EP4511657A1

EP4511657A1 - Methods and kits for predicting liver-related events in patients suffering from alcohol-related cirrhosis

Info

Publication number: EP4511657A1
Application number: EP23720850.9A
Authority: EP
Inventors: Pierre-Emmanuel RAUTOU
Original assignee: Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Cite
Current assignee: Assistance Publique Hopitaux de Paris APHP; Institut National de la Sante et de la Recherche Medicale INSERM; Universite Paris Cite
Priority date: 2022-04-20
Filing date: 2023-04-19
Publication date: 2025-02-26
Also published as: WO2023203083A1

Abstract

In patients with alcohol-related cirrhosis, prognostic indicators include disease activity, liver function, amount of liver fibrosis and alcohol consumption. Reliable prognostic biomarkers are however lacking. Hepatocyte derived large extracellular vesicles (lEVs) concentrations reflect liver disease activity, but their ability to predict liver-related events is unknown. In the present disclosure, the inventors evaluated hepatocyte lEVs concentrations in 500 patients with Child- Pugh class A alcohol-related cirrhosis, from the prospective multicenter CIRRAL cohort. Ability of these hepatocyte-derived biomarkers, alone or combined with MELD and FibroTest, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption. The inventors showed that in patients with active alcohol consumption at enrollment (n=81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEVs concentration >50 U/L and FibroTest>0.74 had a 62% cumulative incidence of liver-related events at 2 years, versus 8% to 13% in other groups. Thus, the present invention relates to methods and kits for predicting liver-related events in patients suffering from alcohol-related cirrhosis.

Description

METHODS AND KITS FOR PREDICTING LIVER-RELATED EVENTS IN PATIENTS SUFFERING FROM ALCOHOL-RELATED CIRRHOSIS

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular hepatology.

BACKGROUND OF THE INVENTION:

Excessive alcohol consumption is one of the most common causes of cirrhosis worldwide (1,2). In patients with alcohol -related cirrhosis, decompensation of cirrhosis is a turning point in terms of risk of mortality (3), quality of life (4) and costs for the healthcare systems (5). Patients at high-risk of decompensation are the target-of-choice population for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials. Hepatic venous pressure gradient (HVPG) predicts decompensation of cirrhosis, since patients with HVPG >10 mm Hg have a 2-year risk of decompensation of 25%, vs. <10% in patients below this threshold (6,7). However, invasiveness - although minimal - and limited availability of HVPG measurement outside expert centers hinders the use of this procedure in routine clinical practice(8). Non-invasive tools have been proposed to substitute for HVPG in its prediction of clinical outcomes, including transient elastography; unfortunately, these tools have not yet been sufficiently validated in prospective cohorts (9). Therefore, noninvasive biomarkers able to accurately predict decompensation in patients with alcohol-related cirrhosis are needed.

In patients with alcohol -related cirrhosis, various prognostic indicators have been identified reflecting (i) disease activity, (ii) liver function, (iii) amount of fibrosis, and (iv) alcohol consumption (10-12).

Disease activity can be estimated on liver biopsy, and refers to the presence of hepatocyte injury and inflammation (10). Histological features of disease activity have been associated with a poor outcome in patients with alcohol -related liver disease (13). Accordingly, plasma keratin- 18 fragments concentrations, reflecting hepatocyte death, have been associated with mortality in patients with alcohol -related liver disease (14,15). Extracellular vesicles (EVs) are vesicles released in the extracellular space by all cell types upon exposure to stress. EVs can be found in all biological fluids including blood. Composition of EVs reflects the type of stress their mother cell was exposed to. Therefore, EVs content holds great potential for predicting disease trajectory (16). In patients with alcohol -related cirrhosis, concentrations of hepatocyte-derived large EVs (lEVs) are significantly higher in patients with alcoholic hepatitis than in those without (17,18); in addition, hepatocyte lEVs have been associated with mortality in patients with cirrhosis, independently of MELD score (19). However, the usefulness of keratin- 18 and hepatocyte lEVs concentrations to predict liver- related events in patients with Child-Pugh class A alcohol -related cirrhosis has not been evaluated.

The Model for End-Stage Liver Disease (MELD), reflecting liver dysfunction, is currently widely used to allocate organs to patients with severe cirrhosis (20,21). In patients with compensated cirrhosis, MELD >10 has been associated with an increased risk of cirrhosis decompensation (12). However, prognostic value of MELD is limited in patients with compensated cirrhosis, since MELD >10 has only 61% sensitivity and 17% positive predictive value for the prediction of 2 year-decompensation or death (3).

The amount of liver fibrosis, measured on liver biopsies using collagen proportionate area, has been related to HVPG and decompensation or liver-related death in patients with cirrhosis (22,23). FibroTest, estimating the amount of fibrosis, has been related to liver-related events and mortality in large cohorts of patients with alcohol -related liver disease of various fibrosis stage, mixing together patients without and with cirrhosis (13,24). Interest of FibroTest to predict cirrhosis decompensation among patients with Child-Pugh class A alcohol -related cirrhosis has not been evaluated.

Finally, even at the stage of cirrhosis, the course of the disease is largely influenced by alcohol consumption (10,25), abstinent patients having a much lower risk of decompensation than patients with active alcohol consumption (25,26). Alcohol consumption status was usually not taken into account in the studies assessing the ability of biomarkers to predict decompensation in patients with alcohol -related cirrhosis.

SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to methods and kits for predicting liver-related events in patients suffering from alcohol -related cirrhosis.

