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Impact of non-invasive biomarkers on hepatology practice: Past, present and future

  • Quentin M. Anstee
    Correspondence
    Corresponding authors. Addresses: Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK
    Affiliations
    Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK

    Newcastle NIHR Biomedical Research Centre, Newcastle Upon Tyne Hospitals NHS Foundation Trust, Newcastle Upon Tyne, UK
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  • Laurent Castera
    Correspondence
    Université de Paris, Service d’Hépatologie, Hôpital Beaujon, F- 92110 Clichy-la-Garenne, France
    Affiliations
    Université de Paris, UMR1149 (CRI), Inserm, F-75018 Paris, France

    Service d’Hépatologie, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Beaujon, F-92110 Clichy-la-Garenne, France
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  • Rohit Loomba
    Correspondence
    NAFLD Research Center, University of California at San Diego, Altman Clinical and Translational Research Institute, 9500 Gilman Drive La Jolla, CA, 92093-0887, United States.
    Affiliations
    NAFLD Research Center, Division of Gastroenterology and Hepatology, University of California at San Diego, La Jolla, CA, United States

    Herbert Wertheim School of Public Health, University of California at San Diego, La Jolla, CA, United States
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      Summary

      Over the last two decades, there have been tremendous advances in the non-invasive diagnosis and risk stratification of chronic liver diseases (CLDs). Non-invasive approaches are based on the quantification of biomarkers in serum samples or on the measurement of liver stiffness, using either ultrasound- or magnetic resonance–based elastography techniques. The fibrosis-4 index (non-patented) and enhanced liver fibrosis test (patented) are the most widely adopted serum markers, whereas vibration-controlled transient elastography is the most widely adopted elastography technique. In this review, we discuss the role of non-invasive tests in the current era, as well as their accuracy and how their use in clinical practice has changed the practice of hepatology, including identification of early cirrhosis in patients with risk factors for CLD, diagnosis of portal hypertension, establishing prognosis in compensated cirrhosis, guiding antiviral treatment, and screening for fibrosis and cirrhosis in primary care.

      Keywords

      Introduction

      Chronic liver diseases (CLDs) represent a major public health problem, accounting for significant morbidity and mortality worldwide.
      • Asrani S.K.
      • Devarbhavi H.
      • Eaton J.
      • Kamath P.S.
      Burden of liver diseases in the world.
      Their prognosis and management greatly depend on the amount and progression of liver fibrosis, and the associated risk of developing cirrhosis. Liver biopsy has been considered the reference standard for staging liver fibrosis since the late fifties when the “one second liver biopsy” technique was introduced by Menghini.
      • Menghini G.
      One-second needle biopsy of the liver.
      Histological staging of fibrosis is a combinatorial assessment of the amount of fibrosis and architectural disorganisation. It is based on semi-quantitative scoring systems, initially developed in viral hepatitis,
      • Knodell R.G.
      • Ishak K.G.
      • Black W.C.
      • Chen T.S.
      • Craig R.
      • Kaplowitz N.
      • et al.
      Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis.
      • Ishak K.
      • Baptista A.
      • Bianchi L.
      • Callea F.
      • De Groote J.
      • Gudat F.
      • et al.
      Histological grading and staging of chronic hepatitis.
      • Bedossa P.
      • Poynard T.
      An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
      and more recently in non-alcoholic fatty fiver disease (NAFLD) and alcohol-related liver disease (ALD).
      • Brunt E.M.
      • Janney C.G.
      • Di Bisceglie A.M.
      • Neuschwander-Tetri B.A.
      • Bacon B.R.
      Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions.
      • Kleiner D.E.
      • Brunt E.M.
      • Van Natta M.
      • Behling C.
      • Contos M.J.
      • Cummings O.W.
      • et al.
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      • Bedossa P.
      • Consortium F.P.
      Utility and appropriateness of the fatty liver inhibition of progression (FLIP) algorithm and steatosis, activity, and fibrosis (SAF) score in the evaluation of biopsies of nonalcoholic fatty liver disease.
      The presence of bridging fibrosis or cirrhosis, coined “advanced fibrosis”, is currently the most clinically relevant target. The presence of cirrhosis is the trigger for surveillance programmes for hepatocellular carcinoma and oesophageal varices in patients with CLD,
      European Association for the Study of the Liver (EASL)
      EASL clinical practice guidelines: management of hepatocellular carcinoma.
      ,
      • de Franchis R.
      • Baveno V.I.F.
      Expanding consensus in portal hypertension: report of the Baveno VI Consensus Workshop: stratifying risk and individualizing care for portal hypertension.
      and the main prognostic driver in patients with NAFLD.
      • Sanyal A.J.
      • Van Natta M.L.
      • Clark J.
      • Neuschwander-Tetri B.A.
      • Diehl A.
      • Dasarathy S.
      • et al.
      Prospective study of outcomes in adults with nonalcoholic fatty liver disease.
      However, liver biopsy has several limitations that should be acknowledged. Its’ accuracy for the assessment of fibrosis has been questioned, in relation to sampling errors and intra- and inter-observer variability that may lead to over- or under-staging.
      • Bedossa P.
      • Dargere D.
      • Paradis V.
      Sampling variability of liver fibrosis in chronic hepatitis C.
      • Davison B.A.
      • Harrison S.A.
      • Cotter G.
      • Alkhouri N.
      • Sanyal A.
      • Edwards C.
      • et al.
      Suboptimal reliability of liver biopsy evaluation has implications for randomized clinical trials.
      • Brunt E.M.
      • Clouston A.D.
      • Goodman Z.
      • Guy C.
      • Kleiner D.E.
      • Lackner C.
      • et al.
      Complexity of ballooned hepatocyte feature recognition: defining a training atlas for artificial intelligence-based imaging in NAFLD.
      Liver biopsy is also a costly procedure, requiring medical expertise as well as an ambulatory facility with periprocedural monitoring by nurses.
      • Tapper E.B.
      • Lok A.S.
      Use of liver imaging and biopsy in clinical practice.
      Finally, it is an invasive procedure associated with complications, some common and mild (transient pain in 30% to 50% of cases),
      • Castera L.
      • Negre I.
      • Samii K.
      • Buffet C.
      Patient-administered nitrous oxide/oxygen inhalation provides safe and effective analgesia for percutaneous liver biopsy: a randomized placebo-controlled trial.
      others rare but potentially life-threatening (serious haemorrhage in 0.6% of cases and mortality in up to 0.1%).
      • Tapper E.B.
      • Lok A.S.
      Use of liver imaging and biopsy in clinical practice.
      For these reasons, acceptability is poor, with many patients refusing to undergo liver biopsy. All these limitations, as well as the availability of powerful antiviral therapies and the epidemic proportion of individuals with NAFLD worldwide, have fuelled the development of alternative non-invasive strategies to improve diagnosis and prognostication in CLD. Non-invasive tests have been an area of intensive research over the past decades with a major impact on hepatology practice, as reflected by several EASL clinical practice guidelines.
      EASL-ALEH Clinical Practice Guidelines
      Non-invasive tests for evaluation of liver disease severity and prognosis.
      ,
      European Association for the Study of the Liver (EASL)
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      Non-invasive strategies rely on distinct but complementary approaches: i) a “biological” approach based on the quantification of biomarkers in serum samples; and ii) a “physical” approach based on the measurement of liver stiffness, using either ultrasound- or magnetic resonance–based elastography techniques. Although complementary, these approaches are based on different rationales: liver stiffness corresponds to a genuine and intrinsic physical property of liver parenchyma, whereas serum biomarkers are combinations of several not strictly liver-specific blood parameters optimised to mimic fibrosis stages, as assessed by liver biopsy. The story of non-invasive tests started in the late nineties when the way was paved for their use by the modelling of fibrosis progression in patients with HCV.
      • Poynard T.
      • Bedossa P.
      • Opolon P.
      Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups.
      Non-invasive approaches, based on the use of individual serum markers, reflecting either the deposition or the removal of extracellular matrix in the liver or simple routine blood tests such as prothrombin index, platelet count, and aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio (AAR) were proposed at that time.
      • Oberti F.
      • Valsesia E.
      • Pilette C.
      • Rousselet M.C.
      • Bedossa P.
      • Aube C.
      • et al.
      Noninvasive diagnosis of hepatic fibrosis or cirrhosis.
      Later on, scores using a combination of these parameters were introduced, either patented or non-patented, for which the pioneers were FibroTest (Biopredictive, Paris, France)
      • Imbert-Bismut F.
      • Ratziu V.
      • Pieroni L.
      • Charlotte F.
      • Benhamou Y.
      • Poynard T.
      Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study.
      and the AST-to-platelet ratio index (APRI).
      • Wai C.T.
      • Greenson J.K.
      • Fontana R.J.
      • Kalbfleisch J.D.
      • Marrero J.A.
      • Conjeevaram H.S.
      • et al.
      A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C.
      The introduction in 2003 of vibration-controlled transient elastography (VCTE), allowing for liver stiffness measurement, using the FibroScan device (Echosens, Paris, France)
      • Sandrin L.
      • Fourquet B.
      • Hasquenoph J.M.
      • Yon S.
      • Fournier C.
      • Mal F.
      • et al.
      Transient elastography: a new noninvasive method for assessment of hepatic fibrosis.
      marked the beginning of a new era. Many other elastography techniques have been developed since, including ultrasound-based elastography techniques integrated into ultrasound devices, such as point-shear wave elastography (pSWE) or 2D-shear wave elastography (2D-SWE), and more recently, magnetic resonance elastography (MRE).
      • Friedrich-Rust M.
      • Poynard T.
      • Castera L.
      Critical comparison of elastography methods to assess chronic liver disease.
      In this review, we discuss the role of non-invasive biomarkers in the current era, as well as their accuracy and how their use in clinical practice has changed hepatology.
      Liver stiffness measurement by VCTE, using the “rule of five”, allows for the early diagnosis of cirrhosis (cACLD) and of portal hypertension (CSPH), and can obviate the need for upper GI endoscopy.

      The lexicon of biomarkers

      The rapid evolution of the biomarker field has been aided by a common lexicon proposed by the FDA-NIH Biomarker Working Group.
      FDA-NIH Biomarker Working Group
      BEST (Biomarkers, EndpointS, and other Tools) Resource.
      Called the BEST (Biomarkers, EndpointS, and other Tools) resource, this has established a framework for describing biomarkers that encompasses biological plausibility (i.e. relevance to the disease or condition), measurement method and context of use (by categorising the purpose/question a biomarker may be used to address as well as in what setting it will be applied). The current BEST context of use category definitions are provided in Table 1.
      Table 1FDA-NIH BEST defined biomarker context of use.
      FDA-NIH Biomarker Working Group
      BEST (Biomarkers, EndpointS, and other Tools) Resource.
      Biomarker context of use categoryBEST definition
      Susceptibility/riskA biomarker that indicates the potential for developing a disease or medical condition in an individual who does not currently have clinically apparent disease or the medical condition.
      DiagnosticA biomarker used to detect or confirm presence of a disease or condition of interest or to identify individuals with a subtype of the disease.
      PrognosticA biomarker used to identify likelihood of a clinical event, disease recurrence or progression in patients who have the disease or medical condition of interest.
      MonitoringA biomarker measured repeatedly for assessing status of a disease or medical condition or for evidence of exposure to (or effect of) a medical product or an environmental agent.
      ResponseA biomarker used to show that a biological response, potentially beneficial or harmful, has occurred in an individual who has been exposed to a medical product or an environmental agent.
      • Pharmacodynamic biomarker: A response biomarker that indicates biologic activity of a medical product or environmental agent without necessarily drawing conclusions about efficacy or disease outcome or necessarily linking this activity to an established mechanism of action.
      • Surrogate endpoint biomarker: A response biomarker that is an endpoint used in clinical trials as a substitute for a direct measure of how a patient feels, functions, or survives.
      PredictiveA biomarker used to identify individuals who are more likely than similar individuals without the biomarker to experience a favourable or unfavourable effect from exposure to a medical product or an environmental agent.
      SafetyA biomarker measured before or after an exposure to a medical product or an environmental agent to indicate the likelihood, presence, or extent of toxicity as an adverse effect.
      Whilst the BEST lexicon was initially developed to support the use of biomarkers in drug development, these definitions are equally applicable to biomarkers in routine clinical practice and will provide a structure to this review.

