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Emerging Biomarkers in Alcohol-associated Hepatitis

Published:August 01, 2022DOI:https://doi.org/10.1016/j.jceh.2022.07.246
      Alcohol-associated hepatitis (AH) is a clinical syndrome of jaundice, abdominal pain, and anorexia due to prolonged heavy alcohol intake. AH is associated with changes in gene expression, cytokines, immune response, and the gut microbiome. There are limited biomarkers to diagnose and prognosticate in AH, but several non-invasive biomarkers are emerging. In this review, clinical risk-stratifying algorithms, promising AH biomarkers like cytokeratin-18 fragments, genetic polymorphisms, and microRNAs will be reviewed.

      Keywords

      Abbreviations:

      AH (Alcohol-associated hepatitis), ALD (alcohol-associated liver disease), ASCA (anti–Saccharomyces cerevisiae antibodies), AUC (area under the curve), FGF (fibroblast growth factor), GAHS (Glasgow alcohol-associated hepatitis score), HCC (hepatocellular carcinoma), MELD (model for end-stage liver disease), miRNAs (MicroRNAs), NASH (non-alcohol-associated steatohepatitis), PPV (positive predictive value), PT (prothrombin time), VCTE (vibration-controlled transient elastography)
      Alcohol-associated hepatitis (AH) is a uniquely severe condition along the spectrum of alcohol-associated liver disease (ALD). It has a somewhat non-specific and nebulous definition as a clinical syndrome of jaundice, anorexia, right upper quadrant pain with tenderness on exam, and features of the systemic inflammatory response syndrome
      • Michelena J.
      • Altamirano J.
      • Abraldes J.G.
      • et al.
      Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcohol-associated hepatitis.
      ,
      • Mendenhall C.
      Alcohol-associated Hepatitis.
      occurring after at least six months, but often decades, of heavy alcohol ingestion.
      • Naveau S.
      • Giraud V.
      • Borotto E.
      • Aubert A.
      • Capron F.
      • Chaput J.
      Excess weight risk factor for alcohol-associated liver disease.
      It is challenging to establish reliable diagnostic and prognostic biomarkers in a disease with variable presentation and non-specific diagnostic criteria, especially with the increasing prevalence of overlapping conditions/confounders like non-alcohol-associated steatohepatitis (NASH). While there have been significant advances in prognostic biomarkers in AH, diagnostic biomarkers specific to AH are lacking and an unmet need. Adding complexity is the requirement of recent alcohol use in AH, which itself (timing of last drink, amount, pattern, duration, and type), is not easily ascertained.
      In an effort to create standardized diagnostic criteria primarily for clinical trials, an expert consortium from the National Institute on Alcohol Abuse and Alcoholism defined AH as definite (biopsy-proven), probable (clinically diagnosed without confounding factors), or possible (clinically diagnosed but confounding factors such as possible ischemic hepatitis, drug-induced liver injury (DILI), unclear alcohol use or atypical laboratory findings).
      • Crabb D.W.
      • Bataller R.
      • Chalasani N.P.
      • et al.
      Standard definitions and common data elements for clinical trials in patients with alcohol-associated hepatitis: recommendation from the NIAAA Alcohol-associated Hepatitis Consortia.
      A recent study of 114 patients with ALD who underwent liver biopsy showed that the positive predictive value of the National Institute on Alcohol Abuse and Alcoholism criteria for AH diagnosis was 81% with a false negative rate of 30%.
      • Crabb D.W.
      • Bataller R.
      • Chalasani N.P.
      • et al.
      Standard definitions and common data elements for clinical trials in patients with alcohol-associated hepatitis: recommendation from the NIAAA Alcohol-associated Hepatitis Consortia.
      ,
      • Avitabile E.
      • Pose E.
      • Graupera I.
      • et al.
      Non-invasive criteria for diagnosis of alcohol-associated hepatitis: use in clinical practice and correlation with prognosis.
      Given its potentially high short-term mortality, AH urgently needs improved diagnostic biomarkers: accurate alcohol histories can be notoriously difficult to obtain and there are no clinical, laboratory, or radiographic features that are completely unique to AH. Thus, a high level of clinical suspicion is required and thorough assessment for covert alcohol use by patient interview and/or alcohol biomarkers (which may not be readily available).

      THE IDEAL BIOMARKER

      As shown in other disease processes, the ideal biomarker is more likely a panel of molecules in combination, which increases the sensitivity and specificity and can both diagnose and prognosticate.
      • Rinck D.
      • Frieling H.
      • Freitag A.
      • et al.
      Combinations of carbohydrate-deficient transferrin, mean corpuscular erythrocyte volume, gamma-glutamyltransferase, homocysteine and folate increase the significance of biological markers in alcohol dependent patients.
      After an episode of AH, approximately 80% of patients will eventually progress to cirrhosis, but even patients who present with end-stage ALD at diagnosis have likely experienced years of “subclinical AH” and inflammation which eventually progressed with fibrosis.
      • Schwartz J.M.
      • Reinus J.F.
      Prevalence and natural history of alcohol-associated liver disease.
      An ideal biomarker would help to identify patients at risk for AH, fibrosis, and cirrhosis prior to a sentinel event.
      Currently available biomarkers in AH fall far short. Laboratory biomarkers currently exist that suggest chronic (such as mean corpuscular volume
      • Koch H.
      • Meerkerk G.-J.
      • Zaat J.O.
      • Ham M.F.
      • Scholten R.J.
      • Assendelft W.J.
      Accuracy of carbohydrate-deficient transferrin in the detection of excessive alcohol consumption: a systematic review.
      and gamma glutamyltransferase
      • Taracha E.
      • Habrat B.
      • Woźiak P.
      • Walkowiak J.
      • Szukalski B.
      The activity of β-hexosaminidase (uHex) and γ-glutamyl-transferase (uGGT) in urine as non-invasive markers of chronic alcohol abuse: I. Alcohol-dependent subjects.
      ) and recent alcohol use (such as urine ethyl glucuronide
      • Kissack J.C.
      • Bishop J.
      • Roper A.L.
      Ethylglucuronide as a biomarker for ethanol detection.
      or serum phosphatidylethanol
      • Comasco E.
      • Nordquist N.
      • Leppert J.
      • et al.
      Adolescent alcohol consumption: biomarkers PEth and FAEE in relation to interview and questionnaire data.
      ). Cytokines (such as tumor necrosis factor (TNF)-alpha, IL-1, IL-6, and IL-8) have been proposed as markers of alcohol-induced tissue damage (and a TNF-alpha promoter polymorphism has been linked to susceptibility to AH),
      • Grove J.
      • Daly A.K.
      • Bassendine M.F.
      • Day C.P.
      Association of a tumor necrosis factor promoter polymorphism with susceptibility to alcohol-associated steatohepatitis.
      but these cytokines are affected by a number of other factors (including non-alcohol-associated hepatitis) and may even normalize in alcohol-associated cirrhosis (AC) (Figure 1).
      • Achur R.N.
      • Freeman W.M.
      • Vrana K.E.
      Circulating cytokines as biomarkers of alcohol abuse and alcoholism.
      When the diagnosis of AH is in question due to atypical presentation or unclear alcohol history, a serum, plasma, stool, saliva, or urine biomarker with rapid turnaround time and high accuracy would be ideal. This biomarker would be specific for AH and would help to distinguish between ischemic hepatitis, DILI, and other causes of acute liver injury.

