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Non-invasive diagnosis and biomarkers in alcohol-related liver disease

  • Christophe Moreno
    Correspondence
    Corresponding author. Address: Department of Gastroenterology, Hepatopancreatology, and Digestive Oncology, CUB Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium.
    Affiliations
    Department of Gastroenterology, Hepatopancreatology, and Digestive Oncology, CUB Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium
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  • Sebastian Mueller
    Affiliations
    Salem Medical Center and Center for Alcohol Research, University of Heidelberg, Zeppelinstraße 11–33, 69121 Heidelberg, Germany
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  • Gyongyi Szabo
    Affiliations
    Department of Medicine, LRB-208, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA
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      Summary

      Even though alcohol-related liver disease (ALD) is a major cause of severe liver disease worldwide, most patients with ALD are diagnosed at the decompensation stage. Liver biopsy is still considered the gold standard for establishing a definite diagnosis and assessing the fibrosis stage of ALD, but it is an invasive procedure, associated with significant morbidity. During the last decade, non-invasive tests have been developed to estimate the severity of liver fibrosis and steatosis. Measurement of liver stiffness by transient elastography has become the most commonly used non-invasive parameter to evaluate fibrosis. In ALD, transient elastography has been demonstrated to have an excellent performance to detect advanced fibrosis and cirrhosis. However, aspartate aminotransferase levels must be considered when interpreting liver stiffness cut-offs. Non-invasive biological tests have also been evaluated to assess liver fibrosis in ALD. The commercially available Enhanced Liver Fibrosis test and FibroTest have comparable performance for the diagnosis of advanced fibrosis in ALD, with studies suggesting that they are better than other biological tests (i.e. FIB-4 and APRI). Although ultrasound is still accepted as an initial screen for fatty liver diagnosis, new methods have recently been developed to detect steatosis. Magnetic resonance spectroscopy and magnetic resonance imaging techniques are highly accurate and reproducible, with superior sensitivities and specificities for detecting histological steatosis than ultrasound. However, low availability and high cost limit the use of magnetic resonance techniques in routine clinical practice. More recently, controlled attenuation parameter was developed as a novel tool to non-invasively assess liver steatosis; performed in combination with transient elastography, it was suggested to be superior to regular ultrasound for detecting steatosis and was shown to have acceptable diagnostic accuracy. New serum biomarkers are under investigation to non-invasively diagnose more severe forms of ALD and to predict prognosis of patients.

      Keywords

      Introduction

      Alcohol-related liver disease (ALD) is the most frequent cause of severe liver disease in Europe. Based on the World Health Organization database, more than 40% of the liver deaths are attributable to alcohol.
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      Management of alcohol-related liver disease.
      The number of liver transplants performed for patients with ALD-related cirrhosis has increased over the past 2 decades, both in Europe and in the US.
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      Despite the high burden of ALD, it is regrettable that the majority of patients with ALD are diagnosed at the decompensation stage. Moreover, a large proportion of patients with newly diagnosed cirrhosis had recent consultations in primary care or emergency units,
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      without any intervention. Since the risk of developing advanced liver disease decreases with abstinence or reduced alcohol consumption, screening for ALD and interventions in at-risk patients should be routinely implemented.
      ALD includes a wide spectrum of lesions ranging from steatosis to steatohepatitis, progressive liver fibrosis, cirrhosis and its complications.
      • EASL Clinical Practice Guidelines
      Management of alcohol-related liver disease.
      Although steatosis is present in almost all heavy drinkers, it is estimated that only 10–20% will eventually develop cirrhosis.
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      it is of clinical importance to diagnose patients with advanced fibrosis before decompensation occurs, in order to promote abstinence and improve survival.
      Liver biopsy is still considered the gold standard for establishing a definite diagnosis of ALD, assessing the stage of fibrosis and excluding alternative causes of liver injury. However, liver biopsy is an invasive procedure, with significant morbidity,
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      and is generally not recommended in routine clinical practice for all patients with suspected ALD.
      Initially developed in viral hepatitis, non-invasive tests are increasingly used to estimate the severity of liver fibrosis in almost all aetiologies of liver disease. Serologic tests, radiographic modalities and liver stiffness (LS) have excellent predictive value for diagnosis of advanced fibrosis, particularly in chronic hepatitis C and non-alcoholic fatty liver disease (NAFLD).
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      Noninvasive evaluation of NAFLD.
      In addition to fibrosis evaluation, recent advances have been made for the diagnosis and staging of steatosis. The aim of the present manuscript is to review the non-invasive methods available for the diagnosis and evaluation of liver fibrosis and steatosis in patients with ALD, while proposing a practical algorithm for the clinician and discussing new biomarkers in development.

      Diagnosis and evaluation of liver steatosis in ALD

      Liver steatosis is characterised by the excessive accumulation of fat-containing vacuoles within the cytoplasm of hepatocytes. Up to 90% of patients with heavy alcohol intake have steatosis, which is usually asymptomatic and rapidly reversible with abstinence.
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      However, 5–10% of patients with simple alcoholic steatosis have been shown to progress to cirrhosis within 5 years,
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      and steatosis has been identified as an independent predictive factor of fibrosis progression in heavy drinkers.
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      Fibrosis progression occurs in a subgroup of heavy drinkers with typical histological features.
      Consequently, reliable non-invasive methods to diagnose and monitor steatosis in patients with ALD are desirable.

      Liver ultrasound

      Ultrasound (US) is accepted as an initial screen for fatty liver because it is non-invasive, inexpensive and widely available. In US images, steatosis appears as a diffuse hyper echogenicity due to increased parenchymal reflectivity that results from intracellular accumulation of fat inclusions.
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      • Schick F.
      Non-invasive assessment and quantification of liver steatosis by ultrasound, computed tomography and magnetic resonance.
      US has a sensitivity of 60–94% and a specificity of 88–95% in detecting steatosis.
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      Noninvasive assessment of liver steatosis using ultrasound methods.
      US sensitivity significantly varies according to degrees of fatty load, with 80% sensitivity at fat accumulation above 30%, as opposed to 55% when the fat content reaches only 10–20%. The US evaluation of steatosis is mainly qualitative, with a grading conveniently classified as mild, moderate or severe.
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      • Schick F.
      Non-invasive assessment and quantification of liver steatosis by ultrasound, computed tomography and magnetic resonance.
      One major weakness of US is its operator dependency.
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      Interobserver and intraobserver variability in the sonographic assessment of fatty liver.
      Another drawback of the technique includes its inaccuracy in differentiating fibrosis from steatosis.
      Ultrasound is accepted as a first screen for steatosis, because it is non-invasive, inexpensive and widely available.

      Magnetic resonance imaging

      Magnetic resonance spectroscopy (MRS) allows for non-invasive studies into the molecular composition of tissues in vivo. MRS quantifies the proton density fat fraction (PDFF), a standardized measure of liver tissue.
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      MRI and MRE for non-invasive quantitative assessment of hepatic steatosis and fibrosis in NAFLD and NASH: Clinical trials to clinical practice.
      MRS is highly accurate and reproducible for measuring hepatic fat. However, MRS has limited availability and is not available on routine scanners. Magnetic resonance imaging (MRI)-based methods have been developed that use MRI-PDFF and routinely available clinical MRI scanners to quantify liver fat without needing spectroscopy. MRI-PDFF is not affected by scanner field strength, patient factors, aetiology of liver disease, and concomitant liver abnormalities such as iron overload or liver inflammation.
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      Correlation between liver histology and novel magnetic resonance imaging in adult patients with non-alcoholic fatty liver disease - MRI accurately quantifies hepatic steatosis in NAFLD.
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      • Hamilton G.
      • Le T.A.
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      Utility of magnetic resonance imaging versus histology for quantifying changes in liver fat in nonalcoholic fatty liver disease trials.
      MRI sensitivities and specificities in detecting histologic steatosis ≥5% were 76.7–90.0% and 80.2–87.0%, respectively.
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      • Park S.H.
      Radiologic evaluation of nonalcoholic fatty liver disease.
      MRI has several advantages: In addition to its high accuracy and reproducibility for measuring hepatic fat, MRI allows follow-up of response after intervention.
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      • Lam J.
      • Peterson M.R.
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      • Hamilton G.
      • Le T.A.
      • et al.
      Utility of magnetic resonance imaging versus histology for quantifying changes in liver fat in nonalcoholic fatty liver disease trials.
      The disadvantages of MRI include its high cost and long examination time.