      Diagnosis and staging of liver fibrosis in chronic liver diseases

      Liver histology remains the reference standard for assessing diagnostic biomarker performance. It should however be noted that, beyond the issues with sampling variability and inter-observer variation already alluded to,
      • Bedossa P.
      • Dargere D.
      • Paradis V.
      Sampling variability of liver fibrosis in chronic hepatitis C.
      • Davison B.A.
      • Harrison S.A.
      • Cotter G.
      • Alkhouri N.
      • Sanyal A.
      • Edwards C.
      • et al.
      Suboptimal reliability of liver biopsy evaluation has implications for randomized clinical trials.
      • Brunt E.M.
      • Clouston A.D.
      • Goodman Z.
      • Guy C.
      • Kleiner D.E.
      • Lackner C.
      • et al.
      Complexity of ballooned hepatocyte feature recognition: defining a training atlas for artificial intelligence-based imaging in NAFLD.
      there are inherent issues with the use of semi-quantitative histological scoring systems as a reference standard. Semi-quantitative scores conflate anatomical distribution of specific lesions with severity and impose a structure whereby continuous variables are assigned into discrete categorical grading bins.
      • Ishak K.
      • Baptista A.
      • Bianchi L.
      • Callea F.
      • De Groote J.
      • Gudat F.
      • et al.
      Histological grading and staging of chronic hepatitis.
      ,
      • Bedossa P.
      • Poynard T.
      An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
      ,
      • Kleiner D.E.
      • Brunt E.M.
      • Van Natta M.
      • Behling C.
      • Contos M.J.
      • Cummings O.W.
      • et al.
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      ,
      • Bedossa P.
      • Consortium F.P.
      Utility and appropriateness of the fatty liver inhibition of progression (FLIP) algorithm and steatosis, activity, and fibrosis (SAF) score in the evaluation of biopsies of nonalcoholic fatty liver disease.
      This process limits accurate quantification of fibrosis burden and leads to misclassification at the margins between two categories. These issues are exemplified by the breadth of disease encompassed within METAVIR stage F3 fibrosis: portal-portal and/or portal-central bridging is the defining feature, yet no weight is given to density of collagen deposition or change in lobular ultrastructure, which may be of greater biological relevance. Although non-invasive biomarkers may in truth provide better estimates of fibrosis burden, the corollary of this is that classification errors in the reference standard are interpreted as biomarker inaccuracy as, within the classic accuracy concept, any discrepancy is assumed to be a failure of the novel test and therefore a “false” positive/negative result.
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Verheij J.
      • Brosnan M.J.
      • et al.
      FibroTest for evaluating fibrosis in non-alcoholic fatty liver disease patients: a systematic review and meta-analysis.

      Serum fibrosis markers

      Serological markers can be divided into ‘indirect’ markers, that reflect hepatic injury, inflammation or alterations in hepatic function and portal hypertension (e.g. prothrombin time, platelet count, and AAR), and ‘direct’ markers, that rely on the measurement of components released into the circulation during fibrogenesis or extracellular matrix remodelling (Table 2). The presence of ‘advanced fibrosis’, histologically characterised by portal-portal bridging fibrosis (modified Ishak stage F4+, METAVIR/NASH-CRN F3+), is considered a clinically relevant milestone in the natural history of CLD.
      • Ishak K.
      • Baptista A.
      • Bianchi L.
      • Callea F.
      • De Groote J.
      • Gudat F.
      • et al.
      Histological grading and staging of chronic hepatitis.
      ,
      • Bedossa P.
      • Poynard T.
      An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
      Therefore, much attention has been focused on distinguishing this from lesser stages of disease. Many fibrosis biomarkers in use today were first developed in hepatitis C but have since been adopted for use in other liver disease areas. A recent series of authoritative systematic reviews and meta-analyses conducted by members of the EU IMI2-funded LITMUS consortium

      LITMUS: Liver Investigation: Testing Marker Utility in Steatohepatitis consortium (European Union IMI2-funded under Grant Agreement 777377). [cited 2022]; Available from: https://www.imi.europa.eu/projects-results/project-factsheets/litmus.

      have collated published evidence on performance in NAFLD for a range of fibrosis biomarkers.
      • Mak A.L.
      • Lee J.
      • van Dijk A.M.
      • Vali Y.
      • Aithal G.P.
      • Schattenberg J.M.
      • et al.
      Systematic review with meta-analysis: diagnostic accuracy of pro-C3 for hepatic fibrosis in patients with non-alcoholic fatty liver disease.
      • Mozes F.E.
      • Lee J.A.
      • Selvaraj E.A.
      • Jayaswal A.N.A.
      • Trauner M.
      • Boursier J.
      • et al.
      Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Verheij J.
      • Brosnan M.J.
      • et al.
      FibroTest for evaluating fibrosis in non-alcoholic fatty liver disease patients: a systematic review and meta-analysis.
      • Van Dijk A.M.
      • Vali Y.
      • Mak A.L.
      • Lee J.
      • Tushuizen M.E.
      • Zafarmand M.H.
      • et al.
      Systematic review with meta-analyses: diagnostic accuracy of FibroMeter tests in patients with non-alcoholic fatty liver disease.
      • Selvaraj E.A.
      • Mozes F.E.
      • Jayaswal A.N.A.
      • Zafarmand M.H.
      • Vali Y.
      • Lee J.A.
      • et al.
      Diagnostic accuracy of elastography and magnetic resonance imaging in patients with NAFLD: a systematic review and meta-analysis.
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Loffler J.
      • Verheij J.
      • et al.
      Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: a systematic review and meta-analysis.
      In general, non-invasive biomarkers are highly effective at ruling out the presence of advanced fibrosis or cirrhosis, exhibiting negative predictive values (NPVs) >90% in many cases; however, their positive predictive values (PPVs) are often more modest, and they are unable to accurately discriminate individual fibrosis stages. Both positive and negative predictive values are heavily influenced by the prevalence of the target condition in the population to which the biomarker is applied.
      • Mozes F.E.
      • Lee J.A.
      • Selvaraj E.A.
      • Jayaswal A.N.A.
      • Trauner M.
      • Boursier J.
      • et al.
      Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.
      ,
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Loffler J.
      • Verheij J.
      • et al.
      Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: a systematic review and meta-analysis.
      Table 2Serum fibrosis biomarkers for detecting advanced fibrosis.
      TestDescriptionRef.
      Indirect fibrosis biomarker panels
      AST:ALT ratio (AAR)AST (IU/L)/ALT (IU/L)
      • Sheth S.G.
      • Flamm S.L.
      • Gordon F.D.
      • Chopra S.
      AST/ALT ratio predicts cirrhosis in patients with chronic hepatitis C virus infection.


      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      AST-to-platelet ratio index (APRI)(AST (IU/L)/(ULN))/platelet count (×109/L) × 100
      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      BARD scoreWeighted sum of BMI ≥28 = 1 point, AST/ALT ratio ≥0.8 = 2 points, T2DM = 1
      • Harrison S.A.
      • Oliver D.
      • Arnold H.L.
      • Gogia S.
      • Neuschwander-Tetri B.A.
      Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease.


      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      Fibrosis-4 indexAge × AST (IU/L)/platelet count (×109/L) × √ ALT (IU/L)
      • Shah A.G.
      • Lydecker A.
      • Murray K.
      • Tetri B.N.
      • Contos M.J.
      • Sanyal A.J.
      • et al.
      Comparison of noninvasive markers of fibrosis in patients with nonalcoholic fatty liver disease.


      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      NAFLD fibrosis score-1.675 + 0.037 × age (years) + 0.094 × BMI (kg/m2) + 1.13 × IFG or T2DM (yes = 1, no = 0) + 0.99 × AST/ALT ratio - 0.013 × platelet count (×109/L) - 0.66 × albumin (g/dl)
      • Angulo P.
      • Hui J.M.
      • Marchesini G.
      • Bugianesi E.
      • George J.
      • Farrell G.C.
      • et al.
      The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD.


      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      Direct fibrosis biomarker panels
      ELFELF = -7.412 + (ln(HA)∗0.681) + (ln(PIIINP)∗0.775) + (ln(TIMP1)∗0.494)
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Loffler J.
      • Verheij J.
      • et al.
      Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: a systematic review and meta-analysis.
      ,
      • Guha I.N.
      • Parkes J.
      • Roderick P.
      • Chattopadhyay D.
      • Cross R.
      • Harris S.
      • et al.
      Noninvasive markers of fibrosis in nonalcoholic fatty liver disease: validating the European Liver Fibrosis Panel and exploring simple markers.
      FibroTestPatented algorithm combining total bilirubin, GGT, α2-macroglobulin, apolipoprotein A1, and haptoglobin, corrected for age and gender
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Verheij J.
      • Brosnan M.J.
      • et al.
      FibroTest for evaluating fibrosis in non-alcoholic fatty liver disease patients: a systematic review and meta-analysis.
      ,
      • Ratziu V.
      • Massard J.
      • Charlotte F.
      • Messous D.
      • Imbert-Bismut F.
      • Bonyhay L.
      • et al.
      Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Munteanu M.
      • Tiniakos D.
      • Anstee Q.
      • Charlotte F.
      • Marchesini G.
      • Bugianesi E.
      • et al.
      Diagnostic performance of FibroTest, SteatoTest and ActiTest in patients with NAFLD using the SAF score as histological reference.
      FibroMeter NAFLDPatented algorithm combining age, body weight, glucose, AST, ALT, ferritin and platelet count
      • Van Dijk A.M.
      • Vali Y.
      • Mak A.L.
      • Lee J.
      • Tushuizen M.E.
      • Zafarmand M.H.
      • et al.
      Systematic review with meta-analyses: diagnostic accuracy of FibroMeter tests in patients with non-alcoholic fatty liver disease.
      ,
      • Cales P.
      • Laine F.
      • Boursier J.
      • Deugnier Y.
      • Moal V.
      • Oberti F.
      • et al.
      Comparison of blood tests for liver fibrosis specific or not to NAFLD.
      HepascoreAlgorithm containing age, gender, α2-macroglobulin, hyaluronic acid and bilirubin
      • Adams L.A.
      • Bulsara M.
      • Rossi E.
      • DeBoer B.
      • Speers D.
      • George J.
      • et al.
      Hepascore: an accurate validated predictor of liver fibrosis in chronic hepatitis C infection.
      ADAPTADAPT = exp(log10((age × PRO-C3)/√(platelet count))) + T2DM
      • Mak A.L.
      • Lee J.
      • van Dijk A.M.
      • Vali Y.
      • Aithal G.P.
      • Schattenberg J.M.
      • et al.
      Systematic review with meta-analysis: diagnostic accuracy of pro-C3 for hepatic fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Boyle M.
      • Tiniakos D.
      • Schattenberg J.M.
      • Ratziu V.
      • Bugianessi E.
      • Petta S.
      • et al.
      Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.
      ,
      • Daniels S.J.
      • Leeming D.J.
      • Eslam M.
      • Hashem A.M.
      • Nielsen M.J.
      • Krag A.
      • et al.
      ADAPT: an algorithm incorporating PRO-C3 accurately identifies patients with NAFLD and advanced fibrosis.
      FIBC3FIBC3 = -5.939 + (0.053∗age) + (0.076∗BMI) + (1.614∗T2DM) – (0.009∗platelet count) + (0.071∗PRO-C3)
      • Mak A.L.
      • Lee J.
      • van Dijk A.M.
      • Vali Y.
      • Aithal G.P.
      • Schattenberg J.M.
      • et al.
      Systematic review with meta-analysis: diagnostic accuracy of pro-C3 for hepatic fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Boyle M.
      • Tiniakos D.
      • Schattenberg J.M.
      • Ratziu V.
      • Bugianessi E.
      • Petta S.
      • et al.
      Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.
      ABC3DAge >50 = 1 point, BMI >30 = 1 point, platelet count <200 = 1-point, PRO-C3 >15.5 = 1 point, T2DM = 2 points
      • Mak A.L.
      • Lee J.
      • van Dijk A.M.
      • Vali Y.
      • Aithal G.P.
      • Schattenberg J.M.
      • et al.
      Systematic review with meta-analysis: diagnostic accuracy of pro-C3 for hepatic fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Boyle M.
      • Tiniakos D.
      • Schattenberg J.M.
      • Ratziu V.
      • Bugianessi E.
      • Petta S.
      • et al.
      Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.
      ALT, alanine aminotransferase; AST, aspartate aminotransferase; ELF, enhanced liver fibrosis; GGT, gamma-glutamyltransferase; HA, hyaluronic acid; IFG, impaired fasting glucose; NPV, negative predictive value; PIIINP, N-terminal peptide of pro-collagen III; PPV, positive predictive value; PRO-C3, pro-collagen 3 neoepitope; Se, sensitivity; Sp, specificity; T2DM, type 2 diabetes mellitus; TIMP1, tissue inhibitor of metalloproteinase 1; ULN, upper limit of normal.

