ADAMTS-13 LEVEL AS PREDICTIVE BIOMARKER FOR DEVELOPMENT OF PORTAL VEIN THROMBOSIS IN LIVER CIRRHOSIS (ADAPTHRO)
ADAMTS-13 LEVEL AS PREDICTIVE BIOMARKER FOR DEVELOPMENT OF PORTAL VEIN THROMBOSIS IN LIVER CIRRHOSIS (ADAPTHRO)
Patients with cirrhosis of viral etiology (HCV/HBV); Patients with cirrhosis of any other
 etiology (alcohol, idiopatic, autoimmune). Planned Number of cirrhotic subjects 200 patients
 Inclusion Criteria Subjects (18 yr old) with liver cirrhosis of any etiology, Exclusion
 Criteria All patients should not have hepatocellular carcinoma or other malignant tumors,
 they should not be treated with anticoagulant / antiplatelet agents, not affected by PVT
 already diagnosed and not suffering from congenital coagulation disorders (haemophilia A / B,
 von disease Willebrand, another congenital deficiency of coagulation factors) or severe
 thrombocytopenia (<30,000 Plt / μL). Subject has participated in another clinical study
 within 30 days prior to study enrollment or is scheduled to participate in another clinical
 study on cirrhosis Primary Objective To describe the prospective modification of ADAMTS-13
 level and other coagulation variables (e.g. FVIII, VWF:Ag/VWF:act) in cirrhotic patients
 during 18 months from the enrolment and to verify their predictive role as biomarker of
 development of portal vein thrombosis (PVT) Secondary Objectives To describe prospectively
 the modification of ADAMTS-13 level as a function of the etiology of cirrhosis Statistical
 analysis The total duration of the study will be of 12 months. The sample size of 200
 subjects will be selected as a feasible number of patients to be recruited in a period of six
 months. The patients will be consecutively enrolled and followed for 18 months. As a result,
 in a follow up period of 18 months about 20-25 cases of PVT are expected. Continuous
 variables will be expressed as means ± standard deviations. In addition to descriptive
 statistics (location parameters), univariate analysis will be performed on each parameter and
 development of PVT during the follow up period. In previous observational studies both 1) a
 reduced PV flow [prospectively] and 2) a reduction of ADAMTS-13 are significantly associated
 with PVT. These associations will be investigated prospectively and analyzed simultaneously
 by a multivariate analysis and ROC curve to establish the sensitivity and specificity of
 these parameters as predictors of PVT development. Analyses will be performed using available
 data
BACKGROUND AND JUSTIFICATION The occurrence of portal vein thrombosis (PVT) radically changes
 the prognosis of cirrhotic patients, particularly those awaiting liver transplantation. If
 this happens, it occurs in about 8-10% / year in patients with cirrhosis awaiting liver
 transplantation. Some studies have suggested the involvement of various risk factors for the
 development of PVT in cirrhotic patients, including the classic congenital and acquired
 thrombophilic conditions [1-6], although contributions of our research group have shown that
 a key element in the development of PVT in these patients is the decreased velocity of portal
 blood flow [7, 8]. The possible contribution of the von Willebrand factor (VWF) and
 ADAMTS-13, produced by the liver stellate cells [9], for the development of PVT is rather
 poorly documented. In a recent observational study of the proponent [10], conducted on 84
 patients with liver cirrhosis, it was found that the level of circulating ADAMTS-13 is
 significantly lower in the group with PVT (median = 16.8% vs. 69.1%, p = 0.0047) and
 independently associated with the presence of portal thrombosis (p = 0.007). However, given
 the non-longitudinal nature of the study, it is possible that the decreased level of
 ADAMTS-13 observed is the consequence and not the cause of PVT. It is also yet to investigate
 the association of ADAMTS-13 level with the specific etiology of cirrhosis (viral, alcoholic,
 idiopathic, etc.) and the resulting inflammation during the course of the disease.

 Duration of Study Period(s) and Subject Participation Two hundred (200) patients,
 consecutively enrolled in a period of about 6 months, will be followed for 18 months from the
 enrolment. As a result, in the subsequent 18 months about 20-25 cases of PVT will be
 expected. All patients will sign an informed consent. The severity of liver function failure
 will be assessed using the Child-Pugh score [11].

