Microvesicles and Monocytes to Predict Mortality of Patients With Cirrhosis
Microvesicles and Monocytes to Predict Mortality of Patients With Cirrhosis
Chronic liver diseases related to viral hepatitis, metabolic syndrome or excessive alcohol
 consumption can evolve towards cirrhosis. Cirrhosis is responsible for 170 000 deaths per
 year in Europe. Initially asymptomatic and called "compensated" it can become "decompensated"
 with the developement of acute complications such as infections, ascites or variceal
 bleeding. The transition from compensated to decompensated cirrhosis is associated with a
 reduction in survival from 95 to 55% at 1 year.

 The only curative treatment for cirrhosis is liver transplantation (LT). Liver transplants
 are allocated according to the severity of the patients. Despite a modest prognostic value
 (area under the ROC curve = 0.7 to predict the risk of death), graft allocation is based on
 the MELD (Model for End-Stage Liver Disease) score including INR, bilirubin and serum
 creatinine. In 2014, 11.5% of registered patients died on the liver transplant waiting list,
 illustrating the need for biomarkers that predict death and improve MELD-based prediction.

 Microvesicles are membrane vesicles released in extracellular space during cell activation or
 apoptosis. Our team showed that circulating levels of hepatocyte microvesicles increase with
 the severity of cirrhosis and predict survival at 6 months independently of MELD score in a
 cohort of 242 patients with cirrhosis.

 Type 1 interferons (IFN-1) are mediators of inflammation, which is excessively activated in
 cirrhosis. Our team has shown that a gene signature (IFN score) measured in the immune cells
 of 101 patients with cirrhosis is able to predict 6 month-survival independently of the MELD
 score.

 Thus, the investigators hypothesize that a composite score combining the level of circulating
 hepatocyte microvesicles, the IFN score and the MELD score could improve the prediction of
 survival in patients with severe cirrhosis.

 The aim of this study is to compare the prognostic performance for the cumulative incidence
 of death at 6 months of a composite score including MELD, hepatocyte microvesicle level and
 IFN score with that of the MELD score alone, in patients with Child B or C cirrhosis,
 considering liver transplantation as a competitive risk.

 To address this question, peripheral blood from 335 patients with Child B or C cirrhosis will
 be obtained and hepatocyte microvesicle levels and IFN score will be measured using
 ELISA/filtration and Real Time-quantitative PCR.
Natural history of cirrhosis

 Cirrhosis is the end stage form of chronic liver disease. It is estimated that 200,000 to
 500,000 people in France have cirrhosis. Cirrhosis is responsible for more than 170,000
 deaths per year in Europe.

 The main causes of cirrhosis include excessive alcohol consumption, the leading cause in
 Europe, and chronic viral hepatitis B and C, which are the leading causes in Asia and Africa.
 In addition, the current epidemic of obesity and type 2 diabetes worldwide has led to a sharp
 increase in the incidence of non-alcoholic steatohepatitis associated with metabolic
 syndrome, particularly in North America.

 Cirrhosis is defined histologically as a diffuse alteration in the architecture of the liver
 by annular fibrosis associated with regeneration nodules. With the emergence of non-invasive
 tests allowing fibrosis measurement, cirrhosis is now considered highly probable when the
 liver elasticity measured by Fibroscan® is > 15 kPa.

 Patients with cirrhosis may be perfectly asymptomatic and have normal liver function. In this
 case, the Child-Pugh score, which rates the severity of cirrhosis, is "A". However, the
 severity of cirrhosis may increase, resulting in the development of signs of liver failure,
 such as jaundice, decreased coagulation factors and hypoalbuminemia, and/or ascites or
 hepatic encephalopathy. Cirrhosis is then classified as Child-Pugh B or C.

 The progression of cirrhosis involves various processes: on the one hand, a slow and
 continuous worsening associated with the persistence of hepatic aggression by one or more
 causal agents (alcohol and/or metabolic syndrome and/or virus) and on the other hand, rapid
 worsening occurring with acute complications of the disease such as variceal bleeding or
 bacterial infections. Indeed, cirrhosis is associated with major alterations in innate
 immunity that promote severe bacterial infections, which can lead to organ failure. Such a
 complication represents a turning point in the natural history of cirrhosis since the
 transition from compensated to decompensated cirrhosis is associated with a reduction in
 1-year survival from 95 to 60%. When these complications are associated with organ failure,
 short term mortality is dramatically increased with one-third of patients dying within one
 month. The association of acute decompensation of cirrhosis with one or more organ failures
 has recently been defined as a new syndrome named Acute-on-Chronic Liver Failure (ACLF).

 The only curative treatment for cirrhosis is liver transplantation (LT). However, each year,
 between 10 and 15% of patients on the French liver transplant waiting list cannot access
 transplant and die prematurely from an acute complication of cirrhosis. In addition, many
 patients die before they are even put on the transplant waiting list, as evidenced by the
 results of a recent French multicenter study showing that the 6-month mortality rate for
 patients with decompensated cirrhosis, Child C, remains equal to 25%[8].