      Indirect fibrosis biomarkers

      As hepatic fibrosis increases, serum ALT typically falls whilst AST remains stable or rises, as a result the AAR increases and can provide a simple, albeit imperfect, method of identifying patients with advanced fibrosis.
      • Williams A.L.
      • Hoofnagle J.H.
      Ratio of serum aspartate to alanine aminotransferase in chronic hepatitis. Relationship to cirrhosis.
      Early studies reported that an AAR >1 could be used to identify cirrhosis in patients with chronic HCV infection.
      • Sheth S.G.
      • Flamm S.L.
      • Gordon F.D.
      • Chopra S.
      AST/ALT ratio predicts cirrhosis in patients with chronic hepatitis C virus infection.
      Although subsequent larger studies in HCV and NAFLD demonstrated lower accuracy, an AAR <0.8 can provide a high negative predictive value to rule-out advanced fibrosis.
      • Imperiale T.F.
      • Said A.T.
      • Cummings O.W.
      • Born L.J.
      Need for validation of clinical decision aids: use of the AST/ALT ratio in predicting cirrhosis in chronic hepatitis C.
      ,
      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      Similarly, APRI was developed to detect advanced fibrosis/cirrhosis in HCV
      • Wai C.T.
      • Greenson J.K.
      • Fontana R.J.
      • Kalbfleisch J.D.
      • Marrero J.A.
      • Conjeevaram H.S.
      • et al.
      A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C.
      ,
      • Borsoi Viana M.S.
      • Takei K.
      • Collarile Yamaguti D.C.
      • Guz B.
      • Strauss E.
      Use of AST platelet ratio index (APRI Score) as an alternative to liver biopsy for treatment indication in chronic hepatitis C.
      but has also been trialled in NAFLD.
      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Cales P.
      • Laine F.
      • Boursier J.
      • Deugnier Y.
      • Moal V.
      • Oberti F.
      • et al.
      Comparison of blood tests for liver fibrosis specific or not to NAFLD.
      ,
      • Loaeza-del-Castillo A.
      • Paz-Pineda F.
      • Oviedo-Cardenas E.
      • Sanchez-Avila F.
      • Vargas-Vorackova F.
      AST to platelet ratio index (APRI) for the noninvasive evaluation of liver fibrosis.
      The BARD score, combining AAR with the presence of raised BMI and type 2 diabetes mellitus (T2DM), was one of the first to be specifically developed in patients with biopsy-proven NAFLD.
      • Harrison S.A.
      • Oliver D.
      • Arnold H.L.
      • Gogia S.
      • Neuschwander-Tetri B.A.
      Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease.
      A score <2 has a high NPV >96%.
      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Harrison S.A.
      • Oliver D.
      • Arnold H.L.
      • Gogia S.
      • Neuschwander-Tetri B.A.
      Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease.
      However, the majority of patients with NAFLD exceed this threshold due to the high prevalence of obesity and T2DM in this population, and so a low PPV limits its utility in practice.
      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      To improve diagnostic performance with optimised positive and negative predictive values, indirect biomarker panels such as fibrosis-4 (FIB-4)
      • Sterling R.K.
      • Lissen E.
      • Clumeck N.
      • Sola R.
      • Correa M.C.
      • Montaner J.
      • et al.
      Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection.
      and the NAFLD fibrosis score (NFS)
      • Angulo P.
      • Hui J.M.
      • Marchesini G.
      • Bugianesi E.
      • George J.
      • Farrell G.C.
      • et al.
      The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD.
      were developed that implemented two discreet thresholds: one with high sensitivity (negative predictive value) used to rule-out advanced fibrosis, and another with high specificity (positive predictive value) to rule-in advanced disease, although at the cost of an ‘indeterminate’ zone where cases cannot be accurately classified. The NFS was developed in an international cohort of patients with histologically characterised NAFLD. Combining age, presence of T2DM, BMI, AST/ALT, albumin and platelet count, the NFS achieves an AUROC of 0.81 (0.71–0.91) with values <-1.455 excluding advanced fibrosis with high accuracy and values >0.676 offering improved positive predictive value.
      • Angulo P.
      • Hui J.M.
      • Marchesini G.
      • Bugianesi E.
      • George J.
      • Farrell G.C.
      • et al.
      The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD.
      This score has been independently validated in several other studies.
      • Cales P.
      • Laine F.
      • Boursier J.
      • Deugnier Y.
      • Moal V.
      • Oberti F.
      • et al.
      Comparison of blood tests for liver fibrosis specific or not to NAFLD.
      ,
      • Qureshi K.
      • Clements R.H.
      • Abrams G.A.
      The utility of the "NAFLD fibrosis score" in morbidly obese subjects with NAFLD.
      ,
      • Wong V.W.
      • Wong G.L.
      • Chim A.M.
      • Tse A.M.
      • Tsang S.W.
      • Hui A.Y.
      • et al.
      Validation of the NAFLD fibrosis score in a Chinese population with low prevalence of advanced fibrosis.
      Consistently found to be one of the best performing ‘simple’ indirect biomarker panels, with an AUROC of 0.86 (0.78–0.94),
      • McPherson S.
      • Stewart S.F.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with non-alcoholic fatty liver disease.
      the FIB-4 score was originally developed for use in patients with HCV/HIV co-infection.
      • Sterling R.K.
      • Lissen E.
      • Clumeck N.
      • Sola R.
      • Correa M.C.
      • Montaner J.
      • et al.
      Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection.
      Values <1.3 effectively rule-out advanced fibrosis in NAFLD, whilst those >2.67 (or >3.25 in some studies) identify patients with advanced fibrosis. In practice, use of the lower (high-sensitivity) threshold alone is generally advocated for serial application with other non-invasive tests as part of a 2- or 3-step triage process given that the negative predictive value is most robust.
      • Anstee Q.M.
      • Lawitz E.J.
      • Alkhouri N.
      • Wong V.W.
      • Romero-Gomez M.
      • Okanoue T.
      • et al.
      Noninvasive tests accurately identify advanced fibrosis due to NASH: baseline data from the STELLAR trials.
      As shown in Table 2, most ‘simple’ indirect biomarker panels incorporate AST, ALT and platelet count along with other readily available components. Whilst test performance remains acceptable irrespective of whether biochemical indices are elevated or within the normal range,
      • McPherson S.
      • Anstee Q.M.
      • Henderson E.
      • Day C.P.
      • Burt A.D.
      Are simple noninvasive scoring systems for fibrosis reliable in patients with NAFLD and normal ALT levels?.
      it should be noted that ALT levels fall with age, leading to a physiological age-related increase in AAR that is unrelated to the presence of liver fibrosis.
      • McPherson S.
      • Hardy T.
      • Dufour J.F.
      • Petta S.
      • Romero-Gomez M.
      • Allison M.
      • et al.
      Age as a confounding factor for the accurate non-invasive diagnosis of advanced NAFLD fibrosis.
      This adversely affects the performance of the FIB-4 and NFS with unacceptably low specificity being observed in patients aged >65-years unless an age-adjusted threshold of <2.0/<0.12 respectively is adopted to control high false-positive rates.
      • McPherson S.
      • Hardy T.
      • Dufour J.F.
      • Petta S.
      • Romero-Gomez M.
      • Allison M.
      • et al.
      Age as a confounding factor for the accurate non-invasive diagnosis of advanced NAFLD fibrosis.
      Non-invasive tests are widely used in clinical practice as first-line tools to assess liver disease severity before antiviral treatment in patients with HBV or HCV.

      Direct fibrosis biomarkers

      Given the imperfect performance of simple biomarker panels, interest focused on direct biomarkers such as hyaluronic acid,
      • Suzuki A.
      • Angulo P.
      • Lymp J.
      • Li D.
      • Satomura S.
      • Lindor K.
      Hyaluronic acid, an accurate serum marker for severe hepatic fibrosis in patients with non-alcoholic fatty liver disease.
      • Yoneda M.
      • Mawatari H.
      • Fujita K.
      • Yonemitsu K.
      • Kato S.
      • Takahashi H.
      • et al.
      Type IV collagen 7s domain is an independent clinical marker of the severity of fibrosis in patients with nonalcoholic steatohepatitis before the cirrhotic stage.
      • Lesmana C.R.
      • Hasan I.
      • Budihusodo U.
      • Gani R.A.
      • Krisnuhoni E.
      • Akbar N.
      • et al.
      Diagnostic value of a group of biochemical markers of liver fibrosis in patients with non-alcoholic steatohepatitis.
      pro-collagen III,
      • Tanwar S.
      • Trembling P.M.
      • Guha I.N.
      • Parkes J.
      • Kaye P.
      • Burt A.D.
      • et al.
      Validation of terminal peptide of procollagen III for the detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty liver disease.
      TIMP1 (tissue inhibitor of metalloproteinase 1)
      • Abdelaziz R.
      • Elbasel M.
      • Esmat S.
      • Essam K.
      • Abdelaaty S.
      Tissue inhibitors of metalloproteinase-1 and 2 and obesity related non-alcoholic fatty liver disease: is there a relationship.
      ,
      • Hemmann S.
      • Graf J.
      • Roderfeld M.
      • Roeb E.
      Expression of MMPs and TIMPs in liver fibrosis - a systematic review with special emphasis on anti-fibrotic strategies.
      and more recently PRO-C3, a collagen neo-epitope generated by ADAMS2-mediated collagen cleavage during the formation of type III collagen.
      • Nielsen M.J.
      • Nedergaard A.F.
      • Sun S.
      • Veidal S.S.
      • Larsen L.
      • Zheng Q.
      • et al.
      The neo-epitope specific PRO-C3 ELISA measures true formation of type III collagen associated with liver and muscle parameters.
      • Niemela O.
      • Risteli L.
      • Parkkinen J.
      • Risteli J.
      Purification and characterization of the N-terminal propeptide of human type III procollagen.
      • Karsdal M.A.
      • Henriksen K.
      • Nielsen M.J.
      • Byrjalsen I.
      • Leeming D.J.
      • Gardner S.
      • et al.
      Fibrogenesis assessed by serological type III collagen formation identifies patients with progressive liver fibrosis and responders to a potential antifibrotic therapy.
      • Boyle M.
      • Tiniakos D.
      • Schattenberg J.M.
      • Ratziu V.
      • Bugianessi E.
      • Petta S.
      • et al.
      Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.
      The use of combined panels incorporating these has proved more accurate than when individual biomarkers are used alone (Table 2).
      The enhanced liver fibrosis (ELF) test is a panel of markers focusing on matrix turnover that comprises hyaluronic acid, pro-collagen III and TIMP1 that was first developed for use in HCV but is now applied more generally.
      • Rosenberg W.M.
      • Voelker M.
      • Thiel R.
      • Becka M.
      • Burt A.
      • Schuppan D.
      • et al.
      Serum markers detect the presence of liver fibrosis: a cohort study.
      This test was reported to marginally outperform the NFS for detection of moderate fibrosis (≥F2, AUROC 0.90 vs. 0.86) and advanced fibrosis (≥F3, AUROC 0.93 vs. 0.89).
      • Guha I.N.
      • Parkes J.
      • Roderick P.
      • Chattopadhyay D.
      • Cross R.
      • Harris S.
      • et al.
      Noninvasive markers of fibrosis in nonalcoholic fatty liver disease: validating the European Liver Fibrosis Panel and exploring simple markers.
      A detailed meta-analysis of 2,665 patients with NAFLD across 11 studies examined performance at a range of thresholds for the detection of advanced fibrosis (ELF values 7.7 and 9.8 [recommended by the manufacturer] and 10.51 [recommended by the UK NICE guideline]).
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Loffler J.
      • Verheij J.
      • et al.
      Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: a systematic review and meta-analysis.
      The AUROC for detecting advanced fibrosis was lower than previously reported at 0.83 (0.71-0.90) with a threshold of 7.7 demonstrating high sensitivity at 0.93 (0.82-0.98) but a low specificity of 0.34 (0.13-0.65). In contrast, a specificity of 0.86 (0.77-0.92) and sensitivity of 0.65 (0.49-0.77) was observed at ELF >9.8, and a specificity of 0.93 (0.85-0.96) and sensitivity of 0.51 (0.31-0.70) at an ELF threshold of >10.51.
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Loffler J.
      • Verheij J.
      • et al.
      Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: a systematic review and meta-analysis.
      Importantly, modelling of test performance across a range of pre-test probabilities demonstrated that, whilst the NPV was good when the prevalence of advanced fibrosis ranged from 5-10% as may be encountered in practice, the PPV was less strong at between 7-52% depending on the threshold applied.
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Loffler J.
      • Verheij J.
      • et al.
      Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: a systematic review and meta-analysis.
      Like ELF, FibroTest was also initially developed in HCV but subsequently validated in NAFLD.
      • Ratziu V.
      • Massard J.
      • Charlotte F.
      • Messous D.
      • Imbert-Bismut F.
      • Bonyhay L.
      • et al.
      Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease.
      ,
      • Munteanu M.
      • Tiniakos D.
      • Anstee Q.
      • Charlotte F.
      • Marchesini G.
      • Bugianesi E.
      • et al.
      Diagnostic performance of FibroTest, SteatoTest and ActiTest in patients with NAFLD using the SAF score as histological reference.
      A meta-analysis of 2,103 patients in 5 studies showed the AUROC for advanced fibrosis was 0.77 (0.64 to 0.86), again with evidence of limited PPV at a prevalence of advanced fibrosis likely to be encountered in routine practice.
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Verheij J.
      • Brosnan M.J.
      • et al.
      FibroTest for evaluating fibrosis in non-alcoholic fatty liver disease patients: a systematic review and meta-analysis.
      More recently, biomarker panels combining measurement of PRO-C3 and indirect biomarkers such as age, T2DM, platelet count with or without BMI (FIBC3
      • Boyle M.
      • Tiniakos D.
      • Schattenberg J.M.
      • Ratziu V.
      • Bugianessi E.
      • Petta S.
      • et al.
      Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.
      and ADAPT
      • Daniels S.J.
      • Leeming D.J.
      • Eslam M.
      • Hashem A.M.
      • Nielsen M.J.
      • Krag A.
      • et al.
      ADAPT: an algorithm incorporating PRO-C3 accurately identifies patients with NAFLD and advanced fibrosis.
      respectively) have been proposed (Table 2). Fewer large cohort studies are available to assess the performance of PRO-C3; however, a recent meta-analysis of 2,058 patients with NAFLD in 35 studies reported that PRO-C3 alone could detect advanced fibrosis with an AUROC of 0.79 (0.73–0.82).
      • Mak A.L.
      • Lee J.
      • van Dijk A.M.
      • Vali Y.
      • Aithal G.P.
      • Schattenberg J.M.
      • et al.
      Systematic review with meta-analysis: diagnostic accuracy of pro-C3 for hepatic fibrosis in patients with non-alcoholic fatty liver disease.
      Both ADAPT and FIBC3 have also been shown to outperform FIB-4 and PRO-C3 alone.
      • Boyle M.
      • Tiniakos D.
      • Schattenberg J.M.
      • Ratziu V.
      • Bugianessi E.
      • Petta S.
      • et al.
      Performance of the PRO-C3 collagen neo-epitope biomarker in non-alcoholic fatty liver disease.
      Published head-to-head comparisons of proprietary biomarkers are lacking although conference reports suggest that, at best, the currently available direct biomarkers only marginally outperform FIB-4 when targeting the presence of advanced fibrosis. Limited availability and cost may also limit their wider adoption. Recent developments including wider proteomic profiling
      • Corey K.E.
      • Pitts R.
      • Lai M.
      • Loureiro J.
      • Masia R.
      • Osganian S.A.
      • et al.
      ADAMTSL2 protein and a soluble biomarker signature identify at-risk non-alcoholic steatohepatitis and fibrosis in adults with NAFLD.
      or the use of novel measures of protease activity
      • Cazanave S.C.
      • Warren A.D.
      • Pacula M.
      • Touti F.
      • Zagorska A.
      • Gural N.
      • et al.
      Peptide-based urinary monitoring of fibrotic nonalcoholic steatohepatitis by mass-barcoded activity-based sensors.
      are at present some way from the clinic but offer the prospect of further improvements in biomarker performance.

