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Two Combined Genetic Markers Identify Hepatitis C

Recipients With a Lower Graft Survival From The Earliest

Post-Transplant Period

Silvia Martini 1 , Renato Romagnoli 2 , Francesco Tandoi 2 , Dominic

Dell’Olio 3 , Paola Magistroni 3,4 , Francesca E. Bertinetto 3,4 , Ennia

Dametto 3,4 , Stefano Mirabella 2 , Giorgia Rizza 2 , Donatella Cocchis

2 , Antonio Ottobrelli 1 , Mario Rizzetto 1 , Mauro Salizzoni 2 ,

Antonio Amoroso 3,4 ; 1 Gastrohepatology - Molinette Hospital,

Turin, Italy; 2 Surgery, Liver Transplant Center - Molinette Hospital,

Turin, Italy; 3 Regional Transplantation Center, Piedmont, Turin,

Italy; 4 Immunogenetics Laboratory - Molinette Hospital, Turin, Italy

Background and Aims. Human Leukocyte Antigen (HLA) variants

and Interleukin 28B (IL28B) gene are associated with

recurrence and progression of hepatitis C virus (HCV) liver

disease in the transplant setting. We investigated the impact

of these immunogenetic factors on graft survival after liver

transplantation (LT) for HCV-related cirrhosis. Methods. This

retrospective study consecutively included all the first adult LTs

performed at a single center from 1999 to 2009. HLA-A/B/

DRB1 frequencies were studied in 1,228 adult Caucasian LT

recipients and their deceased donors. IL28B rs12979860

polymorphism was determined only in the 449 HCV-positive

viremic recipients and their HCV-negative donors, as it does

not affect transplant outcome in non-HCV patients. Graft survival

was the sole endpoint. Results. Median follow-up was 10

years. HLA-DRB1*11 phenotypic frequency was significantly

lower in HCV-positive recipients compared with HCV-negative

recipients (28.2% vs 43.9%, p



Determinants and impact of early viral kinetics in

patients with HCV-recurrence after liver transplantation

treated by sofosbuvir + daclatasvir: results from the


Camille Herve 1 , Audrey Coilly 2 , Claire Fougerou-Leurent 3 , Victor

de Ledinghen 4 , Georges-Philippe Pageaux 5 , Pauline Houssel-Debry

3 , Sylvie Radenne 6 , Christophe Duvoux 7 , Vincent Di

Martino 8 , Nassim Kamar 9 , Louis d’Alteroche 10 , Valerie Canva 11 ,

Pascal Lebray 12 , Jérôme Dumortier 13 , Christine Silvain 14 , Camille

Besch 15 , Danielle Botta-Fridlund 16 , Albert Tran 17 , Helene Montialoux

18 , Emilie Rossignol 3 , Alexandra Rohel 19 , Jean-Charles

Duclos-Vallee 2 , Vincent Leroy 1 ; 1 Gastroenterology, CHU Grenoble,

La Tronche, France; 2 Paul Brousse, Villejuif, France; 3 CHU

Rennes, Rennes, France; 4 CHU Bordeaux, Pessac, France; 5 CHU

Bordeaux, Bordeaux, France; 6 Croix Rousse, Lyon, France; 7 Henri

Mondor, Créteil, France; 8 CHU Besançon, Besançon, France;

9 CHU Toulouse, Toulouse, France; 10 CHU Tours, Tours, France;

11 CHRU Lille, Lille, France; 12 La Pitié Salpétrière, Paris, France;

13 Hôpital Edouard Herriot, Lyon, France; 14 CHU Poitiers, Poitiers,

France; 15 CHU Strasbourg, Strasbourg, France; 16 Conception

Hospital, Marseilles, France; 17 CHU Nice, Nice, France; 18 CHU

Rouen, Rouen, France; 19 ANRS, Paris, France

Early viral kinetic does not seem to impact the chance of SVR12

with the use of new combinations of direct HCV antiviral agents.

However, few data have been reported in HCV-recurrence after

liver transplantation (LT). It could be hypothesized that immunosuppression

might slow down HCV RNA decay. The aims

of our study were to describe in a large cohort of treated LT

patients the HCV RNA kinetics, to identify the predictive factors

of slow response and to evaluate its impact on SVR. Methods:

One hundred and ninety four LT patients prospectively included

in the multi-centric CUPILT cohort and treated by sofosbuvir +

daclatasvir ± ribavirin for 12 or 24 weeks were studied. HCV

RNA was assessed at W0, W2, W4, W8, W12, EOT and

FU12 by real time quantitative PCR with a lower limit of quantification

of 15 IU/ml. Results: The characteristics of patients

were as follows: age: 58.9 ± 8.7 years, baseline HCV RNA:

6.4 log IU/ml, genotype 1: 79%, genotype 3: 11%, treatment

experienced: 47%, HIV co-infection: 7%. The delay between LT

and onset of treatment was 75.8 ± 69.1 months. The majority

(60%) had severe HCV recurrence (F3, F4 or FCH). Immunosuppression

was based on tacrolimus in 62%, cyclosporine in

28% and everolimus in 8% of cases. MMF was administered

in 57% of patients. Treatment duration was 24 weeks in 85%

of cases and 12 weeks in 15% of cases. Ribavirin was used

in 71%) of patients. The percentages of patients with HCV

RNA ≥ 15 IU/ml and HCV RNA < 15 IU/ml but detectable

were distributed as follow: W2: 83% and 13%, W4: 47% and

29%, W8: 13% and 26%, W12 4% and 14%. By multivariate

analysis, independent factors associated with HCV RNA ≥

15 IU/ml at W4 were: use of MMF OR 1.90 (0.99 – 3.66),

p3 treatments.

Intention-to-treat 1- and 5-year overall survival was 84% and

56%, respectively. Among 109 LT recipients, the 1- and 5-year

post-LT survival was 95% and 80%, respectively, after a median

post-LT follow-up of 4.3 years. The Kaplan-Meier probabilities

of treatment failure at 1 and 5 years from first down-staging

procedure were 25% and 44%, respectively. Treatment failure

included dropout due to tumor progression (n=56), liver-related

death without LT (n=12), and HCC recurrence after LT (n=12).

In multivariable Cox proportional hazards regression analy-


sis, factors predicting treatment failure were pre-treatment AFP

>1000 ng/mL (HR 3.3, p1000 ng/mL compared

to 40% for AFP


and Status 1A FHF patients may be over-prioritized according

to current allocation policies.


W. Ray Kim - Advisory Committees or Review Panels: Bristol Myers Squibb,

Gilead Sciences, Abbvie, Merck

Patrick S. Kamath - Advisory Committees or Review Panels: Sequana Medical

The following authors have nothing to disclose: Alina M. Allen, Julie Heimbach,

Joseph J. Larson, Terry M. Therneau


End Stage Liver Disease Patients with MELD Scores

Over 40 Have Significantly Higher Waitlist Mortality

and Lower Probability of Receiving Liver Transplantation

Compared to Status 1A Fulminant Hepatic Failure


Joseph C. Ahn 2 , Taft Bhuket 1 , Sasan Mosadeghi 1 , Catherine T. Frenette

3 , Benny Liu 1 , Robert J. Wong 1 ; 1 Gastroenterology and Hepatology,

Alameda Health System - Highland Hospital, Oakland, CA;

2 School of Medicine, University of California, San Francisco, San

Francisco, CA; 3 Organ Transplantation, Scripps Green Hospital,

San Diego, CA

Background: Current liver transplantation (LT) allocation systems

prioritize Status 1A patients with fulminant hepatic failure

(FHF) over end stage liver disease (ESLD) patients of all MELD

scores. However, ESLD patients with the highest MELD scores

may have higher waitlist mortality than Status 1A FHF patients,

and thereby may require LT more urgently. Aim: Evaluate differences

in LT waitlist mortality and probability of LT between Status1A

FHF patients and ESLD patients with MELD>30. Methods:

Using data from United Network for Organ Sharing registry,

we retrospectively evaluated U.S. adults (age > 18) who were

listed for LT from January 1, 2003 to December 31, 2013.

ESLD patients were stratified into 3 groups of MELD scores (31-

35, 36-40, > 40). Overall waitlist mortality and probability

of LT were stratified by ESLD vs. Status 1A FHF and evaluated

with Kaplan Meier curves and multivariate logistic regression

models. The final multivariate model included adjustments for

age, sex, race/ethnicity, obesity, hepatic encephalopathy,

and ascites. Results: From 2003-2013, 15,049 ESLD patients

with MELD>30 and 3,049 Status 1A FHF patients were listed

for LT. ESLD patients with MELD 31-35 and MELD 36-40 had

significant higher 28-day waitlist survival than Status 1A FHF

patients, whereas MELD >40 patients had similar 28-day survival.