      Controlled attenuation parameter

      Controlled attenuation parameter (CAP) is a novel tool to non-invasively assess liver steatosis, which measures ultrasound attenuation when travelling through fatty liver tissue, compared to normal liver.
      • Sasso M.
      • Beaugrand M.
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      • Marcellin P.
      • Poupon R.
      • et al.
      Controlled attenuation parameter (CAP): a novel VCTE guided ultrasonic attenuation measurement for the evaluation of hepatic steatosis: preliminary study and validation in a cohort of patients with chronic liver disease from various causes.
      The CAP software is incorporated into the Fibroscan® (Echosens, Paris, France) equipment, which enables CAP to be combined with non-invasive liver fibrosis assessment using transient elastography. In an individual data meta-analysis, CAP technology was shown to diagnose moderate and severe steatosis with diagnostic accuracies of between 0.85 and 0.90 in 2,735 patients with mixed liver disease aetiologies (mainly viral hepatitis and NAFLD).
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      • et al.
      Individual patient data meta-analysis of controlled attenuation parameter (CAP) technology for assessing steatosis.
      In a recent European multicentre prospective study including 562 patients with ALD who underwent CAP, regular US and liver biopsy,
      • Thiele M.
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      • Kjaergaard M.
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      • Mueller J.
      • et al.
      Controlled attenuation parameter and alcoholic hepatic steatosis: Diagnostic accuracy and role of alcohol detoxification.
      CAP diagnosed mild, moderate and severe steatosis with an area under the receiver operating characteristic curve (AUROC) of 0.77, 0.78 and 0.82, respectively. A CAP value above 290 dB/m ruled in any steatosis with 88% specificity. Moreover, CAP was shown to be superior to regular US for diagnosing steatosis in patients with ALD. The procedure is non-invasive, non-ionizing, easy to perform and provides immediate results. In addition, CAP can be performed simultaneously with LS measurement, making the simultaneous evaluation of both fibrosis and steatosis possible.
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      • et al.
      Controlled attenuation parameter (CAP) for the diagnosis of steatosis: a prospective study of 5323 examinations.
      For these reasons, CAP technology is an interesting tool for diagnosing steatosis. However, diagnostic accuracy appears to be poorer at low steatosis stages and seems lower in ALD compared to other liver disease aetiologies. Moreover, optimal cut-offs to rule in, rule out and stage steatosis vary in the different studies performed.
      Controlled attenuation parameter is a simple non-invasive technique for the assessment of steatosis, which has been shown to be superior to ultrasound in ALD, although it requires further validation.
      In summary, liver ultrasound remains the first-line screening modality in clinical practice, with acceptable sensitivity and specificity for detection of steatosis. MRI- and MRS-PDFF appear clearly to be the most accurate and reproducible methods for the diagnosis and follow-up of steatosis. However, cost and examination time limit its use in routine clinical practice. CAP is a simple and promising new bedside technique for diagnosing steatosis in patients with ALD, but it requires further validation.