      Elastography

      Elastography techniques measure liver stiffness by quantifying the velocity of an induced shear wave as a surrogate of hepatic fibrosis. These include ultrasound-based VCTE (FibroScan), pSWE (also known as acoustic radiation force impulse), and 2D-SWE; and MRI-based MRE.
      The most widely adopted and validated of these, VCTE, uses pulse-echo ultrasound to quantify the velocity of low frequency vibrations transmitted into the liver.
      • Sandrin L.
      • Fourquet B.
      • Hasquenoph J.M.
      • Yon S.
      • Fournier C.
      • Mal F.
      • et al.
      Transient elastography: a new noninvasive method for assessment of hepatic fibrosis.
      ,
      • Foucher J.
      • Chanteloup E.
      • Vergniol J.
      • Castera L.
      • Le Bail B.
      • Adhoute X.
      • et al.
      Diagnosis of cirrhosis by transient elastography (FibroScan): a prospective study.
      The resulting liver stiffness measurement (LSM) correlates with hepatic fibrosis stage and has been validated in a range of liver diseases including HBV,
      • Papatheodoridi M.
      • Hiriart J.B.
      • Lupsor-Platon M.
      • Bronte F.
      • Boursier J.
      • Elshaarawy O.
      • et al.
      Refining the Baveno VI elastography criteria for the definition of compensated advanced chronic liver disease.
      HCV,
      • Papatheodoridi M.
      • Hiriart J.B.
      • Lupsor-Platon M.
      • Bronte F.
      • Boursier J.
      • Elshaarawy O.
      • et al.
      Refining the Baveno VI elastography criteria for the definition of compensated advanced chronic liver disease.
      primary biliary cholangitis, NAFLD
      • Papatheodoridi M.
      • Hiriart J.B.
      • Lupsor-Platon M.
      • Bronte F.
      • Boursier J.
      • Elshaarawy O.
      • et al.
      Refining the Baveno VI elastography criteria for the definition of compensated advanced chronic liver disease.
      • Siddiqui M.S.
      • Vuppalanchi R.
      • Van Natta M.L.
      • Hallinan E.
      • Kowdley K.V.
      • Abdelmalek M.
      • et al.
      Vibration-controlled transient elastography to assess fibrosis and steatosis in patients with nonalcoholic fatty liver disease.
      • Eddowes P.J.
      • Sasso M.
      • Allison M.
      • Tsochatzis E.
      • Anstee Q.M.
      • Sheridan D.
      • et al.
      Accuracy of FibroScan controlled attenuation parameter and liver stiffness measurement in assessing steatosis and fibrosis in patients with nonalcoholic fatty liver disease.
      • Boursier J.
      • Vergniol J.
      • Guillet A.
      • Hiriart J.B.
      • Lannes A.
      • Le Bail B.
      • et al.
      Diagnostic accuracy and prognostic significance of blood fibrosis tests and liver stiffness measurement by FibroScan in non-alcoholic fatty liver disease.
      and ALD.
      • Papatheodoridi M.
      • Hiriart J.B.
      • Lupsor-Platon M.
      • Bronte F.
      • Boursier J.
      • Elshaarawy O.
      • et al.
      Refining the Baveno VI elastography criteria for the definition of compensated advanced chronic liver disease.
      ,
      • Nguyen-Khac E.
      • Thiele M.
      • Voican C.
      • Nahon P.
      • Moreno C.
      • Boursier J.
      • et al.
      Non-invasive diagnosis of liver fibrosis in patients with alcohol-related liver disease by transient elastography: an individual patient data meta-analysis.
      However, VCTE performance is operator dependant and subject to the confounding effects of other factors that increase liver stiffness or prevent accurate measurements such as congestive cardiac failure, cholestasis, acute hepatitis, recent alcohol or food consumption, and obesity.
      • Castera L.
      • Foucher J.
      • Bernard P.H.
      • Carvalho F.
      • Allaix D.
      • Merrouche W.
      • et al.
      Pitfalls of liver stiffness measurement: a 5-year prospective study of 13,369 examinations.
      Reliability criteria for VCTE (IQR/M <0.30, or IQR/M >0.30 if median reading <7.1 kPa) have been established to assist interpretation of results.
      • Boursier J.
      • Zarski J.P.
      • de Ledinghen V.
      • Rousselet M.C.
      • Sturm N.
      • Lebail B.
      • et al.
      Determination of reliability criteria for liver stiffness evaluation by transient elastography.
      In NAFLD, an individual patient data meta-analysis incorporating 5,735 VCTE measurements from 37 international studies demonstrated AUROCs of 0.85 and 0.90 for the detection of advanced fibrosis (≥F3) and cirrhosis, respectively, outperforming FIB-4.
      • Mozes F.E.
      • Lee J.A.
      • Selvaraj E.A.
      • Jayaswal A.N.A.
      • Trauner M.
      • Boursier J.
      • et al.
      Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.
      This study showed that a VCTE ≤7.4 kPa provided 90% sensitivity for advanced fibrosis and a high NPV >0.95 when the prevalence of advanced fibrosis was <30%, as is the case in most clinical settings.
      • Mozes F.E.
      • Lee J.A.
      • Selvaraj E.A.
      • Jayaswal A.N.A.
      • Trauner M.
      • Boursier J.
      • et al.
      Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.
      VCTE thresholds for liver stiffness may vary somewhat between different aetiologies but, a VCTE cut-off of less than 8 kPa appears adequate to reliably rule-out advanced fibrosis in routine practice.
      European Association for the Study of the Liver (EASL)
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      ,
      • Mozes F.E.
      • Lee J.A.
      • Selvaraj E.A.
      • Jayaswal A.N.A.
      • Trauner M.
      • Boursier J.
      • et al.
      Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: an individual patient data meta-analysis.
      ,
      • Papatheodoridi M.
      • Hiriart J.B.
      • Lupsor-Platon M.
      • Bronte F.
      • Boursier J.
      • Elshaarawy O.
      • et al.
      Refining the Baveno VI elastography criteria for the definition of compensated advanced chronic liver disease.
      One of the most promising and innovative techniques is MRE. By assessing a larger portion of the liver than ultrasound-based elastography, MRE is less prone to sampling error and has been shown to be more accurate than VCTE.
      • Park C.C.
      • Nguyen P.
      • Hernandez C.
      • Bettencourt R.
      • Ramirez K.
      • Fortney L.
      • et al.
      Magnetic resonance elastography vs. transient elastography in detection of fibrosis and noninvasive measurement of steatosis in patients with biopsy-proven nonalcoholic fatty liver disease.
      ,
      • Imajo K.
      • Kessoku T.
      • Honda Y.
      • Tomeno W.
      • Ogawa Y.
      • Mawatari H.
      • et al.
      Magnetic resonance imaging more accurately classifies steatosis and fibrosis in patients with nonalcoholic fatty liver disease than transient elastography.
      It is however more resource intensive, less widely available and not available as a point-of-care assessment. Due to limited availability in some territories, performance has largely been assessed in smaller cohorts and many studies have focused on NAFLD. One of the earliest studies in this disease area reported an AUROC of 0.924 for advanced fibrosis, with a threshold MRE value of >3.63 kPa exhibiting high sensitivity (86% [65-97%]) and specificity (91% [83-96%]).
      • Loomba R.
      • Wolfson T.
      • Ang B.
      • Hooker J.
      • Behling C.
      • Peterson M.
      • et al.
      Magnetic resonance elastography predicts advanced fibrosis in patients with nonalcoholic fatty liver disease: a prospective study.
      More recently a detailed meta-analysis has confirmed these performance characteristics with AUROCs of 0.91 (sensitivity 78%, specificity 89%) for significant fibrosis ≥F2, 0.92 (sensitivity 83%, specificity 89%) for advanced fibrosis ≥F3 and 0.90 (sensitivity 81%, specificity 90%) for cirrhosis in NAFLD.
      • Selvaraj E.A.
      • Mozes F.E.
      • Jayaswal A.N.A.
      • Zafarmand M.H.
      • Vali Y.
      • Lee J.A.
      • et al.
      Diagnostic accuracy of elastography and magnetic resonance imaging in patients with NAFLD: a systematic review and meta-analysis.

      Monitoring disease progression and establishing prognosis

      Emergence of the cACLD concept

      The term “compensated advanced chronic liver disease (cACLD)” has been proposed by the Baveno VI conference
      • de Franchis R.
      • Baveno V.I.F.
      Expanding consensus in portal hypertension: report of the Baveno VI Consensus Workshop: stratifying risk and individualizing care for portal hypertension.
      to reflect the continuum of severe fibrosis and cirrhosis in patients with ongoing CLD. Its definition was pragmatic, based on LSM using VCTE (2 examinations on different days), with LSM values ≥10 kPa, suggestive of cACLD, and values ≥15 kPa, highly suggestive of cACLD. It was aimed at stratifying the risk of clinically significant portal hypertension (CSPH – defined by a hepatic venous pressure gradient [HVPG] ≥10 mmHg) and decompensation at point-of-care, irrespective of histological stage or the ability of LSM to identify these stages.
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      The premise was to raise awareness of cirrhosis outside of the liver field, so that patients with suspected cACLD would be referred to a liver specialist for further work-up.
      MEFIB and FAST scores may be used to detect “at risk” NASH patients, i.e. those with ≥stage 2 fibrosis, and candidates for phase IIb/III clinical trials in NASH.
      LSM values have recently been refined in a validation study that included over 5,500 patients with CLD.
      • Papatheodoridi M.
      • Hiriart J.B.
      • Lupsor-Platon M.
      • Bronte F.
      • Boursier J.
      • Elshaarawy O.
      • et al.
      Refining the Baveno VI elastography criteria for the definition of compensated advanced chronic liver disease.
      This study showed that a LSM cut-off >12 kPa has >90% specificity for diagnosing cACLD, while a cut-off <8 kPa (for NAFLD and ALD) or <7 kPa (for viral hepatitis) has >90% sensitivity for ruling out cACLD. LSM values <10 kPa in the absence of other known clinical/imaging signs rule-out cACLD and these patients have a negligible 3-year risk (≤1%) of decompensation and liver-related death.
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      On the other hand, LSM can lead to false-positive results: an index VCTE showing LSM ≥10 kPa should be repeated in fasting conditions as soon as feasible or complemented with an established serum marker of fibrosis (FIB-4 ≥2.67, ELF ≥9.8, FibroTest ≥0.58 for alcohol-related/viral liver disease, FibroTest ≥0.48 for NAFLD).
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      Finally, VCTE could be repeated every 12 months to monitor changes in patients with cACLD.
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      Other elastography techniques have shown high diagnostic accuracy for cACLD,
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      but cut-offs are system-specific, and no consensual cut-offs have been defined so far. Thus, VCTE remains the reference.