Compared to Status 1A FHF patients, overall probability

of LT was similar among ESLD patients with MELD 36-40 and

MELD>40. On multivariate regression, compared to Status 1A

FHF, ESLD patients with MELD scores >40 had significantly

higher 14-day waitlist mortality (OR 1.92; 95% CI 1.56-2.36;

p40 have significantly higher waitlist mortality compared

to Status 1A FHF patients. Despite the higher waitlist mortality,

ESLD patients with MELD >40 had similar probability of receiving

LT, suggesting that these patients may be under-prioritized


Catherine T. Frenette - Speaking and Teaching: Bayer, Salix, Gilead; Stock

Shareholder: Gilead

The following authors have nothing to disclose: Joseph C. Ahn, Taft Bhuket,

Sasan Mosadeghi, Benny Liu, Robert J. Wong


Enhancing hepatocyte function using an advanced

microfluidic system for clinical and pharmaceutical


Maria Navarro-Zornoza 1,2 , Xavi Illa 2 , Carmen Peralta 3 , Rosa

Villa 2 , Jordi Gracia-Sancho 1 ; 1 Barcelona Hepatic Hemodynamic

Lab, IDIBAPS - Hospital Clinic de Barcelona - CIBEREHD, Barcelona,

Spain; 2 CNM-CSIC, Barcelona, Spain; 3 IDIBAPS - CIBEREHD,

Barcelona, Spain

Background and Aims: Development of an advanced liveron-a-chip

to support liver function in patients with acute liver

failure and for in vitro drug discovery is still challenging. We

hypothesized that representation of the unique architectural

and dynamic features of the hepatic sinusoid using a superior

microfluidic bioreactor system for co-culture of primary liver

sinusoidal endothelial cells (LSECs) and hepatocytes would

mimic the in vivo hepatic metabolism significantly better than

using conventional methods for hepatocytes mono-culture or

co-cultures. Methods: Hepatocytes and LSECs freshly isolated

from Wistar rats were co-cultured in a bioreactor recently

developed by our team. LSECs were grown in the upper area

of the bioreactor on a 1mm pore size membrane with homogeneous

and continuous shear stress stimulation (3dyn/cm 2 );

whereas hepatocytes were plated in the lower area. After

48-72h of co-culture, hepatocyte function and phenotype

were characterized as albumin synthesis (enzymatic method),

urea production (BUN enzymatic method), CYP3A4 (luminescence

assay) and HNF4a expression (qPCR), and compared

to hepatocytes co-cultured with LSECs in the microfluidic system

but under static conditions or in conventional transwells,

and with hepatocytes cultured alone. Results: Hepatocytes

co-cultured in the bioreactor exhibited markedly better phenotype

than those cultured using conventional methods. Indeed,

hepatocytes from the bioreactor maintained polygonal shape

differentiation, had higher albumin and urea production (Albumin:

67-fold higher vs. transwells; 108-fold vs. mono-cultures;

Urea: 79-fold superior vs. transwells; 108-fold vs. mono-cultures),

exhibited markedly higher HNF4a expression (17-fold

vs. transwells; 12-fold vs. mono-cultures), and superior CYP3A4

activity (28-fold vs. transwells; 17-fold vs. mono-cultures). All

differences were p


actions condition). Conclusion: The herein presented bioreactor

allows co-culture of LSECs and hepatocytes, maintaining and

enhancing hepatocyte function long-term significantly better

than using monocultures or conventional co-culture methods.

Our approach provides an overwhelming insight in the importance

of the hepatic sinusoid in the development of technology

for liver support in acute liver failure and for drug discovery.


The following authors have nothing to disclose: Maria Navarro-Zornoza, Xavi

Illa, Carmen Peralta, Rosa Villa, Jordi Gracia-Sancho


Prediction of waitlist mortality in patients with portopulmonary

hypertension (POPH): An analysis of the UNOS


Hilary M. DuBrock 1,2 , David S. Goldberg 4 , Norman L. Sussman 5 ,

Sonja Bartolome 6 , Zakiyah Kadry 7 , Reena Salgia 8 , Andre M.

De Wolf 9 , David C. Mulligan 10 , Steven M. Kawut 4 , Michael J.

Krowka 3 , Richard Channick 2 ; 1 Medicine, Beth Israel Deaconess

Medical Center, Boston, MA; 2 Medicine, Massachusetts General

Hospital, Boston, MA; 3 Mayo Clinic, Rochester, MN; 4 University

of Pennsylvania, Philadelphia, PA; 5 Baylor College of Medicine,

Houston, TX; 6 UT Southwestern, Dallas, TX; 7 Penn State Hershey

Medical Center, Hershey, PA; 8 Henry Ford Hospital, Detroit, MI;

9 Northwester University, Chicago, IL; 10 Yale New Haven Hospital,

New Haven, CT

Purpose: The current United Network for Organ Sharing

(UNOS) system grants Model for End-Stage Liver Disease

(MELD) exception points to patients with POPH and a mean

pulmonary artery pressure (mPAP) 25 and pulmonary vascular

resistance (PVR)>240] was built using a competing risk subdistribution

hazard model and purposeful selection. Significance

was defined as p16 years of age) were approved for

a POPH MELD exception. In patients with true POPH (n=190),

13(6.8%) died on the waitlist, 4(2.1%) died during transplant,

27(14.2%) were removed for being too sick, 100(52.6%)

patients underwent deceased donor liver transplant, 2(1.1%)

underwent living donor transplant, 9(4.7%) were removed for

other reasons and 35(18.4%) remained active on the waitlist.

Initial MELDNa was a significant univariate predictor of waitlist

mortality (HR 1.121, p


1%. Conclusion. In a national cohort of HCC patients undergoing

LT, the recently enacted UNOS criteria improved the accuracy

of radiographic staging but did not reduce false-positive

HCC diagnoses. Overall utilization of HCC MELD exceptions

did not meaningfully change due to a shift from automatic T2

to RRB-approved exceptions.


Jesse M. Civan - Consulting: Merck

The following authors have nothing to disclose: Barry Schlansky, Willscott E.



Treatment with Optifast Reduces Hepatic Steatosis and

Safely Increases Candidacy Rates for Live Donor Liver


Adam Doyle 1 , Jayne Dillon 1 , Oyedele Adeyi 2 , Sandra E. Fischer 2 ,

Meaghan MacArthur 1 , Max Marquez 1 , Robert Smith 1 , David

Grant 1 , Mark S. Cattral 1 , Ian McGilvray 1 , Paul Greig 1 , Anand

Ghanekar 1 , Markus Selzner 1 , Eberhard L. Renner 1 , Les Lilly 1 , Gary

Levy 1 , Nazia Selzner 1 ; 1 Multi Organ Transplant Program, University

Health Network, Toronto, ON, Canada; 2 Department of

Pathology, University Health Network, Toronto, ON, Canada

Background: As a consequence of the increased prevalence

of obesity and associated liver steatosis, the volunteer pool for

live donor liver transplantation is threatened. The Optifast verylow

calorie diet meal replacement system has demonstrated

efficacy in the reduction in obesity and hepatic steatosis in

candidates for bariatric surgery. We studied the ability of

a 6-8 week course of Optifast in potential donors to reduce

body weight and fatty liver converting them to suitable live

liver donors. Methods: Among 447 potential donors evaluated

in our live donor liver transplant program between January

2012 to December 2014, 47 potential donors with a BMI

greater than 30, or evidence of significant steatosis on liver

biopsy, were administered Optifast for 6-8 weeks during their

evaluation for live liver donation. Outcomes including changes

in BMI and degree of steatosis on liver biopsy before and

after Optifast treatment were studied. Post-surgical outcomes

for donors and recipients were also recorded, and compared

to 91 live donors who did not receive Optifast and underwent

donor hepatectomy within the study period. Results: Median

BMI of Optifast potential donors (n=47) was 35.0 (IQR 33.1-

37.3), compared to 25.9 (IQR 23.1-29.1) in all non-Optifast

potential donors (n=400) [p



A simple educational tool for reducing 30-day hospital

readmissions in patients with decompensated cirrhosis

Dennis Kumral, Michael W. Crothers, Stephen H. Caldwell, Zachary

Henry; University of Virginia, Charlottesville, VA

Background: Cirrhosis is a chronic disease with acute on

chronic decompensating events that lead to hospitalization,

similar to congestive heart failure (CHF) and chronic obstructive

pulmonary disease (COPD). While patients with CHF and

COPD have widespread targeted discharge pathways that

help prevent readmissions, patients with cirrhosis have no

standardized discharge programs. We developed a quality

improvement initiative aimed at utilizing a standardized discharge

pathway for patients with cirrhosis. Our goal was to

reduce 30-day readmissions to our inpatient hepatology service

by at least ten percent. Methods: In April 2015 we began a

quality improvement discharge education program for patients

with cirrhosis complicated by either fluid overload or hepatic

encephalopathy. A dedicated hepatology nurse coordinator

performed a teaching session with patients and their families

prior to discharge and provided them with a patient-friendly

cirrhosis management booklet created by the study team. All

patients were provided with a digital scale and pill organizer

if they did not already own one. Within 72-hours of discharge,

a phone call was made to patients to review medications and

reinforce teaching. Patients were monitored for readmission up

to 30 days post discharge as the primary quality outcome. A

control group of cirrhosis patients without a dedicated teaching

program was chosen from hospital discharges during the same

time period one year earlier. Thirty-day readmission rates were

assessed for comparison. Results: A total of 20 patients with

cirrhosis complicated by fluid overload and/or hepatic encephalopathy

went through the discharge pathway and reached

30 days post discharge at the time of this submission. We

identified 25 control patients from the same time period one

year earlier. There was no significant difference in age, gender,

discharge MELD, discharge child pugh score, or length

of stay between the two groups. The 30 day readmission rate

in the study group was 25% compared to 62% in the control

group, p=0.02. This correlates to a relative risk reduction of

60% with a number needed to teach (NNT) of 2.7 to prevent

one 30-day readmission. On multivariate logistic regression

the only significant variable for predicting 30 day readmission

was participating in the educational discharge pathway which

was found to be protective, OR 0.22, p=0.02. Conclusions:

Implementation of a dedicated educational discharge pathway

with detailed teaching of cirrhosis management and a post-discharge

follow-up phone call can reduce 30-day readmissions

for patients with decompensated cirrhosis.


