bwge2017

Authors
S. VAN HEES (1), S. BOURGEOIS (2), H. VAN VLIERBERGHE (3), T. SERSTÉ (4), P. MICHIELSEN (1), H. REYNAERT (5), J. HENRION (6), S. NEGRIN-DASTIS (7), L. LASSER (8), F. JANSSENS (9), G. ROBAEYS (10), P. STÄRKEL (11), C. MORENO (12), F. NEVENS (13), T. VANWOLLEGHEM (1) / [1] Antwerp University Hospital, Edegem, Belgium, Department of Gastroenterology and Hepatology, [2] ZNA Antwerpen, , Belgium, Department of Gastroenterology and Hepatology, [3] Ghent University Hospital, Ghent, Belgium, Department of Gastroenterology and Hepatology, [4] CHU Saint-Pierre, Brussels, Belgium, Department of Gastroenterology and Hepatology, [5] University Hospital Brussels, Vrije Universiteit Brussel, , Belgium, Department of Gastroenterology and Hepatology, [6] Centre Hospitalier de Jolimont-Lobbes., La Louvière, Belgium, Department of Gastroenterology and Hepatology, [7] Grand Hopital de Charleroi, Charleroi, Belgium, Department of Gastroenterology and Hepatology, [8] CHU Brugmann Brussels, Brussels, Belgium, Department of Gastroenterology and Hepatology, [9] Jessa Hospital, Hasselt, Belgium, Department of Gastroenterology and Hepatology, [10] ZOL, Genk, Belgium, Department of Gastroenterology and Hepatology, [11] Cliniques universitaires St-Luc, Brussels, Belgium, Department of Gastroenterology and Hepatology, [12] CUB Hôpital Erasme, Bruxelles, Belgium, Department of Gastroenterology, Hepatopancreatology and Digestive Oncology, [13] University Hospital Leuven, KU Leuven, , Belgium, Department of Gastroenterology and Hepatology

Introduction
Cessation of Nucleo(s)tide analogues (NA) therapy after HBeAg seroconversion is associated with a high degree of relapse, but evidence in Caucasian patients is scarce.

Aim

We investigated relapse rates and clinical outcomes after NA stop in a Belgian cohort of HBeAg positive Chronic Hepatitis B (CHB) patients.

Methods
This is a pooled analysis of non-immune-suppressed HBeAg-positive, mono-infected CHB patients from 13 hospitals in Belgium, treated with different NA for ≥ 3 months. Data were collected between 1998 and 2016. HBeAg seroconversion was defined as the loss of HBeAg and the appearance of anti-HBeAg on two time points ≥1 month apart. Virological relapse was defined as HBV DNA>2000 IU/mL; biochemical relapse as ALT>2xULN (with ULN defined as 40 IU/mL). Clinical events were defined as the appearance of hepatic decompensation, HCC or liver-related death. Cox regression model was used to identify predictive factors for relapse. Follow-up time was calculated as time from HBeAg seroconversion until relapse or end of follow-up.

Results
A total of 326 patients (74.8% male; 63% Caucasian; 17% African) were included; 96 of whom showed HBeAg seroconversion. Treatment was stopped in 57/96 patients (of whom 8 were cirrhotic at baseline) after HBeAg seroconversion with a subsequent median consolidation therapy of 7.5 months. The median follow-up after treatment stop was 2.9 years during which 25 patients showed relapse (14 solely virological, 11 combined biochemical and virological), necessitating retreatment in 15 cases. HBeAg reversion was observed in 3/25 (12%) relapsed patients. Cox regression model showed that neither the presence of cirrhosis (HR 3.386; p=0.116) at start of treatment, nor Caucasian ethnicity (HR 0.509; p=0.133) were significantly associated with relapse after treatment stop. Relapse was accompanied by hepatic failure in two cases leading to liver-related death. Treatment was continued after HBeAg seroconversion in 26 patients (of whom 9 were cirrhotic at baseline) for a median of 4.1 years. Three patients (all cirrhotic) developed ascites in the latter group, but recovered thereafter. No patient died.
Conclusions: Treatment cessation after HBeAg seroconversion led to relapse in 44% of predominantly Caucasian patients within a median follow-up 1056 days. Two relapsed patients showed severe clinical events leading to liver-related death.

Conclusions
Treatment cessation after HBeAg seroconversion led to relapse in 44% of predominantly Caucasian patients within a median follow-up 1056 days. Two relapsed patients showed severe clinical events leading to liver-related death.