      Risk stratification in patients with cACLD

      Screening for portal hypertension and oesophageal varices

      Although the concept of CSPH is HVPG-driven, non-invasive tests (NITs) are sufficiently accurate to identify CSPH in clinical practice. LSM by VCTE is the most validated quantitative individual NIT for portal hypertension in patients with cACLD.
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      At a cut-off of 20–25 kPa, VCTE can identify CSPH with an AUROC of >0.90, as shown in a recent meta-analysis (based on 11 studies including 1,451 patients).
      • You M.W.
      • Kim K.W.
      • Pyo J.
      • Huh J.
      • Kim H.J.
      • Lee S.J.
      • et al.
      A meta-analysis for the diagnostic performance of transient elastography for clinically significant portal hypertension.
      The accuracy of VCTE increases when it is combined with unrelated NITs, in particular platelet count.
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      Accordingly, the latest Baveno VII conference recommended the use of LSM ≤15 kPa and platelet count ≥150 109/L to rule-out CSPH (sensitivity and NPV >90%) in all patients with cACLD.
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      In patients with virus- and/or alcohol-related cACLD and non-obese (BMI <30 kg/m2) patients with non-alcoholic steatohepatitis (NASH)-related cACLD, an LSM value ≥25 kPa is sufficient to rule-in CSPH (specificity and positive predictive value >90%), defining the group of patients at risk of endoscopic signs of portal hypertension and at higher risk of decompensation (Fig. 1). These patients may benefit from beta-blockers, independently of the presence of oesophageal varices, to prevent decompensation.
      • Villanueva C.
      • Albillos A.
      • Genesca J.
      • Garcia-Pagan J.C.
      • Calleja J.L.
      • Aracil C.
      • et al.
      Beta blockers to prevent decompensation of cirrhosis in patients with clinically significant portal hypertension (PREDESCI): a randomised, double-blind, placebo-controlled, multicentre trial.
      Figure thumbnail gr1
      Fig. 1Algorithm for the non-invasive determination of cACLD and CSPH (from ref
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      ).
      Proposed liver stiffness cut-offs, using VCTE, for ruling out (<10 kPa) and ruling in (≥15 kPa) cACLD, ruling out (<15 kPa and plat ≥150) CSPH, avoiding upper GI endoscopy (<20 kPa and plat ≥150), and asuming (≥25 kPa) CSPH in patients with HBV, HCV, ALD and non-obsese NASH. ALD, alcohol-related liver disease; cACLD, compensated advanced chronic liver disease; CSPH, clinically significant portal hypertension; NASH, non-alcoholic steatohepatitis; Plat, platelet count; VCTE, vibration-controlled transient elastography.
      As for ruling out high-risk oesophageal varices, the Baveno VI recommendation to reduce the number of unnecessary endoscopies, based on NIT criteria (LSM <20 kPa and platelet count ≥150 109/L), has led to a significant change in clinical practice over the last 5 years.
      • de Franchis R.
      • Baveno V.I.F.
      Expanding consensus in portal hypertension: report of the Baveno VI Consensus Workshop: stratifying risk and individualizing care for portal hypertension.
      ,
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      The performances of these criteria (about 20% of endoscopies avoided, missing less than 4% of patients with high-risk varices) have since been confirmed by two meta-analyses,
      • Stafylidou M.
      • Paschos P.
      • Katsoula A.
      • Malandris K.
      • Ioakim K.
      • Bekiari E.
      • et al.
      Performance of Baveno VI and expanded Baveno VI criteria for excluding high-risk varices in patients with chronic liver diseases: a systematic review and meta-analysis.
      ,
      • Szakacs Z.
      • Eross B.
      • Soos A.
      • Matrai P.
      • Szabo I.
      • Petervari E.
      • et al.
      Baveno criteria safely identify patients with compensated advanced chronic liver disease who can avoid variceal screening endoscopy: a diagnostic test accuracy meta-analysis.
      and this recommendation is still relevant.
      • de Franchis R.
      • Baveno V.I.F.
      Expanding consensus in portal hypertension: report of the Baveno VI Consensus Workshop: stratifying risk and individualizing care for portal hypertension.
      ,
      • de Franchis R.
      • Bosch J.
      • Garcia-Tsao G.
      • Reiberger T.
      • Ripoll C.
      • Baveno V.I.I.F.
      Baveno VII - renewing consensus in portal hypertension.
      As for other elastography techniques, given the very limited number of studies reporting on their performance, no recommendation can be made.
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      Finally, spleen stiffness (using VCTE or 2D-SWE) may be used as an additional tool to refine risk prediction for high-risk varices in cACLD.
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.

      Screening for hepatocellular carcinoma

      Several risk scores have been developed to predict hepatocellular carcinoma (HCC) in patients with HBV, mostly in Asian cohorts.
      • Voulgaris T.
      • Papatheodoridi M.
      • Lampertico P.
      • Papatheodoridis G.V.
      Clinical utility of hepatocellular carcinoma risk scores in chronic hepatitis B.
      Most scores include simple clinical and biological data (age, male sex, platelet count, and cirrhosis or viral load) but very few include liver stiffness (LSM-HCC and LSPS). The PAGE-B score (age, sex and platelet count) and the modified PAGE-B score (age, sex and platelet count, and albumin) are the only scores that have been externally validated and showed good results in Caucasian and Asian cohorts, with NPVs of 0.95–0.99 at 5 years.
      • Voulgaris T.
      • Papatheodoridi M.
      • Lampertico P.
      • Papatheodoridis G.V.
      Clinical utility of hepatocellular carcinoma risk scores in chronic hepatitis B.
      Thus, data regarding NITs is insufficient to indicate which patients can avoid HCC screening.
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.

      Risk of progression to cirrhosis, risk of hepatic decompensation in cirrhosis, and mortality

      Recent data from natural history studies and randomised, placebo-controlled studies suggest that approximately 10–20% of patients with bridging fibrosis may progress to cirrhosis over a 2-to-3-year period.
      • Sanyal A.J.
      • Harrison S.A.
      • Ratziu V.
      • Abdelmalek M.F.
      • Diehl A.M.
      • Caldwell S.
      • et al.
      The natural history of advanced fibrosis due to nonalcoholic steatohepatitis: data from the simtuzumab trials.
      ,
      • Loomba R.
      • Adams L.A.
      The 20% rule of NASH progression: the natural history of advanced fibrosis and cirrhosis caused by NASH.
      Although change in FIB-4 categories from those that are in the intermediate range (1.3–2.67) to a higher range (>2.67) may be associated with higher risk of progression, these changes occur over a longer time-horizon.
      • Hagstrom H.
      • Talback M.
      • Andreasson A.
      • Walldius G.
      • Hammar N.
      Repeated FIB-4 measurements can help identify individuals at risk of severe liver disease.
      Among patients with bridging fibrosis, an ELF ≥9.8 or LSM ≥16.6 kPa by VCTE has been associated with higher odds of progression to cirrhosis.
      • Loomba R.
      • Adams L.A.
      The 20% rule of NASH progression: the natural history of advanced fibrosis and cirrhosis caused by NASH.
      Among patients with NAFLD, the MEFIB index (LSM ≥3.3 kPa by MRE and FIB-4 ≥1.6) has also been associated with progression of disease and likelihood of clinical decompensation and HCC. MEFIB (negative) has an excellent NPV of >95% in ruling out liver disease progression.
      Among patients with cirrhosis, ELF ≥11.3 or VCTE ≥30 kPa is associated with a higher hazard ratio for hepatic decompensation and mortality.
      • Sanyal A.J.
      • Harrison S.A.
      • Ratziu V.
      • Abdelmalek M.F.
      • Diehl A.M.
      • Caldwell S.
      • et al.
      The natural history of advanced fibrosis due to nonalcoholic steatohepatitis: data from the simtuzumab trials.
      LSM ≥8 kPa by MRE or a 1 kPa increase in LSM by MRE among those who have a baseline LSM ≥5 kPa is associated with higher odds of hepatic decompensation and liver-related mortality.
      • Gidener T.
      • Ahmed O.T.
      • Larson J.J.
      • Mara K.C.
      • Therneau T.M.
      • Venkatesh S.K.
      • et al.
      Liver stiffness by magnetic resonance elastography predicts future cirrhosis, decompensation, and death in NAFLD.
      Higuchi and colleagues have recently shown that the presence of cirrhosis (≥4.67 kPa) vs. minimal fibrosis (<3 kPa), assessed by MRE, is associated with an adjusted hazard ratio of 4.2 (95% CI 2.2-8.2) for the development of incident HCC and 67.5 (95% CI 9.2-492) for the development of hepatic decompensation but is not associated with a higher risk of major atherosclerotic cardiovascular events or extrahepatic cancer.
      • Higuchi M.
      • Tamaki N.
      • Kurosaki M.
      • Inada K.
      • Kirino S.
      • Yamashita K.
      • et al.
      Longitudinal association of magnetic resonance elastography-associated liver stiffness with complications and mortality.

      Treatment decisions

      Treatment decisions in viral hepatitis

      Assessing liver disease severity before antiviral treatment

      In patients with HBV, treatment decisions mainly rely on virological markers (HBeAg and HBV DNA) and transaminase levels, whereas with direct-acting antivirals (DAAs), treatment has become universal in patients with HCV. Nevertheless, international guidelines recommend that all patients with chronic hepatitis B or C should be assessed for liver disease severity before antiviral therapy, using NITs as the first-line tool for assessment.
      EASL Clinical Practice Guidelines on the management of hepatitis B virus infection.
      EASL recommendations on treatment of hepatitis C: final update of the series.
      • Ghany M.G.
      • Morgan T.R.
      • Panel A.-I.H.C.G.
      Hepatitis C guidance 2019 update: American Association for the Study of Liver Diseases-Infectious Diseases Society of America recommendations for testing, managing, and treating hepatitis C virus infection.
      • Terrault N.A.
      • Lok A.S.F.
      • McMahon B.J.
      • Chang K.M.
      • Hwang J.P.
      • Jonas M.M.
      • et al.
      Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance.
      Identifying cirrhosis is of particular importance in patients with HCV as some DAA treatment regimens must be adjusted, whereas in patients with HBV, treatment by nucleos(t)ide analogues should not be stopped in those with cirrhosis. In both cases, surveillance for HCC after viral eradication or suppression is mandatory. VCTE, APRI and FIB-4 are the most used NITs and their combination increases diagnostic accuracy.
      • Castera L.
      • Vergniol J.
      • Foucher J.
      • Le Bail B.
      • Chanteloup E.
      • Haaser M.
      • et al.
      Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C.
      In case of unexplained discordance between the NITs or suspected additional aetiologies of liver disease, a liver biopsy is still recommended in patients with HCV and HBV.
      EASL-ALEH Clinical Practice Guidelines
      Non-invasive tests for evaluation of liver disease severity and prognosis.
      However, the accessibility of elastography, especially in resource-limited settings, needs to be considered. The WHO guidelines for the prevention, care and treatment of persons with chronic HBV infection recommend using APRI in resource-limited settings, and VCTE or patented serum biomarkers when accessible and affordable.
      MRI-PDFF response, defined as ≥ 30% relative reduction in liver fat, is associated with 5-fold higher odds of NASH resolution.

      Surveillance after HCV eradication

      In patients with HCV without cirrhosis, routine use of NITs after sustained virological response (SVR) is not recommended as it does not change clinical disease management.
      EASL recommendations on treatment of hepatitis C: final update of the series.
      ,
      • Ghany M.G.
      • Morgan T.R.
      • Panel A.-I.H.C.G.
      Hepatitis C guidance 2019 update: American Association for the Study of Liver Diseases-Infectious Diseases Society of America recommendations for testing, managing, and treating hepatitis C virus infection.
      In patients with HCV and cACLD or cirrhosis, regression of fibrosis,
      • D'Ambrosio R.
      • Aghemo A.
      • Rumi M.G.
      • Ronchi G.
      • Donato M.F.
      • Paradis V.
      • et al.
      A morphometric and immunohistochemical study to assess the benefit of a sustained virological response in hepatitis C virus patients with cirrhosis.
      improvement in liver-related outcomes and reduced risk of mortality and HCC have been reported after viral eradication with DAAs.
      • D'Ambrosio R.
      • Degasperi E.
      • Anolli M.P.
      • Fanetti I.
      • Borghi M.
      • Soffredini R.
      • et al.
      Incidence of liver- and non-liver-related outcomes in patients with HCV-cirrhosis after SVR.
      ,
      • Carrat F.
      • Fontaine H.
      • Dorival C.
      • Simony M.
      • Diallo A.
      • Hezode C.
      • et al.
      Clinical outcomes in patients with chronic hepatitis C after direct-acting antiviral treatment: a prospective cohort study.
      However, as post-SVR liver biopsies are not the standard of care, the question of whether NITs can capture fibrosis regression and the residual risk of liver-related complications is critical. In a large meta-analysis (24 studies including 2,934 patients with HCV), SVR was associated with a significant decrease in LSM, using VCTE.
      • Singh S.
      • Facciorusso A.
      • Loomba R.
      • Falck-Ytter Y.T.
      Magnitude and kinetics of decrease in liver stiffness after antiviral therapy in patients with chronic hepatitis C: a systematic review and meta-analysis.
      In the absence of paired liver biopsies, it is impossible to conclude whether the observed LSM decrease is related to the resolution of hepatic inflammation or to the regression of liver fibrosis. Also, CSPH persists after SVR and LSM changes do not correlate with changes in HVPG after SVR.
      • Lens S.
      • Baiges A.
      • Alvarado-Tapias E.
      • LLop E.
      • Martinez J.
      • Fortea J.I.
      • et al.
      Clinical outcome and hemodynamic changes following HCV eradication with oral antiviral therapy in patients with clinically significant portal hypertension.
      Even if available evidence
      • Alonso Lopez S.
      • Manzano M.L.
      • Gea F.
      • Gutierrez M.L.
      • Ahumada A.M.
      • Devesa M.J.
      • et al.
      A model based on noninvasive markers predicts very low hepatocellular carcinoma risk after viral response in hepatitis C virus-advanced fibrosis.
      • Ioannou G.N.
      • Beste L.A.
      • Green P.K.
      • Singal A.G.
      • Tapper E.B.
      • Waljee A.K.
      • et al.
      Increased risk for hepatocellular carcinoma persists up to 10 Years after HCV eradication in patients with baseline cirrhosis or high FIB-4 scores.
      • Pons M.
      • Rodriguez-Tajes S.
      • Esteban J.I.
      • Marino Z.
      • Vargas V.
      • Lens S.
      • et al.
      Non-invasive prediction of liver-related events in patients with HCV-associated compensated advanced chronic liver disease after oral antivirals.
      suggests that LSM and FIB-4 after SVR could predict HCC occurrence, more data are needed. Thus, patients with HCV and cACLD before treatment should continue to be monitored for HCC and portal hypertension irrespective of the results of NITs post-SVR.
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      ,
      EASL recommendations on treatment of hepatitis C: final update of the series.
      In those with cirrhosis, surveillance for oesophageal varices by endoscopy should be performed, if varices were present at pre-treatment endoscopy, or if the platelet count falls below 150 G/L and LSM increases to more than 20 kPa.
      • Ghany M.G.
      • Morgan T.R.
      • Panel A.-I.H.C.G.
      Hepatitis C guidance 2019 update: American Association for the Study of Liver Diseases-Infectious Diseases Society of America recommendations for testing, managing, and treating hepatitis C virus infection.