Stephen H. Caldwell - Advisory Committees or Review Panels: Vital Therapy;

Grant/Research Support: Genfit, Gilead Sciences, Immuron, Hyperion, Immuron,


The following authors have nothing to disclose: Dennis Kumral, Michael W.

Crothers, Zachary Henry


Development of a Model to Predict Post-Surgical

Unplanned Readmissions in Patients with Decompensated


Monica Schmidt 1 , Paul H. Hayashi 2 , Alfred S. Barritt 2 ; 1 UNC Liver

Center & Gillings School of Global Public Health, University of

North Carolina Chapel Hill, Chapel Hill, NC; 2 UNC Liver Center,

Chapel Hill, NC

Background:In the U.S., liver cirrhosis is expected to affect more

than 1 million individuals by 2020 and many will require surgery.

Prediction of 30-day readmission will be key in designing

appropriate discharge planning and preventing readmissions.

We developed a predictive model for 30-day readmission that

is specific to patients with decompensated liver cirrhosis undergoing

surgery. Methods:We used the National Surgical Quality

Improvement Program data (NSQIP) from 2011-2013. Patients

with cirrhosis were identified. Predictions were obtained for

the model and the MELD score for comparison. The AUROC,

cut-point, sensitivity/specificity and decision curve analysis

are reported. Results: There were 5,879 patients with 739

readmissions within 30-days (13%). Predictors of 30-day

readmission were insulin dependent diabetes (OR 3.4 CI 1.6-

7.3), discharged home (OR 3.4 CI 1.5-8.7), ASA class 4-life

threatening (OR 4.7 CI 1.8-12.1), and days from surgery to

discharge (OR 0.94 CI 0.91-0.97). The AUROC for the MELD

score was 0.50 while our model reached 0.80 (p



Effect of HCV treatment outcome on hospitalization rate:

Chronic Hepatitis Cohort Study (CHeCS)

Eyasu H. Teshale 1 , Jian Xing 1 , Anne C. Moorman 1 , Scott D. Holmberg

1 , Stuart C. Gordon 2 , Loralee B. Rupp 2 , Mei Lu 2 , Philip R.

Spradling 1 , Joseph A. Boscarino 3 , Connie M. Trinacty 4 , Mark A.

Schmidt 5 , Fujie Xu 1 ; 1 CDC, Atlanta, GA; 2 Henry Ford Health System,

Detroit, MI; 3 Geisinger Health System, Danville, PA; 4 Kaiser

Permanente Hawaii, Honolulu, HI; 5 Kaiser Permanente Northwest,

Portland, OR

Background: Chronic hepatitis C virus (HCV) infection is associated

with high all-cause hospitalization rates. Hospitalization

rates increase with increasing severity of liver disease. Successful

treatment of chronic HCV infection results in reduced

morbidity and mortality. The objective of this study is to determine

the association between HCV treatment outcome and

hospitalization rate. Methods: We used data from HCV-infected

patients seen in four large U.S. healthcare systems from

2006-2012. We included persons who received HCV treatment

during this time and who had follow up before and after

completion of treatment. Treatment outcome was grouped as

sustained virologic response (SVR) or treatment failure (TF). We

determined hospitalization rates [per 100 person years (PY)]

before and after treatment (SVR or TF) and excluded hospitalizations

during treatment. Results: Between 2006 and 2012,

1409 persons received HCV therapy; 680 (48.3%) achieved

SVR and 729 (51.7%) had TF. The overall before treatment

hospitalization rate was 13.2/100 PY; before treatment hospitalization

rates for those who achieved SVR and those with

TF were 10.6/100 PY and 16.0/100 PY, respectively. The

after treatment hospitalization rate decreased to 5.6/100 PY

for those who achieved SVR (p



A Prospective Study of a Protocol to Reduce Early Readmission

after Liver Transplantation

Mark W. Russo 1 , Amit Mori 1 , David Levi 2 , Ruth Pierce 2 , Siddesh

Besur 1 , Vincent P. Casingal 2 , Paul A. Schmeltzer 1 , Philippe J.

Zamor 1 , Andrew Delemos 1 , Lon Eskind 2 ; 1 Hepatology, Carolinas

Healthcare System, Charlotte, NC; 2 Transplant, Carolinas Medical

Center, Charlotte, NC

A Prospective Study of a Protocol to Reduce Early Readmission

after Liver Transplant Introduction: Hospital readmission

is an important marker of quality and delivery of care. Studies

have evaluated risk factors for readmission after liver transplant

(OLT) but few studies evaluated interventions to reduce

readmission. Aim: To reduce 30 day readmission rates after

OLT by implementing a prospective protocol with a multistep

strategy. Methods: In October 2013 a comprehensive strategy

was initiated to address early readmission after OLT. Core

components of the protocol included revising criteria for readmission,

alternatives to readmission, emphasized processes to

improve patient teaching and discharge planning and expansion

of outpatient services. 2014 served as the first full year

the protocol was implemented. Readmission rates for 2014

were compared to 2012 and 2013 with Fisher’s exact test and

ANOVA. Logistic regression was used to control for potential

confounding variables. Results: From January 1st 2012 thru

December 31st 2014 167 adult OLTs were performed at our

center with a mean biologic MELD of 21. The most common

indications were HCV (35%), NAFLD (18%), HCC (17%), and

ETOH (13%). The mean age of the study group was 54 y/o,

63% were male. The mean ICU LOS was 3.4 days and hospital

LOS 12.6 days. Over the study period 30 day readmission

rate was 34% and decreased after implementing the readmission

protocol from 40% in 2012 and 39% in 2013 to 19%

in 2014, p=0.04. The most common reasons for readmission

were biliary complications, infection, and rejection. Discharge

year (before or after implementing protocol) remained significant

after controlling for age, MELD score, indication, dialysis

pre and post liver transplant, distance from transplant center,

length of stay, PRBC transfusion, insurance and weekend discharge

OR=0.40 [95% CI 017,0.94], p=0.04. The top factors

identified as reducing readmission were expanding outpatient

services and alternatives to inpatient readmission. Conclusions:

Early readmission after liver transplantation can be reduced

by expanding outpatient services and implementing alternative

approaches to inpatient readmission.


Mark W. Russo - Grant/Research Support: Merck, Salix; Speaking and Teaching:

janssen, Gilead, ABBVIE, Salix

Philippe J. Zamor - Grant/Research Support: AbbVie, Bristol Myers Squibb,

Gilead, Merck & Co. ; Speaking and Teaching: AbbVie, Bristol Myers Squibb,


The following authors have nothing to disclose: Amit Mori, David Levi, Ruth

Pierce, Siddesh Besur, Vincent P. Casingal, Paul A. Schmeltzer, Andrew Delemos,

Lon Eskind


Reduced Work Productivity (WP), Absenteeism and

Presenteeism of Patients Infected with Hepatitis C Virus

(HCV) are Independently Predicted by Physical Component

of Patient-Reported Outcomes (PROs)

Zobair M. Younossi 1,2 , Maria Stepanova 3 , Linda Henry 3 , Issah

Younossi 3 , Ali A. Weinstein 3 , Fatema Nader 1 , Sharon L. Hunt 3 ;

1 Center For Liver Disease, Department of Medicine, Inova Fairfax

Hospital, Falls Church, VA; 2 Betty and Guy Beatty Center for Integrated

Research, Inova Health System, Falls Church, VA; 3 Center

for Outcomes Research in Liver Disease, Washington, DC

Background: Hepatitis C virus (HCV) infection is associated with

significant impairment of health-related quality of life (HRQOL)

and other PROs. Additionally, HCV infected patients are known

to have reduced work productivity (WP), both in terms of presenteeism

(impairment in work productivity while working) and

absenteeism (productivity loss due to absence from work). Aim:

The aim of this study was to identify PROs which are predictive

of WP in untreated HCV-infected patients. Methods: We

analyzed data from HCV patients prior to intitaion of anti-

HCV regimens in a clinical trial setting. All patients had completed

4 PRO questionnaires (CLDQ-HCV, SF-36, FACIT-F and

WPAI:SHP). In subjects who reported being employed, WP

and its absenteeism and presenteeism components were calculated

using WPAI:SHP instrument. Independent PRO predictors

of WP, absenteeism and presenteeism were assessed using

multiple linear regression. Results: Of 4,121 HCV patients who

completed pre-treatment WPAI:SHP, 2,480 (60.2%) reported

to be employed, and of those, 2,121 had completed all PRO

questionnaires before treatment initiation. The study cohort was

51.1±9.6 years old, 16.7% cirrhotic, 76.1% HCV genotype 1,

and 62.3% treatment-naïve. Average baseline impairment in

WP was 11.0%, including 8.3% (75% of WP impairment) of

presenteeism and 2.7% (25% of WP impairment) of absenteeism.