      CLINICAL SCENARIOS WHERE AH BIOMARKERS ARE NEEDED

      Moderate AH

      A moderate-severity phenotype exists with alcohol-associated steatohepatitis (ASH) on liver biopsy but without clinical symptoms, otherwise known as “walking ASH”.
      • Crabb D.W.
      • Im G.Y.
      • Szabo G.
      • Mellinger J.L.
      • Lucey M.R.
      Diagnosis and treatment of alcohol-associated liver diseases: 2019 practice guidance from the American association for the study of liver diseases.
      This phenotype of patient may have underlying cirrhosis (up to 30–40% of cases
      • Crabb D.W.
      • Im G.Y.
      • Szabo G.
      • Mellinger J.L.
      • Lucey M.R.
      Diagnosis and treatment of alcohol-associated liver diseases: 2019 practice guidance from the American association for the study of liver diseases.
      ) and may progress to clinical AH. This represents an ideal cohort to derive benefit from a risk-stratifying biomarker, fibrosis assessment, and early pharmacologic intervention before patients spiral into the inflammatory cascade of severe AH which is difficult to reverse. One of the challenges is how to identify patients who are misusing alcohol and have “walking ASH” but have not yet reached medical attention. We do not know the true prevalence of “walking ASH”, but ideally, a non-invasive, low-cost biomarker could screen for and detect fibrosis or low-level inflammation in the primary care or alcohol rehabilitation setting.
      A criticism of some of the interventional trials in AH is that these trials target a group with severe AH (modified Maddrey's discriminant function (mDF) > 32), many of whom have irreversible multi-organ dysfunction and in whom most medical treatments are ineffective. Furthermore, patients with non-severe or moderate AH (mDF of <32 or model for end-stage liver disease (MELD) < 20) have been shown to have unexpectedly high mortality rates of 6% at 28 days, 10–13% at 1 year, and 50% at 5 years, with the lack of alcohol abstinence associated with poorer outcomes.,
      • Naveau S.
      • Montembault S.
      • Balian A.
      • et al.
      Biological diagnosis of the type of liver disease in alcohol-associated patients with abnormal liver function tests.
      • Potts J.
      • Goubet S.
      • Heneghan M.
      • Verma S.
      Determinants of long-term outcome in severe alcohol-associated hepatitis.
      • Samala N.
      • Gawrieh S.
      • Tang Q.
      • et al.
      Clinical characteristics and outcomes of mild to moderate alcohol-associated hepatitis.
      • Bennett K.
      • Enki D.G.
      • Thursz M.
      • Cramp M.E.
      • Dhanda A.D.
      Systematic review with meta-analysis: high mortality in patients with non-severe alcohol-associated hepatitis.
      Thus, this represents an ideal group for early pharmacological and/or psychological interventions. Many patients who present with clinical AH and moderate MELD scores (12–19) are not treated pharmacologically, yet are still at risk of fibrosis progression and eventually cirrhosis
      • Delphine D.
      • Stauber R.E.
      • Gaël E.
      • et al.
      Long-term outcome of symptomatic alcohol-associated hepatitis with a Maddrey discriminant function< 32.
      (Figure 2). More studies are needed to better understand the natural history of moderate AH and “walking ASH”.
      Figure 1
      Figure 1Cytokine dysregulation and downstream effects in alcohol-associated hepatitis.
      • Singal A.K.
      • Kodali S.
      • Vucovich L.A.
      • Darley-Usmar V.
      • Schiano T.D.
      Diagnosis and treatment of alcohol-associated hepatitis: a systematic review.
      Figure 2
      Figure 2Typical course of alcohol-associated liver disease. Chronic excess alcohol intake leads to steatohepatitis, fibrosis, and eventually cirrhosis. There may be super-imposed episodes of alcohol-associated hepatitis or steatohepatitis can be subclinical without overt symptoms of inflammation, but eventual progresses to alcohol-associated cirrhosis.
      • Delphine D.
      • Stauber R.E.
      • Gaël E.
      • et al.
      Long-term outcome of symptomatic alcohol-associated hepatitis with a Maddrey discriminant function< 32.
      (from Seminars in Liver Disease, Thieme; with permission).

      AH and Acute-on-chronic Liver Failure

      Acute-on-chronic liver failure (ACLF) is a syndrome characterized by the acute decompensation of cirrhosis, often precipitated by infection or alcohol, and accompanied by failure of at least one other organ system with in-hospital mortality rates of at least 50%.
      • Jalan R.
      • Gines P.
      • Olson J.C.
      • et al.
      Acute-on chronic liver failure.
      Alcohol is thought to be the etiology of approximately 25% of cases of ACLF.
      • Moreau R.
      • Jalan R.
      • Gines P.
      • et al.
      Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis.
      A study by Sersté found that 48% of patients with severe AH met criteria for ACLF; this group had a poor response to steroids and a 28-day mortality rate of 54%, with mortality directly correlating with the number of organs in failure.
      • Sersté T.
      • Cornillie A.
      • Njimi H.
      • et al.
      The prognostic value of acute-on-chronic liver failure during the course of severe alcohol-associated hepatitis.
      For patients with ACLF, the chronic liver failure consortium (CLIF-C) ACLF score (which includes surrogates for organ failure: creatinine, vasopressor use, bilirubin, encephalopathy, INR, and oxygenation parameters) is thought to be the best predictor of short-term mortality.
      • Jalan R.
      • Saliba F.
      • Pavesi M.
      • et al.
      Development and validation of a prognostic score to predict mortality in patients with acute-on-chronic liver failure.
      CLIF-C outperformed MELD-Na and Child-Turcotte-Pugh scores in a Portuguese cohort of 132 male patients, and a score of greater than 64 should trigger consideration for liver transplantation.
      • Barosa R.
      • Roque-Ramos L.
      • Patita M.
      • Nunes G.
      • Fonseca J.
      CLIF-C ACLF score is a better mortality predictor than MELD, MELD-Na and CTP in patients with Acute on chronic liver failure admitted to the ward.
      However, the CLIF-C score can have low accuracy in AH with ACLF, with the Sersté’s study showing that both CLIF-C and mDF have low Harrel's C indices in predicting 28-day mortality (0.68 and 0.64, respectively, where 1 is perfectly accurate).
      • Sersté T.
      • Cornillie A.
      • Njimi H.
      • et al.
      The prognostic value of acute-on-chronic liver failure during the course of severe alcohol-associated hepatitis.
      There are many gaps in knowledge relating to this cohort. It is unknown whether ACLF represents the most severe phenotype of AH associated with high levels of circulating LPS, systemic inflammatory response syndrome, and multi-organ failure, or whether these are two distinct pathophysiologic processes. Does ACLF due to alcohol excess portend a worse prognosis compared to other causes of ACLF? Would biomarkers add prognostic information in this cohort, and would they perform similarly to those validated in non-ACLF severe AH? It remains to be seen how biomarkers and tailored therapies will be adopted for this cohort of patients with the severest form of life-threatening ALD.