      Treatment decisions in NAFLD

      Defining the patient population that is eligible for treatment with a pharmacologic therapy

      Both the FDA and EMA have provided guidance regarding the patient population that is eligible for treatment in a phase III trial with respect to regulatory approval.
      • Loomba R.
      • Ratziu V.
      • Harrison S.A.
      Group NCTDIW
      Expert panel review to compare FDA and EMA guidance on drug development and endpoints in nonalcoholic steatohepatitis.
      Data from several well-conducted meta-analyses suggest that patients with a diagnosis of NASH and stage ≥2 fibrosis are at a higher risk of progression to cirrhosis and long-term liver-related morbidity and mortality.
      • Dulai P.S.
      • Singh S.
      • Patel J.
      • Soni M.
      • Prokop L.J.
      • Younossi Z.
      • et al.
      Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: systematic review and meta-analysis.
      Therefore, both the FDA and EMA recommend that patients with NASH and stage ≥2 fibrosis (also classified as “at risk” NASH) should be considered for treatment with a pharmacologic therapy.
      • Loomba R.
      • Ratziu V.
      • Harrison S.A.
      Group NCTDIW
      Expert panel review to compare FDA and EMA guidance on drug development and endpoints in nonalcoholic steatohepatitis.
      Imaging biomarkers have been utilised to help with a screening strategy to identify patients with “at risk” NASH in the setting of clinical trials.
      • Tamaki N.
      • Ajmera V.
      • Loomba R.
      Non-invasive methods for imaging hepatic steatosis and their clinical importance in NAFLD.
      The FAST score is a combination of LSM by VCTE, liver fat content by controlled attenuation parameter and serum AST. A FAST score of ≥0.67 is associated with a higher likelihood of “at risk” NASH and a score of ≤0.35 is associated with a lower likelihood of having “at risk” NASH.
      • Newsome P.N.
      • Sasso M.
      • Deeks J.J.
      • Paredes A.
      • Boursier J.
      • Chan W.K.
      • et al.
      FibroScan-AST (FAST) score for the non-invasive identification of patients with non-alcoholic steatohepatitis with significant activity and fibrosis: a prospective derivation and global validation study.
      Although helpful and point-of-care, the FAST score has a low PPV. Recently, using a similar logistic regression equation, the MAST score has been proposed, which is a combination of LSM by MRE, liver fat by MRI-proton density fat fraction (PDFF), and serum AST. This score has a higher diagnostic accuracy than FAST.
      • Noureddin M.
      • Truong E.
      • Gornbein J.A.
      • Saouaf R.
      • Guindi M.
      • Todo T.
      • et al.
      MRI-based (MAST) score accurately identifies patients with NASH and significant fibrosis.
      It has an excellent NPV but low PPV. To fill the gap of low PPV, MEFIB has been proposed and validated with a robust PPV for “at risk” NASH of >90%.
      • Jung J.
      • Loomba R.R.
      • Imajo K.
      • Madamba E.
      • Gandhi S.
      • Bettencourt R.
      • et al.
      MRE combined with FIB-4 (MEFIB) index in detection of candidates for pharmacological treatment of NASH-related fibrosis.
      A head-to-head comparative study has shown that MEFIB is superior to FAST.
      • Tamaki N.
      • Imajo K.
      • Sharpton S.
      • Jung J.
      • Kawamura N.
      • Yoneda M.
      • et al.
      Magnetic resonance elastography plus Fibrosis-4 versus FibroScan-aspartate aminotransferase in detection of candidates for pharmacological treatment of NASH-related fibrosis.
      MEFIB could be utilised as a 2-step strategy where FIB-4 is applied initially and only those with a FIB-4 ≥1.6 undergo further testing with MRE.
      • Tamaki N.
      • Imajo K.
      • Sharpton S.R.
      • Jung J.
      • Sutter N.
      • Kawamura N.
      • et al.
      Two-step strategy, FIB-4 followed by magnetic resonance elastography, for detecting advanced fibrosis in NAFLD.
      This strategy is likely to significantly reduce the number of MRE exams needed to identify “at risk” patients and will likely be more cost-effective although detailed cost-effectiveness studies have not yet been reported.
      In addition to imaging biomarkers, the NIS4 circulating biomarker panel has also been developed to specifically target “at risk” NASH.
      • Harrison S.A.
      • Ratziu V.
      • Boursier J.
      • Francque S.
      • Bedossa P.
      • Majd Z.
      • et al.
      A blood-based biomarker panel (NIS4) for non-invasive diagnosis of non-alcoholic steatohepatitis and liver fibrosis: a prospective derivation and global validation study.
      This panel incorporates a circulating micro-RNA (miR34a), which has been independently validated to associate with NASH and fibrosis,
      • Johnson K.
      • Leary P.J.
      • Govaere O.
      • Barter M.J.
      • Charlton S.H.
      • Cockell S.J.
      • et al.
      Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: diagnostic and mechanistic relevance.
      combined with indirect biomarkers of inflammation and glucose tolerance (alpha-2 macroglobulin, YKL-40, and glycated haemoglobin), achieving an AUROC of 0.80 (0.77-0.84) and outperforming simple panels such as FIB-4.
      • Harrison S.A.
      • Ratziu V.
      • Boursier J.
      • Francque S.
      • Bedossa P.
      • Majd Z.
      • et al.
      A blood-based biomarker panel (NIS4) for non-invasive diagnosis of non-alcoholic steatohepatitis and liver fibrosis: a prospective derivation and global validation study.

      Biomarkers of treatment response

      There is a major unmet need to develop biomarkers that will non-invasively predict either NASH resolution or ≥1 stage improvement in fibrosis stage.
      • Castera L.
      • Friedrich-Rust M.
      • Loomba R.
      Noninvasive assessment of liver disease in patients with nonalcoholic fatty liver disease.
      Yet, no biomarkers have been qualified for use as regulatory approved clinical trial endpoints; however, a number of circulating and imaging biomarkers are commonly measured as exploratory endpoints in phase II/III trials. LSM-based biomarkers, such as VCTE and MRE, are routinely utilised in clinical trials although the exact cut-off point, or relative reduction associated with histologic improvements has not yet been documented. Several randomised controlled trials and a recent meta-analysis have demonstrated that MRI-PDFF response, defined as a ≥30% relative reduction in liver fat, is associated with 7-fold higher odds of a ≥2 point improvement in NAFLD activity score, and 5-fold higher odds of NASH resolution.
      • Stine J.G.
      • Munaganuru N.
      • Barnard A.
      • Wang J.L.
      • Kaulback K.
      • Argo C.K.
      • et al.
      Change in MRI-PDFF and histologic response in patients with nonalcoholic steatohepatitis: a systematic review and meta-analysis.
      Further studies are needed to determine if MAST or MEFIB or corrected T1 or other imaging-based biomarkers, whether alone or in combination with serum-based biomarkers, may be used to predict treatment response.
      Circulating biomarkers including the ELF test, PRO-C3 (as a single biomarker or within the ADAPT and FIBC3 panels) are frequently also measured as exploratory endpoints and have shown themselves to be sensitive to change.
      • Harrison S.A.
      • Rossi S.J.
      • Paredes A.H.
      • Trotter J.F.
      • Bashir M.R.
      • Guy C.D.
      • et al.
      NGM282 improves liver fibrosis and histology in 12 weeks in patients with nonalcoholic steatohepatitis.
      ,
      • Rinella M.E.
      • Dufour J.F.
      • Anstee Q.M.
      • Goodman Z.
      • Younossi Z.
      • Harrison S.A.
      • et al.
      Non-invasive evaluation of response to obeticholic acid in patients with NASH: results from the REGENERATE study.
      Although not yet qualified as regulatory approvable endpoints in clinical trials, to advance the field and support transition to such approval there is a need to establish a defined set of biomarkers with accompanying response thresholds that should consistently be measured and reported in all clinical trials. Indeed, arguably the most compelling evidence for disease improvement is when multiple biomarkers that address different aspects of pathophysiology (e.g. histology, direct/indirect fibrosis biomarkers and elastography) are seen to improve in the same individuals.
      Liver stiffness and ELF can be used to predict future risk of decompensation among patients with chronic liver disease.