      Evaluation of liver fibrosis in ALD

      Biological tests

      Enhanced Liver Fibrosis (ELF™) and FibroTest (FT) are the most commonly used blood-based assessment tools for liver fibrosis. The ELF test is commercially available and it combines 3 direct serum markers of extracellular matrix remodelling and fibrogenesis including, hyaluronic acid, tissue inhibitor of metalloproteinase-1 and the N-terminal propeptide for collagen type III.
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      • Schuppan D.
      • et al.
      Serum markers detect the presence of liver fibrosis: a cohort study.
      The FT score uses an algorithm calculated from 6 serum markers, age and gender of the patient. Numerous studies have evaluated the performance of these tests in liver fibrosis. In a meta-analysis, ELF scores showed good performance for prediction of histological stage of fibrosis.
      • Xie Q.
      • Zhou X.
      • Huang P.
      • Wei J.
      • Wang W.
      • Zheng S.
      The performance of enhanced liver fibrosis (ELF) test for the staging of liver fibrosis: a meta-analysis.
      In a Spanish study, ELF was also found to be cost effective for liver fibrosis assessment in patients with ALD.
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      • Mira A.
      • Jimenez W.
      • Navasa M.
      Cost-effectiveness of enhanced liver fibrosis test to assess liver fibrosis in chronic hepatitis C virus and alcoholic liver disease patients.
      A study by Thiele et al. found that ELF and FT had comparable diagnostic accuracy in patients with ALD with an AUROC of 0.92 for ELF and an AUROC of 0.9 for FT.
      • Thiele M.
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      • Hansen J.F.
      • Detlefsen S.
      • Antonsen S.
      • Krag A.
      Accuracy of the enhanced liver fibrosis test vs fibrotest, elastography, and indirect markers in detection of advanced fibrosis in patients with alcoholic liver disease.
      This prospective study concluded that advanced fibrosis can be ruled out in patients with ALD based on an ELF <10.5 or an FT value below 0.58.
      • Thiele M.
      • Madsen B.S.
      • Hansen J.F.
      • Detlefsen S.
      • Antonsen S.
      • Krag A.
      Accuracy of the enhanced liver fibrosis test vs fibrotest, elastography, and indirect markers in detection of advanced fibrosis in patients with alcoholic liver disease.
      Comparison of the performance of the different biological tests suggests that ELF and FT are better than APRI (aspartate aminotransferase [AST] to platelet ratio index) or FIB-4 (Table 1).
      • Xie Q.
      • Zhou X.
      • Huang P.
      • Wei J.
      • Wang W.
      • Zheng S.
      The performance of enhanced liver fibrosis (ELF) test for the staging of liver fibrosis: a meta-analysis.
      • Jin W.
      • Lin Z.
      • Xin Y.
      • Jiang X.
      • Dong Q.
      • Xuan S.
      Diagnostic accuracy of the aspartate aminotransferase-to-platelet ratio index for the prediction of hepatitis B-related fibrosis: a leading meta-analysis.
      • Li Q.
      • Ren X.
      • Lu C.
      • Li W.
      • Huang Y.
      • Chen L.
      Evaluation of APRI and FIB-4 for noninvasive assessment of significant fibrosis and cirrhosis in HBeAg-negative CHB patients with ALT </= 2 ULN: a retrospective cohort study.
      • Salkic N.N.
      • Jovanovic P.
      • Hauser G.
      • Brcic M.
      FibroTest/Fibrosure for significant liver fibrosis and cirrhosis in chronic hepatitis B: a meta-analysis.
      A combination of serum-based fibrosis tests with non-invasive imaging or new biomarkers will likely emerge in future clinical studies to aid patient care.
      Table 1Comparison of the performance of biological tests for fibrosis.
      Biological testsSensitivitySpecificityRef
      Fibrosis
       APRI* threshold 0.549%84%
      • Jin W.
      • Lin Z.
      • Xin Y.
      • Jiang X.
      • Dong Q.
      • Xuan S.
      Diagnostic accuracy of the aspartate aminotransferase-to-platelet ratio index for the prediction of hepatitis B-related fibrosis: a leading meta-analysis.
       FIB-4* cut-off >3.2516%99%
      • Li Q.
      • Ren X.
      • Lu C.
      • Li W.
      • Huang Y.
      • Chen L.
      Evaluation of APRI and FIB-4 for noninvasive assessment of significant fibrosis and cirrhosis in HBeAg-negative CHB patients with ALT </= 2 ULN: a retrospective cohort study.
       FIB-4 cut-off <1.4542%83%
      • Li Q.
      • Ren X.
      • Lu C.
      • Li W.
      • Huang Y.
      • Chen L.
      Evaluation of APRI and FIB-4 for noninvasive assessment of significant fibrosis and cirrhosis in HBeAg-negative CHB patients with ALT </= 2 ULN: a retrospective cohort study.
       APRI >1.508%98%
      • Li Q.
      • Ren X.
      • Lu C.
      • Li W.
      • Huang Y.
      • Chen L.
      Evaluation of APRI and FIB-4 for noninvasive assessment of significant fibrosis and cirrhosis in HBeAg-negative CHB patients with ALT </= 2 ULN: a retrospective cohort study.
       APRI <0.548%72%
      • Li Q.
      • Ren X.
      • Lu C.
      • Li W.
      • Huang Y.
      • Chen L.
      Evaluation of APRI and FIB-4 for noninvasive assessment of significant fibrosis and cirrhosis in HBeAg-negative CHB patients with ALT </= 2 ULN: a retrospective cohort study.
       ELF* moderate83%73%
      • Xie Q.
      • Zhou X.
      • Huang P.
      • Wei J.
      • Wang W.
      • Zheng S.
      The performance of enhanced liver fibrosis (ELF) test for the staging of liver fibrosis: a meta-analysis.
       ELF severe78%76%
      • Xie Q.
      • Zhou X.
      • Huang P.
      • Wei J.
      • Wang W.
      • Zheng S.
      The performance of enhanced liver fibrosis (ELF) test for the staging of liver fibrosis: a meta-analysis.
       FT* (fibrotest)61%80%
      • Salkic N.N.
      • Jovanovic P.
      • Hauser G.
      • Brcic M.
      FibroTest/Fibrosure for significant liver fibrosis and cirrhosis in chronic hepatitis B: a meta-analysis.
      Cirrhosis
       APRI threshold 1.0–1.554%78%
      • Jin W.
      • Lin Z.
      • Xin Y.
      • Jiang X.
      • Dong Q.
      • Xuan S.
      Diagnostic accuracy of the aspartate aminotransferase-to-platelet ratio index for the prediction of hepatitis B-related fibrosis: a leading meta-analysis.
       APRI >2.00%1%
      • Li Q.
      • Ren X.
      • Lu C.
      • Li W.
      • Huang Y.
      • Chen L.
      Evaluation of APRI and FIB-4 for noninvasive assessment of significant fibrosis and cirrhosis in HBeAg-negative CHB patients with ALT </= 2 ULN: a retrospective cohort study.
       APRI <1.035%94%
      • Li Q.
      • Ren X.
      • Lu C.
      • Li W.
      • Huang Y.
      • Chen L.
      Evaluation of APRI and FIB-4 for noninvasive assessment of significant fibrosis and cirrhosis in HBeAg-negative CHB patients with ALT </= 2 ULN: a retrospective cohort study.
       ELF80%71%
      • Xie Q.
      • Zhou X.
      • Huang P.
      • Wei J.
      • Wang W.
      • Zheng S.
      The performance of enhanced liver fibrosis (ELF) test for the staging of liver fibrosis: a meta-analysis.
       FT62%91%
      • Salkic N.N.
      • Jovanovic P.
      • Hauser G.
      • Brcic M.
      FibroTest/Fibrosure for significant liver fibrosis and cirrhosis in chronic hepatitis B: a meta-analysis.
      *APRI = (AST/ULN of AST)/(platelet count × 100). FIB-4 = (age × AST)/(platelet count × (ALT)1/2). Note: ULN of AST: 40 IU/L. ELF = Enhanced Liver Fibrosis score is calculated based on of tissue inhibitor of metalloproteinases 1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP) and hyaluronic acid (HA) levels.
      FT = FibroTest consists of an algorithm of 5 fibrosis markers (alfa2-macroglobulin [g/L], apolipoproteinA1 [g/L], total bilirubin [µmol/L], haptoglobin [g/L], Gamma glutamyltransferase [IU/L]. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ULN, upper limit of normal.