      Fibrosis

      Biomarkers or non-invasive testing for liver fibrosis have tremendous diagnostic potential that are even agnostic to liver disease etiology. Biopsy-based studies have shown that among patients who consume heavy amounts of alcohol, only about 10% will develop cirrhosis.
      • Bellentani S.
      • Saccoccio G.
      • Costa G.
      • et al.
      Drinking habits as cofactors of risk for alcohol induced liver damage.
      Thus, an important question is how to measure underlying fibrosis in patients who consume alcohol to try and predict who will develop AH or AC. The presence of certain gene polymorphisms such as the patatin-like phospholipase domain-containing protein 3 (PNPLA3 gene)
      • Salameh H.
      • Raff E.
      • Erwin A.
      • et al.
      PNPLA3 gene polymorphism is associated with predisposition to and severity of alcohol-associated liver disease.
      and 11β-HSD1 gene induction and mRNA expression
      • Ahmed A.
      • Saksena S.
      • Sherlock M.
      • Olliff S.P.
      • Elias E.
      • Stewart P.M.
      Induction of hepatic 11β-hydroxysteroid dehydrogenase type 1 in patients with alcohol-associated liver disease.
      increases the risk and severity of ALD (Table 1). Genes involved in liver fibrogenesis, inflammation, and oxidative stress are upregulated in AH and may represent targets for diagnosis and therapy.
      • Colmenero J.
      • Bataller R.
      • Sancho–Bru P.
      • et al.
      Hepatic expression of candidate genes in patients with alcohol-associated hepatitis: correlation with disease severity.
      Table 1Genes With Single Nucleotide Polymorphisms Related to Alcohol-associated Liver Disease.
      Genetic polymorphismEffect on risk of ALDYear of discovery of association with ALD
      PNPLA3 (patatin-like phospholipase domain-containing protein 3)↑AC,
      • Buch S.
      • Stickel F.
      • Trépo E.
      • et al.
      A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.
      ↑AH,
      • Salameh H.
      • Raff E.
      • Erwin A.
      • et al.
      PNPLA3 gene polymorphism is associated with predisposition to and severity of alcohol-associated liver disease.
      ,
      • Atkinson S.R.
      • Way M.J.
      • McQuillin A.
      • Morgan M.Y.
      • Thursz M.R.
      Homozygosity for rs738409: G in PNPLA3 is associated with increased mortality following an episode of severe alcohol-associated hepatitis.
      ↑HCC
      • Stickel F.
      • Buch S.
      • Nischalke H.D.
      • et al.
      Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis.
      2010
      MBOAT7 (membrane bound O-acyltransferase domain-containing 6)↑AC,
      • Buch S.
      • Stickel F.
      • Trépo E.
      • et al.
      A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.
      ↑HCC
      • Calès P.
      • Boursier J.
      • Oberti F.
      • et al.
      FibroMeters: a family of blood tests for liver fibrosis.
      2015
      TM6SF2 (transmembrane 6 superfamily member 2)↑AC,
      • Buch S.
      • Stickel F.
      • Trépo E.
      • et al.
      A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.
      ↑HCC
      • Stickel F.
      • Buch S.
      • Nischalke H.D.
      • et al.
      Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis.
      2015
      HSD17B13 (hydroxysteroid 17-beta dehydrogenase 13)↓risk of progression from steatosis to steatohepatitis,
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      ↓risk of severe AH
      • Atkinson S.
      • Buckley T.
      • Strnad P.
      • Thursz M.
      • McQuillin A.
      • Morgan M.
      Genetic variation in HSD17B3 reduces the risk for developing severe alcohol-associated hepatitis.
      2018
      FAF2 (Fas Associated Factor family member 2)↓AC
      • Schwantes-An T.H.
      • Darlay R.
      • Mathurin P.
      • et al.
      Genome-wide association study and meta-analysis on alcohol-related liver cirrhosis identifies novel genetic risk factors.
      2020
      SERPINA1↑AC
      • Schwantes-An T.H.
      • Darlay R.
      • Mathurin P.
      • et al.
      Genome-wide association study and meta-analysis on alcohol-related liver cirrhosis identifies novel genetic risk factors.
      2020
      Abbreviations: AC, alcohol-associated cirrhosis; AH, alcohol-associated hepatitis; ALD, alcohol-associated liver disease; HCC, hepatocellular carcinoma.
      Thiele et al. have shown that the enhanced liver fibrosis™ test (which uses serum biomarkers of extracellular matrix remodeling and fibrogenesis) and ASH FibroTest™ (which uses age, gender, bilirubin, and several other serum biomarkers) can identify advanced liver fibrosis in patients with heavy alcohol consumption with an AUROC of over 0.9 (using liver biopsy as gold standard).
      • 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 alcohol-associated liver disease.
      They outperform the FIB-4 (based on age, platelet count, AST, and ALT) and APRI (AST to platelet ratio) scores.
      • 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.
      These biomarkers may represent an ideal screening tool for the primary care or alcohol rehabilitation setting.
      Measuring liver stiffness with vibration-controlled transient elastography (VCTE) is advantageous over liver biopsy due to its excellent correlation with liver fibrosis, superior safety, and lower cost. VCTE assesses 1/5000 of liver volume compared to 1/50,000 with liver biopsy,
      • Singal A.K.
      • Kodali S.
      • Vucovich L.A.
      • Darley-Usmar V.
      • Schiano T.D.
      Diagnosis and treatment of alcohol-associated hepatitis: a systematic review.
      ,
      • Sandrin L.
      • Fourquet B.
      • Hasquenoph J.-M.
      • et al.
      Transient elastography: a new noninvasive method for assessment of hepatic fibrosis.
      therefore reducing sampling error.
      • Regev A.
      • Berho M.
      • Jeffers L.J.
      • et al.
      Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection.
      However, there are confounders of liver stiffness measurement in patients undergoing alcohol withdrawal. Acute liver inflammation results in falsely elevated liver stiffness scores and an overestimation of fibrosis. As AST elevation decreases, liver stiffness also decreases and below an AST of 100 IU/L, liver stiffness measurement has been found to be more accurate.
      • Mueller S.
      • Sandrin L.
      Liver stiffness: a novel parameter for the diagnosis of liver disease.
      The early detection of fibrosis for VCTE, even in patients with ALD and with concomitant liver diseases like hepatitis C, could improve health and motivation to change with appropriate linkage to care.

      PROGNOSTIC MODELS IN AH

      Predictive models using widely available laboratory biomarkers have been the most studied and successful in AH. One of the earliest and still commonly used clinical tools to prognosticate in AH is the mDF, is based on the patient's prothrombin time (PT) compared to laboratory control PT, and serum bilirubin.
      • Carithers Jr., R.L.
      • Herlong H.F.
      • Diehl A.M.
      • et al.
      Methylprednisolone therapy in patients with severe alcohol-associated hepatitis: a randomized multicenter trial.
      A mDF score of greater than 32 identifies those with 1-month mortality of 50% who may benefit from treatment with corticosteroids but is less helpful at predicting intermediate and long-term outcomes.
      • Carithers Jr., R.L.
      • Herlong H.F.
      • Diehl A.M.
      • et al.
      Methylprednisolone therapy in patients with severe alcohol-associated hepatitis: a randomized multicenter trial.
      Some of other disadvantages of the mDF include the control PT being poorly standardized and not reported by all labs; mDF is also based on patient cohorts from over four decades ago, and supportive medical care has improved significantly since then, thereby reducing its predictive power.
      • Dunn W.
      • Jamil L.H.
      • Brown L.S.
      • et al.
      MELD accurately predicts mortality in patients with alcohol-associated hepatitis.
      However, mDF is universally used as an inclusion criterion in contemporary clinical trials in AH treatment, cementing its role in clinical practice and research.
      Several studies have demonstrated that the MELD score performs at least as well as mDF for the prediction of 30-day and 90-day mortality in AH.
      • Dunn W.
      • Jamil L.H.
      • Brown L.S.
      • et al.
      MELD accurately predicts mortality in patients with alcohol-associated hepatitis.
      ,
      • Sheth M.
      • Riggs M.
      • Patel T.
      Utility of the Mayo End-Stage Liver Disease (MELD) score in assessing prognosis of patients with alcohol-associated hepatitis.
      A larger retrospective cohort study showed that an admission MELD of ≧ 18, first week MELD of ≧ 20, and first week change in MELD ≧ 2 were each independently associated with in-hospital mortality. A week 1 MELD cut-off of 20 had the best test characteristics with 91% sensitivity and 85% specificity in predicting mortality, which significantly outperformed mDF.
      • Srikureja W.
      • Kyulo N.L.
      • Runyon B.A.
      • Hu K.-Q.
      MELD score is a better prognostic model than Child-Turcotte-Pugh score or Discriminant Function score in patients with alcohol-associated hepatitis.
      Other validated prognostic scores in AH include: the Glasgow alcohol-associated hepatitis score (GAHS) which uses age, bilirubin, urea, PT, and white blood cell count
      • Forrest E.H.
      • Evans C.D.J.
      • Stewart S.
      • et al.
      Analysis of factors predictive of mortality in alcohol-associated hepatitis and derivation and validation of the Glasgow alcohol-associated hepatitis score.
      and the age, serum bilirubin, INR and creatinine (ABIC) score.
      • Dominguez M.
      • Rincón D.
      • Abraldes J.G.
      • et al.
      A new scoring system for prognostic stratification of patients with alcohol-associated hepatitis.
      The GAHS is more specific than mDF (at least in the United Kingdom) and may avoid unnecessary treatment with steroids in certain patients.
      • Forrest E.H.
      • Morris A.
      • Stewart S.
      • et al.
      The Glasgow alcohol-associated hepatitis score identifies patients who may benefit from corticosteroids.
      A post-hoc analysis of the STOPAH trial found that the MELD, GAHS, and ABIC scores all individually outperformed mDF.
      • Forrest E.H.
      • Atkinson S.R.
      • Richardson P.
      • et al.
      Application of prognostic scores in the STOPAH trial: discriminant function is no longer the optimal scoring system in alcohol-associated hepatitis.
      Particularly useful appeared to be a baseline “static” score (based on variables at a single timepoint, such as MELD) on admission in combination with either the delta in the “static” score at day 7 or a “dynamic score” such as the Lille score.
      • Forrest E.H.
      • Atkinson S.R.
      • Richardson P.
      • et al.
      Application of prognostic scores in the STOPAH trial: discriminant function is no longer the optimal scoring system in alcohol-associated hepatitis.
      ,
      • Louvet A.
      • Naveau S.
      • Abdelnour M.
      • et al.
      The Lille model: a new tool for therapeutic strategy in patients with severe alcohol-associated hepatitis treated with steroids.
      As patients with AH present at different timepoints in their clinical trajectory, using this algorithm helps to inform the clinician when patients should start, stop, or avoid corticosteroids. Louvet et al. showed that combining MELD plus Lille in a joint-effect model was a well-fitting prognostic model, helping to more accurately predict mortality at 2 and 6 months on a continuum from 0 to 100%, rather than simply classifying patients into responders and non-responders.
      • Louvet A.
      • Labreuche J.
      • Artru F.
      • et al.
      Combining data from liver disease scoring systems better predicts outcomes of patients with alcohol-associated hepatitis.
      This approach may be helpful in future early-phase clinical trials of pharmacological treatment, whereby patients with low baseline static score could be enrolled into a trial and the intervention continued if the patient is not worsening at day 7.
      • Louvet A.
      • Labreuche J.
      • Artru F.
      • et al.
      Combining data from liver disease scoring systems better predicts outcomes of patients with alcohol-associated hepatitis.
      One of the key uses for biomarkers will be identifying patients who will not respond to and who should be excluded from receiving certain treatments, such as steroids.
      • Vergis N.
      • Atkinson S.R.
      • Knapp S.
      • et al.
      In patients with severe alcohol-associated hepatitis, prednisolone increases susceptibility to infection and infection-related mortality, and is associated with high circulating levels of bacterial DNA.
      ,
      • Thursz M.R.
      • Richardson P.
      • Allison M.
      • et al.
      Prednisolone or pentoxifylline for alcohol-associated hepatitis.
      Patients with the most severe AH often have co-morbidities that preclude them from receiving steroids (such as hepatorenal syndrome, gastrointestinal bleeding, or sepsis). It is important to remember that the patients included in randomized controlled trials of treatment in AH were those who had none of the exclusionary co-morbidities and were stable enough to wait for the prerequisite liver biopsy in some trials. This makes obtaining real-world data and finding effective therapies for severe disease more difficult.

