METHODS AND KITS FOR PREDICTING THE TRANSPLANTATION-FREE SURVIVAL TIME OF PATIENTS SUFFERING FROM CIRRHOSIS

Abstract

Following a prospective clinical study that includes 242 patients, the inventors show that hepatocyte-derived MV levels predicted transplantation-free survival at 6 months in univariate analysis. In multivariate analysis, this association was shown to be independent of Child-Pugh and of MELD score. Thus the present invention thus relates to a method of predicting the transplantation-free survival time of a patient suffering from cirrhosis comprising determining the level of hepatocyte-derived microvesicles (e.g. by an ELISA assay) in a blood sample obtained from the patient.

Claims

1. A method of predicting the transplantation-free survival time of a patient suffering from cirrhosis comprising determining the level of hepatocyte-derived microvesicles in a blood sample obtained from the patient, wherein when the level of microvesicles is higher than a predetermined reference value, then the patient will have a short transplantation-free survival time or when the level of microvesicles is lower than the predetermined reference value, then the patient will have a long transplantation-free survival time.

2. The method of claim 1 wherein the level of hepatocytes-derived microvesicles is determined by isolating microvesicles from the blood sample by centrifugation and then contacting the microvesicles with at least one binding partner directed against the specific surface markers of said hepatocytes-derived microvesicles.

3. The method of claim 2 wherein the at least one binding partner is a monoclonal antibody.

4. The method of claim 3 wherein the monoclonal antibody binds to M30 or M65 cytokeratin-18 fragment.

5. The method of claim 2 wherein the level of hepatocytes-derived microvesicles is determined by an ELISA assay.

6-7. (canceled)

8. A method of predicting the transplantation-free survival time of a patient suffering from cirrhosis and treating a patient with a short predicted transplantation-free survival time comprising i) determining the level of hepatocyte-derived microvesicles in a blood sample obtained from the patient, wherein when the level of microvesicles is higher than a predetermined reference value, then the patient will have a short transplantation-free survival time or when the level of microvesicles is lower than the predetermined reference value, then the patient will have a long transplantation-free survival time, and ii) administering a preventive treatment or transplanting a liver into a patient whose measurement is indicative of a short transplantation-free survival time.

9. The method of claim 8, wherein the preventive treatment includes administration of one or more of an antiapoptotic agent, a vasoactive drug and an antifibrotic agent.

Description

FIGURES

[0020] FIG. 1. Circulating hepatocyte MV levels (U/L) according to the Child-Pugh score. A) Test cohort. B) Validation cohort.

[0021] FIG. 2. Cumulative incidence of death according to circulating hepatocyte MV levels (U/L). A) Test cohort. B) Validation cohort.

[0022] FIG. 3. Cumulative incidence of death according to MELD and circulating hepatocyte MV levels (U/L). A) Test cohort. No death occurred in the group of patients with MELD>15 and hepatocyte MV65 U/L so that this group is not represented. B) Validation cohort. No death occurred in the group of patients with MELD15 and hepatocyte MV>65 U/L so that this group is not represented. HMV, hepatocyte microvesicle.

EXAMPLE

Material and Methods

Patients

[0023] All consecutive patients with severe liver fibrosis or cirrhosis undergoing hepatic vein and/or right heart catheterization in two independent centers were prospectively included. The inclusion period ranged from June 2013 to March 2015 for the test cohort (Hpital Beaujon, Clichy, France) and from January 2014 to December 2015 for the validation cohort (Hospital Clinic, Barcelona, Spain). Clinical, laboratory and hemodynamic features were prospectively collected in both centers. Diagnosis of severe liver fibrosis (METAVIR F3) or cirrhosis was based either on histological criteria or on the combination of clinical, laboratory, morphological and hemodynamic features. Non-inclusion criteria were a history of transjugular intrahepatic porto-systemic shunt, liver transplantation, hepatocellular carcinoma (HCC) outside Milan criteria since HCC increases MV levels by itself (Campello Thromb Res 2016; Brodsky J Gastrointestin Liver Dis 2008; Julich-Haertel, J Hepatol 2017), active extra-hepatic cancer, human immunodeficiency virus infection, primary sclerosing cholangitis and primary biliary cirrhosis since HVPG might not reflect portosystemic gradient in patients with cholestatic liver disease (Garcia-Tsao, Hepatology 2017), Budd-Chiari syndrome, or an acute event (hepato-renal syndrome, bacterial infection, alcoholic hepatitis, variceal bleeding) within two weeks prior to hepatic vein catheterization. Patients on the waiting list for liver transplantation and those with HCC were seen every 3 months. Other patients were seen every 6 months. When patients did not attend the follow-up visit, they were called by phone. In the absence of answer, we consulted the liver transplant registry and the national registry of deaths. This study was approved by the Institutional Review Boards of Paris North Hospitals, Paris 7 University, AP-HP (N 11-112) and of Hospital Clinic (Barcelona, Spain). All patients included in this study gave written informed consent. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