 Primary Endpoint To describe in a prospective way the association of both basal ADAMTS-13
 level and portal vein flow with development of PVT in cirrhotic patients during 18 months
 from the enrolment. Secondary Endpoints To describe prospectively the modification of
 ADAMTS-13 level as a function of the etiology of cirrhosis Coagulation and biochemical
 parameters

 The prospective study will measure the following biochemical and coagulation parameters at
 each follow-up visit, scheduled at interval of 4 months according to routine controls:

 Coagulation and thrombophilia factors All blood samples will be drawn under fasting
 conditions in the morning. Determinations of PT, APTT, fibrinogen, antithrombin, protein C,
 protein S, lupus anticoagulant, FVIII (two-stage chromogenic assay) and D-dimer will be
 carried out on an automatic coagulometer (ACL Top 700, IL). VWF-antigen VWF:activity will be
 measured in plasma samples by chemiluminescence assays. VWF multimer (VWFm) pattern was
 measured as previously reported [10]. FV Leiden R506Q and prothrombin G20210A genotyping of
 these polymorphisms was performed by using commercial kits based on PCR-RT-based method (from
 EliTechGroup S.p.A., Torino, Italy) on an automatic instrument (Model 7300, Applied
 Biosystem, Foster City, CA, USA).

 Measurement of ADAMTS-13 activity. ADAMTS-13 activity was measured in citrated plasma samples
 by a fluorescence resonance energy transfer (FRET)-based assay using a VWF86 amino-acid
 peptide substrate contained in a commercial kit from Instrumentation Laboratory (ADAMTS-13
 activity, Werfen Group, Milano, Italy) and a Varian Eclipse spectrofluorometer, as previously
 detailed [10, 13] and expressed as % of normal plasma.

 Clinical Chemistry assays. All clinical chemistry assays (plasma albumin, creatinine,
 alanine-leucine-amino-transferase (ALT) and total bilirubin) will be performed as a part of
 routine diagnostic work-up within 24 hours of clinical observation in the central laboratory
 of the "A. Gemelli" Hospital, using heparinized plasma samples and commercial reagents from
 Siemens.

 Secondary objectives. The secondary objectives of analyzing the levels of ADAMTS-13 and VWF
 as a function of the etiology of cirrhosis will be further assessed only after a certified
 diagnosis of the particular etiologic of cirrhosis. In the case of HCV-associated cirrhosis
 also the genotype of HCV will be analyzed.

 Informed Consent and Enrollment Any patient who will provide informed consent (i.e., signs
 and dates the informed consent form and assent form, if applicable) and will meet all other
 inclusion/exclusion criteria will be considered enrolled in the study.

 Subject Identification Code. The following series of numbers will comprise the subject
 identification code (SIC): protocol acronym (ADAPTHRO), and 2-digit subject number (e.g.,
 01….02) reflecting the order of enrollment (i.e., signing the informed consent form). For
 example, the third subject who signed an informed consent form will be identified as
 ADAPTHRO-03. All study documents (e.g., CRFs, clinical documentation, etc.) will be
 identified with the SIC.

 Screening and Study Visits The study site is responsible for maintaining an
 enrollment/screening log that includes all subjects enrolled. The log also will serve to
 document the reason for screening failure. All screening data will be collected and reported
 in CRFs, regardless of screening outcome.

 Subject Completion/Discontinuation A subject is considered to have completed the study when
 he/she ceases active participation in the study because the subject has, or is presumed to
 have, completed the study according to the protocol, participated for the specified
 observational time and when the study exit form has been completed. Reasons for
 completion/discontinuation will be reported on the Completion/ Discontinuation CRF page,
 including: completed, screen failure, adverse event (e.g., death), discontinuation by subject
 (e.g., lost to follow-up [defined as 3 documented unsuccessful attempts to contact the
 subject], dropout), physician decision (e.g., pregnancy, progressive disease, protocol
 violation(s)), Vital Signs Vital signs will include height (cm) and weight (kg). Height and
 weight will be collected, if available, at screening visit, each annual interval visit, and
 study completion/termination.

