Predicting Acute-on-Chronic Liver Failure in Cirrhosis (PREDICT) study;Addendum ancillary study:
Inflammation, endothelial dysfunction and macro- and microvascular flow in acute decompensation of of cirrhosis and acute-on-chronic liver failure

The CANONIC Study consisted in a 28-day detailed prospective observational
investigation in patients admitted to hospital for the treatment of an acute
decompensation of cirrhosis. The main aim of the CANONIC study was to
characterize acute-on-chronic liver failure (ACLF) regarding diagnostic
criteria, stages and natural history up to one year of follow up. Three
quarters of the ACLF-patients (in total ca. 400) recruited in the CANONIC study
presented with ACLF at enrolment. Therefore, the critical period prior to ACLF
development and possible predictors could not be sufficiently analyzed in these
patients due to the study aim and design. Moreover, the limited knowledge about
the ACLF syndrome itself rendered the prospective and detailed analysis of
predictors for the development of ACLF impossible.

The PREDICT Study is therefore designed to prospectively observe patients with
Acute Decompensation (AD) at risk of developing ACLF within three months and to
discover clinical, laboratory and patho-physiological (using prospective
ancillary studies) predictors and mechanisms involved in the development and
clinical course of ACLF, which might help to prevent and treat ACLF. This
International-European, investigator-initiated, multicenter, prospective,
observational study will be performed in centers that belong to the European
Foundation for the Study of Chronic Liver failure (EF-CLIF
foundation)-EASL-CLIF Consortium.

During the CANONIC study, the largest study so far including prospectively
patients with acute decompensation of cirrhosis (AD), the predisposing factors,
clinical and laboratory predictors for the development of ACLF have been
studied. The CLIF-C AD score was developed to discriminate the patients with
high-risk of ACLF within three months. Taking advantage of this knowledge the
current study PREDICT will screen patients with AD and without ACLF at initial
admission, but will investigate more in detail patients with almost 50% risk of
developing ACLF within 3 months (CLIF-C AD score * 60).

Addendum ancillary study:
Systemic inflammation plays a key role in the development of acute-on-chronic
liver failure (ACLF). Bacterial translocation across the intestinal barrier in
advanced cirrhosis promotes the release of pro-inflammatory molecules, thereby
leading to inflammation. An acute increase in systemic inflammation is thought
to be the primary event in developing ACLF. This results in cardiovascular
dysfunction, organ hypoperfusion and organ inflammation. Patients with
cirrhosis are known to have peripheral vasodilatation and a reduced
responsiveness to exogeneous administered vasoconstrictor agents. Inflammatory
mechanisms like the activation of the host innate immune response may trigger
endothelial molecular mechanisms, which possibly contribute to arterial
vasodilation. It is presently unknown whether inflammation is related to micro-
or macrovascular dysfunction in patients with acute decompensation of cirrhosis
(AD) or ACLF. We aim to measure forearm blood flow and sublingual microvascular
flow and endothelial surface layer (ESL) in order to determine whether blood
flow and ESL are associated with markers of endothelial cell activation,
inflammation and systemic hemodynamics in patients with AD or ACLF.

The aim of this study is to assess prospectively the critical period prior to
the development of ACLF (1), to uncover mechanistic and pathophysiological
processes associated with the development and clinical course of ACLF (2) and
to identify the precipitating events of ACLF (3).
collective of patients will be screened out of cirrhotic patients admitted to
hospital with acute decompensation (ascites, overt encephalopathy,
GI-hemorrhage, new onset of non-obstructive jaundice and/or bacterial
infections) but without the presence of ACLF. Thus this study aims to collect
detailed data (including health trajectory) and diverse biological materials
from these patients in order to uncover the pathophysiological mechanisms
associated with ACLF and the time-line of the development of ACLF. Moreover,
using cutting-edge technologies and prospective ancillary studies PREDICT will
identify predictors and potential biomarkers for the development of ACLF.