Platelet-Free Plasma Preparation

[0024] Platelet-free plasma was prepared at each centre following a standardized protocol proposed by Lacroix and colleagues. The principal investigator of this study trained the investigators responsible for plasma preparation to this procedure. Briefly, peripheral venous blood was collected within 2 hours prior to liver catheterization from the cubital vein of the patients, with a 21-gauge tourniquet needle, in 0.129 mol/L citrated tubes, after having discarded the first mL of blood. Tubes then remained motionless in the up-right position at room temperature for a maximum of 2 hours until platelet free plasma preparation, consisting in two successive centrifugations, each of 15 min at 2500 g at 20 C. with a light brake. Aliquots of platelet free plasma were then stored at 80 C. until use.

[0025] In some patients, hepatic venous blood was collected from the median or the right hepatic vein using a tip-curved catheter (Cook, HNB7.0-38-100-P-NS-MPA) and was prepared as mentioned above.

Characterization of Circulating Microvesicle Levels

[0026] Circulating levels of annexin V+, platelet (CD41+), leuko-endothelial (CD31+/41), pan-leukocyte (CD11a+) and endothelial (CD62e+ and CD144+) MVs were determined on a Gallios flow cytometer (Beckman Coulter, Villepinte, France) using a technique previously described. Regions corresponding to MVs were identified in forward light scatter and side-angle light scatter intensity dot plot representation set at logarithmic gain. MV gate was defined, using calibration beads (Megamix plus FSC, Biocytex, France), as events having a 0.1-1 m diameter. This gate was separated into large (0.5 to 1 m) and small (<0.5 m) MVs. Events were then plotted on a fluorescence/forward light scatter dot plot to determine MV counts positively labeled by specific antibodies. Anti-CD41-Phycoerythrin-Cyanin7, anti-CD31-Phycoerythrin, anti-CD11a-Phycoerythrin, and anti-CD144-Phycoerythrin antibodies as well as their matched isotype controls were obtained from Beckman-Coulter. Anti-CD62E-fluoroisothiocyanate antibodies as well as their matched isotype controls were obtained from R&D Systems Europe. Annexin V fluoroisothiocyanate was purchased from Beckman-Coulter. MV concentration was assessed by comparison with a known amount of flowcount calibrator beads (AccuCount Fluorescent Particles, Spherotech, Chicago, 20 L) added to each sample just before performing flow cytometry analysis. To limit variability, all measurements of MV levels by flow cytometry were performed using a unique batch for each antibody.

[0027] We determined plasma levels of hepatocyte-derived MVs using a technique previously described. Briefly, we measured circulating cytokeratin-18 levels (M65 EpiDeath ELISA, Peviva, Bromma, Sweden) before and after filtration of the plasma through two 0.2 m filters (Ceveron MFU 500, Technoclone, Austria). The difference between soluble cytokeratin-18 levels in initial and in 0.2 m filtrated platelet-free plasma reflected the concentration in hepatocyte MVs (data not shown). We also determined plasma levels of extracellular vesicles having a size ranging from 0.02 to 0.2 m, corresponding to small MVs and/or exosomes, further referred to as hepatocyte exosomes. These levels corresponded to the difference between cytokeratin-18 levels in 0.2 m filtrated and in 0.02 um filtrated (Whatman 6809-1002 Anotop Syringe Filter) platelet-free plasma. Hepatocyte MV and exosome levels were expressed as units per liter (U/L), according to the manufacturer's instructions. All ELISA were performed in duplicate with determination of the coefficient of variation between samples from the same patient. The results were considered adequate when the coefficient of variation was less than 20%. Otherwise, samples were measured again.