      Transient elastography

      Diagnosis of fibrosis by transient elastography: general findings

      Since the introduction of transient elastography (TE, Fibroscan®) in 2003,
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      LS measurement has become the best non-invasive parameter to screen for liver cirrhosis.
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      Non-invasive diagnosis of alcoholic liver disease.
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      Noninvasive screening for liver fibrosis and portal hypertension by transient elastography–a large single center experience.
      This success has largely been driven by the fact that a) no extensive training is required, b) TE is non-invasive and rapid and, c) TE is reproducible with a lower sampling error than liver biopsy.
      • Mueller S.
      • Sandrin L.
      Liver stiffness: a novel parameter for the diagnosis of liver disease.
      Due to these promising findings other competing technologies are now available on the market such as acoustic radiation force impulse imaging (ARFI),
      • Palmeri M.L.
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      Noninvasive evaluation of hepatic fibrosis using acoustic radiation force-based shear stiffness in patients with nonalcoholic fatty liver disease.
      shear wave elastography (SWE)
      • Poynard T.
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      Liver fibrosis evaluation using real-time shear wave elastography: applicability and diagnostic performance using methods without a gold standard.
      and magnetic resonance elastography (MRE).
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      • Patterson A.J.
      • Beddy P.
      • Bowden D.
      • et al.
      Magnetic resonance elastography: feasibility of liver stiffness measurements in healthy volunteers at 3T.
      In particular, the latter holds great promise for three-dimensional assessment of stiffness in various organs not restricted to the liver. So far, most published LS studies have been performed with TE.
      In general, LS is an excellent surrogate marker of advanced fibrosis (F3) and cirrhosis (F4) in ALD and superior to all serum markers.
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      Assessment of asymptomatic liver fibrosis in alcoholic patients using fibroscan: prospective comparison with seven non-invasive laboratory tests.
      LS strongly correlates with histological fibrosis stage, independently of the underlying liver disease (r >0.8).
      • Mueller S.
      • Sandrin L.
      Liver stiffness: a novel parameter for the diagnosis of liver disease.
      During fibrosis progression, LS increases continuously from ca. 2 kPa up to 75 kPa (upper detection limit of the Fibroscan device). A threshold of 12.5 kPa is widely considered a cut-off value of histological cirrhosis, F4 stage, although cut-off values are aetiology dependent.
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      Performance of transient elastography for the staging of liver fibrosis: a meta-analysis.
      A normal LS measurement (<6 kPa) is thought to exclude liver pathology and liver fibrosis.
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      • Friedrich S.
      • Reimann F.M.
      • Pritsch M.
      • et al.
      Increased liver stiffness in alcoholic liver disease: differentiating fibrosis from steatohepatitis.
      Finally, LS values strongly correlate with portal pressure and complications such as oesophageal varices and hepatocellular carcinoma and are likely at LS values >20 kPa.
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      Liver stiffness: a novel parameter for the diagnosis of liver disease.
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      • Castera L.
      Biopsy and non-invasive methods for the diagnosis of liver fibrosis: does it take two to tango?.
      In addition, LS has been shown to be an excellent short- and long-term predictor of survival in various settings ranging from the emergency room up to treatment response in viral hepatitis
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      • Chermak F.
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      Non-invasive tests for fibrosis and liver stiffness predict 5-year survival of patients chronically infected with hepatitis B virus.
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      Increase in liver stiffness after transjugular intrahepatic portosystemic shunt is associated with inflammation and predicts mortality.
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      • Christensen P.B.
      Liver stiffness and 30-day mortality in a cohort of patients admitted to hospital.
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      Liver stiffness measurements and short-term survival after left ventricular assist device implantation: a pilot study.
      although data on LS as a long-term survival predictor in ALD are still not available. Interestingly, a decrease of LS either during a pharmacological intervention or transjugular intrahepatic portosystemic shunt seems to predict a favourable outcome.
      • Jansen C.
      • Moller P.
      • Meyer C.
      • Kolbe C.C.
      • Bogs C.
      • Pohlmann A.
      • et al.
      Increase in liver stiffness after transjugular intrahepatic portosystemic shunt is associated with inflammation and predicts mortality.
      • Piecha F.
      • Mandorfer M.
      • Peccerella T.
      • Ozga A.K.
      • Poth T.
      • Vonbank A.
      • et al.
      Pharmacological decrease of liver stiffness is pressure-related and predicts long term clinical outcome.
      Based on the relation of LS with portal hypertension, LS has also been evaluated as a prognostic tool to predict adverse events and clinical outcome including survival. Thus, patients with higher baseline LS values have worse clinical outcomes including decreased 5-year survival
      • Vergniol J.
      • Foucher J.
      • Terrebonne E.
      • Bernard P.H.
      • le Bail B.
      • Merrouche W.
      • et al.
      Noninvasive tests for fibrosis and liver stiffness predict 5-year outcomes of patients with chronic hepatitis C.
      and increased risk of developing hepatocellular carcinoma.
      • Jung K.S.
      • Kim S.U.
      • Ahn S.H.
      • Park Y.N.
      • Kim D.Y.
      • Park J.Y.
      • et al.
      Risk assessment of hepatitis B virus-related hepatocellular carcinoma development using liver stiffness measurement (FibroScan).
      • Masuzaki R.
      • Tateishi R.
      • Yoshida H.
      • Goto E.
      • Sato T.
      • Ohki T.
      • et al.
      Prospective risk assessment for hepatocellular carcinoma development in patients with chronic hepatitis C by transient elastography.
      Longitudinal studies in patients undergoing treatment for chronic hepatitis B and C infection showed that patients were less likely to experience liver-related events if LS had decreased over time.
      • Kim B.K.
      • Fung J.
      • Yuen M.F.
      • Kim S.U.
      Clinical application of liver stiffness measurement using transient elastography in chronic liver disease from longitudinal perspectives.
      • Vergniol J.
      • Boursier J.
      • Coutzac C.
      • Bertrais S.
      • Foucher J.
      • Angel C.
      • et al.
      Evolution of noninvasive tests of liver fibrosis is associated with prognosis in patients with chronic hepatitis C.
      In fact, patients whose LS increased by more than 1 kPa/year presented a worse overall survival compared to patients whose LS remained unchanged or even decreased after hepatitis C treatment.
      • Vergniol J.
      • Boursier J.
      • Coutzac C.
      • Bertrais S.
      • Foucher J.
      • Angel C.
      • et al.
      Evolution of noninvasive tests of liver fibrosis is associated with prognosis in patients with chronic hepatitis C.
      Liver stiffness measurement, as measured by transient elastography, is an excellent surrogate marker of fibrosis and can act as a prognostic marker of clinical outcomes, including survival.

      Confounders of LS other than cirrhosis

      LS is affected by many (patho)physiological conditions that should be considered when assessing fibrosis stage (see Fig. 1).
      • Mueller S.
      • Sandrin L.
      Liver stiffness: a novel parameter for the diagnosis of liver disease.
      Major confounders of LS elevation encompass hepatic necroinflammation,
      • Sagir A.
      • Erhardt A.
      • Schmitt M.
      • Haussinger D.
      Transient elastography is unreliable for detection of cirrhosis in patients with acute liver damage.
      • Dechene A.
      • Sowa J.P.
      • Gieseler R.K.
      • Jochum C.
      • Bechmann L.P.
      • El Fouly A.
      • et al.
      Acute liver failure is associated with elevated liver stiffness and hepatic stellate cell activation.
      congestion,
      • Millonig G.
      • Friedrich S.
      • Adolf S.
      • Fonouni H.
      • Golriz M.
      • Mehrabi A.
      • et al.
      Liver stiffness is directly influenced by central venous pressure.
      mechanic cholestasis,
      • Millonig G.
      • Reimann F.M.
      • Friedrich S.
      • Fonouni H.
      • Mehrabi A.
      • Buchler M.W.
      • et al.
      Extrahepatic cholestasis increases liver stiffness (FibroScan) irrespective of fibrosis.
      alcohol
      • Millonig G.
      • Mueller S.
      • Sarovska L.
      • Friedrich S.
      • Reimann F.M.
      • Pritsch M.
      • et al.
      Increased liver stiffness in alcoholic liver disease: differentiating fibrosis from steatohepatitis.
      and food intake.
      • Mederacke I.
      • Wursthorn K.
      • Kirschner J.
      • Rifai K.
      • Manns M.P.
      • Wedemeyer H.
      • et al.
      Food intake increases liver stiffness in patients with chronic or resolved hepatitis C virus infection.
      In addition, arterial
      • Piecha F.
      • Peccerella T.
      • Bruckner T.
      • Seitz H.-K.
      • Rausch V.
      • Mueller S.
      Arterial pressure suffices to increase liver stiffness.
      and portal pressure are independent factors of LS elevation.
      • Piecha F.
      • Paech D.
      • Sollors J.
      • Seitz H.K.
      • Rossle M.
      • Rausch V.
      • et al.
      Rapid change of liver stiffness after variceal ligation and TIPS implantation.
      Figure thumbnail gr1
      Fig. 1Factors influencing liver stiffness measurement.
      Based on these observations, an inflow/outflow model (see Fig. 1) has been developed recently that includes all known conditions that affect LS at the molecular, cellular and vascular level.
      • Mueller S.
      Does pressure cause liver cirrhosis? The sinusoidal pressure hypothesis.
      Accordingly, the hepatic outflow (bile flow, hepatic veins) and inflow (hepatic artery and portal vein) are important determinants of the sinusoidal pressure ultimately underlying LS. Pharmacological modulation in rodent models of cirrhosis demonstrated a sophisticated effect of the inflow/outflow components on LS depending not only on central venous, arterial and portal pressure but also on heart rate.
      • Piecha F.
      • Mandorfer M.
      • Peccerella T.
      • Ozga A.K.
      • Poth T.
      • Vonbank A.
      • et al.
      Pharmacological decrease of liver stiffness is pressure-related and predicts long term clinical outcome.
      While cardiac circulation is tightly connected to these components by dynamic pressure, static pressure also strongly affects LS through water retention resulting from hormonal, osmotic or albumin-related conditions.
      • Mueller S.
      Does pressure cause liver cirrhosis? The sinusoidal pressure hypothesis.
      Finally, although not all subcellular factors associated with LS have been clarified yet, it is well established that histological features such as fibrosis, ballooning and inflammation are associated with LS elevation.
      • Rausch V.
      • Peccerella T.
      • Lackner C.
      • Yagmur E.
      • Seitz H.K.
      • Longerich T.
      • et al.
      Primary liver injury and delayed resolution of liver stiffness after alcohol detoxification in heavy drinkers with the PNPLA3 variant I148M.
      • Mueller S.
      • Nahon P.
      • Rausch V.
      • Peccerella T.
      • Silva I.
      • Yagmur E.
      • et al.
      Caspase-cleaved keratin-18 fragments increase during alcohol withdrawal and predict liver-related death in patients with alcoholic liver disease.
      The role of fat, which has been shown to lower LS in the absence or presence of inflammation, remains controversial.
      • Rausch V.
      • Peccerella T.
      • Lackner C.
      • Yagmur E.
      • Seitz H.K.
      • Longerich T.
      • et al.
      Primary liver injury and delayed resolution of liver stiffness after alcohol detoxification in heavy drinkers with the PNPLA3 variant I148M.