      EMERGING BIOMARKERS IN AH

      Most of the biomarkers in AH that will be discussed are only available in research settings; those that are clinically available are outlined in Table 2.
      Table 2Emerging Biomarkers in Alcohol-associated Hepatitis.
      BiomarkerStudy typeClinically available?Sensitivity/specificity (if available)Study
      Diagnostic Biomarkers
      PNPLA3 gene polymorphismHumanYesN/aSalameh
      • Salameh H.
      • Raff E.
      • Erwin A.
      • et al.
      PNPLA3 gene polymorphism is associated with predisposition to and severity of alcohol-associated liver disease.
      White blood count >11K and platelets <148KHumanYesSensitivity = 44%, specificity = 100% for diagnosis of AH (PPV 100%, NPV 38%)Hardy
      ANI scoreHumanYesSensitivity = 93.5%, specificity of 92% to distinguish AH from NASHDunn
      • Dunn W.
      • Angulo P.
      • Sanderson S.
      • et al.
      Utility of a new model to diagnose an alcohol basis for steatohepatitis.
      Breath trimethylamine, acetone and pentaneHumanNoSensitivity = 90%, specificity = 80% to distinguish AH from healthy control or alcohol-associated cirrhosisHanouneh

      Hanouneh IA, Zein NN, Cikach F, et al. The breathprints in patients with liver disease identify novel breath biomarkers in alcohol-associated hepatitis. Clin Gastroenterol Hepatol. 2014;12:516-523.

      AshTestHumanYesSensitivity = 80%, specificity of 84% for diagnosis of AHThabut,
      • Thabut D.
      • Naveau S.
      • Charlotte F.
      • et al.
      The diagnostic value of biomarkers (AshTest) for the prediction of alcohol-associated steato-hepatitis in patients with chronic alcohol-associated liver disease.
      Rudler
      • Rudler M.
      • Mouri S.
      • Charlotte F.
      • et al.
      Validation of AshTest as a non-invasive alternative to transjugular liver biopsy in patients with suspected severe acute alcohol-associated hepatitis.
      Cytokeratin-18 M65 level: ALT ratioHumanNoSensitivity = 97.1%, specificity = 82.9% to distinguish AH from NASHVatsalya
      • Vatsalya V.
      • Cave M.C.
      • Kong M.
      • et al.
      Keratin 18 is a diagnostic and prognostic factor for acute alcohol-associated hepatitis.
      Cytokeratin-18 M65 levelHumanNoM65 > 2000 IU/L: sensitivity = 67%, specificity = 92% (PPV of 91%)

      M65 < 641 IU/L: sensitivity = 93%, specificity = 62% (NPV of 88%) for diagnosis of AH.
      Bissonnette
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcohol-associated hepatitis.
      Cytokeratin-18 M65 and M30 levelsHumanNoN/aWoolbright
      • Woolbright B.L.
      • Bridges B.W.
      • Dunn W.
      • Olson J.C.
      • Weinman S.A.
      • Jaeschke H.
      Cell death and prognosis of mortality in alcohol-associated hepatitis patients using plasma keratin-18.
      Fibroblast growth factor (FGF) 21MouseNoN/aGrigorios
      • Grigorios C.
      • Karatayli E.
      • Hall R.
      • Weber S.N.
      • Lammert F.
      • Karatayli S.C.
      Fibroblast growth factor 21 response in a preclinical model of alcohol induced acute-on-chronic liver injury.
      miRNA-192,

      miRNA-122,

      miRNA-30a
      MouseNomiR-192

      AUC = 0.96, miRNA-122 AUC = 0.92,

      miRNA-30a AUC = 0.85 to distinguish alcohol-fed mice from controls
      Momen-Heravi
      • Momen-Heravi F.
      • Saha B.
      • Kodys K.
      • Catalano D.
      • Satishchandran A.
      • Szabo G.
      Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcohol-associated hepatitis.
      miRNA-192, miRNA-30aHumanNomiRNA-192 AUC = 0.95, miRNA-30a AUC = 0.58 for diagnosis of AH (vs. control)Momen-Heravi
      • Momen-Heravi F.
      • Saha B.
      • Kodys K.
      • Catalano D.
      • Satishchandran A.
      • Szabo G.
      Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcohol-associated hepatitis.
      miRNA-155 in Kupffer cellsMouseNoHigher in alcohol-fed vs. control mice and correlates with TNF-alpha inductionBala
      • Bala S.
      • Csak T.
      • Momen-Heravi F.
      • et al.
      Biodistribution and function of extracellular miRNA-155 in mice.
      Prognostic and Theragnostic Biomarkers
      Cytokeratin-18 M30/M65 ratio (cut-off of 0.3884)HumanNoSensitivity = 90%, specificity = 86% for 30-day mortalityWoolbright
      • Woolbright B.L.
      • Bridges B.W.
      • Dunn W.
      • Olson J.C.
      • Weinman S.A.
      • Jaeschke H.
      Cell death and prognosis of mortality in alcohol-associated hepatitis patients using plasma keratin-18.
      Cytokeratin-18 M65 levels (cut-off of 8403 U/L)HumanNoSensitivity = 80%, specificity = 79% for 30-day mortalityWoolbright
      • Woolbright B.L.
      • Bridges B.W.
      • Dunn W.
      • Olson J.C.
      • Weinman S.A.
      • Jaeschke H.
      Cell death and prognosis of mortality in alcohol-associated hepatitis patients using plasma keratin-18.
      Cytokeratin-18 M65 & M30 levelsHumanNoAUC 0.652 for M65, AUC 0.744 for M30 for 90-day mortalityVatsalya
      • Vatsalya V.
      • Cave M.C.
      • Kong M.
      • et al.
      Keratin 18 is a diagnostic and prognostic factor for acute alcohol-associated hepatitis.
      Cytokeratin-18 M30 level (cut-off of >5000 U/L)HumanNoPredicts patients who will benefit from prednisolone at 90 daysAtkinson
      • Atkinson S.R.
      • Grove J.I.
      • Liebig S.
      • et al.
      In severe alcohol-associated hepatitis, serum cytokeratin-18 fragments are diagnostic, prognostic and theragnostic biomarkers.
      ASCA levelsHumanYesSensitivity = 97%, specificity = 47%. PPV = 49%, NPV = 97% for 90-day mortalityLang
      • Lang S.
      • Duan Y.
      • Liu J.
      • et al.
      Intestinal fungal dysbiosis and systemic immune response to fungi in patients with alcohol-associated hepatitis.
      ASCA levels + MELD scoreHumanYesSensitivity = 74%, specificity = 81%, PPV = 67%, NPV = 86% for 90-day mortalityLang
      • Lang S.
      • Duan Y.
      • Liu J.
      • et al.
      Intestinal fungal dysbiosis and systemic immune response to fungi in patients with alcohol-associated hepatitis.
      MicroRNA-182 expression in the liverHumanNoSensitivity = 79%, specificity = 74% for 90-day mortalityBlaya
      • Blaya D.
      • Coll M.
      • Rodrigo-Torres D.
      • et al.
      Integrative microRNA profiling in alcohol-associated hepatitis reveals a role for microRNA-182 in liver injury and inflammation.
      MELD cut-off of 11HumanYesSensitivity = 81%, specificity = 86% for 30-day mortalitySheth
      • Sheth M.
      • Riggs M.
      • Patel T.
      Utility of the Mayo End-Stage Liver Disease (MELD) score in assessing prognosis of patients with alcohol-associated hepatitis.
      Week 1 MELD cut-off of 20HumanYesSensitivity = 91%, specificity = 85% for in-hospital mortalitySrikureja
      • Srikureja W.
      • Kyulo N.L.
      • Runyon B.A.
      • Hu K.-Q.
      MELD score is a better prognostic model than Child-Turcotte-Pugh score or Discriminant Function score in patients with alcohol-associated hepatitis.
      ABIC score cut-off of 8.555HumanYesSensitivity = 80%, specificity = 79% for 30-day mortality (AUC = 0.8)Woolbright
      • Woolbright B.L.
      • Bridges B.W.
      • Dunn W.
      • Olson J.C.
      • Weinman S.A.
      • Jaeschke H.
      Cell death and prognosis of mortality in alcohol-associated hepatitis patients using plasma keratin-18.
      Maddrey's modified DF cut-off of 32HumanYesSensitivity = 83%, specificity = 60% for in-hospital mortality; AUC of 0.673 and 0.670 for 28-day and 90-day mortality respectivelySrikureja
      • Srikureja W.
      • Kyulo N.L.
      • Runyon B.A.
      • Hu K.-Q.
      MELD score is a better prognostic model than Child-Turcotte-Pugh score or Discriminant Function score in patients with alcohol-associated hepatitis.