C Reactive Protein and Interleukin 6 Concentrations Measurements

[0028] C reactive protein (DY1707, R&D Systems Europe, France), and Interleukin-6 (DY008, R&D Systems Europe, France) concentrations were measured in patients' plasma samples according to the manufacturer's instructions.

Hemodynamic Evaluation

[0029] In both centers, HVPG was assessed using a technique previously described. The principal investigator of this study went to Barcelona to homogenize HVPG measurement between both centres. Briefly, after an overnight fasting, local anesthesia was performed and an introducer was placed under ultrasound guidance using the Seldinger technique. A 7 French balloon catheter (Lemaitre Vascular, for the test cohort; Edwards Lifesciences, Irvine, Calif., USA for the validation cohort) was inflated in the right or median hepatic vein and wedge hepatic venous pressure was measured. Then, free hepatic venous pressure was obtained. HVPG was calculated as the difference between wedged and free hepatic venous pressures. Adequate occlusion was confirmed by injection of 5 mL of iodinated radiologic contrast medium. Permanent tracings were recorded. Clinically significant portal hypertension was defined as a HVPG10 mm Hg. When indicated, right heart hemodynamic measurements including pulmonary artery pressure, right atrial pressure, and pulmonary capillary wedge pressure were also performed using a Swan-Ganz catheter (Edwards Life Sciences). Cardiac index was measured by the thermodilution method and obtained by the average of 3 to 5 consecutive measurements.

Histological Analysis

[0030] Liver tissue samples obtained from patients of the test cohort within 3 months before or after venous blood collection for MV measurement were retrospectively reviewed by an expert pathologist unaware of the results of MV measurements. The following features were analyzed on hematein and eosin, and on picrosirius stained tissue sections using semi-quantitative scoring defined a priori. Fibrosis was evaluated using picrosirius staining and scored according to Metavir Hepatology 1996;24:289-93; in case of cirrhosis (F4), the Laennec scoring system was used (stage 4a, mild/definitive cirrhosis with marked septation and visible nodules although most septa are thin; stage 4b, moderate cirrhosis with at least two broad septa and less than half of the tissue section composed of micronodules (<3 mm); stage 4c, severe cirrhosis with at least one very broad septum or more than half of the tissue section composed of micronodules) (J Hepatol 2011;55:1004-9). Activity was classified as absent to moderate vs. severe. Presence of apoptotic hepatocytes was also evaluated.

Statistical Analyses

[0031] Quantitative variables were expressed as median (interquartile range) and categorical variables as frequencies. Comparisons of independent quantitative variables between groups were performed using the Mann-Whitney test. Comparisons of hepatocyte MV levels between hepatic and peripheral vein were performed using the Wilcoxon test. Spearman correlation analyses were used to evaluate the relationships between MV circulating levels, clinical and hemodynamic features. Follow-up time was defined as the period from the date of liver catheterization to 6 months after this procedure. Study outcome was evaluated using a multistate model as recommended in cirrhotic patients: data for patients who had not died were censored at the date of the last follow-up visit and were coded 0; data for patients who died before liver transplantation were coded 1; liver transplantation was considered to be a competing risk event and data were coded 2. A cumulative incidence function of death was calculated to describe the probability of death at a given time and was reported at 6 months with a 95% confidence interval. Univariate regression analyses were conducted using the Fine and Gray proportional hazards models to identify whether MV levels at baseline were associated with 6-month mortality. The value of MV level with the best sensitivity and specificity in area under the receiver operating characteristic curve analysis (Youden's Index) for death was chosen for further analyses. Each MV subpopulation achieving a P value <0.05 was introduced into a multivariable Fine and Gray proportional hazards model with Child-Pugh score or MELD to adjust our analyses for these severity scores and determine whether these MV levels had a prognostic value independently of these scores. All statistical tests were two-sided. P values <0.05 were considered to be statistically significant. Statistical analyses and figures were performed using the SPSS statistical package 16.0 software (SPSS Inc., Chicago, Ill., United States) and GraphPad Prism 5 software, respectively. Survival analyses were performed using SAS 9.4 statistical software.