      Elastographic assessment of fibrosis in ALD

      In contrast to other common liver diseases such as viral hepatitis, the performance of LS in ALD was assessed rather late. An actual meta-analysis
      • 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.
      has carefully analysed biopsy-proven studies on LS in patients with ALD based on various quality measures (Table 2). Early direct comparison with serum fibrosis markers showed a better performance of TE in patients with ALD
      • Nguyen-Khac E.
      • Chatelain D.
      • Tramier B.
      • Decrombecque C.
      • Robert B.
      • Joly J.P.
      • et al.
      Assessment of asymptomatic liver fibrosis in alcoholic patients using fibroscan: prospective comparison with seven non-invasive laboratory tests.
      and AUROCs are typically >0.9 to detect F4 cirrhosis. Although an excellent performance was shown in all studies, the cut-off values used differed drastically, ranging from 11.5–25.8 kPa. Mueller et al. could then demonstrate that this was primarily related to the presence of inflammation as assessed by aminotransferase levels.
      • Mueller S.
      • Sandrin L.
      Liver stiffness: a novel parameter for the diagnosis of liver disease.
      In this study, it was shown that LS decreases in patients with ALD during alcohol withdrawal.
      • Mueller S.
      • Sandrin L.
      Liver stiffness: a novel parameter for the diagnosis of liver disease.
      The decrease of LS was best estimated based on AST levels. When only considering patients with low or normal aminotransferase levels, cut-off values were comparable to those observed in patients with viral hepatitis.
      • Mueller S.
      • Sandrin L.
      Liver stiffness: a novel parameter for the diagnosis of liver disease.
      In addition, the diagnostic accuracy of LS could be improved when considering the AST levels. These data have also been confirmed by others
      • Trabut J.B.
      • Thepot V.
      • Nalpas B.
      • Lavielle B.
      • Cosconea S.
      • Corouge M.
      • et al.
      Rapid decline of liver stiffness following alcohol withdrawal in heavy drinkers.
      and resuming alcohol drinking was associated with an increased LS value.
      • Gelsi E.
      • Dainese R.
      • Truchi R.
      • Marine-Barjoan E.
      • Anty R.
      • Autuori M.
      • et al.
      Effect of detoxification on liver stiffness assessed by fibroscan((R)) in alcoholic patients.
      In our present cohort of 365 patients undergoing alcohol withdrawal (Centre for Alcohol Research Heidelberg), the overall mean decrease of LS was 10%, which led to an underestimation of fibrosis stage in 27% of patients. In some patients, fibrosis stage changed by up to 3 degrees after alcohol withdrawal (unpublished). For these reasons, we require actual laboratory testing to correctly interpret LS values (see Fig. 2).
      Table 2Liver stiffness and fibrosis stages in ALD (biopsy-proven studies).
      Number of patientsCorrelationAUROC, F4Cut-off, F4Ref
      1740.70, p <0.00010.8722.6Nahon et al., 2008
      1030.72, p <0.0140.9219.5Nguyen-Khac et al., 2008
      450.9725.8Kim et al., 2009
      2170.87, p <0.020.9117.3Boursier et al., 2009
      1010.72, p <0.0010.9211.5Mueller et al., 2010
      490.8621.1Janssens et al., 2010
      150.9318.0Fernandez et al., 2015
      1990.9416.9Thiele et al., 2016
      2170.73, p <0.00010.9320.8Voican et al., 2017
      AUROC, area under the receiver operating characteristic curve; ALD, alcohol-related liver disease.
      Figure thumbnail gr2
      Fig. 2Practical algorithm in patient with excessive alcohol consumption. AST, aspartate aminotransferase; HCC, hepatocellular carcinoma.
      It is not yet completely clear why AST is best correlated with LS, however a recent multicentre study on both ALD and HCV confirmed the impact of AST on LS elevation.
      • Mueller S.
      • Englert S.
      • Seitz H.K.
      • Badea R.I.
      • Erhardt A.
      • Bozaari B.
      • et al.
      Inflammation-adapted liver stiffness values for improved fibrosis staging in patients with hepatitis C virus and alcoholic liver disease.
      Based on 677 patients with ALD and 1,391 with HCV, AST correlated best with LS in both diseases (HCV: r = 0.54, p <0.0001 and ALD: r = 0.34, p <0.0001) (Fig. 3). In the absence of elevated aminotransferases, cut-off values were almost identical between HCV and ALD for F1-2, F3 and F4 (HCV 5.1, 9.0 and 11.9 kPa vs. ALD 4.9, 8.1 and 10.5 kPa). These cut-off values increased exponentially as a function of median AST level. The impact of AST on LS was higher in lobular-pronounced ALD compared to portal tract-localised HCV. Most notably, Cohen's weighted Kappa displayed an improved agreement of the novel AST-dependent cut-off values with histological fibrosis stage both for HCV (0.68 vs. 0.65) and ALD (0.80 vs. 0.76).
      Figure thumbnail gr3
      Fig. 3Influence of AST elevation on liver stiffness measurement in ALD patients. ALD, alcohol-related liver disease; AST, aspartate aminotransferase.
      In ALD, AST levels are typically higher than alanine aminotransferase (ALT) and in ca. 70% of patients the AST/ALT ratio is higher than 2.
      • Mukai M.
      • Ozasa K.
      • Hayashi K.
      • Kawai K.
      Various S-GOT/S-GPT ratios in nonviral liver disorders and related physical conditions and life-style.
      However, AST levels higher than 300 IU/L are rarely detected. In cirrhotic stages, aminotransferases may normalise while AST levels may be continuously increased despite the absence of alcohol consumption.
      • Mueller S.
      • Englert S.
      • Seitz H.K.
      • Badea R.I.
      • Erhardt A.
      • Bozaari B.
      • et al.
      Inflammation-adapted liver stiffness values for improved fibrosis staging in patients with hepatitis C virus and alcoholic liver disease.
      Novel markers such as caspase-cleaved cytokeratin 18 fragments (M30) and M65 are more sensitive than aminotransferases and more specifically detect apoptotic cell death.
      • Mueller S.
      • Nahon P.
      • Rausch V.
      • Peccerella T.
      • Silva I.
      • Yagmur E.
      • et al.
      Caspase-cleaved keratin-18 fragments increase during alcohol withdrawal and predict liver-related death in patients with alcoholic liver disease.
      Notably, and in contrast to M65 and AST levels, M30 levels significantly increase during alcohol withdrawal, which highlights the specific role of apoptosis in ALD.
      • Mueller S.
      • Nahon P.
      • Rausch V.
      • Peccerella T.
      • Silva I.
      • Yagmur E.
      • et al.
      Caspase-cleaved keratin-18 fragments increase during alcohol withdrawal and predict liver-related death in patients with alcoholic liver disease.
      Finally, in the aforementioned meta-analysis
      • 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.
      of more than 1,000 patients, AST as well as bilirubin concentrations had a significant effect on LS. The presence of histological features of asymptomatic and non-severe alcoholic hepatitis was associated with increased LS (p <0.0001). In a multivariate analysis, AST (p <0.0001) and bilirubin (p = 0.0002) concentrations, and prothrombin activity (p = 0.01), were independently associated with the presence of histological features of asymptomatic and non-severe alcoholic hepatitis. It remains to be confirmed whether bilirubin levels really add to the overall performance of LS, since patients with ALD develop jaundice at end-stage cirrhosis, where LS is normally higher than 30 kPa and the status of cirrhosis is unquestionable. In contrast, patients with clinical alcoholic hepatitis may develop high levels of bilirubin in the absence of drastic LS elevation.