      Forrest
      • Forrest E.H.
      • Atkinson S.R.
      • Richardson P.
      • et al.
      Application of prognostic scores in the STOPAH trial: discriminant function is no longer the optimal scoring system in alcohol-associated hepatitis.
      Dynamic score of baseline MELD and Lille scoreHumanYesAkaike Information Criterion score (measure derived from the likelihood model) of 1305, c-statistic of 0.75 for 2- and 6-month mortalityLouvet
      • Louvet A.
      • Labreuche J.
      • Artru F.
      • et al.
      Combining data from liver disease scoring systems better predicts outcomes of patients with alcohol-associated hepatitis.
      Total and conjugated bile acidsHumanYesCorrelates with MELD scoreBrandl
      • Brandl K.
      • Hartmann P.
      • Jih L.J.
      • et al.
      Dysregulation of serum bile acids and FGF19 in alcohol-associated hepatitis.
      Bile acid profileHumanNoIncrease in C4 from healthy controls to moderate AH to severe AH (P < 0.005 for both); decrease in intestinal microbial metabolite isoursodeoxycholic acid also correlates with severity.Muthiah
      • Muthiah M.
      • Smirnova E.
      • Puri P.
      • et al.
      The development and severity of alcohol-associated hepatitis is associated with decreased intestinal microbiome-derived secondary bile acid isoursodeoxycholic acid.
      Serum fibroblast growth factor (FGF) 19HumanYesCorrelates with fibrosis stage and in patients with MELD≥29, correlates with 30-day mortality126Brandl
      • Brandl K.
      • Hartmann P.
      • Jih L.J.
      • et al.
      Dysregulation of serum bile acids and FGF19 in alcohol-associated hepatitis.
      E. faecalis cytolysinHumanNoAUC of 0.81 for 90-day mortalityDuan
      • Duan Y.
      • Llorente C.
      • Lang S.
      • et al.
      Bacteriophage targeting of gut bacterium attenuates alcohol-associated liver disease.
      Plasma metabolome signaturesHumanNoBaseline levels of l-kynurenine, indoleacrylic acid, lecithin, and prostaglandin B1 are predictors of non-response to steroids and non-survival (AUROC>0.80; P < 0.05)Maras
      • Maras J.
      • Bhat A.
      • Yadav G.
      • Thakur A.
      • Sarin S.K.
      Temporal change in the plasma metabolome profile is indicative of outcome in severe alcohol-associated hepatitis.
      Abbreviations: ASCA, anti-Saccharomyces cerevisiae antibodies; AUC, area under the curve; FGF, fibroblast growth factor; MELD, model for end-stage liver disease; N/a, not available; NPV, negative predictive value; PNPLA3, patatin-like phospholipase domain-containing protein 3; PPV, positive predictive value.

      Diagnostic Biomarkers

      ANI Score

      Dunn et al. developed the ALD/NAFLD Index (ANI score) to distinguish between ASH and NASH, independent of recent alcohol consumption. It incorporates several widely available variables: BMI, gender, MCV, and AST:ALT ratio.
      • Dunn W.
      • Angulo P.
      • Sanderson S.
      • et al.
      Utility of a new model to diagnose an alcohol basis for steatohepatitis.
      Gamma-glutamyl transferase (GGT) itself also showed promise in differentiating between ASH and NASH, but GGT values were available for only 31% of patients in this study. In clinical practice, the ANI score has significant value for its ease of use, especially in patients with heavy alcohol use where MCV and transaminase ratios would be more altered. However, its reliance on transaminase ratios however would limit its use in patients with NASH and with advanced fibrosis, where the ratios more resemble patients with ASH.

      AshTest

      The AshTest is proprietary panel of 10 serum biomarkers to diagnose ASH in a cohort of patients with heavy alcohol use (>50 g/day).
      • Thabut D.
      • Naveau S.
      • Charlotte F.
      • et al.
      The diagnostic value of biomarkers (AshTest) for the prediction of alcohol-associated steato-hepatitis in patients with chronic alcohol-associated liver disease.
      At a cut-off of 0.50, it could identify ASH with a sensitivity of 0.80 and a specificity of 0.84. The AshTest correlated strongly with a histologic grade of AH, with particularly high predictive values in severe histological cases, and is proposed as an alternative to mDF for determining which patients would benefit from corticosteroids.
      • Thabut D.
      • Naveau S.
      • Charlotte F.
      • et al.
      The diagnostic value of biomarkers (AshTest) for the prediction of alcohol-associated steato-hepatitis in patients with chronic alcohol-associated liver disease.
      In 2015, the test was validated in a cohort of 123 patients. It correlated better with a histological grade of AH than MELD or mDF, identified patients who should be treated with steroids with similar accuracy to liver biopsy but did not have any prognostic value.
      • Rudler M.
      • Mouri S.
      • Charlotte F.
      • et al.
      Validation of AshTest as a non-invasive alternative to transjugular liver biopsy in patients with suspected severe acute alcohol-associated hepatitis.
      AshTest is commercially available as the ASH FibroSure test from LabCorp and also provides quantitative fibrosis and steatosis data.

      Breathprints

      An emerging diagnostic biomarker is the “breathprint” in patients with AH, characterized by high levels of trimethylamine (TMA), acetone, and pentane, described as the TAP score. Using a cut-off of 36 distinguishes patients with AH compared to healthy controls or patients with decompensated AC with 90% sensitivity and 80% specificity.

      Hanouneh IA, Zein NN, Cikach F, et al. The breathprints in patients with liver disease identify novel breath biomarkers in alcohol-associated hepatitis. Clin Gastroenterol Hepatol. 2014;12:516-523.

      Elevated TMA levels in the breath and urine of patients with ALD was discovered in the 1950s when it was observed that these patients had a characteristic unpleasant smell from their breath and urine.
      • Wranne L.
      Urinary excretion of trimethylamine and trimethylamine oxide following trimethylamine-administration to normals and to patients with liver disease.
      It is believed that elevated TMA levels may be due to decreased ability of the liver to convert TMA to trimethylamine oxide in liver disease,
      • Wranne L.
      Urinary excretion of trimethylamine and trimethylamine oxide following trimethylamine-administration to normals and to patients with liver disease.
      and it has been shown that TMA levels correlate with MELD score.