SPECIFIC GOALS:
* To identify early clinical predictors, biomarkers, mechanisms and
precipitating events during the critical period prior to and involved in the
development and clinical course of ACLF (with special emphasis to medical
trajectory and drug history) in patients admitted to hospital with acute
decompensation of cirrhosis (ascites, GI-hemorrhage, overt encephalopathy, new
onset of non-obstructive jaundice and/or bacterial infections) and the
chronological relationship of the events with occurrence and dynamics of ACLF
development.
* To develop a score predicting ACLF development (CLIF-PREDICT score) and
assess 28-day, 90-day, 6-month and 1-year all-cause mortality in cirrhotic
patients *with acute AD, but without ACLF.
* To serve as a core (hub) study for prospective ancillary studies regarding
diagnosis, prognosis and pathogenesis of AD and ACLF.

Addendum ancillary study:
Protocol 1) To determine endothelial, microvascular and macrovascular function
in patients with (high-risk CLIF-C * 60) AD to assess the relationship between
vascular function and outcome (development of ACLF, mortality)
Protocol 2) To assess markers of endothelial cell activation, systemic
vasoconstrictor systems and inflammation in the critical period prior to the
development of ACLF to predict the development of vascular dysfunction, ACLF
and outcome.

1. This International-European, investigator-initiated, multicenter,
prospective, observational study will be performed in centers that belong to
the European Foundation for the Study of Chronic Liver failure (EF-CLIF
foundation)-EASL-CLIF Consortium.
2. After the enrolment visit, the patients will be stratified into two groups:
Group 1 patients with high risk of ACLF development (CLIF-C AD score * 60) and
in Group 2 patients with low risk of ACLF (CLIF-C AD score <60). The whole
cohort will be followed for 3 months, while Group 1 will be followed more
closely. Development of ACLF is an end-point and in this case a final visit
7-10 days after ACLF development is planned. Data on liver transplantation,
mortality and causes of mortality 3 months, 6 months and 12 months will be
collected in the whole cohort.
3. Prospective collection of biological material and performance of ancillary
studies investigating predictors for development and pathogenesis of ACLF.

Addendum ancillary study:
Protocol 1) Two-dimensional Doppler echocardiography and vascular function
measurements will be obtained by a dedicated radiologist. The average of 5
subsequent measurements will be used at every time point. Sublingual
microcirculatory density and flow will be monitored using sidestream dark field
(SDF) imaging. Briefly, a hand-held video microscope system that
epi-illuminates with stroboscopic green (530 nm) light-emitting diodes will be
positioned gently on the sublingual mucosa on the mouth at the base of the
tongue. Video*s, during at least 10 seconds, from five different sites will be
recorded. Repeated assessments will be performed between 5 and 10 days after
the day of inclusion in the Predict study. If a patient develops ACLF according
to the criteria from the CANONIC study (16), assessment of microvascular and
macrovascular blood flow is repeated at time of development of ACLF, and
between day 5 and 10 after development of ACLF. Twelve weeks after inclusion in
the PREDICT study the Doppler ultrasound measurements will be repeated.

Protocol 2) A pilot study consisting of 200 patients participating in the
PREDICT study (including the 40 patients from protocol 1) will be performed.
Centrally randomly selected patients admitted for AD>60 with (n=100) and
without (n=100) ACLF development during follow-up will be included in study
protocol 2. VWF:Ag and VWFpp, ADMA and markers of endogenous vasoconstrictor
systems will be measured in samples drawn at hospital admission. In total, 900
µl blood (50 µl serum, 600 µl EDTA plasma, 200 µl 3.2% sodium citrate plasma
and 50 µl heparin plasma) will be sampled at baseline.In the group of patients
who developed ACLF during follow-up a second measurement of these biomarkers
will be performed at time of onset of ACLF and 5-8days after development of
ACLF.

There are no anticipated risks associated with this study. The patients will
not receive any direct benefit from participation. There is no guarantee or
promise that patients will receive any benefits from this study.

Addendum ancillary study:
There are no anticipated risks associated with this ancillary study. The
patients will not receive any direct benefit from participation. There is no
guarantee or promise that patients will receive any benefits from this study.