Using multivariate regression analysis, WP was predicted

by the activity/energy domain of CLDQ-HCV (beta=4.55±0.56

per 1 point, p



Administration of an Ileal Apical Sodium-dependent Bile

Acid Transporter Inhibitor Protects Against Non-alcoholic

Fatty Liver Disease in High Fat Diet-fed Mice

Anuradha Rao 1 , Astrid Kosters 1 , Jamie Mells 1 , Bradley T. Keller 4 ,

Hong Yin 2 , Sophia Banton 2 , Shuzhao Li 2 , Dean P. Jones 2 , Hao

Wu 3 , Paul Dawson 1 , Saul J. Karpen 1 ; 1 Pediatrics, Emory University,

Atlanta, GA; 2 Medicine, Emory University, Atlanta, GA; 3 Public

Health, Emory University, Atlanta, GA; 4 Vasculox, Inc, St. Louis,


Non-alcoholic fatty liver disease (NAFLD) is the most common

chronic liver disease in the Western world. The mechanisms

underlying NAFLD development and progression are not

fully elucidated, and safe and effective NAFLD therapies are

needed. Bile acids (BA) and their receptors have important

roles in regulating whole body metabolism, including multiple

hepatic targets coordinating lipid handling. We hypothesized

that interruption of the enterohepatic BA circulation via

a non-absorbable Apical Sodium-dependent BA Transporter

(ASBT) inhibitor (ASBTi; SC435) would modify signaling in

the gut-liver axis and diminish the development of NAFLD in

High Fat Diet (HFD) fed mice. Methods: Male C57Bl/6 mice

(n=12-16/group) were fed for 16 weeks with A) chow, B) HFD

composed of 45% fat and 0.2% cholesterol, with 4% sucrose

water (HFD), and C) HFD plus ASBTi (SC435; 60 ppm; HFD/

ASBTi). Body weight, food intake, glucose and insulin tolerance,

liver histology, and liver lipids were measured. Molecular

mechanisms were examined using RNASeq, metabolomics,

and real-time PCR analyses. Results: In HFD versus HFD/ASBTi

mice, administration of the ASBTi increased fecal BA excretion,

and mRNA expression for colonic Ibabp (7.0±2.0 fold;



Inflammasomes are new molecular platforms that respond to

microbial products through the synthesis of pro-inflammatory

cytokines. The Nlrp3 inflammasome activation is known to

determine Il-1β and Il-18 synthesis and release, thereby modulating

cell biology and eliciting pro-inflammatory signals in

different cell types. We sought to verify whether the inflammasome

is activated in sclerosing cholangitis and which are its

effects in reactive cholangiocytes. Methods: Expression levels

of the NLRP3 inflammasome were tested in cholangiocytes of

normal and cholestatic livers. The effects of Nlrp3 activation

induced by incubation with LPS/ATP were studied in vitro in

normal and siRNA Nlrp3 knockdown cholangiocytes. In vivo,

wild type (WT) and Nlrp3 A350VneoR

(Nlrp3 -/- ) mice were fed

with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, a model

of sclerosing cholangitis) for 4 weeks. Results: Expression of

Nlrp3 and its components is increased in in cholangiocytes

of mice subjected to DDC and in patients affected by PSC

compared to normal conditions. LPS/ATP-induced activation

of Nlrp3 in cholangiocytes stimulates the expression of Il-18

and Il-6, but not of Il-1β. Nlrp3 activation also significantly

decreased the expression of Zonulin-1 and E-cadherin but

had no effect on cholangiocyte proliferation. The effects of

LPS/ATP-induced activation of Nlrp3 in cholangiocytes were

neutralized by knockdown of Nlrp3 by siRNA. In vivo, the

increases in the liver CK-19-positive parenchyma induced by 4

week DDC in WT animals were reduced in Nlrp3 -/- mice and

expression of Zonulin-1 tended to be re-established. Conclusions:

Nlrp3 is expressed in reactive cholangiocytes, both in

murine models and in PSC. The activation of Nlrp3 leads to

the synthesis of pro-inflammatory cytokines and influences the

epithelial integrity of cholangiocytes. These findings suggest

that microbial products may participate to the development of

certain cholangiopathies by activating the inflammasome in

cholangiocytes and altering the barrier function of the biliary



The following authors have nothing to disclose: Luca Maroni, Laura Agostinelli,

Stefania Saccomanno, Eleonora Mingarelli, Chiara Rychlicki, Samuele De Minicis,

Jesus M. Banales, Antonio Benedetti, Gianluca Svegliati Baroni, Marco



Taurocholate Activates YAP via Sphingosine-1 Phosphate

Receptor 2 in Cholangiocytes

Kesong Peng 1 , Yunzhou Li 2 , Runping Liu 1,2 , Jing Yang 1 , Yongqing

Wang 3,1 , Guang Liang 4 , Phillip B. Hylemon 1,2 , Huiping Zhou 1,2 ;

1 Microbiology and Immunology, Virginia Commonwealth University,

Richmond, VA; 2 McGuire Veterans Affairs Medical Center,

Richmond, VA; 3 Nanjing Medical University, Nanjing, China;

4 School of Pharmacy, Wenzhou Medical University, Wenzhou,


Introduction: The transcription co-activators YAP/TAZ are the

core components of the Hippo pathway. Recent studies demonstrated

that activation of several G protein coupled receptors

(GPCRs), including sphingosine-1-phosphate receptor 2

(S1PR2), causes dephosphorylation and nuclear translocation

of YAP. Cholangiocytes are continuously exposed to high concentrations

of conjugated bile acids. Obstruction of bile flow

is a potent stimulus for cholangiocyte proliferation. We have

recently reported that taurocholate (TCA) promotes cholangiocyte

proliferation via activation of S1PR2, which is the predominant

S1P receptor in cholangiocytes. Recently, it was reported

that bile acids promote liver carcinogenesis through activation

of YAP. However, whether activation of S1PR2 by conjugated

bile acids induces YAP activation in cholangiocytes has not

been examined and is the focus of this study Methods: A mouse

cholangiocyte cell line was used for in vitro studies. Bile duct

ligation (BDL) mouse models were used to examine the role

of S1PR2 in YAP activation in vivo. A chemical antagonist of

S1PR2, JTE-013, was used to inhibit S1PR2 activation. S1P

was used as positive control. YAP activation was determined

by Western blot analysis. The mRNA levels of the YAP target

genes were determined by real-time RT-PCR. Results: Both

TCA- and S1P-induced cell migration and proliferation were

inhibited by JTE-013 in cholangiocytes. TCA- and S1P-induced

activation of YAP was indicated by decreasing of phosphorylated

YAP in cytosol and increasing of nuclear translocation of

YAP, which was markedly inhibited by JTE-013. In addition,

TCA and S1P also significantly up-regulated the expression

of the down-stream target genes of YAP including: IQGAP1,

cyclin D1, p21 and CTGF (connective tissue growth factor),

in cholangiocytes. YAP activation was significantly reduced in

BDL S1PR2 -/- mice as compared to wild-type mice. Discussion/

Conclusion: Elevated conjugated bile acid levels are correlated

with cholangiocyte proliferation and cholangiocarcinoma by

unknown mechanism(s). YAP-activation has been linked to

tumour growth. This study identified TCA/S1PR2/YAP/TAZ as

a cell signalling pathway involved in TCA-mediated cholangiocyte

proliferation and possibly transformation.


The following authors have nothing to disclose: Kesong Peng, Yunzhou Li, Runping

Liu, Jing Yang, Yongqing Wang, Guang Liang, Phillip B. Hylemon, Huiping



SRT1720 protects against cholestatic liver injury in mice

by altering hepatic bile acid composition and enhancing

urinary excretion of bile acids

Supriya Kulkarni 1 , Carol J. Soroka 1 , Lee R. Hagey 2 , James L.