Authors
B. DELIRE (1), P. HENRIET (2), P. LEMOINE (2), I. LECLERCQ (1), P. STÄRKEL (3) / [1] Institut de Recherche Expérimentale et Clinique (IREC), Catholic University of Louvain (UCL), , Belgium, Laboratory of Hepato-Gastroenterology, [2] De Duve Institute, , Belgium, Cell Biology Unit, [3] Saint-Luc Academic Hospital and Institute of Clinical Research, Catholic University of Louvain, , Belgium, Department of Gastroenterology
Introduction Liver fibrosis is the main risk factor for hepatocarcinoma (HCC). Mechanisms linking fibrosis and hepatocarcinogenesis remain however poorly understood. In many malignant diseases, inflammatory cells that infiltrate the tumor are key players in cancer development.
Aim
Our aim was to study in a mouse orthotopic transplantation model the impact of fibrosis on HCC development and local tumor infiltration and explore potential roles of macrophages and neutrophils.
Methods The HCC cell line Hepa 1-6 is syngenic with the C57Bl/6 mouse strain. Hepa 1-6 cells were injected into non-fibrotic livers (normal liver group-NLG) and in severe fibrotic livers (severe fibrosis group-SFG) without immunosuppressive therapy. Severe fibrosis was induced by CCl4 for 7 weeks. Mice were sacrificed 2 weeks post HCC cell injection. The liver was sliced and examined for the presence of tumor (nodule ≥ 1mm). The tumor volume and the liver to body weight ratio (LW/BW) were used as parameters of tumor burden. A part of each tumor was used for histological analysis, proteins and RNA preparation.
Results A tumor nodule was observed in 60% of animals in the NLG but in 100% of them in SFG. The tumor volume and the LW/BW were significantly higher in the SFG (p<0.0001; p=0.005) compared to the NLG. Tumor macrophages infiltration was evaluated by F4/80 immunohistochemistry: while F4/80 positive cells were mainly located around the tumor in NLG livers, macrophages infiltrated deeper the HCC nodules in SFG livers. F4/80 mRNA expression (p<0.0003) as well as CD11b expression (p<0.0003), a marker of recruited macrophages, were higher in SFG than in NFG tumors. Similarly, we observed a higher NIMP-R14+ neutrophils infiltration in tumors that developed in SFG compared to those in NLG (p=0.0289). Many tumor-associated macrophages and neutrophils-derived molecules such as matrix metalloproteinase (MMP-2) and MMP-9 are involved in tumor progression and invasiveness. Compared to NLG, tumors in SFG livers expressed higher levels of Mmp2 (p=0.0019) and Mmp9 (p=0.0047). Mmp2 mRNA was significantly higher in the tumor compared to adjacent liver parenchyma in both groups (NLG: p=0.0006;SFG: p=0.0002) while high Mmp9 expression in tumor compared to adjacent parenchyma was only seen in SFG livers (p=0.0006) but not in NLG livers. Furthermore, tumor volume positively and significantly correlated with intra-tumor Mmp2 (rS=.571, p=0.026) as well as with intra-tumor Mmp -9 (rS=.741, p 0.002) mRNAs. Zymography evaluates pro- and active MMP-2/ -9: pro- and active MMP-2 and -9 were significantly higher in SFG tumors compared to NLG tumors (pro-MMP-2:p=0.0007, active MMP-2:p=0.008; pro-MMP-9:p=0.008, active MMP-9: p<0.05). Similarly to gene expression, MMP-2 and -9 enzymes were significantly more active in tumor than in adjacent parenchyma (MMP-2: p<0.05; MMP-9: p<0.05). MMP-2 and -9 are known activators of transforming growth factor β (TGFβ), an inflammatory cytokine that promotes tumor cells growth. TGFβ mRNA expression was higher in SFG than in NLG tumors (p=0.0012). Moreover, there were higher amounts of active (cleaved) TGFβ protein, measured by ELISA, in the SFG tumors compared to the NLG tumors.
Conclusions Liver fibrosis promotes HCC development in a mouse orthotopic transplantation model. Our results suggest that a fibrotic liver background favors a higher infiltration of tumor associated macrophages and neutrophils in the developing tumor. These secrete and activate molecules such as MMP-2, MMP-9 and TGFβ that promote tumor progression.