 STATISTICAL ANALYSIS. Planned Statistical Analysis Continuous variables will be expressed as
 means ± standard deviations. Categorical variables will be expressed as frequencies and
 percentages. Fisher's exact tests and/or Chi-Square tests will be mainly used to compare
 qualitative variables and Student's t-test for quantitative unpaired data. A p-value < 0.05
 will be considered statistically significant in an exploratory sense. In addition to
 descriptive statistics (location parameters), and univariate analysis, multivariate analyses
 are planned. Independent variables that will be found associated with PVT in univariate
 analysis with p<0.05 will be used as independent covariates associated with PVT in the next
 18 months in the multivariable logistic regression analysis. A two-sided p <0.05 was defined
 as statistically significant. Analyses will be performed using SPSS software (version 21,
 Chicago, IL, USA) and GraphPad Prism software (GraphPad Software, Inc, La Jolla, CA, USA).

 Informed Consent. Investigators will choose patients for enrollment based on the study
 eligibility criteria.The investigator will exercise no selectivity so that no bias is
 introduced from this source. All patients must sign an informed consent form before entering
 into the study according to applicable regulatory requirements. Before use, the informed
 consent form will be reviewed by the EC. By signing the informed consent form, patients agree
 to take part in the study.

 Confidentiality Policy The investigator will comply with the confidentiality policy as
 described in the Non- Interventional Trial Agreement.

 Study Documents and Case Report Forms The investigator will maintain complete and accurate
 study documentation in a separate file. Documentation may include medical records, records
 detailing the progress of the study for each subject, signed informed consent forms,
 correspondence with the EC, enrollment and screening information, CRFs and laboratory
 reports. The investigator will comply with the procedures for data recording and reporting.

 Document and Data Retention The investigator will retain study documentation and data in
 accordance with applicable regulatory requirements and the document and data retention
 policy, as described in the Non-Interventional Study Agreement.
Inclusion Criteria:

 Inclusion criteria:

 - Patients with liver cirrhosis of any etiology, diagnosed using imaging and/or
 histological criteria and observed in the Liver Unit of the "A. Gemelli" University will be
 included in this study. All patients will sign an informed consent. The severity of liver
 function failure will be assessed using the Child-Pugh score [11]. Upper gastrointestinal
 endoscopy will be performed in all patients; the size of esophageal varices will be
 recorded as well as the presence of previous episodes of gastrointestinal bleeding. All the
 subjects will be outpatients and the time interval between the follow up visits will be
 approximately 120 days. The ultrasound criteria for the diagnosis of PVT will be the
 detection of echogenic material within one or more of the following vessels: portal trunk,
 right portal branch, left portal branch, splenic vein and superior mesenteric vein. Color
 and pulsed Doppler analysis will allow to confirm the diagnosis and to distinguish partial
 from complete obstructive thrombosis and in all cases the Doppler ultrasound diagnosis of
 PVT will be confirmed by contrast enhanced abdomen computed tomography. The presence and/or
 extension of the thrombosis to the other major vessels of the portal venous system
 (mesenteric or splenic vein) will registered in all patients. PVT will be classified
 according to the extension of thrombosis, as previously reported: grade I is referred to as
 portal thrombosis confined to the portal vein beyond the confluence of the splenic vein;
 grade II was a PVT extended to the superior mesenteric vein, but with patent mesenteric
 vessels; grade III is a PVT extended to the whole splanchnic venous system, but with large
 collaterals; grade IV is a thrombosis extended to the whole splanchnic venous system with
 only fine collaterals [12]. All the biochemical and coagulation parameters described below,
 including ADAMTS-13 and VWF levels, will be measured at each follow up visit.

 Exclusion Criteria:

 -  Exclusion Criteria All patients should not have hepatocellular carcinoma or other
 malignant tumors, they should not be treated with anticoagulant / antiplatelet agents,
 not affected by PVT already diagnosed and not suffering from congenital coagulation
 disorders (haemophilia A / B, von disease Willebrand, another congenital deficiency of
 coagulation factors) or severe thrombocytopenia (<30,000/µl).