Boyer 1 ; 1 Liver Center, Internal Medicine, Digestive Diseases, Yale

University School of medicine, New Haven, CT; 2 Division of Gastroenterology,

Department of Medicine, University of California at

San Diego, La Jolla, CA

BACKGROUND: Sirtuin1 (SirT1, mammalian homolog of S. Cerevisiae

enzyme Sir2) is a critical transcriptional and transactivational

regulator of murine Fxr. Liver specific knockout of Sirt1

deregulates Fxr activity. Our previous studies demonstrated that

bile duct ligation (BDL), or cholic acid (CA) feeding decrease

hepatic Sirt1 expression and that a Sirt1 activator SRT1720

improves cholestatic liver injury. AIM: To determine the mechanisms

by which Sirt1 activation may alleviate cholestatic liver

injury. METHODS: C57Bl/6 mice (n=11-13, male, 8-9 weeks

old) were fed standard rodent chow or chow supplemented

with 1% CA for 5 days. Both groups (n=5-6) were administered

either vehicle or Sirt1 activator, SRT1720 (50mg/kg/

day) for duration of the diet. Liver injury (ALT, ALP), BA levels in

plasma, liver, ileum, urine and the bile acid pool size as well

as gene and protein expression of BA transporter and synthesis

genes were determined. Immunoprecipitation assays were

used to determine Sirt1 acetylation and Fxr activity. RESULTS:

Sirt1 mRNA, protein and acetylation activity were significantly

reduced (75%) in CA fed mouse livers. SRT1720 administration

in CA fed mice reduced plasma ALT (40%) and plasma

BA (50%) levels while hepatic and total BA pool remained

unchanged. SRT1720 significantly increased tetra- hydroxylated

BA by 1.96x and decreased di-hydroxylated BA fraction

to 21% over CA fed mice and increased Cy2b10 (1.75x)

expression. SRT1720 increased ileal Fgf15 (30x) and hepatic

Fgfr4 (2X), decreased hepatic Cyp7a1 (99%), 27a1 (75%)

expression. SRT1720 also increased renal Mrp2 and Mrp4

expression (2-4.5x) along with increased urinary BA concen-


tration (2x) relative to CA fed mice, thus increasing BA excretion

in the urine. Hepatic Jnk was activated by CA feeding,

a finding that that was reversed by SRT1720 administration

which restored Sirt1 and Fxr mRNA, protein and activity levels

to normal. CONCLUSION: Sirt1 protein levels and acetylation

activity are decreased in CA feeding model of cholestasis Sirt1

activator SRT1720 decreased cholestatic liver injury by several

mechanisms: SRT1720 increased the hepatic concentration of

hydrophilic hydroxylated BA likely via increased expression

of Cyp2b10. In addition, SRT1720 decreased BA synthesis

via the Fgf15-Fgfr-Cyp7a1 pathway and finally SRT1720

decreased serum bile acid levels by increasing renal Mrp2

and Mrp4 expression and urinary BA excretion. These observations

could be attributed to SRT1720 mediated reversal of Jnk

activation and hence restoration of Sirt1 and Fxr expression in

the CA fed mice.


James L. Boyer - Advisory Committees or Review Panels: Pfizer; Consulting:


The following authors have nothing to disclose: Supriya Kulkarni, Carol J.

Soroka, Lee R. Hagey


FXR-β-catenin complex and bile acid homeostasis: therapeutic

implications in cholestatic liver disease

Kari Nejak-Bowen, Satdarshan (Paul) S. Monga; University of Pittsburgh,

Pittsburgh, PA

Cholestatic liver diseases are characterized by bile acid (BA)

accumulation in the liver, which can lead to inflammation

and fibrosis, and eventually result in end-stage liver disease.

We utilize the bile duct ligation (BDL) model and identify a

dramatic reduction in liver injury and fibrosis in liver-specific

β-catenin knockout (KO) mice. This is due to a previously unrecognized

role of β-catenin in regulation of BA synthesis and

transport through association with farnesoid X receptor (FXR).

We confirm this association through immunoprecipitation (IP)

of FXR and β-catenin from wild-type (WT), β-catenin KO, and

FXR KO livers and showed association of the two proteins in

WT but not in either KO. Further, the association with FXR

occurs at the C-terminal of β-catenin, since IP of β-catenin and

FXR from HepG2 cells reveals association with both the fulllength

and N-terminal truncated β-catenin forms. Additionally,

the β-catenin-FXR pool is unresponsive to BA stimulation as

demonstrated by IP studies on GW4064-treated Hep3B cells.

To address the functional relevance of this complex, we show

that absence of β-catenin allows FXR activation to occur more

readily, both in vitro and in vivo. In fact, a more pronounced

FXR activation in KO is the major reason of decreased total

hepatic BA and injury after BDL. Conversely, when Hep3B cells

were transfected with S33Y-mutated stable β-catenin, we find

increased FXR-β-catenin association by IP. This coincides with

loss of FXR-RXRα association, which normally causes activation

of SHP and suppresses BA synthesis. Expression of S33Y-βcatenin

also abrogates the GW4064-induced increase in SHP

reporter activity seen in Hep3B cells treated with control plasmid,

confirming the inhibitory role of this association. This was

validated in HepG2 cells, which show less SHP induction after

β-catenin silencing than Hep3B cells, as the truncated/stable

form of β-catenin is still present in these cells after β-catenin suppression.

The complex interplay between β-catenin, FXR and

RXRα was further validated when overexpression of RXRα led

to a switch in association of FXR from β-catenin to RXRα. Thus,

we have identified a novel FXR-β-catenin complex and propose

its manipulation may provide a novel therapeutic opportunity

for alleviating BA-associated hepatic injury during cholestasis.


Satdarshan (Paul) S. Monga - Advisory Committees or Review Panels: Abbvie;

Grant/Research Support: Dicerna Pharmaceuticals

The following authors have nothing to disclose: Kari Nejak-Bowen


HIV and HCV co-infection in hepatocytes and stellate

cells reveals cooperative transcriptional activation

between viruses and cell types

Shadi Salloum 1 , Cynthia Brisac 1 , Rohit Jindal 2 , Shyam Sundhar

Bale 2 , Nadia Alatrakchi 1 , Annie Kruger 1 , Anna Lidofsky 1 , Martin

L. Yarmush 2 , Raymond T. Chung 1 ; 1 GI, MGH, Boston, MA; 2 Center

for Engineering in Medicine, Shriner, Boston, MA

Background: HIV/HCV co-infection accelerates progression to

liver fibrosis, a major public health problem. Currently, the

molecular and cellular mechanisms underlying progression of

this dynamic disease remain incompletely understood. We previously

demonstrated, in monoculture experiments, that HCV

and HIV independently led to the production of profibrogenic

TGFβ through an increase in reactive oxygen species (ROS)

and NFkB activation in hepatocytes as well as in hepatic stellate

cells (HSCs), all of which are associated with the development

of hepatic fibrosis. Aim: We sought to create a co-culture

model combining hepatocytes and HSCs to effectively simulate

HCV and HIV co-infection in vitro. This model will be of

great utility to evaluate the mechanistic contributions of HIV

and HCV infection to hepatic fibrogenesis in a multicellular

context. Methods: We first constructed stable monoclonal

green fluorescent protein (GFP) reporter cells expressing functional

antioxidant response element (ARE), NFkB, and SMAD3

driven reporter transgenes, which enables these transcriptional

regulators to be studied dynamically in living cells. Then, we

adapted the reporter cells to create a model of co-infection

that allows investigation of transcriptional dynamics associated

with HIV/HCV co-infection. Finally, we used the reporter cells

to create co-cultures of hepatocytes and stellate cells to further

investigate the effect of HIV/HCV co-infection. Results: The

efficiency of reporter cell lines was validated by using positive

controls which activated 85% to 60% of total cells versus only

2 to 10% mock activated cells. In In monoculture, HIV and

HCV induced ARE, and SMAD3 in both cell lines (suggesting

an increase in cellular oxidative stress and TGF-β production).

In contrast, NFkB was activated only in Huh7.5.1 but not in

LX2 cells. Co-infection with HCV and HIV led to a significant

increase (2-3 fold) in the activation of these reporters in comparison

to monoinfection. The co-culture model confirmed HCV

and/or HIV effects, on SMAD, ARE, and NFkB activation.

Interestingly, the viruses’ effects on these reporters increased

additively compared to monoculture in Huh7.5.1 cells. Conclusions:

HIV accentuates the HCV-related profibrogenic program

in HSCs and hepatocytes through initial ROS induction, NF-kB,

and TGF-B1 upregulation. Co-culture studies indicated additive

effects compared with monoculture. Our novel co-culture

reporter cell model represents an efficient system for studying

transcriptional responses and has the potential to provide

important insights into the progression of fibrosis liver disease,

as well as study of potential therapeutic interventions.