      Use of non-invasive tests beyond the liver clinic

      Screening for liver disease and cirrhosis in the general population

      NITs represent an attractive strategy for screening fibrosis and cirrhosis in the general population, where the prevalence of CLDs is high and will likely increase, particularly due to the rising prevalence of NAFLD.
      • Gines P.
      • Castera L.
      • Lammert F.
      • Graupera I.
      • Serra-Burriel M.
      • Allen A.M.
      • et al.
      Population screening for liver fibrosis: toward early diagnosis and intervention for chronic liver diseases.
      Most patients with CLD are asymptomatic for many years and generally do not seek medical attention until they develop complications of cirrhosis. Early diagnosis of CLD would enable initiation of measures and treatments to prevent disease progression and improve survival. In their pioneer proof-of-concept studies, Poynard
      • Poynard T.
      • Lebray P.
      • Ingiliz P.
      • Varaut A.
      • Varsat B.
      • Ngo Y.
      • et al.
      Prevalence of liver fibrosis and risk factors in a general population using non-invasive biomarkers (FibroTest).
      and Roulot
      • Roulot D.
      • Costes J.L.
      • Buyck J.F.
      • Warzocha U.
      • Gambier N.
      • Czernichow S.
      • et al.
      Transient elastography as a screening tool for liver fibrosis and cirrhosis in a community-based population aged over 45 years.
      have shown that NITs (FibroTest or VCTE), were acceptable and accurate tools for detecting liver fibrosis and cirrhosis in presumably healthy individuals without known liver disease. Several population-based studies, performed in different areas of the world, using the same design, have since confirmed their findings
      • Caballeria L.
      • Pera G.
      • Arteaga I.
      • Rodriguez L.
      • Aluma A.
      • Morillas R.M.
      • et al.
      High prevalence of liver fibrosis among European adults with unknown liver disease: a population-based study.
      • Cheng P.N.
      • Chiu Y.C.
      • Chiu H.C.
      • Chien S.C.
      The application of liver stiffness measurement in residents without overt liver diseases through a community-based screening program.
      • Hagstrom H.
      • Talback M.
      • Andreasson A.
      • Walldius G.
      • Hammar N.
      Ability of noninvasive scoring systems to identify individuals in the population at risk for severe liver disease.
      • Koehler E.M.
      • Plompen E.P.
      • Schouten J.N.
      • Hansen B.E.
      • Darwish Murad S.
      • Taimr P.
      • et al.
      Presence of diabetes mellitus and steatosis is associated with liver stiffness in a general population: the Rotterdam study.
      • Wong V.W.
      • Chu W.C.
      • Wong G.L.
      • Chan R.S.
      • Chim A.M.
      • Ong A.
      • et al.
      Prevalence of non-alcoholic fatty liver disease and advanced fibrosis in Hong Kong Chinese: a population study using proton-magnetic resonance spectroscopy and transient elastography.
      (Table 3). These studies focused on populations aged >40 (mainly in their fifth decade), without known liver disease, either seen in primary care for a medical check-up or randomly selected from large existing cohorts. VCTE was the most used NIT, far ahead of serum biomarkers (FibroTest and FIB-4). Overall, their findings were consistent with around 6-7% of patients having undetected significant liver fibrosis (LSM ≥8 kPa), around 3% having advanced fibrosis (LSM ≥9.6 kPa), and less than 1% having cirrhosis (LSM ≥13 kPa). The main limitation of these studies is the small number of liver biopsies performed (<300)
      • Poynard T.
      • Lebray P.
      • Ingiliz P.
      • Varaut A.
      • Varsat B.
      • Ngo Y.
      • et al.
      Prevalence of liver fibrosis and risk factors in a general population using non-invasive biomarkers (FibroTest).
      • Roulot D.
      • Costes J.L.
      • Buyck J.F.
      • Warzocha U.
      • Gambier N.
      • Czernichow S.
      • et al.
      Transient elastography as a screening tool for liver fibrosis and cirrhosis in a community-based population aged over 45 years.
      • Caballeria L.
      • Pera G.
      • Arteaga I.
      • Rodriguez L.
      • Aluma A.
      • Morillas R.M.
      • et al.
      High prevalence of liver fibrosis among European adults with unknown liver disease: a population-based study.
      ,
      • Hagstrom H.
      • Talback M.
      • Andreasson A.
      • Walldius G.
      • Hammar N.
      Ability of noninvasive scoring systems to identify individuals in the population at risk for severe liver disease.
      (Table 3). When considering aetiologies of underlying liver disease, NAFLD was, as expected, the leading cause, followed by ALD. Age (>40), components of the metabolic syndrome (particularly obesity and T2DM), and harmful alcohol use were independent risk factors for liver fibrosis and cirrhosis. Interestingly, in studies in populations with known risk factors, rates of significant fibrosis (LSM ≥8 kPa) were much higher, ranging from 12.4% to 25.6%.
      • Harman D.J.
      • Ryder S.D.
      • James M.W.
      • Wilkes E.A.
      • Card T.R.
      • Aithal G.P.
      • et al.
      Obesity and type 2 diabetes are important risk factors underlying previously undiagnosed cirrhosis in general practice: a cross-sectional study using transient elastography.
      • Harris R.
      • Card T.R.
      • Delahooke T.
      • Aithal G.P.
      • Guha I.N.
      Obesity is the most common risk factor for chronic liver disease: results from a risk stratification pathway using transient elastography.
      • Llop E.
      • Iruzubieta P.
      • Perello C.
      • Fernandez Carrillo C.
      • Cabezas J.
      • Escudero M.D.
      • et al.
      High liver stiffness values by transient elastography related to metabolic syndrome and harmful alcohol use in a large Spanish cohort.
      • Petta S.
      • Di Marco V.
      • Pipitone R.M.
      • Grimaudo S.
      • Buscemi C.
      • Craxi A.
      • et al.
      Prevalence and severity of nonalcoholic fatty liver disease by transient elastography: genetic and metabolic risk factors in a general population.
      • Zhang X.
      • Heredia N.I.
      • Balakrishnan M.
      • Thrift A.P.
      Prevalence and factors associated with NAFLD detected by vibration controlled transient elastography among US adults: results from NHANES 2017-2018.
      Finally, in the largest study to date,
      • Llop E.
      • Iruzubieta P.
      • Perello C.
      • Fernandez Carrillo C.
      • Cabezas J.
      • Escudero M.D.
      • et al.
      High liver stiffness values by transient elastography related to metabolic syndrome and harmful alcohol use in a large Spanish cohort.
      the prevalence of significant fibrosis did not differ between patients with normal (85% of the cohort) and abnormal transaminase levels. These findings confirm that conventional liver function tests have poor sensitivity and specificity for detecting fibrosis
      • Udell J.A.
      • Wang C.S.
      • Tinmouth J.
      • FitzGerald J.M.
      • Ayas N.T.
      • Simel D.L.
      • et al.
      Does this patient with liver disease have cirrhosis?.
      and that NITs perform better. Altogether these results suggest that focusing on populations aged >40 with metabolic risk factors is the best strategy for detecting CLD with significant fibrosis and might be cost-effective.
      • Serra-Burriel M.
      • Graupera I.
      • Toran P.
      • Thiele M.
      • Roulot D.
      • Wai-Sun Wong V.
      • et al.
      Transient elastography for screening of liver fibrosis: cost-effectiveness analysis from six prospective cohorts in Europe and Asia.
      Such a strategy is currently being investigated by the LiverScreen consortium, which aims to screen 30,000 European adults for liver fibrosis using VCTE.
      • Gines P.
      • Graupera I.
      • Lammert F.
      • Angeli P.
      • Caballeria L.
      • Krag A.
      • et al.
      Screening for liver fibrosis in the general population: a call for action.
      Table 3Adult population-based studies evaluating screening for liver fibrosis and cirrhosis using NITs worldwide.
      CountrySettingDesignPatients, NAge, years (mean)NITCut-offsF≥2Cut-offsF≥3Cut-offsF4LB (n)Aetiology
      Poynard 2010
      • Poynard T.
      • Lebray P.
      • Ingiliz P.
      • Varaut A.
      • Varsat B.
      • Ngo Y.
      • et al.
      Prevalence of liver fibrosis and risk factors in a general population using non-invasive biomarkers (FibroTest).
      FranceHeath check-up in 2 primary care centresP7,463≥40 (-)FT≥0.482.8%---0.1%4NAFLD/ALD
      Roulot 2011
      • Roulot D.
      • Costes J.L.
      • Buyck J.F.
      • Warzocha U.
      • Gambier N.
      • Czernichow S.
      • et al.
      Transient elastography as a screening tool for liver fibrosis and cirrhosis in a community-based population aged over 45 years.
      FranceHeath check-up in 1 primary care centreP1,190>45 (58 ± 9)TE≥8 kPa7.5%--≥13 kPa0.76%27NAFLD/ALD
      Wong 2012
      • Wong V.W.
      • Chu W.C.
      • Wong G.L.
      • Chan R.S.
      • Chim A.M.
      • Ong A.
      • et al.
      Prevalence of non-alcoholic fatty liver disease and advanced fibrosis in Hong Kong Chinese: a population study using proton-magnetic resonance spectroscopy and transient elastography.
      Hong KongNationwide population-based, randomly selectedP75918-70 (48 ± 11)TE--≥9.6 kPa2.0%---NAFLD
      Koehler 2016
      • Koehler E.M.
      • Plompen E.P.
      • Schouten J.N.
      • Hansen B.E.
      • Darwish Murad S.
      • Taimr P.
      • et al.
      Presence of diabetes mellitus and steatosis is associated with liver stiffness in a general population: the Rotterdam study.
      NetherlandsRotterdam cohort, population-based, randomly selectedP3,041≥45 (66 ± 8)TE≥8 kPa5.6%--≥13 kPa0.6%-NAFLD
      Cheng 2016
      • Cheng P.N.
      • Chiu Y.C.
      • Chiu H.C.
      • Chien S.C.
      The application of liver stiffness measurement in residents without overt liver diseases through a community-based screening program.
      TaiwanPopulation-based study in 2 villagesP559- (56 ± 16)TE≥7 kPa7.2%-----NAFLD
      Caballeria 2018
      • Caballeria L.
      • Pera G.
      • Arteaga I.
      • Rodriguez L.
      • Aluma A.
      • Morillas R.M.
      • et al.
      High prevalence of liver fibrosis among European adults with unknown liver disease: a population-based study.
      SpainPopulation-based, randomly selectedP3,01418-75 (54 ± 12)TE≥8 kPa5.8%≥9.0 kPa3.6%--92NAFLD/ALD
      Hagstrom 2020
      • Hagstrom H.
      • Talback M.
      • Andreasson A.
      • Walldius G.
      • Hammar N.
      Ability of noninvasive scoring systems to identify individuals in the population at risk for severe liver disease.
      SwedenAMORIS cohort, population-based, sub-analysisR126,94135-79 (52)FIB-4--≥2.671.4%----
      Llop 2021
      • Llop E.
      • Iruzubieta P.
      • Perello C.
      • Fernandez Carrillo C.
      • Cabezas J.
      • Escudero M.D.
      • et al.
      High liver stiffness values by transient elastography related to metabolic syndrome and harmful alcohol use in a large Spanish cohort.
      SpainPREVEP-ETHON cohort, population-based, randomly selectedP11,44020-79 (50)TE≥8 kPa5.6%≥10.0 kPa2.9%≥15 kPa1.2%170NAFLD/ALD
      ALD, alcohol-related liver disease; FT, FibroTest; LB, liver biopsy; NAFLD, non-alcoholic fatty liver disease; NIT, non-invasive test; P, prospective ; R, retrospective; TE, transient elastography.

      Referral pathways for NAFLD in primary care and diabetology

      Once patients with suspected CLD and fibrosis have been identified, their linkage to care is critical. There is thus an urgent need for an integrated management plan between primary and secondary care, with robust pathways for fibrosis testing and subsequent referrals.
      • Tsochatzis E.A.
      • Newsome P.N.
      Non-alcoholic fatty liver disease and the interface between primary and secondary care.
      However, despite its high prevalence in primary care, NAFLD remains largely unknown outside Hepatology and Gastroenterology and is overlooked by most physicians.
      • Younossi Z.M.
      • Ong J.P.
      • Takahashi H.
      • Yilmaz Y.
      • Eguc Hi Y.
      • El Kassas M.
      • et al.
      A global survey of physicians knowledge about nonalcoholic fatty liver disease.
      As a result, less than 10% of patients with NAFLD are referred to a specialist and opportunities for early interventions are missed.
      • Blais P.
      • Husain N.
      • Kramer J.R.
      • Kowalkowski M.
      • El-Serag H.
      • Kanwal F.
      Nonalcoholic fatty liver disease is underrecognized in the primary care setting.
      The large amount of evidence regarding the diagnostic accuracy of NITs for detecting advanced fibrosis in patients with NAFLD contrasts with the paucity of data on their applied use in referral pathways.
      • Castera L.
      • Friedrich-Rust M.
      • Loomba R.
      Noninvasive assessment of liver disease in patients with nonalcoholic fatty liver disease.
      ,
      • Castera L.
      Non-invasive tests for liver fibrosis in NAFLD: creating pathways between primary healthcare and liver clinics.
      For instance, several algorithms, combining serum biomarkers (FIB-4 and NFS) and VCTE, either paired
      • Petta S.
      • Wong V.W.
      • Camma C.
      • Hiriart J.B.
      • Wong G.L.
      • Vergniol J.
      • et al.
      Serial combination of non-invasive tools improves the diagnostic accuracy of severe liver fibrosis in patients with NAFLD.
      or sequentially,
      • Anstee Q.M.
      • Lawitz E.J.
      • Alkhouri N.
      • Wong V.W.
      • Romero-Gomez M.
      • Okanoue T.
      • et al.
      Noninvasive tests accurately identify advanced fibrosis due to NASH: baseline data from the STELLAR trials.
      ,
      • Boursier J.
      • Guillaume M.
      • Leroy V.
      • Irles M.
      • Roux M.
      • Lannes A.
      • et al.
      New sequential combinations of non-invasive fibrosis tests provide an accurate diagnosis of advanced fibrosis in NAFLD.
      have been developed but only validated in tertiary care centres, where the prevalence of advanced fibrosis is much higher than in primary care (20 to 30% vs. 5%). Context of use is critical and a key challenge when using NITs in low prevalence settings is that their sensitivity and PPV will be lower.
      • Vali Y.
      • Lee J.
      • Boursier J.
      • Spijker R.
      • Loffler J.
      • Verheij J.
      • et al.
      Enhanced liver fibrosis test for the non-invasive diagnosis of fibrosis in patients with NAFLD: a systematic review and meta-analysis.
      ,
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      Thus, the best strategy is to start with a first-line widely available, simple and cheap blood test (such as FIB-4) with high NPV to rule-out advanced fibrosis, followed, if positive, by a second-line confirmatory test with high specificity (VCTE or patented serum markers)
      • Castera L.
      • Boursier J.
      Non-invasive algorithms for the case finding of “at-risk” patients with NAFLD.
      (Fig. 2). Such a strategy (FIB-4 followed by ELF in patients with an indeterminate FIB-4 between 1.3–3.25) has recently been evaluated in a large population of patients with suspected NAFLD (n = 1,452) seen in primary care in the UK.
      • Srivastava A.
      • Gailer R.
      • Tanwar S.
      • Trembling P.
      • Parkes J.
      • Rodger A.
      • et al.
      Prospective evaluation of a primary care referral pathway for patients with non-alcoholic fatty liver disease.
      As a result, 81% of patients were deemed at low risk of advanced fibrosis (FIB-4 <1.3 or ELF <9.5) and remained in primary care, while the remaining 19% were referred to liver clinics. The use of this algorithm not only resulted in reduced unnecessary referrals to liver specialists but also in a markedly increased (5-fold) rate of accurate referrals for advanced fibrosis. However, it should be noted that 87% of the cases triaged as being at low risk of advanced fibrosis in this pathway were excluded by use of the FIB-4 test alone. Another strategy, recommended by EASL guidelines
      EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis - 2021 update.
      (FIB-4 followed by VCTE in patients with FIB-4 >1.3), has been assessed in 440 patients with T2DM seen in primary care.
      • Mansour D.
      • Grapes A.
      • Herscovitz M.
      • Cassidy P.
      • Vernazza J.
      • Broad A.
      • et al.
      Embedding assessment of liver fibrosis into routine diabetic review in primary care.
      Twenty (4.5%) patients were found to have advanced liver disease following specialist review, compared to 3 patients previously identified through standard care (odds ratio [OR] 6.71; 2.0–22.7; p = 0.002). Such sequential approaches have been suggested to be cost-effective in healthcare systems in the UK and US,
      • Vilar-Gomez E.
      • Lou Z.
      • Kong N.
      • Vuppalanchi R.
      • Imperiale T.F.
      • Chalasani N.
      Cost effectiveness of different strategies for detecting cirrhosis in patients with nonalcoholic fatty liver disease based on United States health care system.
      • Crossan C.
      • Majumdar A.
      • Srivastava A.
      • Thorburn D.
      • Rosenberg W.
      • Pinzani M.
      • et al.
      Referral pathways for patients with NAFLD based on non-invasive fibrosis tests: diagnostic accuracy and cost analysis.
      • Srivastava A.
      • Jong S.
      • Gola A.
      • Gailer R.
      • Morgan S.
      • Sennett K.
      • et al.
      Cost-comparison analysis of FIB-4, ELF and fibroscan in community pathways for non-alcoholic fatty liver disease.
      but further studies in other countries are needed.
      Figure thumbnail gr2
      Fig. 2Proposed NAFLD patient pathway (adapted from ref
      • Castera L.
      • Boursier J.
      Non-invasive algorithms for the case finding of “at-risk” patients with NAFLD.
      ).
      Patients with risk factors for NAFLD (age >40 years, type 2 diabetes, obesity, metabolic syndrome) seen in primary care should undergo first-line testing by non-patented blood tests (FIB-4 or NAFLD fibrosis score), followed, if positive, by VCTE in liver clinics, or ELF test or MRE if VCTE is not available. ELF, enhanced liver fibrosis; MRE, magnetic resonance elastography; NAFLD, non-alcoholic fatty liver disease; VCTE, vibration-controlled transient elastography.
      As for patients with T2DM seen in diabetes clinics, the prevalence of advanced fibrosis may be much higher (17%) than in primary care and closer to that seen in liver clinics.
      • Younossi Z.M.
      • Golabi P.
      • de Avila L.
      • Paik J.M.
      • Srishord M.
      • Fukui N.
      • et al.
      The global epidemiology of NAFLD and NASH in patients with type 2 diabetes: a systematic review and meta-analysis.
      Evidence to support screening of patients with T2DM for advanced fibrosis is still limited and international guidelines are conflicting.
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
      American Diabetes A. 4
      Comprehensive medical evaluation and assessment of comorbidities: standards of medical care in diabetes-2021.
      • Chalasani N.
      • Younossi Z.
      • Lavine J.E.
      • Charlton M.
      • Cusi K.
      • Rinella M.
      • et al.
      The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases.
      Thus, no specific algorithm has been developed in diabetes clinics so far. Given the high prevalence of advanced fibrosis, the direct use of a more specific test such as VCTE seems more suited to this setting. Such a strategy has been assessed in populations from diabetes clinics, reporting a prevalence of advanced fibrosis (LSM ≥9.6 kPa) of around 15-20%,
      • Kwok R.
      • Choi K.C.
      • Wong G.L.
      • Zhang Y.
      • Chan H.L.
      • Luk A.O.
      • et al.
      Screening diabetic patients for non-alcoholic fatty liver disease with controlled attenuation parameter and liver stiffness measurements: a prospective cohort study.
      • Lai L.L.
      • Wan Yusoff W.N.I.
      • Vethakkan S.R.
      • Nik Mustapha N.R.
      • Mahadeva S.
      • Chan W.K.
      Screening for non-alcoholic fatty liver disease in patients with type 2 diabetes mellitus using transient elastography.
      • Lomonaco R.
      • Godinez Leiva E.
      • Bril F.
      • Shrestha S.
      • Mansour L.
      • Budd J.
      • et al.
      Advanced liver fibrosis is common in patients with type 2 diabetes followed in the outpatient setting: the need for systematic screening.
      • Mikolasevic I.
      • Domislovic V.
      • Turk Wensveen T.
      • Delija B.
      • Klapan M.
      • Juric T.
      • et al.
      Screening for nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus using transient elastography - a prospective, cross sectional study.
      • Roulot D.
      • Roudot-Thoraval F.
      • NKontchou G.
      • Kouacou N.
      • Costes J.L.
      • Elourimi G.
      • et al.
      Concomitant screening for liver fibrosis and steatosis in French type 2 diabetic patients using Fibroscan.
      but further data are needed.
      The next step is the use of NITs beyond the liver clinics, to establish pathways between primary care and liver clinics for patients “at-risk” of chronic liver disease.