DETAILED DESCRIPTION OF THE INVENTION:

In patients with alcohol -related cirrhosis, prognostic indicators include disease activity, liver function, amount of liver fibrosis and alcohol consumption. Reliable prognostic biomarkers are however lacking. Hepatocyte derived large extracellular vesicles (lEVs) concentrations reflect liver disease activity, but their ability to predict liver-related events is unknown.

In the present disclosure, the inventors evaluated hepatocyte lEVs concentrations in 500 patients with Child-Pugh class A alcohol -related cirrhosis, from the prospective multicenter CIRRAL cohort. Ability of these hepatocyte-derived biomarkers, alone or combined with MELD and FibroTest, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption.

The inventors show that in patients with active alcohol consumption at enrollment (n=81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEVs concentration >50 U/L and FibroTest>0.74 had a 62% cumulative incidence of liver-related events at 2 years, versus 8% to 13% in other groups.

Therefore, in patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte- derived biomarkers with FibroTest or MELD score identifies patients at high-risk of liver- related events, and could be used for risk stratification and patient selection in clinical trials.

Accordingly, the present invention relates to a method of predicting a liver-related event in a patient suffering from alcohol -related cirrhosis comprising determining the level of hepatocyte derived large extracellular vesicles (lEVs) in a blood sample obtained from the patient wherein said level indicates the risk that the patient will have a liver-related event.

As used herein, the term "cirrhosis" refers to a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue and regenerative nodules (lumps that occur as a result of a process in which damaged tissue is regenerated), leading to loss of liver function. The term “compensated advanced chronic liver disease” or “cACLD” has been introduced to describe asymptomatic patients with either severe fibrosis or compensated cirrhosis who are at high risk of complications. The term “alcohol-related cirrhosis” indicates that cirrhosis is mostly caused by excessive alcohol consumption.

In some embodiments, the patient has a Child-Pugh class A alcohol-related cirrhosis. As used herein, the term the “Child-Pugh score” has its general meaning in the art and refers to the score used to assess the prognosis of chronic liver disease, mainly cirrhosis as described by Child CG, Turcotte JG (1964). "Surgery and portal hypertension". In Child CG (ed.). The liver and portal hypertension. Philadelphia: Saunders, pp. 50-64. Although it was originally used to predict mortality during surgery, it is now used to determine the prognosis, as well as the required strength of treatment and the necessity of liver transplantation. The score employs five clinical measures of liver disease including total bilirubin, serum albumin, prothrombin time prolongation (or INR), ascites and hepatic encephalopathy. Each measure is scored 1-3, with 3 indicating most severe derangement. Chronic liver disease is classified into Child-Pugh class A to C, as depicted in Table A.

Table A: Child-Pugh score and significance.

In some embodiments, the patient has an active alcohol consumption. The term “active alcohol consumption” is defined as either hazardous or harmful of alcohol use, i.e. a consumption of 7 glasses per week or more when the method is carried out (ReidMC, Fiellin DA, O ’Connor PG. Hazardous and Harmful Alcohol Consumption in Primary Care. Archives of Internal Medicine. 1999;159:1681-1689).

As used herein, the term “liver-related event” refers to any event selected from the group consisting of cirrhosis decompensation, clinical ascites, overt hepatic encephalopathy, variceal bleeding, spontaneous bacterial peritonitis, acute kidney injury, increase in Child-Pugh score >2 points, liver transplantation in the absence of hepatocellular carcinoma and/or liver-related deaths in the absence of hepatocellular carcinoma. The method of the present invention is particularly suitable for predicting the liver-related event at 2 years.

As used herein, the term “predicting” refers to the determination of the risk that the patient will develop a liver-related event. Especially, the term “prediction”, as used herein, relates to an individual assessment of any parameter that can be useful in determining the evolution of a patient with respect to the risk of having a liver-related event.

As used herein, the term "risk" in the context of the present invention, relates to the probability that a liver-related event will occur over a specific time period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event may occur, the rate of occurrence of the event. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population.

As used herein, the term “blood sample” means a whole blood, serum, or plasma sample obtained from the patient. Preferably the blood sample, according to the invention, is a plasma sample. A plasma sample may be obtained using methods well known in the art. For example, blood may be drawn from the patient following standard venipuncture procedure on tri-sodium citrate buffer. Plasma may then be obtained from the blood sample following standard procedures including but not limited to, centrifuging the blood sample at about 2500*g for about 15 minutes (room temperature), followed by pipeting of the plasma layer. Platelet-free plasma (PFP) is obtained following a second centrifugation at about 2500*g for 15 min. Analyses can be performed directly on this PFP. Alternatively, extracellular vesicles may be more specifically isolated by further centrifuging the PFP at about 15,000 to about 25,000*g at 4°C or room temperature. Different buffers may be considered appropriate for resuspending the pelleted cellular debris which contains the MPs. Such buffers include reagent grade (distilled or deionized) water and phosphate buffered saline (PBS) pH 7.4. Preferably, PBS buffer (Sheath fluid) or NaCl 0.9% is used.

As used herein the term “extracellular vesicle” or “EV” has its general meaning in the art and denotes a plasma membrane vesicle shed from an apoptotic or activated cell. The surface markers of extracellular vesicles are the same as the cells from they originated. As used herein, the term “large extracellular vesicle” or “lEVs” refers to EVs sedimenting at 2,000 x g (Mateescu B, Kowal EJ, van Balkom BW, Bartel S, Bhattacharyya SN, Buzds El, et al. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper. J Extracell Vesicles (2017) 6: 1286095. )

As sued herein the term “hepatocyte-derived large extracellular vesicle” refers to a extracellular vesicle that derive from hepatocyte. Hepatocytes-derived extracellular vesicles are characterized by the expression of keratin-18.