      Hanouneh IA, Zein NN, Cikach F, et al. The breathprints in patients with liver disease identify novel breath biomarkers in alcohol-associated hepatitis. Clin Gastroenterol Hepatol. 2014;12:516-523.

      Another theory is that alcohol-induced intestinal bacteria overgrowth, increased gut permeability, and translocation of LPS to the liver results in increased TMA levels.
      • Rao R.
      Endotoxemia and gut barrier dysfunction in alcohol-associated liver disease.
      It is unknown whether TMA represents a therapeutic target in AH, or whether it is just an innocent bystander and marker of damage. The “breathprint” is an attractive non-invasive marker but larger studies are needed to validate and determine its prognostic value.

      Diagnostic and Prognostic Biomarkers

      Cytokeratin-18

      Cytokeratin-18 is an intermediate filament protein, present in simple epithelial tissues, the main component of Mallory bodies, and previously used as a tumor marker.
      • Omary M.B.
      • Ku N.-O.
      • Strnad P.
      • Hanada S.
      Toward unraveling the complexity of simple epithelial keratins in human disease.
      It is the most promising diagnostic and prognostic biomarker to date. Over 20 years ago, circulating cytokeratin-18 levels were shown to correlate with liver cell necrosis and hyaline degeneration on biopsy in AH.
      • Gonzalez-Quintela A.
      • Mella C.
      • Perez L.
      • Abdulkader I.
      • Caparrini A.
      • Lojo S.
      Increased serum tissue polypeptide specific antigen (TPS) in alcohol-associateds: a possible marker of alcohol-associated hepatitis.
      ,
      • Gonzalez-Quintela A.
      • Garcia J.
      • Campos J.
      • et al.
      Serum cytokeratins in alcohol-associated liver disease: contrasting levels of cytokeratin-18 and cytokeratin-19.
      The theory is that excess serum CK fragments are derived from hepatocyte necrosis.
      • Kramer G.
      • Erdal H.
      • Mertens H.J.
      • et al.
      Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18.
      Bissonnette et al. used a test cohort of 151 consecutive patients undergoing transjugular liver biopsy for the clinical suspicion of AH: 83 in the test cohort with median MELD of 20 and mDF of 46, and 68 in the validation cohort. They showed that specific circulating fragments of CK-18, M30, and M65 epitopes could be a very useful non-invasive diagnostic tool in AH. M65 was particularly valuable marker, where a cut-off of over 2000 IU/L had a positive predictive value of 91% and less than 641 IU/L had a negative predictive value of 88% for the diagnosis of AH. Using their proposed algorithm with the aforementioned cut-offs could obviate the need for liver biopsy in two-thirds of cases.
      • Bissonnette J.
      • Altamirano J.
      • Devue C.
      • et al.
      A prospective study of the utility of plasma biomarkers to diagnose alcohol-associated hepatitis.
      A study of 87 patients who underwent liver biopsy for suspicion of AH, showed that a specific plasma N-glycomic profile (log NA3Fb/NG1A2F) combined with CK-18 M65 had a diagnostic accuracy for AH of 97% and could avoid liver biopsy in 55% of patients with ABIC ≥ 6.71.
      • Xavier V.
      • Geerts A.
      • Meuris L.
      • et al.
      Plasma protein glycomics combined with circulating fragments of cytokeratin-18 are reliable biomarkers to diagnose alcohol-associated hepatitis.
      Woolbright et al. found that levels of M30 and M65 could differentiate AH from AC, and M65 levels and the M30/M65 ratio were predictive of 30-day mortality in AH, with the M30/M65 ratio outperforming MELD and ABIC scores.
      • Woolbright B.L.
      • Bridges B.W.
      • Dunn W.
      • Olson J.C.
      • Weinman S.A.
      • Jaeschke H.
      Cell death and prognosis of mortality in alcohol-associated hepatitis patients using plasma keratin-18.
      Another study showed that M30 and M65 outperformed four biomarkers (mDF, MELD, ABIC, and GAHS) in predicting 90-day mortality.
      • Vatsalya V.
      • Cave M.C.
      • Kong M.
      • et al.
      Keratin 18 is a diagnostic and prognostic factor for acute alcohol-associated hepatitis.
      A more recent post-hoc analysis of 824 patients from the STOPAH trial showed that using CK-18 fragments could have rendered diagnostic biopsy unnecessary in 84% of all AH cases and that CK-18 strongly predicted 90-day mortality and responsiveness to steroids.
      • Atkinson S.R.
      • Grove J.I.
      • Liebig S.
      • et al.
      In severe alcohol-associated hepatitis, serum cytokeratin-18 fragments are diagnostic, prognostic and theragnostic biomarkers.
      In those with M30 > 5000 U/L, prednisolone was associated with a significant reduction in mortality at 90 days (odds ratio 0.43, 95% CI 0.19–0.95, P = 0.040).
      • Atkinson S.R.
      • Grove J.I.
      • Liebig S.
      • et al.
      In severe alcohol-associated hepatitis, serum cytokeratin-18 fragments are diagnostic, prognostic and theragnostic biomarkers.
      This suggests that M30 may be able to identify a subset of patients with significant inflammation who will benefit from steroids. Of note, CK-18 levels are also elevated in NASH, where levels seem to correlate with fibrosis.
      • Feldstein A.E.
      • Wieckowska A.
      • Lopez A.R.
      • Liu Y.C.
      • Zein N.N.
      • McCullough A.J.
      Cytokeratin-18 fragment levels as noninvasive biomarkers for nonalcohol-associated steatohepatitis: a multicenter validation study.
      The M65:ALT ratio may help differentiate patients with AH from those with NASH.
      • Vatsalya V.
      • Cave M.C.
      • Kong M.
      • et al.
      Keratin 18 is a diagnostic and prognostic factor for acute alcohol-associated hepatitis.

      Genetic Markers

      There are several significant genetic factors relating to prognosis in AH. The gene signature MELD (gs-MELD) score used the expression pattern of 123 genes on liver biopsy plus MELD score to predict 90-day survival in AH with an area under the curve of 0.86.
      • Trépo E.
      • Goossens N.
      • Fujiwara N.
      • et al.
      Combination of gene expression signature and model for end-stage liver disease score predicts survival of patients with severe alcohol-associated hepatitis.
      Variants in genes such as PNPLA3, transmembrane 6 superfamily member 2 (TM6SF2), and membrane bound O-acyltransferase domain-containing 7 (MBOAT7) have been shown to increase the risk of ALD (PNPLA3 more associated with steatosis and inflammation; MBOAT7 more with fibrosis and cirrhosis).
      • Salameh H.
      • Raff E.
      • Erwin A.
      • et al.
      PNPLA3 gene polymorphism is associated with predisposition to and severity of alcohol-associated liver disease.
      ,
      • Buch S.
      • Stickel F.
      • Trépo E.
      • et al.
      A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.
      ,
      • Mueller J.
      • Rausch V.
      • Peccerella T.
      • Elshaarawy O.
      • Mueller S.
      The role of PNPLA3, MBOAT7 and TM6SF2 during alcohol detoxification: different mechanisms of fibrosis and steatosis development.
      Polymorphisms in PNPLA3 and TM6SF2 are associated with a higher risk of HCC in persons with AC.
      • Stickel F.
      • Buch S.
      • Nischalke H.D.
      • et al.
      Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis.
      Persons of Hispanic ethnicity are at a higher risk for ALD, including AH, than non-Hispanic white and African-American persons, which is at least partially related to genetics.
      • Levy R.E.
      • Catana A.M.
      • Durbin-Johnson B.
      • Halsted C.H.
      • Medici V.
      Ethnic differences in presentation and severity of alcohol-associated liver disease.
      PNPLA3 increases the predisposition to developing ALD and is associated with more severe disease.
      • Sanyal A.J.
      • Gao B.
      • Szabo G.
      Gaps in knowledge and research priorities for alcohol-associated hepatitis.
      A genome-wide association study from the STOPAH trial found that homozygosity for rs738409:G in PNPLA3 independently conferred significant additional risk of medium-term mortality in patients with severe AH with hazard ratio of 1.69.
      • Salameh H.
      • Raff E.
      • Erwin A.
      • et al.
      PNPLA3 gene polymorphism is associated with predisposition to and severity of alcohol-associated liver disease.
      ,
      • Atkinson S.R.
      • Way M.J.
      • McQuillin A.
      • Morgan M.Y.
      • Thursz M.R.
      Homozygosity for rs738409: G in PNPLA3 is associated with increased mortality following an episode of severe alcohol-associated hepatitis.
      Polymorphisms in the genes encoding members of the alcohol dehydrogenase (ADH) family are associated with an increased risk of alcohol use disorder and ALD in Asian populations, but do not correlate with the risk of ALD in Caucasians.
      • Crabb D.W.
      • Matsumoto M.
      • Chang D.
      • You M.
      Overview of the role of alcohol dehydrogenase and aldehyde dehydrogenase and their variants in the genesis of alcohol-related pathology.
      ,
      • Charatcharoenwitthaya P.
      • Liangpunsakul S.
      • Piratvisuth T.
      Alcohol-associated liver disease: east versus west.
      Patients with variants encoding for high levels of enzyme activity (ADH1B∗2 and ADH1C∗1 alleles) are believed to be at risk for ALD because of the accumulation of a toxic alcohol metabolite, acetaldehyde. Recently, a polymorphism encoding for the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) was shown to be protective against ALD, AH, AC, and non-ALD.
      • Atkinson S.
      • Buckley T.
      • Strnad P.
      • Thursz M.
      • McQuillin A.
      • Morgan M.
      Genetic variation in HSD17B3 reduces the risk for developing severe alcohol-associated hepatitis.
      Interestingly, the HSD17B13 variant mitigated the risk of liver injury associated with the PNPLA3 variant.
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      There is a strong genetic component to ALD and AH, and the discovery of biomarkers like PNPLA3 has just scratched the surface. Hopefully in the future, we will be able to use a patient's genomics to prognosticate, predict response to therapy, and eventually tailor therapy.

