose.

In WP7, discussions have started and an initial meeting has been held with both the EMA and FDA regarding regulatory qualification of biomarkers and a plan has been drafted. A dissemination plan has been written and some general conference presentations have been given which mention the LITMUS project. The project website has been set up. A Twitter account has also been set up and is regularly posting information related to the project the NAFLD in general.

WP8 collected the existing historical ethical approvals to provided them to IBBL as the basis for work carried out in WP3 and WP6. The main achievements are related to: (1) completion of the assessment of the historical consents; (2) obtaining a central approval for the prospective study protocol and ethical approval at the lead ethics in UNEW including sub-studies; and (3) rollout at the national level by supporting the national leads and collecting information on the status of the prospective approval.

Final results

The first year of the project has been focused on work to enable successful delivery of the wider objectives. However, we have already achieved the two following important advances:
1. European NAFLD Registry and LITMUS Biobank - the largest international registry of histologically characterised NAFLD patients
2. Validation of 7-tier histological staging system - an important advance that will improve granularity of patient assessment

By the end of the project, we expect to have validated a number of biomarkers that diagnose, risk stratify and/or monitor NAFLD/NASH progression and fibrosis stage.

Website & more info

More info: http://www.litmus-project.eu.