As used herein the term “keratin-18” or “CK18” has its general meaning in the art and refers to the protein encodes by KRT18 gene (Gene ID 3875). An exemplary human amino acid sequence of cytokeratin-18 is represented by the NCBI reference sequence NP_000215.1.

Standard methods for determining the level of hepatocytes-derived extracellular vesicles in a blood sample are well known in the art. For instance, circulating extracellular vesicles can be isolated from the blood sample by coupling filtration (e.g. through 0.2 pm pore membranes) and contacting them with a set of binding partners directed against the specific surface markers of said extracellular vesicles (Gastroenterology. 2012 Jul; 143(1): 166-76.e6f.

In some embodiments, the binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal, directed against the specific surface marker of extracellular vesicles. Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique; the human B-cell hybridoma technique; and the EBV- hybridoma technique.

In some embodiments, the binding partner is antibody which binds to M65 cytokeratin-18 fragment. As used herein, the term “M65 cytokeratin-18 fragment” refers to the soluble human keratin 18 protein (or “cytokeratin-18,” “CK-18,” “keratin- 18,” “KI 8”) encoded by the KRT18 gene, and is a serum indicator of cellular death. The fragment is specifically recognized by M65 antibody which detects a common epitope present in the full-length protein as well as the 21-kDa caspase cleaved fragment and is thus believed to measure, in addition to apoptosis, intact CK18 that is released from cells undergoing necrosis (Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, Marberger M, Biven K, Shoshan MC, Linder S. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res. 2004;64: 1751 1756.).

In some embodiments, the binding partner of the invention is labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as I¹²³, I¹²⁴, In¹¹¹, Re¹⁸⁶, Re¹⁸⁸. Preferably, the antibodies against the surface markers are already conjugated to a fluorophore (e.g. FITC- conjugated and/or PE-conjugated).

The aforementioned assays may involve the binding of the binding partners to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. The solid surfaces are preferably beads. Since large extracellular vesicles have a diameter of roughly 0.1 to 1 pm, the beads for use in the present invention should have a diameter larger than 1 pm. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic. In addition, the beads are preferably fluorescently labelled.

In some embodiments, an ELISA method is used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said the extracellular vesicle of interest. The blood sample is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

In some embodiments, the method of the present invention comprises i) determining the level of hepatocyte-derived large extracellular vesicles in the blood sample obtained from the patient and ii) comparing the level determined at step i) with a predetermined reference value wherein a difference between the level determined at step i) and the predetermined reference value is indicative of risk of having a liver-related event.

Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of expression levels in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the expression level in a group of reference, one can use algorithmic analysis for the statistic treatment of the determined levels in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is Receiver Operator Characteristic Curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer.

In some embodiments, the predetermined reference value is 50 U/L.

Typically, when the level of hepatocyte-derived large extracellular vesicles is higher than the predetermined reference value, then it is concluded that the risk of having a liver-related event is high. On contrary, when the level of hepatocyte-derived large extracellular vesicles is lower than the predetermined reference value, then it is concluded that that the risk of having a liver- related event is high. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in some embodiments, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off" value as described above. For example, according to this specific embodiment of a "cut-off" value, the outcome can be determined by comparing the expression level with the range of values which are identified. In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centred on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. For example, a patient may be assessed by comparing values obtained by determining the level of hepatocyte-derived large extracellular vesicles, where values greater than 5 reveal a low risk of having a related-liver event and values less than 5 reveal a high risk of having a related-liver event. In some embodiments, a patient may be assessed by comparing values obtained by measuring the level of extracellular vesicles and comparing the values on a scale, where values above the range of 4-6 indicate a low risk of having a liver-related event and values below the range of 4-6 indicate a high risk of having a liver-related event, with values falling within the range of 4-6 indicating an intermediate risk.

In some embodiments, the method of the present invention further comprises determining the MELD score wherein the combination between the level of level of hepatocyte-derived large extracellular vesicle and MELD score indicates the risk of having a liver-related event.

As used herein, the “MELD score” has its general meaning in the art and refers to a scoring system for assessing the severity of chronic liver disease as described in Kamath, Patrick S; Kim, W. Ray (2007). "The model for end-stage liver disease (MELD)". Hepatology. 45 (3): 797-805. doi: 10.1002/hep.21563. PMID 17326206. The score uses the patient's values for serum bilirubin, serum creatinine, and the international normalized ratio for prothrombin time (INR) to predict survival. It is calculated according to the following formula:

MELD = 3.78><ln[serum bilirubin (mg/dL)] + l L2xln[INR] + 9.57><ln[serum creatinine (mg/dL)] + 6.43

Typically, when the MELD score is above 10 (>10) and the level of hepatocyte-derived large extracellular vesicles is higher than the predetermined reference value (e.g. 50 U7L), then it is concluded that the patient is at high risk of having a liver-related event. In some embodiments, the method of the present invention further comprises determining the FibroTest score wherein the combination between the level of level of hepatocyte-derived large extracellular vesicles and FibroTest indicates the risk of having a liver-related event.