      Transfer into clinical practice

      Fibrosis assessment with TE in combination with ultrasound and alcohol withdrawal

      Fig. 2 shows a typical interpretation of LS if ultrasound and laboratory testing is available. If ALD is suspected based on patient reporting, alongside clinical and/or laboratory signs, then TE is performed directly after the abdominal ultrasound and routine blood tests. Patients should be kept in a horizontal position for a minimum of 5 minutes to stabilise haemodynamics. During the ultrasound, liver size, spleen size, morphology, abnormalities such as congestion, cholestasis, morphological signs of cirrhosis, the presence of ascites and the diameter of the lower caval vein are assessed. TE is then performed either with the M probe or in cases of M probe failure, obvious obesity or ascites with the XL probe.
      • Kohlhaas A.
      • Durango E.
      • Millonig G.
      • Bastard C.
      • Sandrin L.
      • Golriz M.
      • et al.
      Transient elastography with the XL probe rapidly identifies patients with non-hepatic ascites.
      • Durango E.
      • Dietrich C.
      • Seitz H.K.
      • Kunz C.U.
      • Pomier-Layrargues G.T.
      • Duarte-Rojo A.
      • et al.
      Direct comparison of the FibroScan XL and M probes for assessment of liver fibrosis in obese and nonobese patients.
      Ascites is no contraindication for the XL probe, which performs well in such cases.
      • Kohlhaas A.
      • Durango E.
      • Millonig G.
      • Bastard C.
      • Sandrin L.
      • Golriz M.
      • et al.
      Transient elastography with the XL probe rapidly identifies patients with non-hepatic ascites.
      If LS is elevated and patients have AST >100 U/ml, alcohol withdrawal for at least 2 weeks is recommended followed by a second LS measurement. In patients with LS >30 kPa, the diagnosis of cirrhosis is confirmed, irrespective of steatohepatitis as measured by elevated aminotransferase levels. At these levels, the development of ascites is very likely.
      This approach enables definitive non-invasive assessment of fibrosis stage in ca. 95% of patients. Compared to conventional routine ultrasound, TE identifies twice as many patients with advanced fibrosis/cirrhosis (Mueller S, unpublished) and has a smaller sample error than histology (3–5% vs. 20–50%). In a recent French elastography screening study on more than 1,000 apparently healthy people older than 45 years, 7.5% had a pathologically increased liver stiffness >8 kPa, which in 36% of cases was eventually linked to ALD.
      • 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.
      Therefore, it is anticipated that these novel non-invasive screening tools will improve the early recognition and follow-up of patients with ALD, the most common and unfortunately too often underestimated liver disease. Whether in addition AST-adapted cut-off values should be used e.g. for ad hoc decisions in patients with no time or option to withdraw from alcohol, remains a matter of debate.

      Fibrosis assessment with TE using inflammation adapted cut-off values

      We have recently developed an algorithm to avoid repetitive re-assessment of LS in patients with ALD and elevated AST levels (Fig. 3). In this multicentre study of more than 2,000 biopsy-proven patients with ALD and HCV, cut-off values for fibrosis increased exponentially as a function of median AST level.
      • Mueller S.
      • Englert S.
      • Seitz H.K.
      • Badea R.I.
      • Erhardt A.
      • Bozaari B.
      • et al.
      Inflammation-adapted liver stiffness values for improved fibrosis staging in patients with hepatitis C virus and alcoholic liver disease.
      While AST-adapted cut-off values allow an immediate assessment of fibrosis stage, even in patients with pronounced steatohepatitis, and avoid overestimation of fibrosis stages, it remains unclear why AST is so strongly correlated with LS. Moreover, AST may not only be derived from hepatocytes but also myocytes and erythrocytes. It also remains to be studied whether indeed all patients with elevated AST levels will necessarily develop elevated LS.

      LS follow-up in patients with ALD

      LS measurement enables drinking activity and ALD progression to be monitored, as LS encompasses the sum of all pathological features from inflammation, ballooning to fibrosis. LS has been shown to improve, shortly after alcohol withdrawal, in more than 80% of patients.
      • Millonig G.
      • Mueller S.
      • Sarovska L.
      • Friedrich S.
      • Reimann F.M.
      • Pritsch M.
      • et al.
      Increased liver stiffness in alcoholic liver disease: differentiating fibrosis from steatohepatitis.
      Preliminary unpublished mortality data from a 10-year survey in Heidelberg indicate that LS predicts mortality independently of bilirubin and international normalized ratio.

      Comparison of various elastographic techniques

      Several studies have been performed to directly compare the performance of TE with ARFI or SWE, however, no robust data are available on ALD. Thus, ARFI had a similar predictive value as TE in both chronic hepatitis B and C.
      • Sporea I.
      • Sirli R.
      • Bota S.
      • Popescu A.
      • Sendroiu M.
      • Jurchis A.
      Comparative study concerning the value of acoustic radiation force impulse elastography (ARFI) in comparison with transient elastography (TE) for the assessment of liver fibrosis in patients with chronic hepatitis B and C.
      In an Asian non-alcoholic steatohepatitis (NASH) population, AUROCs of TE, SWE, and ARFI were 0.757, 0.759, and 0.657 for significant fibrosis and 0.870, 0.809, and 0.873 for advanced fibrosis. Thus, these elastographic methods had similar diagnostic performance for staging fibrosis in patients with NAFLD.
      • Lee M.S.
      • Bae J.M.
      • Joo S.K.
      • Woo H.
      • Lee D.H.
      • Jung Y.J.
      • et al.
      Prospective comparison among transient elastography, supersonic shear imaging, and ARFI imaging for predicting fibrosis in nonalcoholic fatty liver disease.
      Similar data were found in a large study of 349 consecutive patients with various chronic liver diseases who underwent liver biopsy and SWE, ARFI and TE.
      • Cassinotto C.
      • Lapuyade B.
      • Mouries A.
      • Hiriart J.B.
      • Vergniol J.
      • Gaye D.
      • et al.
      Non-invasive assessment of liver fibrosis with impulse elastography: comparison of Supersonic Shear Imaging with ARFI and FibroScan(R).
      Although larger trials are required to finally settle advantages and limitations of the various elastographic methods, they seem to perform similarly.
      Larger trials are required to conclusively determine the comparative advantages and limitations of various elastographic methods, although their performances seem similar.