Results

Patients' Characteristics in the Test Cohort

[0032] One hundred and thirty nine patients were included in the test cohort. Their characteristics are presented in Table 1. The main cause of liver disease was excessive alcohol consumption. Indications for hepatic vein, with or without right heart, catheterization were evaluation before liver transplantation in 70 (50%) patients or before liver surgery in 7 (5%) patients or assessment of the severity and/or the cause of liver disease using a liver biopsy in 62 (45%) patients. During the 6 months follow-up, 20 (14%) patients underwent liver transplantation and 9 (6%) patients died. Causes of death were HCC related in 2; liver related in 5, including variceal bleeding in 1, Klebsiella oxytoca pneumonia in 1, acute on chronic liver failure in 1, acute alcoholic hepatitis complicated with pneumonia in 1 and complicated refractory hepatic hydrothorax in 1; and unknown in 2.

Circulating MVs Levels According to the Severity of the Liver Disease

[0033] As shown in FIG. 1A (and data not shown), hepatocyte MV levels were 4.0 and 2.2 fold higher in patients with Child-Pugh C than in those with Child-Pugh A or B liver disease, respectively. Similar results were obtained when restricting the analysis to patients without HCC (data not shown). Hepatocyte MV levels weakly correlated with HVPG (r=0.22; p=0.011), and could not discriminate patients with HVPG >10 mm Hg from those having an HVPG below this threshold. Hepatocyte MV levels did not correlate with right heart hemodynamic values or with cardiac index, but correlated with markers of systemic inflammation (leukocytes, C-reactive protein, and interleukin 6) and with MELD and its components, and inversely correlated with serum sodium levels (data not shown). Severe liver necro-inflammatory activity and abundant liver fibrosis were associated with higher circulating levels of hepatocyte MVs (data not shown). There was also a trend towards higher hepatocyte MVs levels in patients with apoptotic hepatocytes. In ten additional patients (data not shown), we compared hepatocyte MV levels in hepatic vs. peripheral vein and observed 78% (27-233%, p=0.037) higher levels in hepatic than in peripheral vein from the same patients.

[0034] Total soluble cytokeratin 18 levels (bound and unbound to MVs) were slightly higher in patients with Child Pugh C than in those with Child Pugh B liver disease (data not shown for overall cohort and for patients without HCC), correlated weakly with HVPG (r=0.21; p=0.017), and did not significantly differ between patients with HVPG10 mm Hg and those with HVPG<10 mm Hg (data not shown).

[0035] Neither circulating levels of hepatocyte exosomes nor circulating levels of annexin V+, platelet (CD41+), leuko-endothelial (CD31+/41), pan-leukocyte (CD11a+) and endothelial (CD62e+, CD144+) MVs measured by flow cytometry, were influenced by the severity of the liver disease, except for CD144+ MV levels found slightly higher in patients with Child-Pugh C liver disease in the overall cohort (data not shown) and CD31+/41 MV levels mildly higher in patients with Child-Pugh C liver disease without HCC (data not shown). Annexin V+, platelet, leuko-endothelial, pan-leukocyte and endothelial MV levels did not correlate with HVPG (data not shown) and could not identify patients with HVPG10 mm Hg (data not shown).

Factors Associated with Six-Month Mortality

[0036] By univariate analysis, hepatocyte MV levels were strongly associated with 6-month mortality (Table 2). A cut-off value of 65 U/L yielded the most accurate sensitivity and specificity to identify patients' mortality. As shown in FIG. 2A, patients having hepatocyte MV levels>65 U/L had a 6-month cumulative incidence of death of 18% (8-31%) vs. 1% (1-5%) for patients having hepatocyte MV levels below this threshold. Hepatocyte MV level>65 U/L still predicted 6-month mortality after adjustment on Child-Pugh score or on MELD (Table 3). To further explore the added prognostic value of hepatocyte MV levels to MELD, we evaluated 6-month mortality according to the cut-off of 65 U/L and to MELD below or above 15, this threshold being recommended to list patients with end-stage liver disease for liver transplantation (1). As shown in FIG. 3A, patients with hepatocyte MV levels>65 U/L and MELD>15 were clearly at higher risk for 6-month mortality than the other patients (23% vs. 3%; p=0.001). Hepatocyte MV levels were also associated with 6-month liver-related mortality by univariate analysis (data not shown). Analyses adjusted on MELD and Child-Pugh score could not be performed since no liver-related death occurred in the group of patients with hepatocyte MV <65 in the test cohort.