As used herein, the term “FibroTest” has its general meaning in the art and refers to a biomarker test to generate a score that is correlated with the degree of liver damage. The FibroTest score is calculated from the results of a six-parameter blood test, combining six serum markers with the age and gender of the patient: Alpha-2-macroglobulin, Haptoglobin, Apolipoprotein Al, Gamma-glutamyl transpeptidase (GGT), Total bilirubin, and Alanine transaminase (ALT). The equation for calculating the FibroTest score regression coefficient (logistic regression) is described in U.S. patent 6,631,330 that is incorporated by reference.

Typically, when the FibroTes score is above 0.74 (>0.74) and the level of hepatocyte-derived large extracellular vesicles is higher than the predetermined reference value (e.g. 50 U/L), then it is concluded that the patient is at high risk of having a liver-related event.

The result given by the method of the invention may be used as a guide in selecting a therapy or treatment regimen for the patient. For example, when the patient has been determined as having a poor prognosis he can be eligible for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials testing new drugs aiming at preventing liver-related events.

A further object of the invention relates to a kit for performing the method of the invention comprising means for determining the level of hepatocyte-derived large extracellular vesicles in a blood sample obtained from said patient. The kit may include filtration means (e.g. filters) and a set of antibodies as above described. In some embodiments, the antibody or set of antibodies are labelled as above described. The kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards. Typically, the kits described above will also comprise one or more other containers, containing for example, wash reagents, and/or other reagents capable of quantitatively detecting the presence of bound antibodies. Typically compartmentalised kit includes any kit in which reagents are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of reagents from one compartment to another compartment whilst avoiding cross-contamination of the samples and reagents, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion. Such kits may also include a container which will accept the blood sample, a container which contains the antibody(s) used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and like), and containers which contain the detection reagent.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Flow chart and number of events

Figure 2. Distribution of keratin-18 concentrations (Fig 2A) and hepatocyte large extracellular vesicles (lEVs) concentrations (Fig 2B) according to alcohol consumption at enrollment

Figure 3. Cumulative incidence of liver-related events within 2 years according to keratin-18 concentrations in patients without active alcohol consumption (Fig 3A) or according to large extracellular vesicles (lEVs) concentrations in patients with active alcohol consumption (Fig 3B) at enrollment

Figure 4. Cumulative incidence of liver-related events within 2 years according to hepatocyte biomarkers concentrations and Fibrotest or MELD. Cumulative incidence of liver-related events within 2 years in patients without active alcohol consumption according to keratin-18 concentrations and FibroTest are displayed in Fig 4A, and according to keratin-18 and MELD in fig 4B. Cumulative incidence of liver-related events within 2 years in patients with active alcohol consumption according to hepatocyte large extracellular vesicles (lEVs) concentrations and FibroTest are displayed in Fig 4C, and according to hepatocyte large extracellular vesicles (lEVs) concentrations and MELD in Fig 4D.

Figure 5. Stratification model for prediction of liver-related events at 2 years of follow-up, based on either FibroTest <0.74 vs >0.74 (Fig 5A and C) or MELD <10 vs >10 (Fig 5 B and D) and either keratin-18 concentrations <285 U/L vs >285 U/L in patients without active alcohol consumption (Fig 5A and B) or hepatocyte large extracellular vesicle concentrations <50 U/L vs >50 U/L in patients with active alcohol consumption (Fig 5C and D). 1EV, large extracellular vesicles.

EXAMPLE:

Methods:

We used STROBE (27) as a reporting guideline to ensure a clear and complete report of the study design, conduct and findings.

Design

This study was an ancillary study of the institutional CIRRAL multicenter cohort study (28), promoted by Assistance Publique des Hopitaux de Paris. This study was approved by the local institutional review board (Comite de Protection des Personnes, Aulnay-sous-Bois, France). Written informed consent was obtained from each patient before enrollment.

Patients

Patients with Child-Pugh class A alcohol -related cirrhosis were included from the French multicentric prospective CIRRAL cohort between October, 2010 and April, 2016 (28). All patients had histologically proven cirrhosis with a history of excessive alcohol consumption according to the World Health Organization criteria (more than 21 glasses per week for female and more than 28 glasses per week for male) (29) that occurred for at least ten years and was considered the cause of chronic liver disease. Patients did not have associated hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. All patients had Child-Pugh class A cirrhosis at enrollment, but some had a previous history of decompensated cirrhosis before enrollment. Patients with either a history of primary liver cancer or detectable liver nodule at enrollment were not included. From this cohort, we specifically selected patients with the following criteria: (i) available plasma samples for Keratin-18, hepatocyte lEVs and FibroTest™ measurements; (ii) Child-Pugh class A at enrollment, and (iii) no history of transjugular intrahepatic portosystemic shunt at enrollment (Figure 1). Active alcohol consumption at enrollment was defined as either hazardous or harmful of alcohol use, i.e. a consumption of 7 glasses per week or more at enrollment (30). Metabolic syndrome was defined as the presence of at least 3 of the following features: abdominal obesity with increased waist circumference; serum lipids triglycerides >150 mg/dL (1.7 mmol/L), serum HDL-Cholesterol <40 mg/dL (1.03 mmol/L) in men or <50 mg/dL (1.29 mmol/L) in women, blood pressure >130 mm Hg systolic or >85 mm Hg diastolic, fasting serum glucose >100 mg/dL (5.6 mmol/L) (31). Model for endstage liver disease was calculated according to LINOS (32). Date of entry was the date of enrollment in the CIRRAL study. Patients were then followed until the reference date of January 11^th 2017.