Authors
S. FRANCQUE (1), P. LEFEBVRE (2), F. LALLOYER (2), M. PAWLAK (2), E. BAUGÉ (2), C. GHEERAERT (2), H. DEHONDT (2), J. VANHOUTTE (2), N. HENNUYER (2), C. CLAIRE MAZUY (2), B. DERUDAS (2), A. DRIESSEN (3), G. HUBENS (4), L. VONGHIA (1), W. KWANTEN (1), T. VANWOLLEGHEM (1), P. MICHIELSEN (1), J. EECKHOUTE (2), A. VERRIJKEN (5), L. VAN GAAL (5), B. STAELS (2) / [1] ANTWERP UNIVERSITY HOSPITAL, Edegem, Belgium, Gastroenterology Hepatology, [2] Univ. Lille, CHU-Lille, Institut Pasteur de Lille, Lille, France, Inserm, [3] ANTWERP UNIVERSITY HOSPITAL, Edegem, Belgium, Pathology, [4] ANTWERP UNIVERSITY HOSPITAL, Edegem, Belgium, Abdominal Surgery, [5] ANTWERP UNIVERSITY HOSPITAL, Edegem, Belgium, Endocrinology, Diabetology and Metabolic Diseases

Introduction
Pathogenic mechanisms leading to progression from simple steatosis towards active non-alcoholic steatohepatitis (NASH) and fibrosis are poorly defined.

Aim

We investigated the liver transcriptome in a human cohort of histologically staged NASH patients both at baseline and follow-up to identify key components of progression of disease and hence potential targets for therapy.

Methods
Obese patients were prospectively screened for presence of NASH and if suspected, liver biopsy was proposed. Patients entered a weight management program, including bariatric surgery (BarSur) in some, and were re-evaluated after 1 year including biopsy. Liver biopsy was scored using the NASH CRN scoring system. Gene profiling (Affymetrix GeneChip arrays + functional annotation and enrichment) was performed. Paired analysis of the liver transcriptome before and 1 year after BarSur identified genes dysregulated in NASH and fibrosis and whose expression was normalized upon regression of lesions. A meta-analysis with publicly available datasets with comparable histology was carried out to even more stringently identify genes dysregulated in NASH and fibrosis. Data were further crossed with transcriptomic data from NASH and fibrosis mouse models.

Results
Analysis was performed in 87 patients with paired biopsies. Progressive baseline histological damage from steatosis to NASH to NASH+fibrosis were characterized by gene expression patterns successively reflecting altered functions in metabolism, inflammation and epithelial-mesenchymal transition. The molecular signature for active NASH+fibrosis contained 193 upregulated genes (immune responses and ECM homeostasis) and 58 downregulated (metabolic pathways). Of these, 103 and 36 were normalized after BarSur, leading to a 139-gene signature of NASH+fibrosis normalized upon resolution. Comparison with existing databases led to a 24 BarSur-sensitive human NASH+fibrosis signature strongly enriched with ECM matrix formation and inflammatory responses. Comparison with NASH and fibrosis gene signatures of MCD and CCl4 mouse models respectively resulted in a 16-gene set of NASH+fibrosis with normalisation upon regression. This analysis pointed towards dermatopontin (DPT) as an important player.

Conclusions
Liver damage during NASH progression is characterized by deregulated expression of a restricted set of inflammation- and ECM-related genes. Targeting DPT may be a valuable strategy to reverse the hepatic fibrotic process.