 Predicting of the outcome is therefore a major challenge in the management of cirrhotic
 patients.

 Prediction of the outcome of patients with cirrhosis: clinical scores.

 Currently, prediction of the outcome of cirrhotic patients is based on the MELD (Model for
 End-Stage Liver Disease) score. MELD score includes three objective laboratory criteria (INR,
 serum bilirubin and serum creatinine). It was established in 2000 in an American cohort to
 predict the risk of death at 3 months after TIPS placement (Transjugular Intrahepatic
 Portosystemic Shunt). Its use was very quickly extended to the prediction 3-month survival of
 cirrhotic patients on the transplant waiting list. Since 2007, in France, graft allocation
 has been based on the MELD score, with patients with the highest score being transplanted
 first.

 Since that time, the predictive value of MELD has been studied in large cohorts, showing that
 MELD's performance in predicting death is modest, with areas under the ROC curves (AUROC)
 ranging from 0.64 to 0.72[3, 7, 19, 20]. In 2014, 7 years after the introduction of the MELD
 score, it was found that 11.5% of patients on the liver transplant waiting list still died
 before they could be transplanted. These data illustrate the need to improve our ability to
 predict death of patients with advanced cirrhosis.

 In this perspective, some authors have tried to improve the MELD score by adding one or more
 variables, such as serum sodium (MELD-Na=MELD-Na-[0.025×MELD× (140 - Na)] + 140). The
 contribution of these modified MELD scores was not considered sufficient to promote their use
 in clinical practice, leaving the objective of improving MELD unmet.

 Prediction of the outcome of patients with cirrhosis: novel biomarkers

 Beside clinical scores, some biomarkers look promising. Indeed, as part of the close
 collaboration between the clinical departments of Beaujon Hospital (Hepatology and
 Anesthesia-Resuscitation) and the Inserm teams (Dr E Weiss, Dr R Moreau, Inserm U1149, Centre
 de recherche sur l'inflammation, Paris) and Pr PE Rautou, Dr C Boulanger, Inserm U970 ;
 Centre de recherche cardiovasculaire, Paris), two types of biomarkers have recently been
 identified that predict the survival of patients with cirrhosis.

 Microvesicles are vesicles released into the extracellular medium during cell activation or
 apoptosis, two phenomena strongly involved in cirrhosis. The Inserm U970 team (Pr PE Rautou,
 Dr C Boulanger) showed in 2012 that the circulating concentrations of hepatocyte
 microvesicles increase with the severity of cirrhosis. In addition, microvesicles in patients
 with child B or C cirrhosis promote portal hypertension by inducing arterial hyporeactivity
 to vasoconstrictive agents. Based on these results, a prospective study including 242
 patients with cirrhosis showed that circulating concentrations of hepatocyte microvesicles
 are able to predict the survival at 6 months of patients with cirrhosis independently of the
 MELD score.

 The susceptibility of cirrhotic patients to severe infections is related to abnormalities in
 their innate immune response and in particular to the occurrence of an excessive inflammatory
 response, responsible for organ failure. Type 1 interferons (IFN 1) are inflammatory
 mediators whose impact on the prognosis of sepsis could be harmful. To determine whether type
 1 IFN pathway activity was involved in the innate immune response deregulation and in the
 susceptibility to sepsis of patients with cirrhosis, the Inserm U1149 team measured, using
 Real-Time quantitative PCR, a gene signature (IFN score) reflecting the activation of this
 pathway in the immune cells of 99 patients with decompensated cirrhosis hospitalized at the
 liver department at Beaujon Hospital. The investigators have thus shown that the IFN score is
 able to predict the risk of death of cirrhotic patients independently of the MELD score with
 AUROC equal to 0.79 (0.7 for MELD).

 It should be noted that these two biomarkers (hepatocyte microvesicles and IFN score) reflect
 different and complementary pathophysiological processes: hepatocyte microvesicles reflect
 persistent liver injury while IFN score reflects innate immune response. It should also be
 noted that these biomarkers, protected by two international patents (EP121528133.5 and
 EP2016/077129) can be measured on peripheral blood using simple and readily available
 techniques (ELISA/filtration, qPCR).

 Hypotheses

 In this project, the investigators hypothesize that prediction of death of patients with
 advanced cirrhosis (Child-Pugh B or C) by a composite score combining circulating
 concentrations of hepatocyte microvesicles, IFN score and MELD score will be better than that
 assessed using the MELD score alone.

 This composite score will help physicians to judge the emergency of liver transplantation and
 thus to refine criteria for the allocation of liver transplants. This composite score may
 also identify a patient population at particular risk of death that should be subjected to
 increased surveillance while waiting for liver transplantation. Through these two approaches
 (better prioritization of patients for liver transplantation and increased monitoring of
 high-risk patients), this new composite score will reduce mortality in cirrhosis patients
 waiting for liver transplantation.