[0037] We did similar prognostic analyses using total soluble cytokeratin 18 levels and found less obvious differences (Table 2 and data not shown). Hepatocyte MVs being a reflection of hepatocyte injury, we investigated the prognostic value of serum transaminase levels, but neither AST nor ALT levels were associated with 6-month mortality (data not shown). Other MV subpopulations did not predict mortality except for small CD.sup.31/41.sup.MV levels that were associated with mortality by univariate (Table 2). When adjusting for severity scores of cirrhosis, CD.sup.31/41.sup. small MV levels predicted 6-month mortality independently of Child-Pugh score, but not of MELD (data not shown).

Validation cohort

[0038] Characteristics of the 103 patients with cirrhosis included in the validation cohort are presented in Table 1. The main cause of liver disease was hepatitis C virus infection. Indications for hepatic vein, with or without right heart, catheterization were evaluation before liver transplantation in 4 (4%) patients or before liver surgery in 19 (18%) patients or assessment of the severity and/or the cause of liver disease by a liver biopsy in 80 (78%) patients. During the 6-month follow-up, 4 (4%) patients underwent liver transplantation, and 7 (7%) died. Causes of death were acute on chronic liver failure in 2 patients, septic shock in 1, variceal bleeding in 1, hemorrhagic stroke in 1 and HCC related in 2. The main results obtained in the test cohort were confirmed in the validation cohort, namely higher levels of hepatocyte MV levels in patients with Child-Pugh C liver disease (FIG. 1B; data not shown for the overall cohort and for patients without HCC), and a correlation with HVPG, but not with other hemodynamic values (data not shown). In the validation cohort, hepatocyte MVs levels were higher in patients with HVPG10 mm Hg than in those with an HVPG below this threshold [15 (0-63) vs. 5 (0-15); p=0.015]. Hepatocyte MV levels were associated with 6-month mortality by univariate analysis (Table 2, FIG. 2B) and after adjustment on Child-Pugh score (Table 3). Again, patients with hepatocyte MV levels>65 U/L and MELD>15 had 6-month mortality significantly higher than other groups (FIG. 3B). Hepatocyte MV levels were also associated with 6-month liver-related mortality by univariate analysis (data not shown). Analyses adjusted on MELD and Child-Pugh score were not performed due to the low number of events.

[0039] As CD144.sup.+ and CD31.sup.+/41.sup.MV levels predicted patients mortality either in the test or in the validation cohort, we performed additional analyses to get further insight into the variables influencing circulating concentrations of these subpopulations of MVs in the 242 patients with cirrhosis. CD31.sup.+/41.sup. large MVs levels correlated with markers of systemic inflammation (C reactive protein and leukocytes) (data not shown).