Raymond T. Chung - Grant/Research Support: Gilead, Mass Biologics, Abbvie,

Merck, BMS

The following authors have nothing to disclose: Shadi Salloum, Cynthia Brisac,

Rohit Jindal, Shyam Sundhar Bale, Nadia Alatrakchi, Annie Kruger, Anna

Lidofsky, Martin L. Yarmush



Elevation of Galectin-9 is associated with the cytotoxicity

of NK cells in chronic hepatitis

Akira Nishio, Tomohide Tatsumi, Takatoshi Nawa, Takahiro

Suda, Atsuo Takigawa, Tadashi Kegasawa, Yoshiki Onishi, Seiichi

Tawara, Teppei Yoshioka, Satoshi Aono, Minoru Shigekawa,

Hayato Hikita, Ryotaro Sakamori, Naoki Hiramatsu, Tetsuo Takehara;

Department of Gastroenterology and Hepatology, Osaka

University Graduate School of Medicine, Suita, Japan

Background & Aims: NK cells play an essential role in the

pathogenesis of chronic hepatitis C (CHC). NK cells are activated

in CHC and activated NK cells exhibit elevated cytotoxicity

leading to liver injury. Recently, the activated NK cells

contribute to exhaustion of virus-specific T cells, which is associated

with persistent viral infection. However, the mechanism

of NK cell activation has not been clearly understood. The

immunoregulatory protein Galectin-9 (Gal-9) is elevated in the

serum and liver in CHC patients, suggesting that Gal-9 might

be involved in the pathogenesis of CHC. In this study, we investigated

the effect of Gal-9 on the activation of NK cells in CHC.

Methods: Serum Gal-9 levels were analyzed in 39 healthy

donors, 50 CHC patients and 24 IFN-treated patients achieving

sustained viral response (SVR) by ELISA. The cytotoxicity of

NK cells and Gal-9 levels in the supernatant were evaluated by

flow cytometry and ELISA. Immunofluorescence staining was

performed in CHC liver tissue. Results: Serum Gal-9 levels in

CHC patients were significantly higher than those in healthy

donors, and those in SVR patients were significantly lower

than those in CHC patients (median, 0-90 percentile, CHC:

146 pg/ml, 0-508, healthy donors: 0, 0-55.4, SVR: 53.6,

0-185.6). In CHC patients, serum levels of Gal-9 in elevated

ALT patients were significantly higher than those in normal ALT

patients, suggesting that elevated Gal-9 might be associated

with liver injury. Serum Gal-9 levels were not associated with

HCV viral loads. In vitro study revealed that addition of recombinant

Gal-9 resulted in significant increase of the cytotoxicity

of naïve NK cells in a dose dependent manner, independent of

Tim-3. Gal-9-activated NK cells showed the cytotoxicity against

CD4+ or CD8+ T cells, which might contribute to persistent

infection by depleting T cell. We found that a large amount of

Gal-9 production was induced in the co-culture of PBMCs with

JFH-1/Huh7.5.1 cells, but not with uninfected Huh7.5.1 cells.

A transwell system revealed that cell-cell contact was required

for Gal-9 production. In vitro depletion study revealed CD14+

monocytes were required for Gal-9 in the co-culture, and both

Gal-9 and CD14 positive cells were observed in immunofluorescence

staining, indicating that Gal-9 was produced by

Kupffer cells in the liver. The cytotoxicity of NK cells was significantly

elevated when co-cultured with JFH-1 cells compared

with control. Conclusion: Monocytes-derived Gal-9 induces the

cytotoxicity of NK cells, which might be involved in liver injury

and persistent HCV infection. These results suggest the role of

Gal-9 in the pathogenesis of chronic hepatitis C.


Hayato Hikita - Grant/Research Support: Bristol-Myers Squibb

Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD

K.K., Bristol-Meyer Squibb, Mitsubishi Tanabe Pharma Corparation, Toray Industories

Inc. ; Speaking and Teaching: MSD K.K., Bristol-Meyer Squibb, Janssen

Pharmaceutical Companies

The following authors have nothing to disclose: Akira Nishio, Tomohide Tatsumi,

Takatoshi Nawa, Takahiro Suda, Atsuo Takigawa, Tadashi Kegasawa, Yoshiki

Onishi, Seiichi Tawara, Teppei Yoshioka, Satoshi Aono, Minoru Shigekawa,

Ryotaro Sakamori, Naoki Hiramatsu


De novo formation and maturation of hepatitis C virus

replication membranes visualized by pulse-chase imaging

Hongliang Wang 1 , Andrew W. Tai 1,2 ; 1 Internal Medicine, University

of Michigan, Ann Arbor, MI; 2 VA Ann Arbor Healthcare

System, Ann Arbor, MI

Hepatitis C virus (HCV) replicates on an altered host membrane

structure called the “membranous web” (MW). Little is known

regarding temporal events in MW formation and association

with putative sites of virus assembly at or near lipid droplets

(LDs). For example, it is not known whether membranous webs

are stable structures that are continually renewed by newly

synthesized viral proteins, or whether newly synthesized viral

proteins are directed to form new MWs de novo. The HCV

nonstructural protein NS5A is believed to be a component of

MWs. We have developed pulse-chase fluorescent labeling

and imaging approaches that allow us to discriminate ‘old’

from ‘new’ NS5A-positive membranous structures (NS5A foci).

With this system, we find that ‘new’ NS5A-positive foci form at

sites distinct from ‘old’ NS5A foci, supporting a model of de

novo MW formation instead of renewal of previously formed

MWs. We also find that the phosphatidylinositol 4-kinase

PI4KA and oxysterol-binding protein, which are known to be

required for normal MW morphogenesis, are required to maintain

the normal morphology of both ‘old’ and ‘new’ foci, and

that these two proteins appear to play a role in de novo NS5A

focus formation. Finally, we observe increasing colocalization

of NS5A-positive foci with HCV core protein and LDs over time.

Overall, these data support a model in which HCV MWs are

formed de novo rather than renewed. We hypothesize that

newly-formed NS5A foci undergo a process of maturation and

movement towards sites of putative virion assembly at or near

LDs. This may have implications for our understanding of how

replication membranes are formed for other positive-sense RNA



The following authors have nothing to disclose: Hongliang Wang, Andrew W.



Rapid normalization of antiviral and pro-inflammatory

transcriptional signatures in peripheral blood of patients

and livers of humanized mice treated with DAAs

Matthew A. Burchill 1 , Lucy Golden-Mason 1 , Megan Wind-Rotolo 2 ,

Michael Gale 3 , Hugo R. Rosen 1 ; 1 GI-Hepatology, University of

Colorado-Denver, Aurora, CO; 2 Exploratory Clinical and Translational

Research, Bristol-Myers Squibb, Lawrenceville, NJ; 3 Immunology,

University of Washington, Seattle, WA

Chronic HCV infection results in sustained immune activation

in both the periphery and hepatic tissue. Recently, novel

direct acting antiviral (DAA) regimens have been shown to

induce rapid and sustained clearance of HCV. DAAs have

provided the unique opportunity to determine whether successful

treatment-induced eradication of viral RNA normalizes

the dysregulated antiviral innate immune response in patients

chronically infected with HCV. Methods: We used both a

prospective cohort of patients and a novel humanized mouse

model. Results: First, in 35 patients receiving DAAs, we found

that a regimen of daclatasvir, asunaprevir, and BMS-791325

induced not only rapid viral clearance, but also a re-setting

of antiviral responses in the peripheral blood. Specifically,

we observed that following treatment with DAAs, there was

a significant reduction in IFITM1 and the chemokines CXCL10


and CXCL11 in the peripheral blood by Affymetrix microarray

and confirmatory RT-PCR. Next, using a novel humanized

mouse model (FRG), we assessed the transcriptional changes

that occur in the hepatic tissue following DAA treatment. After

establishing a stable chronic HCV infection in the mice, they

were treated with daclatasvir, asunaprevir, and sofosbuvir that

induced the clearance of circulating HCV within 48 hours.

Despite the rapid clearance of circulating viral particles, we

observed HCV NS3 protein within infected hepatocytes 14

days after initiation of DAA therapy. The presence of residual

NS3 protein did not affect the blunting of the expression

of proinflammatory responses as we observed a significant

reduction in the chemokines CXCL10 and CXCL11 in the liver

of treated mice. These data suggested that the transcriptional

changes that occur in the peripheral blood of patients treated

with DAAs may partially reflect intrahepatic transcriptional

changes. However, in livers of DAA-treated humanized mice

we observed a restoration of mitochondrial anti-viral signaling

protein (MAVS) and an increase in the protein IFITM1, the

latter a tight junction protein typically induced only by IFN.

Conclusions: These findings highlight how viral clearance elicited

by DAA treatment leads to the rapid restoration of innate

signaling moieties that are suppressed by HCV infection. Collectively,

our data demonstrate that the rapid viral clearance

following treatment with DAAs results in the rebalancing of

innate antiviral response in both the peripheral blood and the

liver as well as enhanced antiviral signaling within previously

infected hepatocytes.