Study endpoints

All the following outcomes were considered from the date of enrollment in the CIRRAL study, and were prospectively collected as part of the case report form. All extrahepatic events occurring during follow-up were also recorded. Likely cause(s) of death were established.

The primary endpoint of the present study was liver-related events at 2 years, defined as a composite endpoint including at least one liver-related event among clinical ascites, overt hepatic encephalopathy, variceal bleeding, spontaneous bacterial peritonitis, acute kidney injury, increase in Child-Pugh score >2 points, liver transplantation in the absence of hepatocellular carcinoma and/or liver-related deaths in the absence of hepatocellular carcinoma. Secondary endpoints included (i) liver-related death and liver transplantation in the absence of hepatocellular carcinoma within 2 years, (ii) bacterial infection (including spontaneous bacterial peritonitis, bacteriemia, pneumonia, urinary tract infection and skin infection), within 2 years; and (iii) hepatocellular carcinoma, within 2 years. Since Baveno 7 guidelines recently refined definition of cirrhosis decompensation (33), we secondarily added cirrhosis decompensation as a post-hoc composite endpoint, defined as time to occurrence of ascites, overt hepatic encephalopathy, or variceal bleeding, whichever occurred first.

Plasma preparation

Peripheral blood was collected inEDTA-treated tubes at enrollment into the study and prepared at each center following a standardized protocol. Plasma preparation consisted of one centrifugation at 3000g for 10 minutes. Aliquots of 500 pL of plasma were then stored at -80°C until use.

Measurement of hepatocyte-derived biomarkers concentrations

We determined plasma concentrations of hepatocyte-derived lEVs using a technique previously described (17,19,34). Briefly, we measured keratin-18 concentrations (M65 Epi- Death ELISA; Peviva, Bromma, Sweden) before and after filtration of the plasma through two 0.2-pm filters (Ceveron MFU 500; Technoclone, Austria). The difference between keratin-18 concentrations in initial and in 0.2 pm-filtrated plasma reflected the concentration on lEVs. Keratin-18 as well as hepatocyte 1EV concentrations were expressed as units per liter (U/L), according to the manufacturer’s instructions.

Measurement of FibroTest score

Measurements were performed on the same plasma samples collected in EDTA-treated tubes at enrollment (-80°C). FibroTest (Biopredictive, Paris, France) was determined as previously published (35). FibroTest includes a2-macroglobulin, apolipoprotein Al, haptoglobin, total bilirubin, and GGT, adjusted for age and gender. Standard manufacturer algorithms were used to exclude high risk profile of false negative/positive (36). FibroTest score ranges from zero to 1.00, with higher scores indicating a greater probability of significant fibrosis.

Statistical analysis

Results are presented by summary statistics, namely median (interquartile range) or absolute frequency (percentage) unless specified otherwise, with comparisons between baseline groups based on the Wilcoxon rank-sum test, or the exact Fisher exact, respectively. Correlations between laboratory values and hepatocyte-derived biomarkers were assessed using nonparametric Spearman correlation coefficient. Cumulative incidence of liver-related events was estimated using the nonparametric cumulative incidence function, considering hepatocellular carcinoma and liver-unrelated deaths as competing events; in patients without active alcohol consumption, alcohol relapse was considered as an additional competing event. Cumulative incidence of liver-related death was estimated using the cumulative incidence function, considering hepatocellular carcinoma and liver-unrelated deaths as competing events and liver transplantation as a liver-related death. Cumulative incidence of hepatocellular carcinoma and bacterial infections were estimated using the cumulative incidence function, considering death and liver transplantation as competing events. Cumulative incidence of cirrhosis decompensation was estimated using the cumulative incidence function, considering hepatocellular carcinoma, death and liver transplantation as competing events. Since alcohol consumption is a main driver for a poor outcome in patients with alcohol -related cirrhosis (25,26), all analyses were stratified on alcohol consumption, distinguishing patients without (0 to 6 glasses per week) from those with an active (7 glasses per week or more) alcohol consumption at enrollment, separately. To assess the ability of hepatocyte-derived biomarkers, including keratin-18 and hepatocyte 1EV concentrations, to predict outcome at 2 years, cause-specific Cox models were fitted, with effects quantified by hazards ratio (HR) with 95% confidence interval (95% CI). Proportional hazards were assessed using the Grambsch and Themeau test (37). Univariable models were fitted, then multivariable models were performed with the Cox proportional hazards model, including hepatocyte-derived biomarkers, MELD and FibroTest.

To assess the discriminatory performance of biomarkers to predict liver-related events, taking into account competing events, time-dependent receiver operating characteristic (ROC) curves using the cumulative sensitivity and dynamic specificity definitions measured at time t=24 months, and resulting area under the curve (AUC), were used (38). This allowed to select the best cut-off value of keratin-18 and hepatocyte 1EV concentrations according the Youden index. Previously reported cutoffs were also considered, namely 65 IU/L for hepatocyte lEVs (19), 10 for MELD (12) or 0.74 for the FibroTest (39,40). Missing data were imputed by imputation of the median on the sample. Multiple imputations with chained equation was also used as sensitivity analyses. All tests were two-sided with p-values <0.05 considered significant. Analyses were performed using open-source R software version 4.0.3 (http://www.R- project.org/), and LATEX.

Results

Patients

Between October 7 2010 and April 30 2016, 650 patients were included in the CIRRAL cohort in 22 centers. Among them, 500 fulfilled the inclusion criteria, including 419 without and 81 patients with active alcohol consumption at enrollment (Figure 1). The main characteristics of the patients are summarized in Table 1. Among the 343 patients completely abstinent from alcohol at enrollment, median duration of alcohol abstinence was 24 (9-69) months. Plasma biomarkers concentrations are shown in Table 1.