      Non-invasive diagnosis of alcoholic hepatitis

      Alcoholic hepatitis is a clinical syndrome characterised by the recent onset of jaundice in patients with ongoing alcohol abuse. Other signs of liver decompensation (i.e. ascites and/or encephalopathy) can be present, particularly in severe forms of the disease.
      • EASL Clinical Practice Guidelines
      Management of alcohol-related liver disease.
      Underlying this clinical syndrome is steatohepatitis, a disease defined histologically by steatosis, hepatocyte ballooning and infiltrates by polymorphonuclear neutrophils.
      • MacSween R.N.
      • Burt A.D.
      Histologic spectrum of alcoholic liver disease.
      Laboratory findings in patients with alcoholic hepatitis reveal neutrophilia, hyperbilirubinemia (>3 mg/dl), AST >50 IU/L (but rarely above 300 IU/L), with an AST/ALT ratio typically greater than 1.5–2. Diagnosis of alcoholic hepatitis is based on clinical and laboratory findings, and ideally confirmed by a transjugular liver biopsy. Liver biopsy is also useful to rule out other diagnoses and is of prognostic interest.
      • Altamirano J.
      • Miquel R.
      • Katoonizadeh A.
      • Abraldes J.G.
      • Duarte-Rojo A.
      • Louvet A.
      • et al.
      A histologic scoring system for prognosis of patients with alcoholic hepatitis.
      However, in routine clinical practice, liver biopsy is restricted by the lack of availability in some countries, the risk of the procedure, and the costs. Therefore, it appears reasonable that a liver biopsy should be mandatory when there is diagnostic uncertainty, and that stringent clinical and biological criteria should be applied in the absence of liver biopsy, in order to avoid misdiagnosis. Recently, the NIAAA Alcoholic Hepatitis consortia proposed a classification of alcoholic hepatitis diagnosis into 3 degrees of confidence:
      • Crabb D.W.
      • Bataller R.
      • Chalasani N.P.
      • Kamath P.S.
      • Lucey M.
      • Mathurin P.
      • et al.
      Standard definitions and common data elements for clinical trials in patients with alcoholic hepatitis: recommendation from the NIAAA Alcoholic Hepatitis Consortia.
      Definite alcoholic hepatitis, clinically diagnosed and biopsy-proven; Probable alcoholic hepatitis, clinically diagnosed, and without confounding factors (including 1 or more of the following: presence of auto-antibodies, sepsis, shock, cocaine use, recent use of a drug with potential hepatotoxicity within 30 days, uncertain alcohol use assessment, atypical laboratory tests); Possible alcoholic hepatitis, clinically diagnosed but with 1 of the confounding factors listed before. We strongly recommend performing a liver biopsy in this last case to confirm the diagnosis of alcoholic hepatitis.
      Very little attention has been given to patients with a non-severe form of the disease (i.e. Maddrey discriminant function [MDF] <32, see below), probably related to the low risk of death in the short term (1 month) in this group of patients. However, our group recently reported (in an abstract form) a 2-year mortality rate of 35% in patients with non-severe alcoholic hepatitis, which suggests that those patients should be identified and could benefit from specific therapies in the future, while alcohol abstinence is a key target to achieve in this population.

