ADAMTS-13 LEVEL AS PREDICTIVE BIOMARKER FOR DEVELOPMENT OF PORTAL VEIN THROMBOSIS IN LIVER CIRRHOSIS (ADAPTHRO)

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.