Project description - abstract

Introduction

Non alcoholic fatty liver disease (NAFLD) and non alcoholic steatohepatitis (NASH) are emerging as the most prevalent liver diseases in Western world due to their close association with the epidemics of diabetes and obesity. While the epidemiological burden of NAFLD/NASH is now widely recognized, there is still a debate about its severity and long-term outcomes.

Objectives

The overall objective of this proposal is to better understand the relationship between obesity, diabetes and liver injury.

Specifically, this proposal will deal with three aspects:

1) the current epidemiological burden and characteristics of hepatocarcinoma (HCC) occurring in patients with NAFLD/NASH;

2) the impact of features of the metabolic syndrome and of histologically defined NAFLD/NASH on the clinical course of chronic hepatitis C (HCV) and B (HBV);

3) the optimization and validation of non-invasive diagnostic algorithms of severe NASH in patients at risk of NAFLD.
- To collect both retrospectively and prospectively well phenotyped cases of patients with histologically documented NAFLD.
- To describe clinical, biological and metabolic correlates of advanced NASH in these patients.
- To devise non-invasive algorithms for the diagnosis of advanced NASH based on serum markers of adipocytokines, apoptosis, inflammatory and fibrosis markers and selected imaging procedures.
- To cross-validate results from the Romanian NAFLD/NASH cohort with those from the multicentric European FLIP cohort.

Methods and approach

This is a single center, 3-year prospective, observational study.
The clinical research on HCC, chronic HCV and HBV and NAFLD/NASH will be conducted prospectively in a single academic center in Bucharest, Romania. For the first and the third objectives the results will be compared with those collected in a European registry of HCC arising in NASH and with those from the multinational prospective cohort of patients with steatosis, metabolic risk factors and histologically documented NAFLD. Both these cohorts are being currently developed as part of an Framework Program 7 funded European Consortium called FLIP (Fatty Liver Inhibition of Progression) which is coordinated by Prof Vlad Ratziu.

1. HCC cohort:

Inclusion criteria: (i) all incident cases of HCC (according to EASL/AASLD diagnostic recommendations), (ii) adult patients ≥ 18 years old

Exclusion criteria: (i) primary non HCC liver cancer, (ii) secondary liver tumors

Study Design: For all incident cases of HCC we will first identify the underlying liver disease based on standard diagnostic tests (e.g. alcoholic liver disease, HCV or HBV infection, hemochromatosis, autoimmune hepatitis etc.). If none of these “classical” causes of CLD can be identified the following items will be recorded : a) the presence of steatosis (on ultrasound); b) the presence of metabolic risk factors (see Table); c) an historical biopsy documented NAFLD. If at least one of these items is confirmed those cases will be labeled NAFLD-related. If none of these items can be confirmed the case will be labeled cryptogenic.
Staging of HCC (number and size of the nodules, portal vein thrombosis, extrahepatic spread, BCLC class), the presence or absence of cirrhosis, hepatic liver function and cirrhosis complications. Patient demographic, anthropometric, clinical, biological and metabolic features will be recorded, thus allowing a better characterization of tumoral and host factors in NAFLD-related HCC compared to HCC of other etiologies (viral and alcoholic). Initial treatment options will be recorded allowing a comparison of accessibility to curative treatments in NAFLD-related HCC compared to HCC of other etiologies (viral and alcoholic).
A follow-up until death, liver transplantation or the end of the grant program will allow to determine survival and time to progression thus gaining a better insight into the neoplastic course of NAFLD-related HCC.

2. IR and fibrosis progression in CLD others than NAFLD/NASH

Inclusion criteria: (i) consecutive patients with CHC (detectable HCV RNA) and CHB (positive hepatitis B surface antigen and detectable serum HBV DNA), (ii) adults ≥ 18 years old, (iii) liver biopsy (maximum 6 month before inclusion).

Exclusion criteria: (i) advanced cirrhosis (Child Pugh B and C), (ii) hepatocellular carcinoma, (iii) other causes of CLD or mixed etiologies, (iv) alcohol consumption > 20 g/day, (v) HIV co-infection, (vi) previous antiviral therapy one year preceding liver biopsy, (vii) previous type 1 diabetes.