Megan Wind-Rotolo - Employment: Bristol-Myers Squibb; Stock Shareholder:

Bristol-Myers Squibb

The following authors have nothing to disclose: Matthew A. Burchill, Lucy Golden-Mason,

Michael Gale, Hugo R. Rosen


Activation of Hepatic Stellate Cells Drives the Rapid

Development of Liver Fibrosis During Acute HCV Infection

in HIV-infected Men

Robert D. Gillies 1,2 , Daniel S. Fierer 1 , Marcus Yip 1,2 , Scott L. Friedman

3 , Andrea D. Branch 3 , M. Isabel Fiel 4 ; 1 Infectious Diseases,

Mount Sinai School of Medicine, New York, NY; 2 Monash University,

Melbourne, VIC, Australia; 3 Liver Diseases, Icahn School

of Medicine at Mount Sinai, New York, NY; 4 Pathology, Icahn

School of Medicine at Mount Sinai, New York, NY

Background The international epidemic of sexually-transmitted

HCV infection among HIV-infected men is characterized by

rapid development of liver fibrosis of unknown mechanism(s).

The purpose of this study is to determine whether increased

fibrogenesis during acute HCV infection is associated with

activation of hepatic stellate cells (HSC), the principal collagen-producing

cell in liver injury. Methods Liver biopsies were

evaluated from HIV-infected men with acute and recent HCV

infection in New York City. Date of HCV diagnosis was set

as the date of the first-noted ALT elevation. The presence of

alpha-smooth muscle actin (ASMA), indicative of activated

HSC, was determined by counting stained foci in 10 random

lobular areas per high-powered field (hpf) (40x magnification)

on slides immunostained with antibodies to ASMA. Fibrosis

stage (Scheuer, 0-4) was determined by examining Masson

trichrome-stained slides. Collagen-proportionate area (CPA)

was determined by computer digital image analysis of Sirius

red-stained slides. The effect of time to biopsy on these three

measurements was examined as a dichotomous variable, using

1 year after HCV diagnosis. The Chi-Square test

for trend was used to compare Scheuer stage of fibrosis; and

the Mann-Whitney U Test was used to compare the median

CPA and median activated HSC/hpf between groups. Results

Liver biopsies from 20 men were evaluated. Median age at

HCV diagnosis was 42 years, with a median of 9 years since

HIV diagnosis; and median CD4 count was 501 cells/uL (all

had >200). The median number of activated HSC/hpf was

significantly higher in biopsies obtained 1 year after HCV diagnosis (p = 0.002, Table), indicating

greater HSC activation early in HCV infection and lower

HSC activation later in infection. Despite this lower activation

of HSC in biopsies >1 year after HCV diagnosis, fibrosis

stage (p = 0.01), and CPA (p = 0.06) were both higher in

those with biopsy >1 year after HCV infection. Conclusion The

rapid development of fibrosis that occurs in HIV-infected men

during the first year after acute HCV infection is associated

with ASMA-staining fibrogenic myofibroblasts. The number of

activated HSC was lower after the first year of HCV infection

while the stage of fibrosis was higher, indicating that once

fibrogenesis has been set in motion, this process continues,

although possibly at a slower pace. This rapid development of

fibrosis can therefore not be considered to be benign.


Daniel S. Fierer - Stock Shareholder: Gilead

Scott L. Friedman - Advisory Committees or Review Panels: Pfizer Pharmaceutical;

Consulting: Conatus Pharm, Exalenz, Genfit, Exalenz Biosciences, Eli Lilly PHarmaceuticals,

Fibrogen, Boehringer Ingelheim, Nitto Corp., Immune Therapeutics,

Synageva, Roche/Genentech Pharmaceuticals, DeuteRx, Abbvie, Novartis,

RuiYi, Kinemed, Sanofi Aventis, Takeda Pharmaceuticals, Nimbus Therapeutics,

Bristol Myers Squibb, Astra Zeneca, Sandhill Medical Devices, Galmed, Northern

Biologics, Enanta Pharmaceuticals, Regado Bioscience, Raptor Pharmaceuticals,

Teva Pharmaceuticals, Zafgen Pharmaceuticals, Merck Pharmaceuticals,

Debio Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira

Pharm; Stock Shareholder: Angion Biomedica, Intercept Pharma

Andrea D. Branch - Grant/Research Support: Gilead, Janssen

The following authors have nothing to disclose: Robert D. Gillies, Marcus Yip,

M. Isabel Fiel


Alcohol increases the production of hepatitis C virus

(HCV) lipo-viro-particles in primary human hepatocytes

Veronique Pene 1 , Céline Hernandez 1 , Etienne Blanc 2 , Lynda

Aoudjehane 3,4 , Béatrice Le Grand 2 , Arnaud Carpentier 1 , Jean-

François Méritet 1,5 , Filomena Conti 4,6 , Yvon Calmus 4,6 , Hélène

Rouach 2 , Philippe Podevin 1,7 , Arielle R. Rosenberg 1,5 ; 1 EA 4474

“Hepatitis C Virology”, Université Paris Descartes, Paris, France;

2 UMRS 1124 “Toxicology Pharmacology and Cellular Signaling”,

Université Paris Descartes, Inserm, Paris, France; 3 Human HepCell,

Hôpital Saint-Antoine, Paris, France; 4 UMRS 938 “CDR Saint-Antoine”,

Inserm, Paris, France; 5 Service de Virologie, AP-HP, Hôpital

Cochin, Paris, France; 6 Unité de Transplantation Hépatique, AP-HP,

Hôpital Pitié-Salpêtrière, Paris, France; 7 Centre de Référence en

addictologie, AP-HP, Hôpital Pitié-Salpêtrière, Paris, France

BACKGROUND & AIM: Alcoholism is a major aggravating

factor of HCV infection, yet the molecular mechanisms of this

comorbidity remain elusive. Clinical studies found higher viral

loads in patients with chronic excessive alcohol consumption.

However, the so-called “viral load” reflects a wide heterogeneity

of HCV particles, the most infectious ones being those

of lowest buoyant density due to their association with verylow-density

lipoproteins (VLDL, the apolipoprotein B-containing

triglyceride-rich lipoproteins secreted by hepatocytes) in hybrid

structures termed lipo-viro-particles (LVP). With the aim of investigating

the impact of alcohol on HCV infectious cycle, we took


advantage of our HCV culture system in primary human adult

hepatocytes (PHH), which, contrary to the widely used hepatocarcinoma-derived

Huh-7 sublines, retain the liver metabolism

of ethanol and secrete authentic VLDL and LVP. METHODS:

PHH were infected with the HCV strain JFH1 or a chimeric

virus derived thereof and treated with increasing doses of ethanol

(0-100 mM) for 2 weeks. Cultures were monitored for

HCV genome replication (negative strand RT-qPCR), viral load

(clinically used test), production of infectious virus (titration by

focus-formation assay), and VLDL secretion (apolipoprotein B

ELISA). The density of viral particles was assessed by isopycnic

iodixanol ultracentrifugation. RESULTS: Ethanol exposure of

HCV-infected PHH caused a time- and dose-dependent increase

in the viral load, comparable to that reported in clinical studies.

Most strikingly, it caused an even greater increase in the

infectious titer but did not significantly affect the viral genome

replication, thus pointing to an effect on virus morphogenesis.

This effect was not seen in Huh-7.5.1 cells treated in parallel,

suggesting that it involves the liver metabolism of ethanol. The

higher specific infectivity of HCV particles produced by PHH

in the presence of ethanol correlated with lower mean buoyant

density, consistent with triglyceride enrichment. Finally, in

either HCV-infected or naïve PHH, addition of ethanol caused

a time-dependent increase in VLDL secretion. CONCLUSION:

This study in the relevant PHH model reveals that ethanol via its

metabolites increases the production of HCV particles of lowest

buoyant density and highest infectivity, i.e., LVP, most likely

due to the impact of ethanol on triglyceride metabolism that

results in increased VLDL secretion. Drugs targeting this host

metabolic pathway may be useful in difficult-to-treat alcoholic

patients, often poorly compliant with therapy and therefore at

risk for resistance if treated by direct antivirals only. VP & CH:

equal contribution.