      Prognostic Biomarkers and Therapeutic Targets

      MicroRNAs

      MicroRNAs (miRNAs) are small non-coding RNAs of ∼22 nucleotides, found in circulating extracellular vesicles (EVs) or exosomes, and regulate a significant portion of the human genome. miRNAs, such as miRNA-155, regulate genes involved in TNF-alpha regulation, inflammatory response to endotoxin, and various other immune responses, as well as LPS and oxidative stress signaling pathways.
      • Bala S.
      • Szabo G.
      MicroRNA signature in alcohol-associated liver disease.
      Alcohol-fed mice demonstrate increased levels of EVs and human subjects with AH have been shown to have higher levels of miRNA-192 and miRNA-30a than healthy individuals.
      • Alexander M.
      • Hu R.
      • Runtsch M.C.
      • et al.
      Exosome-delivered microRNAs modulate the inflammatory response to endotoxin.
      Animal models suggest that miRNA-182 is associated with cholangiocyte damage and ductular reaction, both of which are poor prognostic factors in AH.
      • Momen-Heravi F.
      • Saha B.
      • Kodys K.
      • Catalano D.
      • Satishchandran A.
      • Szabo G.
      Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcohol-associated hepatitis.
      In vivo human studies show that miRNA-182 is highly expressed in AH, and that its expression on liver biopsy but not in the serum correlates with disease severity and short-term mortality, so it may not be a practical biomarker.
      • Sancho-Bru P.
      • Altamirano J.
      • Rodrigo-Torres D.
      • et al.
      Liver progenitor cell markers correlate with liver damage and predict short-term mortality in patients with alcohol-associated hepatitis.
      Alcohol affects the expression of other miRNAs, such as miRNA-212 which regulates gut permeability and sensitizes macrophages to LPS-driven pro-inflammatory response,
      • Blaya D.
      • Coll M.
      • Rodrigo-Torres D.
      • et al.
      Integrative microRNA profiling in alcohol-associated hepatitis reveals a role for microRNA-182 in liver injury and inflammation.
      ,
      • Tang Y.
      • Banan A.
      • Forsyth C.B.
      • et al.
      Effect of alcohol on miR-212 expression in intestinal epithelial cells and its potential role in alcohol-associated liver disease.
      and miRNA-199 which up-regulates endothelin-1
      • Momen-Heravi F.
      • Bala S.
      • Kodys K.
      • Szabo G.
      Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.
      and hypoxia-inducible factor-1α, thus increasing inflammation and steatosis.
      • Yeligar S.
      • Tsukamoto H.
      • Kalra V.K.
      Ethanol-induced expression of ET-1 and ET-BR in liver sinusoidal endothelial cells and human endothelial cells involves hypoxia-inducible factor-1α and microRNA-199.
      Circulating miRNAs, together with cytokines or other biomarkers, may eventually compose a powerful prognostication tool and possible therapeutic target, but the studies remain preliminary and small in size.

      EVs as Tailored Treatment

      EVs which originate from hepatocytes have the ability to modulate liver macrophages and regulate liver regeneration.
      • Yáñez-Mó M.
      • Siljander P.R.-M.
      • Andreu Z.
      • et al.
      Biological properties of extracellular vesicles and their physiological functions.
      EVs present an exciting opportunity for drug delivery, having the ability to deliver tissue-targeted miRNA which can regulate gene expression in the host target cell, and early studies have shown promise in using EVs to deliver therapy in liver disease.
      • Bala S.
      • Csak T.
      • Momen-Heravi F.
      • et al.
      Biodistribution and function of extracellular miRNA-155 in mice.
      For example, Szabo et al. found that EVs could deliver a miRNA-155 inhibitor in vivo resulting in decreased TNF-alpha levels in alcohol fed-mice.
      • Momen-Heravi F.
      • Bala S.
      • Bukong T.
      • Szabo G.
      Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages.
      ,
      • Bala S.
      • Marcos M.
      • Kodys K.
      • et al.
      Up-regulation of microRNA-155 in macrophages contributes to increased tumor necrosis factor α (TNFα) production via increased mRNA half-life in alcohol-associated liver disease.
      The eventual hope is that miRNA manipulation in human subjects with AH could down-regulate the inflammatory pathways. EVs can be extracted and stored for years, so potentially stem-cell-derived EVs could be isolated from patients with early stage liver disease and manipulated for future “personalized medicine” if the patient develops more advanced liver disease.
      • Fleury A.
      • Martinez M.C.
      • Le Lay S.
      Extracellular vesicles as therapeutic tools in cardiovascular diseases.
      Although a very attractive concept, it is important to remember that the safety of EVs is not fully known, particularly relating to carcinogenesis, and regulation standards still need to be established.
      • Szabo G.
      • Momen-Heravi F.
      Extracellular vesicles in liver disease and potential as biomarkers and therapeutic targets.

      Microbiome as Biomarker

      The microbiome is an emerging biomarker with largely unrealized potential, especially given the close relationship between the gut and the liver. Schnabl et al. performed a transcriptome and metabolomic analysis of chronic and binge ethanol-fed mice and found that ethanol up-regulated and down-regulated genes which controlled immune response, inflammation, fatty acid oxidation, apoptosis, and glucose, triglyceride and bile acid metabolism.
      • Jiang L.
      • Chu H.
      • Gao B.
      • et al.
      Transcriptomic profiling identifies novel hepatic and intestinal genes following chronic plus binge ethanol feeding in mice.
      They found that the Prok2 gene which encodes for prokineticin 2, an inflammatory cytokine-like molecule, was down-regulated in ethanol-fed mice. Prok2 correlated negatively with abundance of the bacteria Allobaculum in the gut, opening up exciting possibilities of manipulating Prok2 expression in patients with ALD, as a method of controlling inflammation and/or the gut microbiome.
      The gut microbiome is significantly altered in AH, and the microbiome contributes to the susceptibility to and severity of AH (Figure 3).
      • Llopis M.
      • Cassard A.
      • Wrzosek L.
      • et al.
      Intestinal microbiota contributes to individual susceptibility to alcohol-associated liver disease.
      Specifically, cytolytic E. fecalis is increased and correlates directly with mortality in AH. A mouse study found that bacteriophages which target and reduce cytolytic E. faecalis in the gut could abolish ethanol-induced liver disease in mice, presenting an exciting future therapeutic target.
      • Duan Y.
      • Llorente C.
      • Lang S.
      • et al.
      Bacteriophage targeting of gut bacterium attenuates alcohol-associated liver disease.
      Additionally, a pilot study in humans has shown that fecal microbiota transplant in AH is safe and may improve clinical outcomes.
      • Philips C.A.
      • Pande A.
      • Shasthry S.M.
      • et al.
      Healthy donor fecal microbiota transplantation in steroid-ineligible severe alcohol-associated hepatitis: a pilot study.
      The intestinal virome is also significantly altered in AH, with certain viral taxa associated with higher MELD scores and decreased 90-day mortality.