 Finally, new therapeutic approaches to improve survival of patients with severe cirrhosis
 awaiting liver transplantation (e. g. by reducing the occurrence of complications) are
 regularly tested. The composite score the investigators propose could also be used as a
 "companion" to monitor the effectiveness of these new treatments.

 Population

 Patients with Child-Pugh B or C cirrhosis (proven by liver biopsy, by non invasive method of
 fibrosis quantification or by a combination of clinical, biological and radiological signs,
 see above) will be included during a two-year period.

 Patients will be included during follow-up outpatient visits in hepatology or during
 scheduled hospitalization for example for digestive endoscopy to screen signs of portal
 hypertension or for ascites removal. They will therefore be in a stable condition.

 Presence of acute events or events that may modify the risk of death or liver
 transplantation, and/or the inflammatory response or release of microvesicles will be among
 non-inclusion criteria (see above).

 Biomarker measurements

 After informed consent, fifteen to twenty milliliters of peripheral venous blood (3 EDTA
 tubes and 3 citrate tubes) will be collected from patients participating in the study

 For hepatocyte microvesicles, citrated tubes will be centrifuged twice and then frozen at
 -80°C until analysis.

 For the IFN score, blood contained in the 3 EDTA tubes will be used to isolate Peripheral
 Blood Mononuclear Cells (PBMCs). PBMCs will be lysed to obtain RNA that will be stored at
 -80°C until the IFN score is measured.

 At the end of the 2-year inclusion period, measurements of hepatocyte microvesicles and IFN
 score will be performed for all patients during a 1-year period. Plasma concentrations of
 hepatocyte microvesicles will be determined on platelet-free plasma using ELISA/filtration.
 To measure IFN score, RNA from previously isolated PBMCs will first be reverse transcribed.
 Then, the expression of five mRNAs of interest will be measure to calculate the IFN score.

 Primary endpoint

 The primary endpoint is the area under the ROC curve of the composite score including MELD,
 hepatocyte microvesicle concentration and IFN score for predicting the cumulative incidence
 of death at 6 months (considering liver transplantation as a competitive risk) compared to
 the area under the ROC curve of the MELD score alone.

 Measurement of the primary endpoint will be performed according to the recommendations
 specific for survival analysis in patients with cirrhosis, using a survival analysis that
 considers liver transplantation as a competitive risk. Data from patients who did not die or
 have not been transplanted will be censored at the end of follow-up or on the date of last
 news (if the patient is lost to follow-up) or at the date of termination of participation (in
 the event of termination of participation). Follow-up period will be one year after inclusion
 (maximum 13 months for patients who have been transplanted in the 12th month after
 inclusion). A follow-up visit 6 and 12 months after inclusion will be scheduled. Data on
 possible deaths or transplants will be obtained during this visit, using medical records, or
 following telephone calls to patients or their relatives. For subjects who are lost to
 follow-up, requests will be made to the CépiDc to obtain their vital status. Data concerning
 possible transplants will be obtained via a request for access to the "Cristal" database
 (https://www.sipg.sante.fr) developed by the Agence de la biomédecine.
Inclusion Criteria:

 -  Age between 18 and 90 years

 -  Child-Pugh B or Child-Pugh C cirrhosis diagnosed on the basis of one or several of the
 following elements :

 -  Liver Biopsy

 -  Liver stiffness>15kPa measured by Fibroscan

 -  Combination of clinical, laboratory and imaging criteria characteristic of
 cirrhosis (association of signs of portal hypertension, liver failure and
 abnormal liver morphology in a patient with at least one cause of cirrhosis)

 Exclusion Criteria:

 -  Acute renal failure (increase in serum creatinin level by more than 1.5 times the
 baseline value or by more than 26 μmol/l from the baseline value) within 15 days
 before inclusion

 -  Bacterial infection proven or highly suspected on the basis of clinical-laboratory
 features within 15 days of inclusion

 -  Digestive bleeding within 15 days before inclusion

 -  Alcoholic hepatitis in the previous month

 -  History of porto-systemic shunt or liver transplant

 -  Primary sclerosing cholangitis

 -  Primary biliary cirrhosis

 -  Budd-Chiari Syndrome

 -  Hepatocellular carcinoma outside the Milan criteria contraindicating transplantation

 -  Active extrahepatic neoplasia

 -  HIV or known immune deficiency or immunosuppressive treatment

 -  Pregnant or breastfeeding woman

 -  Protected populations: persons under guardianship or curatorship

 -  Patient not affiliated to social security

 -  Patient who did not signed consent

 -  Ongoing participation in an intervention research whose protocol could, according to
 the literature, modify the release of hepatocyte microvesicles or the IFN score