      Prognosis and new biomarkers in ALD

      Currently there are few prognostic markers or biomarkers in ALD.
      • Rahimi E.
      • Pan J.J.
      Prognostic models for alcoholic hepatitis.
      However, there is a major need for biomarkers in ALD and in alcoholic hepatitis, particularly of diagnostic markers, indicators of response to therapy, prognostic markers, early indicators of inflammation and markers of liver regeneration.
      • Sanyal A.J.
      • Gao B.
      • Szabo G.
      Gaps in knowledge and research priorities for alcoholic hepatitis.
      Recent studies attempted to address these questions, however, larger studies validating emerging biomarkers have not yet been performed. Biomarker discovery focusses on easily accessible specimens such as circulating blood markers (serum, plasma, blood cell populations), urine, hair, saliva or stool.
      Originally described prognostic calculators include the MDF, model for end-stage liver disease (MELD), ABIC (age, serum bilirubin, international normalized ratio, serum creatinine) and other less frequently used scoring models in alcoholic hepatitis.
      • Papastergiou V.
      • Tsochatzis E.A.
      • Pieri G.
      • Thalassinos E.
      • Dhar A.
      • Bruno S.
      • et al.
      Nine scoring models for short-term mortality in alcoholic hepatitis: cross-validation in a biopsy-proven cohort.
      An MDF >32 and MELD >20 are defined as severe alcoholic hepatitis.
      • Crabb D.W.
      • Bataller R.
      • Chalasani N.P.
      • Kamath P.S.
      • Lucey M.
      • Mathurin P.
      • et al.
      Standard definitions and common data elements for clinical trials in patients with alcoholic hepatitis: recommendation from the NIAAA Alcoholic Hepatitis Consortia.
      However, a recent study suggests that combining data from scoring systems is better for predicting outcomes in alcoholic hepatitis.
      • Louvet A.
      • Labreuche J.
      • Artru F.
      • Boursier J.
      • Kim D.J.
      • O'Grady J.
      • et al.
      Combining data from liver disease scoring systems better predicts outcomes of patients with alcoholic hepatitis.
      A recent study suggested that MELD performs better than MDF in alcoholic hepatitis.
      • Louvet A.
      • Labreuche J.
      • Artru F.
      • Boursier J.
      • Kim D.J.
      • O'Grady J.
      • et al.
      Combining data from liver disease scoring systems better predicts outcomes of patients with alcoholic hepatitis.
      However, new biomarkers are needed to better evaluate individual responses to steroids and other emerging therapies under investigation.
      In severe alcoholic hepatitis, infection, sepsis and multi-organ dysfunction syndrome have been shown to correlate with poor survival.
      • Forrest E.H.
      • Evans C.D.
      • Stewart S.
      • Phillips M.
      • Oo Y.H.
      • McAvoy N.C.
      • et al.
      Analysis of factors predictive of mortality in alcoholic hepatitis and derivation and validation of the Glasgow alcoholic hepatitis score.
      • Louvet A.
      • Wartel F.
      • Castel H.
      • Dharancy S.
      • Hollebecque A.
      • Canva-Delcambre V.
      • et al.
      Infection in patients with severe alcoholic hepatitis treated with steroids: early response to therapy is the key factor.
      Consistent with this, the Glasgow coma scale and Glasgow alcoholic hepatitis score (GAHS) may help to stratify patients and identify those who will benefit from corticosteroids in alcoholic hepatitis.
      • Forrest E.H.
      • Morris A.J.
      • Stewart S.
      • Phillips M.
      • Oo Y.H.
      • Fisher N.C.
      • et al.
      The Glasgow alcoholic hepatitis score identifies patients who may benefit from corticosteroids.
      Close monitoring and supportive care in the intensive care setting are the most important factors in improving survival in acute alcoholic hepatitis. Current treatment in acute alcoholic hepatitis is prednisolone for 28 days. The Lille score is a commonly used and validated prognostic marker of response or lack of thereof to corticosteroid therapy.
      • Louvet A.
      • Naveau S.
      • Abdelnour M.
      • Ramond M.J.
      • Diaz E.
      • Fartoux L.
      • et al.
      The Lille model: a new tool for therapeutic strategy in patients with severe alcoholic hepatitis treated with steroids.
      In the setting of chronic, otherwise stable, ALD or cirrhosis, acute alcohol binge has been identified as a major trigger of acute-on-chronic liver failure (ACLF).
      • Mehta G.
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      Systemic inflammation is associated with increased intrahepatic resistance and mortality in alcohol-related acute-on-chronic liver failure.
      ACLF is characterised by a rapid increase in bilirubin.
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      Acute alcoholic binge can also trigger ACLF in advanced liver disease of other aetiologies such as viral hepatitis.
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      Alcoholic hepatitis and HCV interactions in the modulation of liver disease.
      It has been proposed that alcohol-related increased gut permeability, increased circulating endotoxin and induction of pro-inflammatory cytokines mediate the ACLF event.
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      The pathogenesis of ACLF: the inflammatory response and immune function.
      Clinical prognosis of ACLF is poor with development of infection, sepsis and multi-organ failure. The CLIF-C ACLF score has been proposed as the best currently available predictor of mortality in patients with ACLF.
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      The characteristic AST >ALT ratio greater than 1.5 is considered as a classic diagnostic biomarker in ALD and alcoholic hepatitis. However, increased aminotransferases do not distinguish between key components of the disease pathology such as hepatocyte damage and inflammation. Recent investigations focussed on circulating small non-coding RNA (miRNA) and long-non-coding RNAs (lncRNAs). Increased levels of serum miR-122 have been found in mouse models of ALD and in human patients.
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      MicroRNA signature in alcoholic liver disease.
      Because miR-122 is a miRNA primarily found in hepatocytes, its increase in the serum is believed to represent hepatocyte damage. Indeed, circulating miR-122 is increased in many different forms of liver disease, including NASH, viral hepatitis and drug-induced liver injury. Therefore, it cannot serve as a disease-specific marker in ALD.
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      MicroRNAs in liver disease.
      Another miRNA, miR-155 is a master regulator of inflammation. Increased levels of circulating miR-155 were found in a mouse model of ALD and both miR-155 and miR-122 were enriched in circulating exosomes.
      • Szabo G.
      • Bala S.
      Reply: to PMID 22684891.
      Further analysis of exosome miRNA content revealed that miR-192 and miR-30a are also highly abundant in exosomes in mice with ALD and more importantly, in patients with alcoholic hepatitis compared to normal controls.
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      Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcoholic hepatitis.
      (Fig. 4). Unique lncRNAs were shown to be expressed in the serum and liver of patients with alcoholic cirrhosis.
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      • Zhao S.
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      LncRNA AK054921 and AK128652 are potential serum biomarkers and predictors of patient survival with alcoholic cirrhosis.
      Among those, AK128652 and AK054921 were the most abundantly increased lncRNAs in patients with alcoholic cirrhosis. Furthermore, in 480 prospectively followed patients, AK128652 and AK054921 inversely correlated with survival in patients with alcoholic cirrhosis.
      • Yang Z.
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      • Wang L.
      LncRNA AK054921 and AK128652 are potential serum biomarkers and predictors of patient survival with alcoholic cirrhosis.
      Prospective analysis of these emerging diagnostic and prognostic biomarkers in larger patient populations is awaited.
      Figure thumbnail gr4
      Fig. 4MicroRNAs as emerging biomarkers in ALD. In ALD, miR-155 expression is increased in the liver in hepatocytes and particularly in Kupffer cells and macrophages. Hepatocyte expression of miR-122 is reduced in ALD in mice. These changes in the liver occur while circulating levels of several miRNAs increase including miR-122, miR-155, miR-192 and miR-30a. Most of these miRNAs a found in the circulation packaged into exosomes and/or extracellular vesicles. ALD, alcohol-related liver disease; miRNAs, small non-coding RNAs.
      A number of miRNAs have been found to associated with ALD and could prove to be useful diagnostic or prognostic markers in the future.
      Because of the presence of systemic increase in inflammation in alcoholic hepatitis, circulating cytokine levels have been evaluated as biomarkers.
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      • Freeman W.M.
      • Vrana K.E.
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      Significantly increased circulating levels of IL-1β, TNF and IL-8 were found in severe alcoholic hepatitis in several independent studies.
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      However, it remains to be evaluated whether the dynamics of increases/decreases in specific cytokines have potential as disease-specific or prognostic biomarkers. Cytokeratins have emerged as markers of liver, particularly hepatocyte, damage. Mallory bodies, the hallmark of alcoholic hepatitis, contain cytokeratin-18 (CK-18) and cytokeratin-19 (CK-19).
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      Serum cytokeratins in alcoholic liver disease: contrasting levels of cytokeratin-18 and cytokeratin-19.
      Serum levels of CK-18 and CK-19 were increased in patients with alcoholic hepatitis compared to fatty liver or controls.
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      Serum cytokeratins in alcoholic liver disease: contrasting levels of cytokeratin-18 and cytokeratin-19.
      In a recent study, Bissionette et al. reported higher levels of total and microvesicle-bound M65 and M30, circulating fragments of CK-18 in the circulation in biopsy-proven alcoholic hepatitis.
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      A cut-off of 2,000 IU/L for M65 has a positive predictive value of 91% and a cut-off of 642 IU/L has a negative predictive value of 88%.
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      Serum levels of keratin-18 fragments [tissue polypeptide-specific antigen (TPS)] are correlated with hepatocyte apoptosis in alcoholic hepatitis.
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      • et al.
      New potential biomarker proteins for alcoholic liver disease identified by a comparative proteomics approach.
      Interestingly, most of these proteins were also increased in NASH livers and only ornithine aminotransferase, vitamin D binding protein and phosphatidylethanolamine-binding protein were higher in ALD compared to NASH and normal controls.
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      In a mouse model of ALD, proteomic analysis of circulating extracellular vesicles (EVs) revealed a unique cluster or EV-associated proteins that were increased compared to control EVs.
      • Saha B.
      • Momen-Heravi F.
      • Furi I.
      • Kodys K.
      • Catalano D.
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      • et al.
      Extracellular vesicles from mice with alcoholic liver disease carry a distinct protein cargo and induce macrophage activation through heat shock protein 90.
      Among these was heat-shock protein-90 (hsp90) that was also associated with a biological effect in macrophages after exosome transfer both in vitro and in vivo, indicating both the biomarker potential of EV-associated proteins as well as the potential role of EVs in cell-to-cell communication.
      • Saha B.
      • Momen-Heravi F.
      • Furi I.
      • Kodys K.
      • Catalano D.
      • Gangopadhyay A.
      • et al.
      Extracellular vesicles from mice with alcoholic liver disease carry a distinct protein cargo and induce macrophage activation through heat shock protein 90.
      In addition to serum-based biomarkers, new approaches include identification of urine-based biomarkers by targeting urinary metabolomics.
      • Manna S.K.
      • Patterson A.D.
      • Yang Q.
      • Krausz K.W.
      • Li H.
      • Idle J.R.
      • et al.
      Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse.
      The clinical prognosis in alcoholic cirrhosis is affected by ongoing alcohol use.
      • Orntoft N.W.
      • Sandahl T.D.
      • Jepsen P.
      • Vilstrup H.
      Short-term and long-term causes of death in patients with alcoholic hepatitis in Denmark.
      While early survival in alcoholic hepatitis at 28 and 90 days is largely determined by liver-related causes, infection and organ failure, longer-term survival is related to non-hepatic causes including a return to alcohol use.
      • Orntoft N.W.
      • Sandahl T.D.
      • Jepsen P.
      • Vilstrup H.
      Short-term and long-term causes of death in patients with alcoholic hepatitis in Denmark.
      Cessation of alcohol use can improve long-term survival while continued alcohol use accelerates decompensation in alcoholic cirrhosis and increases other causes of death related to trauma and alcohol use. In the alcoholic patient, alcohol cessation is a key therapeutic goal and also a determinant of long-term survival. Thus, biomarkers of alcohol use are useful in the management of alcoholic hepatitis and alcoholic cirrhosis, particularly in the pre- and post-transplant settings.
      • Cabezas J.
      • Lucey M.R.
      • Bataller R.
      Biomarkers for monitoring alcohol use.

      Conclusions and future directions

      While the topics of non-invasive diagnosis and biomarkers in ALD have been the focus of many clinical and translational research studies, reliable and validated approaches that are practical in general practice are still awaited. Many issues still need to be addressed in the application of elastographic techniques to patients with ALD. These include better comparison of various elastographic techniques, clarification of inflammation-induced LS and its molecular basis, defining inflammation markers other than AST to prevent overestimation of LS in the presence of steatohepatitis, data on LS as a long-term survival predictor in patients with ALD, and clarification of the role of fat on LS in patients with ALD.
      There is a continued need for studies with large and clinically well-characterised patient populations to address the above list of highly clinically relevant questions. Consortium-based projects within the EU and United States, as well as international collaborations will help to assess larger sets of data and diverse patient populations.

      Conflict of interest

      Christophe Moreno and Gyongyi Szabo have no conflict of interest, Sebastian Mueller is adviser for Echosens.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      All authors contributed equally to the production of this manuscript.

      Supplementary data

      The following are the Supplementary data to this article:

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