Authors
R. MANCO (1), L. CLERBAUX (1), I. LECLERCQ (1) / [1] Université Catholique de Louvain, Brussels, Belgium, Laboratory of Hepato-Gastroenterology
Introduction Self-renewal of mature hepatocytes supports homeostasis and regeneration of adult liver. Recent studies indicate that liver progenitor cells (LPC) are recruited upon injury as a facultative reservoir for generation of hepatocytes, although only a small number of mature hepatocytes were shown to derive from LPC in vivo. Models used for these studies do not recapitulate long lasting chronic hepatocellular damage and fibrosis seen in human chronic liver disease and cirrhosis.
Aim
Our aim is therefore to follow the dynamics of ductular reaction (DR) and the LPC’s fate during chronic liver injury in mice.
Methods We used tamoxifen-inducible Osteopontin-Cre (OPN-CreERT2) mice crossed with yellow fluorescent protein (YFP) reporter mice to follow the fate of LPC and biliary cells with an efficiency >85%. Long-term chronic injury was induced by repeated carbon tetrachloride (CCl4) injections 3x/week for 4, 6, 8, 16 and 24 weeks, resulting in chronic fibrosis and eventually cirrhosis. Livers from 8 and 16 weeks were also analysed after 4 weeks and 2 and 4 weeks of CCl4-free recovery period, respectively.
Results After 4 weeks CCl4, DR is minimal with few ck19+/YFP+ positive cells in periportal area and LPC-derived hepatocytes (traced as YFP+ hepatocytes) are inconspicuous. After 6 weeks, DR is similar in intensity but small foci of YFP+ hepatocytes adjacent to portal area are readily seen; these have a median size of 3010µm². As fibrotic disease increases in severity, the DR is negligible while patches of YFP+ hepatocytes become larger (median size of 3850µm² and 7040µm² at 8 and 16weeks, respectively) and extend to into the parenchyma. In the cirrhotic liver (24 weeks CCl4) some regenerative nodules are entirely composed of YFP+ hepatocytes. The number of YFP+ hepatocytes does no rise accordingly to the size of the patches as they represent 4.2 ± 2.4% of the lobule area in 6 weeks’ samples, increases up to 11.5 ± 3.8 % in 8 weeks’ samples and stabilizes around 5% thereafter, suggesting that not all YFP+ hepatocytes expand into growing patches. At 6 weeks, YFP+ hepatocytes are significantly smaller cells than YFP- native hepatocytes (750 vs 981 µm²) but in 16 weeks’ samples YFP+ and YFP- hepatocytes have the same size (996 and 1001 µm²). The dynamic of the YFP+ hepatocytes was also evaluated upon recovery: in the 4 weeks following 8 weeks of CCl4, the area occupied by YFP+ hepatocytes has a tendency to decrease from 11.5 ± 3.8% to 5.03 ± 3.8% (p=NS), while in the 2 and 4 weeks of recovery after 16 weeks of CCl4 the area significantly increases from 4.58 ± 1.7% to 7.7 ± 3.3% (p=NS), up to 13.8 ± 0.7 % (p<0.001), respectively. Whereas, upon recovery the size of the YFP+ hepatocytes, in all the different time points, is the same of the native hepatocytes.
Conclusions Our data demonstrate a significant contribution of LPC to the hepatocytes regeneration in a model of chronic liver injury leading to cirrhosis. The kinetic study supports that when DR is present, LPC differentiate into small hepatocytes, some of these subsequently increase in number, to form growing patches, and in size, becoming undistinguishable from the native hepatocytes. Upon recovery the growth of the patches of the LPC-derived hepatocytes depends on the severity of the underlying injury. Clonality studies are ongoing to test this hypothesis.

Authors
E. VERLY (1), X. VERHELST (1), H. VAN VLIERBERGHE (1), A. GEERTS (1) / [1] Ghent University Hospital, Ghent, Belgium, Department of hepatology & gastroenterology

Introduction
Variceal bleeding is a severe complication of cirrhosis. The treatment of variceal bleeding is based on proper supportive care, vasoactive medication and endoscopic therapy. Since 2010, early TIPS placement has shown improved survival in patients variceal bleeding with a high risk for rebleeding as defined by Garcia Pagan et al. (NEJM 2010).

Aim

The aim of this study was to retrospectively review the use of TIPS in variceal bleeding in our centre.

Methods
This retrospective monocentric study was performed in a tertiary referral centre for liver disease and liver transplantation (Ghent University Hospital). All patients admitted with variceal bleeding between January 2010 and December 2014 were included. Clinical data and results were retrieved from the medical files. Outcome was assessed at hospitalisation, 3 and 12 months after variceal bleeding.
Statistical analysis was performed using SPSS (version 23).

Results
In this cohort 56 patients were identified with variceal bleeding, 16 female and 40 male patients between the ages of 22 and 84. Forty-nine (87,5%) patients survived the hospitalisation, 48 (85,7%) were alive after 3 months and 1-year survival was 73,2% (41 patients). 17 patients had a CHILD-PUGH classification of A, 24 CHILD-PUGH B and 12 were CHILD-PUGH C. Of 3 patients, the CHILD-PUGH score could not be calculated due to missing variables. All patients received supportive care, vasoactive medication and endoscopy within 12 hours of admission.
In this cohort, 20 patients were treated with TIPS placement. 6 of these patients were classified as CHILD-PUGH A, 9 as CHILD-PUGH B and 5 as CHILD-PUGH C. Eleven patients (19.6%) received TIPS in the early-TIPS strategy after initial bleeding, 7 (12.5%) due to a rebleeding episode. In two patients (3.6%) TIPS placement was postponed after the 72 hours time window but was given as an early-TIPS placement, and not due to rebleeding. In the early-TIPS group, 3 month and one year survival was respectively 92,3% and 84,6%. Transient encephalopathy after TIPS placement was observed in 7 patients (35,0%). In the early TIPS group, 4 patients (30,8%) had transient encephalopathy.

Conclusions
The implementation of the early TIPS protocol for variceal bleeding is safe and shows excellent one-year survival rates in this high-risk population. Serious Adverse events were rare and manageable in the majority of patients.

Invited lecture by Thierry Gustot (Erasme, ULB)