Hepatocyte biomarker concentrations according to clinical and laboratory features

Keratin-18 concentrations were significantly higher in patients with active alcohol consumption than in those without, and also higher in patients who declared a consumption of 1-6 glasses than in fully abstinent patients (Table 1 and Figure 2A). Hepatocyte lEVs concentrations were significantly higher in patients with active alcohol consumption than in those without (Table 1 and Figure 2B).

Ability of hepatocyte biomarkers concentrations to predict liver-related events at 2 years Median follow-up was 48 months (IQR, 34 to 71) and 52 months (IQR, 34 to 74) in patients without and with active alcohol consumption at enrollment. Events occurring during the 2 years follow-up period after enrollment are detailed in Figure 1. Cumulative incidence (95% CI) of liver-related events at 2 years was 11.7% (8.8-15.1) and 19.9% (11.8-29.7) in patients without and with active alcohol consumption, respectively. Patients’ characteristics, as well as serum biomarkers concentrations, at enrollment associated with liver-related events are shown in Table 2

Keratin-18 concentrations

Keratin- 18 concentrations were highly predictive of liver-related events in patients without active alcohol consumption, but not in those with active alcohol consumption (Table 2). Timedependent AUC analysis found that a cut-off value of keratin-18 concentration >285 U/L yielded the most accurate sensitivity and specificity to predict liver-related events at 2 years (not shown).

In patients without active alcohol consumption, incidence of liver-related events at 2 years was 20.1% (13.3-28.0) in patients with keratin-18 concentrations >285 U/L versus 8.3% (5.4-11.9) in patients having keratin-18 concentrations below this threshold (Figure 3A). After adjustment on MELD and FibroTest, total keratin-18 concentrations > 285 U/L still predicted liver-related events at 2 years (Table 3). As shown in Figure 4A and 4B, patients with both high keratin- 18 and either high FibroTest or high MELD had a higher risk of liver-related events at 2 years (24.2%, 95%CI, 15.1 to 34.5 and 28.7%, 95%CI, 16.6 to 41.9, respectively) than other groups of patients.

When restricting the analysis to the 343 patients with null alcohol consumption at enrollment, keratin-18 concentrations > 285 U/L still predicted liver-related events at 2 years (HR 1.70 (95% CI 1.04-2.77), p=0.03), after adjustment on MELD and FibroTest (data not shown).

Hepatocyte IEV concentrations Hepatocyte lEVs concentrations were highly predictive of liver-related events in patients with active alcohol consumption, but not in patients without. Time-dependent AUC analysis found that a cut-off value of hepatocyte lEVs >50 U/L yielded the most accurate sensitivity and specificity to predict liver-related events at 2 years (not shown).

In patients with active alcohol consumption, incidence of liver-related events at 2 years was 39.2% (95% CI 21.2-56.8) in patients with hepatocyte 1EV concentrations >50 U/L versus 8.5% (95% CI 2.7-18.7) in patients having hepatocyte lEVs concentrations below this threshold (Figure 3B). After adjustment on MELD andFibroTest, hepatocyte EV > 50 U/L still predicted liver-related events at 2 years (Table 3). We did similar analyses using hepatocyte lEVs >65 U/L threshold. After adjustment on MELD and FibroTest, hepatocyte 1EV > 65 U/L still predicted liver-related events, but the predictive accuracy was less obvious (Table 3). As shown in Figure 4C and 4D, patients with both high hepatocyte lEVs concentrations and either high FibroTest or high MELD had a higher risk of liver-related events at 2 years (62.4% (33.0-81.8) and 56.2% (11.6-85.8), respectively) than other groups of patients. Hepatocyte lEVs concentrations >50 U/L still predicted liver-related events at 2 years after adjustment on MELD, FibroTest and alcohol consumption (HR 4.42 (1.80-10.88), p= 0.0012) (not shown). The NRI (95% CI) of adding hepatocyte lEVs >50 U/L to FibroTest or MELD in patients with liver- related event at 24 months was 0.62 (0.00; 0.88) over FibroTest alone, and 0.30 (0.00; 0.78) over MELD alone. During the 2 years follow-up, 14 (17%) cirrhosis decompensations occurred in patients with active alcohol consumption, according to recent Baveno VII criteria. Patients’ characteristics at enrollment associated with cirrhosis decompensation (not shown ). By multivariate analysis, only hepatocyte lEVs concentrations tended to be independent predictors of cirrhosis decompensation at 2 years (not shown).

Ability of hepatocyte biomarkers to predict other events at 2 years after enrollment.

During the follow-up, 51 (12%) and 11 (14%) liver-related death or liver transplantation without hepatocellular carcinoma occurred in patients without and with active alcohol consumption, respectively. Independent predictors of death or liver transplantation at 2 years were MELD and FibroTest -but not Keratin-18 concentrations - in patients without active alcohol consumption, and MELD and hepatocyte lEVs concentrations in patients with active alcohol consumption (not shown). Bacterial infections

Thirteen (3%) and 7 (9%) bacterial infections occurred in patients without and with active alcohol consumption, respectively. In patients without active alcohol consumption, no independent predictor of bacterial infection was identified. In patients with active alcohol consumption, only hepatocyte lEVs concentrations independently predicted bacterial infection within 2 years (not shown).