      Future personalised medicine and non-invasive tests

      Over the last decade, the development of high-throughput analytic platforms, coupled with major advances in data science, have provided ever richer genomic, epigenetic, transcriptomic, proteomic, metabolomic and metagenomic datasets describing inter-individual variation and helping to elucidate the factors and mechanisms that contribute to disease evolution (Fig. 3). These new insights into the underlying heterogeneity of many CLD processes suggest that future management strategies may be tailored to an individual’s unique physiological and pathophysiological profile – offering the prospect of a knowledge-driven “precision medicine” paradigm.
      Figure thumbnail gr3
      Fig. 3A knowledge-driven paradigm for biomarker use in liver disease.
      “Omic” technologies provide new insights into the underlying heterogeneity of many chronic liver disease processes. These offer the prospect of a knowledge-driven “precision medicine” paradigm. First, greater understanding of an individual’s a priori genetic/epigenetic disease propensity may guide primary prevention strategies. Second, once disease is established, these insights, coupled with transcriptomic, proteomic and metabolomic profiling could inform prognosis and guide selection of tailored therapeutics to optimize treatment response.
      Genome-wide association studies (GWAS) have provided fundamental insights into genetic variations that modify disease severity or, in the case of IL-28 in hepatitis C, treatment response.
      • Ge D.
      • Fellay J.
      • Thompson A.J.
      • Simon J.S.
      • Shianna K.V.
      • Urban T.J.
      • et al.
      Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance.
      GWAS have also highlighted areas of common pathophysiology between NAFLD
      • Anstee Q.M.
      • Darlay R.
      • Cockell S.
      • Meroni M.
      • Govaere O.
      • Tiniakos D.
      • et al.
      Genome-wide association study of non-alcoholic fatty liver and steatohepatitis in a histologically characterised cohort.
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • Pertsemlidis A.
      • Cox D.
      • Pennacchio L.A.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • Xin Y.
      • Schurmann C.
      • Stevis P.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      and ALD.
      • Schwantes-An T.H.
      • Darlay R.
      • Mathurin P.
      • Masson S.
      • Liangpunsakul S.
      • Mueller S.
      • et al.
      Genome-wide association study and meta-analysis on alcohol-associated liver cirrhosis identifies genetic risk factors.
      ,
      • Buch S.
      • Stickel F.
      • Trepo E.
      • Way M.
      • Herrmann A.
      • Nischalke H.D.
      • et al.
      A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.
      Harmful genetic polymorphisms in genes including PNPLA3 (rs738409), TM6SF2 (rs58542926), GCKR (rs1260326), and MBOAT7 (rs641738), as well as a protective variant in HSD17B13 (rs72613567), have been widely replicated and linked to not only hepatic fat content but also severity of steatohepatitis, stage of fibrosis
      • Liu Y.L.
      • Reeves H.L.
      • Burt A.D.
      • Tiniakos D.
      • McPherson S.
      • Leathart J.B.
      • et al.
      TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.
      ,
      • Valenti L.
      • Al-Serri A.
      • Daly A.K.
      • Galmozzi E.
      • Rametta R.
      • Dongiovanni P.
      • et al.
      Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease.
      and risk of HCC for some variants.
      • Liu Y.L.
      • Patman G.L.
      • Leathart J.B.
      • Piguet A.C.
      • Burt A.D.
      • Dufour J.F.
      • et al.
      Carriage of the PNPLA3 rs738409 C>G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.
      ,
      • Donati B.
      • Dongiovanni P.
      • Romeo S.
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      Once disease is established, the ability to deeply characterise how normal physiological processes become perturbed during disease progression, using large scale transcriptomic, proteomic and metabolomic technologies, has provided new insights into CLD and revealed novel biomarkers. Whilst these putative biomarkers will require further independent validation, the strategies adopted serve as useful exemplars of the approach.
      Transcriptomic profiling of liver tissue across the full NAFLD spectrum suggests that gene expression profiles can identify disease subgroups distinct from those defined by standard histological indices.
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      Transcriptomic profiling across the nonalcoholic fatty liver disease spectrum reveals gene signatures for steatohepatitis and fibrosis.
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      Emerging role of the gut microbiome in nonalcoholic fatty liver disease: from composition to function.

      Conclusion

      Over the last two decades there have been tremendous advances in the non-invasive diagnosis and risk stratification of CLDs. Clinical prediction rules such as FIB-4 are now routinely utilised in early risk stratification for population-level health and typically followed by either a serum-based fibrosis test or an elastography-based test. Several advanced MRI-based biomarkers are now routinely utilised for the assessment of treatment response in early phase NASH trials. We are entering a new era in which precision medicine-based biomarkers will be able to more precisely diagnose those who are at an increased risk of morbidity and mortality due to liver disease. The next step is the application of NITs beyond the liver clinic.

      Abbreviations

      2D-SWE, 2D-shear wave elastography; AAR, AST-to-ALT ratio; ALD, alcohol-related liver disease; ALT, alanine aminotransferase; APRI, AST-to-platelet ratio index; AST, aspartate aminotransferase; cACLD, compensated advanced chronic liver disease; CLD, chronic liver disease; ELF, enhance liver fibrosis; FIB-4, fibrosis-4; GWAS, genome-wide association studies; HCC, hepatocellular carcinoma; HVPG, hepatic venous pressure gradient; LSM, liver stiffness measurement; MRE, magnetic resonance eleastography; MRI-PDFF, MRI-proton density fat fraction; NAFLD, non-alcoholic fatty fiver disease, NASH, non-alcoholic steatohepatitis; NFS, NAFLD fibrosis score; NITs, non-invasive tests; NPV, negative predictive value; PPV, positive predictive value; pSWE, point-shear wave elastography; SVR, sustained virological response; VCTE, vibration-controlled transient elastography.

      Financial support

      QMA is funded by the LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis) consortium funded by the Innovative Medicines Initiative (IMI2) Program of the European Union under Grant Agreement 777377, which receives funding from the EU Horizon 2020 programme and EFPIA; the EPoS (Elucidating Pathways of Steatohepatitis) consortium funded by the Horizon 2020 Framework Program of the European Union under Grant Agreement 634413; and the Newcastle NIHR Biomedical Research Centre. RL receives funding support from NCATS ( 5UL1TR001442 ), NIDDK ( U01DK061734 , U01DK130190 , R01DK106419 , R01DK121378 , R01DK124318 , P30DK120515 ), NHLBI ( P01HL147835 ), and NIAAA ( U01AA029019 ).

      Authors’ contributions

      All authors contributed equally to the production of this manuscript.

      Conflicts of interest

      Quentin Anstee is Coordinator of the EU IMI-2 LITMUS consortium, which is funded by the EU Horizon 2020 programme and EFPIA. This multi-stakeholder consortium includes industry partners. He reports research Grant Funding: Allergan/Tobira, AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Glympse Bio, Intercept, Novartis Pharma AG, Pfizer Ltd. Consultancy: 89Bio, Abbvie/Allergan, Akero, Altimentiv, Altimmune, AstraZeneca, Axcella, Blade, BMS, BNN Cardio, Boehringer-Ingelheim, Cirius, CymaBay, EcoR1, E3Bio, Eli Lilly & Company Ltd., Galmed, Genentech, Genfit SA, Gilead, Grunthal, HistoIndex, Indalo, Intercept Pharma Europe Ltd., Inventiva, IQVIA, Janssen, Johnson & Johnson, Madrigal, MedImmune, Medpace, Merck, Metacrine, NGMBio, North Sea Therapeutics, Novartis, Novo Nordisk A/S, PathAI, Pfizer Ltd., Poxel, ProSciento, Raptor Pharma, Roche, Servier, Shionogi, Terns, The Medicines Company, Viking Therapeutics. Speaker:
      Abbott Laboratories, Allergan/Tobira, BMS, Clinical Care Options, Falk, Fishawack, Genfit SA, Gilead, Integritas Communications, Kenes, Medscape. Royalties: Elsevier Ltd. Laurent Castera has received consultancy fees from Alexion, Echosens, Gilead, Intercept, MSD, Novo Nordisk, and Pfizer, and lecture fees from Abbvie, Echosens, Gilead, Intercept and Novo Nordisk. Rohit Loomba serves as a consultant to Aardvark Therapeutics, Altimmune, Anylam/Regeneron, Amgen, Ar Pharmaceuticals, AstraZeneca, Bristol-Myer Squibb, CohBar, Eli Lilly, Galmed, Gilead, Glympse bio, Hightide, Inipharma, Intercept, Inventiva, Ionis, Janssen Inc., Madrigal, Metacrine, Inc., NGM Biopharmaceuticals, Novartis, Novo Nordisk, Merck, Pfizer, Sagimet, Theratechnologies, 89 bio, Terns Pharmaceuticals and Viking Therapeutics. In addition his institutions received research grants from Ar Pharmaceuticals, Astrazeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, Eli Lilly, Galectin Therapeutics, Galmed Pharmaceuticals, Gilead, Intercept, Hanmi, Intercept, Inventiva, Ionis, Janssen, Madrigal Pharmaceuticals, Merck, NGM Biopharmaceuticals, Novo Nordisk, Merck, Pfizer, Sonic Incytes and Terns Pharmaceuticals. Co-founder of LipoNexus Inc.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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