      Jiang L, Lang S, Duan Y, et al. Intestinal virome in patients with alcohol-associated hepatitis. Hepatology. 2020 Dec;72:2182-2196. doi: 10.1002/hep.31459. Epub 2020 Oct 10. PMID: 32654263; PMCID: PMC8159727.

      Figure 3
      Figure 3Pathophysiology of alcohol-associated hepatitis changes in the gut microbiome mediate liver inflammation.
      • Delphine D.
      • Stauber R.E.
      • Gaël E.
      • et al.
      Long-term outcome of symptomatic alcohol-associated hepatitis with a Maddrey discriminant function< 32.
      (from Seminars in Liver Disease, Thieme; with permission).
      Patients with ALD have decreased gut fungal diversity with overgrowth of Candida species. Serum anti–Saccharomyces cerevisiae antibodies (ASCA) can be used as a surrogate of systemic immune response to fungi. Patients with AH have higher ASCA levels than controls or patients with alcohol use disorder without AH, and higher ASCA levels were associated with lower 90-day survival in AH.
      • Lang S.
      • Duan Y.
      • Liu J.
      • et al.
      Intestinal fungal dysbiosis and systemic immune response to fungi in patients with alcohol-associated hepatitis.

      Cytokines

      Cytokines are mediators of cellular communication, which are produced by many cells in the liver. It has been shown that abnormal cytokine metabolism is a key feature of AH, and that cytokines contribute to inflammation (such as IL-6 and IL-8), increase oxidative stress (TNF-alpha), regulate liver regeneration (IL-6, TNF-alpha, hepatocyte growth factor, tissue growth factor-alpha), control fibrinogenesis (TGB-beta-1), and induce hepatocyte death.
      • McClain C.J.
      • Song Z.
      • Barve S.S.
      • Hill D.B.
      • Deaciuc I.
      Recent advances in alcohol-associated liver disease IV. Dysregulated cytokine metabolism in alcohol-associated liver disease.
      Levels of TNF-alpha and pro-inflammatory cytokines seem to correlate with clinical outcomes in ALD so cytokine manipulation presents an ideal therapeutic target.
      • McClain C.J.
      • Barve S.
      • Deaciuc I.
      • Kugelmas M.
      • Hill D.
      Cytokines in alcohol-associated liver disease.
      Steroids work in AH by inhibiting the proinflammatory cytokine production. Ideally, future pharmacologic therapies could neutralize circulating cytokines or cytokine receptors, administer anti-inflammatory cytokines, or inhibit cytokine production.
      • McClain C.J.
      • Song Z.
      • Barve S.S.
      • Hill D.B.
      • Deaciuc I.
      Recent advances in alcohol-associated liver disease IV. Dysregulated cytokine metabolism in alcohol-associated liver disease.
      Initially, there was hope that anti-TNF agents, used commonly in autoimmune conditions, could reduce liver inflammation and mortality in AH. However, early studies of high-dose infliximab with prednisone
      • Naveau S.
      • Chollet-Martin S.
      • Dharancy S.
      • et al.
      A double-blind randomized controlled trial of infliximab associated with prednisolone in acute alcohol-associated hepatitis.
      and etanercept
      • Menon K.N.
      • Stadheim L.
      • Kamath P.S.
      • et al.
      A pilot study of the safety and tolerability of etanercept in patients with alcohol-associated hepatitis.
      showed increased rates of infection, gastrointestinal bleeding, and mortality resulting in early discontinuation. This could have been due to the inhibition of liver regeneration by biologic therapy. Cytokines remain an attractive pharmacological target in AH, but may require specific patient selection, and perhaps measurement of cytokine levels to identify the patients who could benefit.

      Newest Biomarkers

      A myriad of new AH biomarker studies have been recently published. Lipidomics have begun to emerge as a promising class of biomarkers in many disease entities, and ALD is no exception. Thiele et al. showed that sphingomyelins decrease significantly in the peripheral circulation and the liver, and negatively correlate with fibrosis stage.
      • Thiele M.
      • Trost K.
      • Suvitaival T.
      • et al.
      Lipidomics profiling reveals a distinct pattern of selective lipid depletion in the circulation and liver tissue in patients with progressive alcohol-associated liver fibrosis: a biopsy-controlled study in 400 people.
      Lipid metabolites (plasma oxylipin levels) have been shown to differ between severe AH, moderate AH, and socially drinking healthy controls, which may represent future therapeutic targets.
      • Warner J.
      • Zirnheld K.
      • Vatsalya V.
      • et al.
      Alterations and role of bioactive lipid metabolites (oxylipins) in human and experimental ALD: associations with disease severity.
      Another new biomarker showing promise is the bromodeoxyuridine (BrdU) incorporation in lymphocyte steroid sensitivity (BLISS) assay which was able to differentiate between AH survivors and non-survivors with AUROC of 0.95 (95% CI 0.84–1.0) and a sensitivity of 90% in identifying steroid responsive patients with a negative predictive value of 86%.
      • Yates E.
      • Tan H.
      • Dhanda A.
      A novel lymphocyte proliferation assay accurately predicts 90-day survival in severe alcohol-associated hepatitis patients.
      Molecular ellipticity of the albumin-bilirubin complex (measured using circular dichroism spectroscopy) may be correlated with short- and intermediate-term outcomes in severe AH, but the study only evaluated men who were treated with steroids, and has not been externally validated.
      • Das S.
      • Maras J.S.
      • Maiwall R.
      • et al.
      Molecular ellipticity of circulating albumin-bilirubin complex associates with mortality in patients with severe alcohol-associated hepatitis.
      Lastly, dysregulation of plasma and stool bile acids, including serum fibroblast growth factor 19 (FGF19), a major regulator of bile acid synthesis, have been recently discovered in patients with AH to differ compared to heavy drinking controls and AC.
      • Brandl K.
      • Hartmann P.
      • Jih L.J.
      • et al.
      Dysregulation of serum bile acids and FGF19 in alcohol-associated hepatitis.
      ,
      • Muthiah M.
      • Smirnova E.
      • Puri P.
      • et al.
      The development and severity of alcohol-associated hepatitis is associated with decreased intestinal microbiome-derived secondary bile acid isoursodeoxycholic acid.

      CURRENTLY AVAILABLE BIOMARKERS

      At the time of writing this review, biomarkers available for commercial use include PNPLA3 gene testing (via Arup labs) which is associated with increased predisposition to and severity of ALD. For determination of fibrosis, available biomarkers include the blood tests FibroTest™ (FibroSure® in the US) or the enhanced liver fibrosis™ test, and imaging with VCTE (FibroScan® in the US). FibroSure® can be sent via LabCorp but it must be specified whether the patient's diagnosis is that of ASH, NASH, or hepatitis C, and in real-world patients these diagnoses may co-exist. FibroMeter™ combines age, gender, and 5 blood parameters (platelets, GGT, prothrombin ratio, alpha-2-macroglobulin, and AST) and is validated in viral hepatitides, NAFLD, and ALD.
      • Calès P.
      • De Ledinghen V.
      • Halfon P.
      • et al.
      Evaluating the accuracy and increasing the reliable diagnosis rate of blood tests for liver fibrosis in chronic hepatitis C.
      • Dincses E.
      • Yilmaz Y.
      Diagnostic usefulness of FibroMeter VCTE for hepatic fibrosis in patients with nonalcohol-associated fatty liver disease.
      • Calès P.
      • Boursier J.
      • Oberti F.
      • et al.
      FibroMeters: a family of blood tests for liver fibrosis.
      AH is a unique and severe manifestation of ALD that is associated with significant morbidity and mortality, and often is superimposed on undiagnosed liver fibrosis due to years of excess alcohol use. Over the past two decades, studies have revealed more about the pathophysiology of AH, and we now understand that it is associated with cytokine dysregulation, alterations in gene expression, immune response, and the gut microbiome. While lab-based clinical prediction models have improved, significant knowledge gaps remain. There is a need to develop reliable, non-invasive biomarkers with rapid turnaround times for diagnosis and prognosis at a molecular level and develop a “precision medicine” approach, where the specific treatment is tailored to a patient's individual metabolomics, genomics, lipidomics and microbiomics. Further investment in research and large multi-center studies will be needed with a diverse population of well-characterized patients representing the full spectrum of diverse presentations and severity in AH.

      Conflicts of interest

      The author has none to declare.

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