Hepatocellular carcinoma

Thirty-nine (9%) and 10 (12%) patients developed hepatocellular carcinoma in patients without and in those with active alcohol consumption, respectively. In patients without or with active alcohol consumption, FibroTest was the only independent predictor of hepatocellular carcinoma (not shown). Hepatocyte biomarkers were not independent predictors of hepatocellular carcinoma.

During the follow-up, 63 (15%) and 14 (17%) cirrhosis decompensations occurred in patients without and with active alcohol consumption, according to recent Baveno VII criteria, respectively. In patients without active alcohol consumption, MELD and keratin-18 concentrations were independent predictors of cirrhosis decompensation at 2 years. In patients with active alcohol consumption, only hepatocyte lEVs concentrations tended to be independent predictors of cirrhosis decompensation at 2 years (not shown).

Discussion:

Patients with compensated alcohol -related cirrhosis are the target-of choice population for an intensive surveillance, intensive management of alcohol use disorders, or new drugs aiming at preventing liver-related events. The present study demonstrates, in a large prospective cohort of patients with Child-Pugh class A alcohol -related cirrhosis, that combining hepatocyte- derived biomarkers to usual biomarkers, namely MELD or FibroTest, allows estimating the individual risk of liver-related events at 2 years (Figures 5A to 5D).

The first major finding of the present study is that circulating concentrations of the hepatocyte biomarkers keratin-18 and hepatocyte-lEVs improve ability of FibroTest and of MELD to predict the risk of liver-related events at 2-year and allow the identification of patients at high risk. Indeed, in patients without active alcohol consumption, those with both keratin-18 concentration > 285 U/L and MELD >10 had a 28.7% cumulative incidence of liver-related events at 2 years vs. 5 to 14% in patients without this combination. In patients with active alcohol consumption, those with both total hepatocyte-EVs concentration > 50 U/L and Fibrotest>0.74 had a 62% cumulative incidence of liver-related events at 2 years, vs. 8 to 13% in patients without this combination. This synergy between hepatocyte biomarkers and FibroTest or MELD is likely accounted for by the different pathophysiological processes they reflect, namely liver cell stress or injury, liver fibrosis and liver function, respectively. A limitation of the current study is the lack of a validation cohort to confirm our results. However, the large number of patients prospectively included and followed-up in multiple centers strengthens our results. Even though no study assessed so far the ability of hepatocyte-derived biomarkers to predict liver-related events in patients with Child-Pugh class A cirrhosis, our results are in line with previous studies. Keratin-18 fragments concentrations predicted mortality in a cohort of 175 patients (39% with ascites) with alcohol-related cirrhosis followed for more than 2 years (14), as well as 90-day mortality in patients with alcoholic hepatitis in the STOP AH trial (15). Hepatocyte-derived lEVs concentrations also predicted 6-months mortality independently of Child-Pugh and MELD scores in a previous study including patients with compensated and decompensated cirrhosis (19). Of note, in the present study, hepatocyte lEVs concentrations not only predicted liver-related events, but also bacterial infection and 2-year risk of death or liver transplantation. Liver stiffness measurement > 20 or 25 kPa using transient or shearwave elastography is increasingly used to predict decompensation and death in patients with compensated cirrhosis( 12,41). Combining hepatocyte biomarkers with liver stiffness measurement would be very attractive, but was not possible in the present study, because liver stiffness measurement was available at inclusion in only a minority of the patients in the CIRRAL cohort (42).

The second major result of the present study is that the choice of the hepatocyte biomarker to be used should take into account alcohol consumption, a finding allowed by the prospective design of the CIRRAL trial including careful alcohol consumption analysis. Although alcohol withdrawal is a cornerstone of the management of patients with alcohol-related cirrhosis, with a high impact on long-term prognosis (26,43), most studies evaluating biomarkers of diagnosis and prognosis in patients with alcohol -related liver diseases cirrhosis did not take into account the alcohol consumption status (24,44). The different prediction ability of Keratin-18 and hepatocyte-lEVs according to alcohol consumption status suggests that these hepatocyte biomarkers reflect different pathophysiological processes driving patients’ outcome. Several lines of evidence suggest that hepatocyte lEVs capture individual susceptibility to alcohol by reflecting hepatocyte apoptosis in patients with active alcohol consumption. Indeed, alcohol induces hepatocytes apoptosis following caspase-dependent and caspase- independent pathways (45,46). Apoptosis is a well-known stimulus for lEVs release and several studies showed that alcohol stimulates release of extracellular vesicles from hepatocytes in vitro and in vivo (47,48). Furthermore, circulating concentrations of hepatocyte-lEVs were associated with apoptotic hepatocytes on liver biopsies of patients with cirrhosis of various causes (19). In the CIRRAL cohort, although all included patients had biopsy proven alcohol -related cirrhosis, liver biopsies were not performed at enrollment in the majority of the patients, so that we were not able to evaluate the association between hepatocyte 1EV concentrations and histological lesions. Keratin-18 concentrations predicted 2-years liver-related events in patients without active alcohol consumption better than hepatocyte-lEVs. Keratin-18 is a member of the intermediate filament family of cytoskeletal proteins (49). The M65 fragment of keratin-18 is a marker of overall cell death, including both apoptosis and other forms of cell death (50). These results suggest that, in patients without active alcohol consumption, other types of cell stress or cell death than apoptosis drive patients’ outcome.

