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Liver Investigation: Testing Marker Utility in Steatohepatitis

Periodic Reporting for period 4 - LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis)

Période du rapport: 2020-11-01 au 2021-10-31

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, and ultimately facilitate drug development and novel treatments.

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.
WP2 continued the statistical analysis of the performance of markers for the diagnostic and diagnostic screening context of use. The analysis focused on the diagnostic accuracy in detecting fibrotic NASH.

WP3 highlights include: (i) Continuing to build the largest international cohort of histologically characterised NAFLD patients: the “European NAFLD Registry”, including the Metacohort (1a) and LITMUS Study cohort (1b), with biological samples held at a central biobank; (ii) Creation of an Atlas of digital images for the harmonisation of NAFLD histological evaluation and future AI-based analysis; (iii) Central histological interpretation and scoring of liver biopsies progressing well; (iv) Further development of the NASH-CHECK PROM to cover NASH F1-3 and F4 patients completed.

In WP4, a range of blood-based biomarkers have been tested at the central labs for both 1a and 1b cases, with data passed to WP2 for analysis. Biomarker discovery activities have identified a range of promising novel markers including miRNA miR193a, GDF15 and TSP-2 . A comprehensive review of metabolomics/lipidomics biomarker literature in NAFLD was published and an inter-laboratory ring study completed to measure the candidate lipidomics markers across the participating LITMUS laboratories, with good agreement.

In WP5, imaging data acquisition is underway in 10 countries. The study design has been modified to mitigate the effects of slow recruitment due to COVID-19 and broaden the range of data captured to include vendor-specific PDFF. We have conducted comprehensice individual patient data metaanalyses and systematic reviews of diagnostic accuracy of imaging biomarkers.

WP6 has collected phenotype and RNA sequencing data on numerous pre-clinical models. A coherent pipeline for data analysis to compare the different models to the progression of human disease was finalised. A biobank of FFPE liver and snap frozen tissues from these retrospective pre-clinical models was created. The second study on Göttingen Minipigs fed CDAHFD plus Fructose was also completed.

In WP7, two Letters of Intent were accepted by the FDA (covering Diagnostic Screening and Prognostic Enrichment), qualification advice has also been successfully received from the EMA. Despite the impacts of COVID19 on travel, dissemination activities have continued to increase in P4, with multiple publications in peer reviewed journals and participating in virtual conferences.

WP8 has collected ethical approvals for the prospective patient recruitment as these became available in the various different countries.
The 4th period has been focused on gathering prospective samples and analysing the performance of various wet and imaging biomarkers, both in the literature and in our own cohorts. We have already achieved the following important advances:
1. European NAFLD Registry and LITMUS Biobank - the largest international registry of histologically characterised NAFLD patients under longitudinal follow-up
2. LITMUS Demonstrator: Detailed analysis of biomarker performance using 1a and 1b cases, with results presented at international conferences/journals (WP2).
3. LITMUS Accelerator: Use of detailed transcriptomics (gene expression) and proteomic analysis (SOMAScan) to identify a range of promising novel circulating biomarkers.
4. Meta-analyses of diagnostic accuracy studies which will provide insight on the different cut-off values of the biomarkers for detecting different target conditions in NAFLD
5. Established the proof-of-principle that NMR-based metabolomics can be used to find non-invasive metabolic biomarkers to measure NASH onset and progression
6. Atlas of histological images available online for educational purposes, including training of general pathologists in diagnosing human NAFLD/NASH, grading necro-inflammatory activity and staging fibrosis in liver biopsies
7. Comparative biology assessments (utilising histology, phenotype and transcriptomic profiles) and ranking of animals models of NASH for fidelity to human disease and initiation of in vivo validation studies


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.

Twitter: @LITMUS_IMI
The Imperative for Biomarkers in NAFLD