Study design: Patients with chronic HBV or HCV infection and available liver biopsy will be evaluated for the presence of NAFLD (histologically defined steatosis or steatohepatitis), phenotypic complications of the metabolic syndrome and surrogate markers of IR. We will analyze the correlations between these items and the fibrotic severity and inflammatory activity of viral hepatitis, and the viral replication level. The overall objective is to determine if histological NAFLD or manifestations of the MS or IR impact the severity of viral associated liver disease and the level of viral replication. In particular we will determine if these factors have a similar impact on the context of HCV and HBV infection or if the interaction is restricted to HCV infection only. Disease severity will be determined based on METAVIR classification (e.g. staging of fibrosis and grading of necroinflammatory activity). Associated metabolic risk factors will be recorded and insulin resistance assessed by the HOMA-IR index = fasting glucose (mmol/l) x fasting insulin (microUI/ml) /22.5. Patient’s demographic, anthropometric, clinical and biological data, viral load and serum markers of fibrosis and transient elastometry of the liver will be recorded. This will allow to analyze the impact of IR and moderate-severe steatosis on the severity of fibrosis and fibrosis progression (METAVIR stage/duration of disease) according to host (age, presence/absence of metabolic risk factors) and viral (viral load, viral genotype) factors in CHB and CHC patients and to identify those factors independently associated with disease severity.

3. NAFLD/NASH cohort

Patients with histologically defined NAFLD/NASH will be included and those with advanced disease will be identified and compared to those with minimal disease in order to understand basic clinical and biological risk factors for advanced disease. We will also evaluate the diagnostic potential of a combination of well identified (FibroTest) or novel serum markers and liver elastometry to predict the presence/absence of advanced disease.

Inclusion criteria: (i) patients with histological proven NAFLD (Brunt, Kleiner), (ii) adults ≥18 years old. The indication for liver biopsy will be established based on: (i) presence of steatosis on ultrasound examination and (ii) the presence of 1 major or 2 minor metabolic risk factors, or (iii) elevated transaminases levels > 2xN.

Exclusion criteria: (i) others causes of chronic liver disease (viral hepatitis B and C, autoimmune hepatitis, hemochromatosis, Wilson disease, alpha1-antitripsine deficiency), (ii) alcohol consumption (more than 30g/d in men and 20 g/d in women), (iii) causes of secondary NASH.

Study design: In patients with well defined NAFLD (based on the above described clinical and histological criteria), demographic, anthropometric, metabolic risk factors, biological data (liver function tests, prothrombin time, albumin, serum glucose and insulin, HOMA-IR, lipidic profile, etc), adipocytokines, serum markers of steatosis, inflammation, fibrosis and apoptosis will be recorded. This will allow a better description of clinical, biological and metabolical profile of NAFLD/NASH patients according to disease severity (simple steatosis vs steatohepatitis), a better insight into the mechanisms involved in fibrosis progression and the validation of new markers and algorithms to simplify the diagnosis work-up and to allow early identification of those patients at risk for disease progression. Finally, in regards with all these data, better screening and monitoring strategies could be proposed for NAFLD/NASH patients.

Sub-study NAFLD and chronic kidney disease

Background

NAFLD and chronic kidney disease (CKD) shares many common metabolic risk factors and pathophysiological pathways. Given the health burden of both diseases it is of crucial interest to understand the mechanism’s interplay linking NAFLD and CKD. Several cross-sectional and large-prospective studies have already documented a higher prevalence of CKD in NAFLD patients but only a few included patients with histological well-documented NAFLD. However, several aspects need further clarification. First, the causal relationship between NAFLD and CKD (above and beyond known risk factors), it is not clearly established because of cross-sectional design of previous studies. Second, it is important to state if the risk for CKD is also increased in patients with simple steatosis or is only seen in NASH patients. Finally, none of these studies explored if NAFLD selectively contributes to pathogenesis of different types (glomerular or tubular) of CKD.

Aim

The aim of our study is to analyze the prevalence, type and mechanisms responsible of CKD in a large cohort of patients with histological well-defined NAFLD.

Patients and methods

Patients with histological proven NAFLD, which met the inclusion and exclusion criteria. Estimation of renal function based on the following parameters: urea, creatinine, estimated GFR, microalbuminuria, KIM, beta 2 microglobulin, NGAL.

Presumed results

This project is intended to explore the impact of IR in liver injury with special emphasis on NAFLD/NASH-related liver fibrosis and liver carcinogeneis. It will allow a better characterization of the most severe forms of liver damage (advanced fibrosis, cirrhosis and primary liver cancer) in patients with features of the metabolic syndrome, currently the top cause of chronic liver disease in developed countries.
A particular emphasis will be placed on primary liver cancer related to NASH, diabetes and obesity as no comprehensive study has been conducted in Romania on this emerging cause of liver cancer in Western European countries and in the United States. The results generated by this proposal will help the identification of those with advanced liver disease (hence in need for specific treatment and sustained monitoring) among the large number of patients exposed to metabolic risk factors. It will also help optimize non-invasive diagnostic methods, which will simplify diagnosis and reduce the need for liver biopsy, a procedure unsuitable for the large number of patients at risk, and often ill-accepted by patients. Because the project leader for this project is also the overall coordinator of the FP7 -funded European FLIP consortium on NAFLD, there is a unique opportunity for cross-validation of the results obtained in Romanian patients with those obtained in a large European multinational consortium.