In the present study, neither keratin-18 nor hepatocyte lEVs concentrations predicted hepatocellular carcinoma at 2 years. This result is in line with a previous study of 175 patients with alcohol -related cirrhosis, where keratin-18 concentrations did not predict hepatocellular carcinoma-related death (14). Conversely, the present study further validates accuracy of FibroTest in patients with compensated alcohol -related cirrhosis to predict hepatocellular carcinoma development (39). Furthermore, this study validates that using FibroTest or its derived tests (such as the LCR 1 - LCR 2 algorithm) could be useful for risk -stratification and personalized strategies of hepatocellular carcinoma screening (51).

In conclusion, hepatocyte-derived biomarkers combined with FibroTest or MELD allow the identification of a population of patient with Child-Pugh A cirrhosis at high risk of liver-related events at 2 years. The usefulness of these hepatocyte-derived biomarkers to predict liver-related events in patients with non-alcohol -related cirrhosis should be investigated in further studies.

TABLES: Table 1. Characteristics and serum biomarkers levels of the included patients according to the alcohol status

Abbreviations: AST, aspartate amino transferase, ALT, alanine amino transferase, GGT, gammaglutamyl transpeptidase, MELD, model for end stage liver disease; lEVs, large extracellular vesicles

* MELD was available in 386/419 patients with past alcohol consumption and in 78/81 patients with active alcohol consumption ** Coffee consumption was available in 366/419 patients with past alcohol consumption and in 64/81 patients with active alcohol consumption

Data are presented as median (interquartile range) or absolute number (percentage)

Table 2. Univariable cause-specific Cox regression analysis of the variables associated with liver-related events at 2 years according to alcohol status at enrollment

In patients with past alcohol consumption, hepatocellular carcinoma, liver-unrelated deaths and alcohol relapse were treated as competing events; In patients with active alcohol consumption, hepatocellular carcinoma and liver-unrelated deaths were treated as competing events

Abbreviations AST, aspartate amino transferase, ALT, alanine amino transferase, GGT, gammaglutamyl transpeptidase, MELD, model for end stage liver disease; lEVs, large extracellular vesicles

** Coffee consumption was available in 366/419 patients with past alcohol consumption and in 64/81 patients with active alcohol consumption

*** MELD was available in 386/419 patients with past alcohol consumption and in 78/81 patients with active alcohol consumption

Bold indicate statistically significant results

Table 3. Multivariate cause-specific Cox regression analyses of the ability of keratin-18 and hepatocyte large extravesicles (IE Vs) concentrations to predict liver-related events within 2 years, adjusted on MELD and FibroTest

Abbreviations: MELD, model for end stage liver disease; lEVs, large extracellular vesicles

*MELD was available in 386/419 patients with past alcohol consumption and in 78/81 patients with active alcohol consumption

§In patients with past alcohol consumption, hepatocellular carcinoma, liver-unrelated deaths and alcohol relapse were treated as competing events; In patients with active alcohol consumption, hepatocellular carcinoma and liver-unrelated deaths were treated as competing events.

Multivariate analysis was performed in 386 patients and 90 events in patients with past alcohol consumption and 78 patients and 25 events in patients with active alcohol consumption.

Bold indicate statistically significant results.

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Claims

CLAIMS:

1. A method of predicting a liver-related event in a patient suffering from alcohol -related cirrhosis comprising determining the level of hepatocyte derived large extracellular vesicles (lEVs) in a blood sample obtained from the patient wherein said level indicates the risk that the patient will have a liver-related event.

2. The method of claim 1 wherein the patient has a Child-Pugh class A alcohol -related cirrhosis.

3. The method according to claim 1 or 2 wherein the patient has an active alcohol consumption.

4. The method of claim 1 wherein the liver-related event is selected from the group consisting of cirrhosis decompensation, clinical ascites, overt hepatic encephalopathy, variceal bleeding, spontaneous bacterial peritonitis, acute kidney injury, increase in Child-Pugh score >2 points, liver transplantation in the absence of hepatocellular carcinoma and/or liver-related deaths in the absence of hepatocellular carcinoma.

5. The method of claim 1 for predicting the liver-related event at 2 years.

6. The method of claim 1 wherein the blood sample is a plasma sample.

7. The method of claim 1 wherein the level of hepatocyte derived large extracellular vesicles is determined by isolating them from the blood sample by centrifugation and then contacting them with a set of binding partners directed against the specific surface markers of said extracellular vesicles.

8. The method of claim 6 wherein the binding partner is a monoclonal antibody.

9. The method of claim 7 wherein the monoclonal antibody binds M65 cytokeratin-18 fragment.

10. The method of claim 1 that comprises i) determining the level of hepatocyte-derived large extracellular vesicles in the blood sample obtained from the patient and ii) comparing the level determined at step i) with a predetermined reference value wherein a difference between the level determined at step i) and the predetermined reference value is indicative of risk of having a liver-related event.

11. The method of claim 10 wherein when the level of hepatocyte-derived large extracellular vesicles is higher than the predetermined reference value, then it is concluded that the risk of having a liver-related event is high or when the level of hepatocyte-derived large extracellular vesicles is lower than the predetermined reference value, then it is concluded that that the risk of having a liver-related event is high.

12. The method of claim 1 that further comprises determining the MELD score wherein the combination between the level of hepatocyte-derived large extracellular vesicle and MELD score indicates the risk of having a liver-related event.

13. The method of claim 1 comprises determining the FibroTest score wherein the combination between the level of hepatocyte-derived large extracellular vesicles and FibroTest indicates the risk of having a liver-related event.