TABLE-US-00001 TABLE 1 Baseline characteristics of the 242 patients with advanced chronic liver disease Test cohort Validation cohort (n = 139) (n = 103) p value Clinical features Age (years) 56 (50-62) 58 (51-66) 0.019 Male gender-N (%) 107 (77) 68 (66) 0.076 Body Mass Index (kg/m.sup.2) 26 (23-30) 26 (23-29) 0.985 Fibrosis-N (%) 0.005 Advanced fibrosis (F3) 10 (7) 0 (0) Cirrhosis (F4) 129 (93) 103 (100) Cardiovascular risk factors-N (%) Hypertension 50 (36) 23 (22) 0.022 Smoking 50 (36) 18 (18) 0.002 Diabetes 45 (32) 23 (22) 0.086 Dyslipidemia 13 (9) 8 (8) 0.665 Causes of liver disease-N (%) Alcohol 59 (42) 19 (18) <0.001 Non alcoholic steatohepatitis 37 (27) 3 (3) <0.001 Hepatitis C 41 (29) 77 (75) <0.001 Hepatitis B 10 (7) 4 (4) 0.2754 Other 16 (12) 2 (2) 0.005 Ascites-N (%) 76 (55) 23 (22) <0.001 Hepatocellular carcinoma-N (%) 43 (31) 20 (19) 0.044 Child Pugh Class A/B/C 43 (31)/52 (37)/ 66 (64)/29 (28)/ <0.001 44 (32) 8 (8) Model for end-stage liver disease (MELD) 13 (9-17) 11 (8-14) 0.004 Large varices esophageal or history of 26 (23) 4 (5) band ligation-N (%) <0.001 Laboratory data Serum sodium (mmol/L) 136 (134-138) 141 (138-143) <0.001 Serum creatinine (mol/L) 70 (63-86) 68 (53-81) 0.015 Serum aspartate aminotransferase (ULN) 1.81 (1.3-2.8) 1.63 (1-2.75) 0.465 Serum alanine aminotransferase (ULN) 1.1 (0.6-1.98) 1.3 (0.8-2.15) 0.143 Serum bilirubin (mol/L) 33 (14-64) 24 (14-43) 0.039 Leukocytes (10.sup.9/L) 5.1 (3.7-7.1) 4.4 (3.3-6) 0.011 Hemoglobin (g/dL) 12.0 (10.5-14.0) 13.2 (12.0-14.8) 0.014 Platelet count (G/L) 93 (68-138) 85 (59-134) 0.271 C Reactive protein (mq/L) 4.5 (1.6-8.8) 1.8 (0.4-4.1) <0.001 Interleukin 6 (pg/mL) 9 (0-27) 0 (0-24) 0.108 Hemodynamic data Hepatic venous pressure gradient (mm Hg) 16 (12-20) 16 (13-19) 0.100 10 mm Hg-N (%) 114 (85) 83 (81) Wedge hepatic venous pressure (mm HQ) 24 (18-29) 23 (18-27) 0.231 Free hepatic venous pressure (mm Hq) 7 (5-10) 8 (6-9) 0.664 Heart rate (bpm) 72 (65-84) 63 (55-75) <0.001 Mean arterial pressure (mm Hg) 91 (85-102) 90 (80-98) 0.012 Right atrial pressure (mm Hg) 4 (2-5) 5 (3.5-6.5) <0.001 Mean pulmonary artery pressure (mm Hg) 13 (11-17) 15 (12-19) 0.009 Cardiac index (L/min/m.sup.2) 3.5 (2.8-4.4) 3.4 (2.8-4.5) 0.964 Beta-blockers treatment-N (%) 69 (50) 45 (44) 0.359 Data are expressed as median (interquartile range) or number (%) as appropriate.
Some patients had several causes of cirrhosis.

[0040] Esophageal varices data were available in 111 patients in the test cohort and in 83 patients in the validation cohort. Hepatic venous pressure gradient, heart rate, mean arterial pressure, right atrial pressure, mean pulmonary artery pressure and cardiac index were available in the test cohort for 134, 139, 139, 134, 125, 125 patients respectively and in the validation cohort for 103, 101, 102,102, 47, 46 patients, respectively.

[0041] Abbreviations: bpm, beat per minute; ULN, upper limit of normal values.

TABLE-US-00002 TABLE 2 Univariate analyses evaluating the association of circulating microvesicle (MV) subpopulation and soluble cytokeratin 18 levels with 6-month transplantation-free survival using Gray's test (transplantation counted as competing risk, death counted as event). Test cohort Validation cohort Hazard 95% confidence Hazard 95% confidence Variable ratio interval P value ratio interval P value Univariate analysis Annexin V .sup.+ 10.sup.3 0.877 0.748-1.029 0.108 1.049 0.977-1.127 0.190 MVs/L Annexin V.sup.+ 0.831 0.656-1.053 0.125 1.068 0.985-1.159 0.112 small 10.sup.3 MVs/L Annexin V.sup.+ large 0.751 0.442-1.274 0.289 1.134 0.674-1.909 0.636 10.sup.3 MVs/L CD11a.sup.+ 10.sup.3 0.787 0.551-1.124 0.188 0.189 0.004-9.225 0.401 MVs/L CD11a.sup.+ small 0.704 0.326-1.521 0.798 0.064 0.000-18.559 0.342 10.sup.2 MVs/L CD11a.sup.+ large 0.611 0.288-1.298 0.200 0.031 0.000-985.24 0.512 10.sup.2 MVs/L CD144.sup.+ 10.sup.2 0.667 0.156-2.855 0.585 5.060 2.326-10.838 <0.001 MVs/L CD144.sup.+ small 0.592 0.027-12.890 0.739 23.358 1.425-382.801 0.027 10.sup.2 MVs/L CD144.sup.+ large 0.470 0.052-4.231 0.501 9.238 4.566-18.69 <0.001 10.sup.2 MVs/L CD62E.sup.+ 10.sup.3 1.029 0.731-1.449 0.868 0.337 0.071-1.591 0.170 MVs/L CD62E.sup.+ small 1.047 0.638-1.720 0.855 0.186 0.006-5.369 0.327 10.sup.3 MVs/L CD62E.sup.+ large 1.043 0.361-3.014 0.938 0.109 0.007-1.800 0.121 10.sup.3 MVs/L CD41.sup.+ 10.sup.3 0.966 0.795-1.174 0.729 1.036 0.698-1.538 0.861 MVs/L CD41.sup.+ small 10.sup.3 0.871 0.623-1.219 0.421 0.972 0.475-1.991 0.939 MVs/L CD41.sup.+ large 10.sup.3 1.042 0.611-1.776 0.881 1.386 0.503-3.818 0.528 MVs/L CD31.sup.+/41.sup. 10.sup.3 0.086 0.003-2.678 0.1622 7.434 0.952-58.018 0.056 MVs/L CD31.sup.+/41.sup. small 0.010 0.000-0.732 0.036 20.211 0.254-1606.147 0.178 10.sup.3 MVs/L CD31.sup.+/41.sup. large 0.024 0.000-44.352 0.331 61.075 1.500-2486.37 0.030 10.sup.3 MVs/L Hepatocyte 1.000 0.997-1.002 0.735 Non available exosomes (U/L) Hepatocyte MVs 1.998 1.543-2.588 <0.001 17.440 5.862-51.884 <0.001 10.sup.3 (U/L) Hepatocyte MVs 17.560 2.157-143 0.0074 6.997 1.614-30.335 0.0093 (U/L) >65 vs. 65 U/L * Total soluble 1.544 1.353-1.761 <0.001 1.544 1.330-1.792 <0.001 cytokeratine 18.sup.+ 10.sup.3 (U/L) Total soluble 11.457 1.405-93.412 0.0227 4.299 0.986-18.744 0.0522 cytokeratine 18.sup.+ (U/L) >300 vs 300 ** Child-Pugh 1.451 0.918-2.292 0.1108 2.054 1.566-2.694 <0.0001 score MELD 1.140 1.019-1.274 0.0216 1.635 1.376-1.943 <0.0001 * 65 U/L: cut-point found with Youden index (with hepatic transplantation considered as censored); ** 300 U/L: cut-point found with Youden index (with hepatic transplantation considered as censored) Bold indicates significant associations with survival.

TABLE-US-00003 TABLE 3 Analyses of the ability of hepatocyte MV levels to predict 6-month mortality adjusted on Child Pugh Score and MELD (Gray's test, transplantation counted as competing risk, death counted as event). Test cohort Validation cohort 95% 95% Hazard confidence Hazard confidence Variable ratio interval P value ratio interval P value Hepatocyte MVs (U/L) 12.616 1.922-82.810 0.0083 4.892 1.283-18.662 0.0201 >65 vs 65 Child Pugh Score 1.244 0.852-1.818 0.2583 2.009 1.472-2.740 <0.0001 Hepatocyte MVs (U/L) 12.229 1.691-88.428 0.0131 3.484 0.972-12.482 0.0502 >65 vs 65 MELD 1.087 0.978-1.208 0.1233 1.604 1.341-1.919 <0.0001 Bold indicates significant associations with survival.

REFERENCES

[0042] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.