The following authors have nothing to disclose: Veronique Pene, Céline Hernandez,

Etienne Blanc, Lynda Aoudjehane, Béatrice Le Grand, Arnaud Carpentier,

Jean-François Méritet, Filomena Conti, Yvon Calmus, Hélène Rouach, Philippe

Podevin, Arielle R. Rosenberg

followed by 15 weeks of combined therapy with 250mg REP

2139-Ca and 180ug Pegasys® and then 33 weeks with

Pegasys® monotherapy. Viremia (HDV RNA and HBV DNA),

HBsAg and anti-HBs are followed every two weeks (Robogene

RT-PCR, Abbott RealTime HBV, Abbott Architect) performed at

the Institute of Virology, University of Duisburg-Essen (Essen,

Germany). HDV RNA is validated on separate test platforms

at the National Genetics Institute (Los Angeles, USA) and the

Institute of Virology, Technische Universität München (Munich,

Germany). Results: REP 2139-Ca treatment is well tolerated

with mild and quickly resolving IV infusion reactions. Serum

HBsAg is currently reduced 1-6 log in 11/12 patients (5 with

serum HBsAg < 1 IU / ml) and HDV RNA is currently reduced

1.5-7 log in 12 /12 patients (undetectable in 6 patients). Anti-

HBs is detected in 10/12 patients, with 6 patients < 10mIU /

ml and after combined exposure to Pegasys®, 5 patients have

substantially increased anti-HBs titers from 50 to > 800 mIU /

ml. In all patients with pre-treatment HBV DNA < 10 IU / ml,

de-repression of serum HBV DNA is observed. Conclusions:

Updated interim data from the REP 301 protocol assessing the

safety and antiviral efficacy of REP 2139 (first in monotherapy

and then with add on Pegasys® at week 16) in 12 Caucasian

patients with chronic HBV / HDV co-infection demonstrated

substantial reductions in serum HBsAg and HDV RNA as well

as appearance of anti-HBs. HDV RNA reductions appear stronger

than HBsAg reductions, suggesting an additional antiviral

mechanism other than the inhibition of subviral particle assembly

may affect HDV directly. REP 2139-Ca may become an

important new therapeutic option for patients with chronic HBV

/ HDV infection.


Michel Bazinet - Board Membership: REPLICor Inc.; Employment: REPLICor Inc.;

Management Position: REPLICor Inc.; Patent Held/Filed: REPLICor Inc.; Stock

Shareholder: REPLICor Inc.

Andrew Vaillant - Employment: REPLICor; Stock Shareholder: REPLICor

The following authors have nothing to disclose: Victor Pantea, Valentin

Cebotarescu, Lilia Cojuhari, Paulina Jimbei, Jeffrey Albrecht, Peter Schmid, Hadi

Karimzadeh, Michael Roggendorf


Update on the safety and efficacy of REP 2139 monotherapy

and subsequent combination therapy with

pegylated interferon alpha-2a in chronic HBV / HDV

co-infection in Caucasian patients

Michel Bazinet 1 , Victor Pantea 2 , Valentin Cebotarescu 2 , Lilia

Cojuhari 3 , Paulina Jimbei 3 , Jeffrey Albrecht 4 , Peter Schmid 4 , Hadi

Karimzadeh 5 , Michael Roggendorf 5 , Andrew Vaillant 1 ; 1 Replicor

Inc., Montreal, QC, Canada; 2 N. Testemitanu State University of

Medicine and Pharmacy, Chisinau, Moldova (the Republic of);

3 Toma Ciorba Infectious Clinical Hospital, Chisinau, Moldova

(the Republic of); 4 National Genetics Institute, Los Angeles, CA;

5 Institure of Virololgy, Technische Universität München, Munich,


Introduction: HBV / HDV co-infection causes rapid progression

of liver disease and with no approved therapy, presents a

significant unmet medical need. Nucleic acid polymers (NAPs)

block HBV subviral particle assembly and release from infected

hepatocytes and can eliminate serum HBsAg. As the NAP REP

2139 was previously been shown to clear serum HBsAg and

improve the ability of immunotherapy to elicit SVR in Asian

patients with HBV, its activity in combination with Pegasys® in

HBV / HDV co-infected Caucasian patients is currently being

examined. Methods: In a phase II proof of concept trial (REP

301; NCT02233075), patients with chronic HBV / HDV co-infection

received once weekly dosing of 500mg REP 2139-Ca

(calcium chelate complex) by 2h IV infusion for 15 weeks,


Reductions in cccDNA under NUC and ARC-520 therapy

in chimpanzees with chronic hepatitis B virus infection

implicate integrated DNA in maintaining circulating


Christine I. Wooddell 1 , Deborah Chavez 2 , Jason E. Goetzmann

3 , Bernadette Guerra 2 , Ryan M. Peterson 1 , Helen Lee 2 ,

Julia O. Hegge 1 , Robert Gish 4 , Stephen Locarnini 5 , Christopher

R. Anzalone 1 , Robert E. Lanford 2 , David L. Lewis 1 ; 1 Arrowhead

Madison, Arrowhead Research Corporation, Madison, WI; 2 Texas

Biomedical Research Institute, San Antonio, TX; 3 New Iberia

Research Center, University of Louisiana at Lafayette, New Iberia,

LA; 4 Department of Medicine, Stanford University Medical Center,

San Diego, CA; 5 Victorian Infectious Diseases Reference Laboratory,

Melbourne, VIC, Australia

Background: RNAi therapeutic ARC-520 designed to target

all cccDNA-derived transcripts reduces viral antigenemia for

>1 month after single doses in HBV patients. Here we report

the effect of multiple ARC-520 doses on hepatic HBV DNA

and RNA in HBV chimps. Methods: 9 chimps (5 M, 4 F; 9-37

yrs) received 6-11 monthly injections of ARC-520 concurrent

with NUC therapy. 5 were HBeAg-positive (HBeAg+), baseline

DNA 8-9 log 10

IU/mL serum, and 4 were HBeAg-negative

(HBeAg-), ≤3 log 10

IU/mL. Chimps received NUCs for

8-24 weeks prior to ARC-520 dosing. Liver biopsies from 8

chimps were taken at baseline, completion of NUC lead-in

and on study. HBV DNA, +/- plasmid-safe DNase digestion to


enrich cccDNA, was measured by qPCR. Pre-core/core RNA

(C probe) and total HBV RNA (Total probe) were measured

by RT-qPCR. The Guide for the Care and Use of Laboratory

Animals was strictly adhered to. Results: During NUC lead-in,

total liver HBV DNA decreased 1.1-2.5 log 10

in HBeAg+ but

not appreciably in HBeAg- chimps. cccDNA in HBeAg+ chimps

decreased 0.7 ± 0.6 log 10

. Following addition of ARC-520 in

HBeAg+, total liver DNA decreased from baseline by 1.5–2.9

log 10

and cccDNA by 1.4 ± 0.7 log 10

, the degree of reduction

generally correlating with duration of treatment. Neither

total HBV DNA nor cccDNA levels changed remarkably in

HBeAg- during the study, which at baseline had 2-4 orders of

magnitude less cccDNA than HBeAg+ chimps. HBeAg- chimps

had 50-fold more DNase-sensitive HBV DNA, possibly indicating

the majority is integrated DNA rather than cccDNA.

HBV RNA was not reduced by NUCs, but with addition of

ARC-520 RNA reductions tracked qHBsAg reductions. In

HBeAg+, Total probe detected 1-2x as many transcripts as

the C probe, suggesting similar levels of core/pre-core and

S transcripts. In HBeAg-, the Total probe detected 37x more

transcripts than the C probe, supporting a greater proportion of

HBsAg transcripts being produced from integrated HBV DNA

in HBeAg- chimps. Integration between DR1 and DR2 would

result in HBV RNA lacking ARC-520 target sites, consistent

with greater HBsAg reduction in HBeAg+ (1.7 ± 0.5 log 10


than HBeAg- chimps (0.7 ± 0.1 log 10

). Administration of siRNA

targeted to integrant-produced transcripts resulted in HBsAg

reductions up to 2.3 log 10

beyond those produced by ARC-520

in HBeAg-. Conclusions: 1) ARC-520 reduced total liver DNA

and cccDNA beyond levels achieved in HBeAg+ with NUCs

during lead-in; 2) ARC-520 but not NUCs reduced HBV RNA

and antigens; 3) integrated HBV DNA may be important in

maintaining HBsAg in chronic HBV, especially in HBeAg-. This

finding, if confirmed, has important implications for development

of new HBV therapies.


Christine I. Wooddell - Employment: Arrowhead Research Corporation

Ryan M. Peterson - Employment: Arrowhead Research Corporation

Julia O. Hegge - Employment: Arrowhead Research Corp

Robert Gish - Advisory Committees or Review Panels: Gilead, AbbVie, Arrowhead;

Consulting: Eiger, Isis, Genentech; Speaking and Teaching: Gilead, Abb-

Vie; Stock Shareholder: Arrowhead

Stephen Locarnini - Consulting: Gilead, Arrowhead; Employment: Melbourne


Robert E. Lanford - Grant/Research Support: Arrowhead Research

David L. Lewis - Employment: Arrowhead Research Corporation

The following authors have nothing to disclose: Deborah Chavez, Jason E. Goetzmann,

Bernadette Guerra, Helen Lee, Christopher R. Anzalone


Inhibition of Hepatitis B Virus Replication by the HBV

Core Inhibitor NVR 3-778

Angela Lam, Suping Ren, Robert Vogel, Christine Espiritu, Mollie

Kelly, Vincent Lau, George D. Hartman, Lalo Flores, Klaus Klumpp;

Novira Therapeutics Inc., Doyleston, PA

Background: Hepatitis B virus (HBV) chronically infects more

than 350 million people worldwide, and is a major cause of

severe liver disease including cirrhosis and hepatocellular carcinoma.

NVR 3-778 represents a new class of HBV core inhibitors

and is currently in clinical development for the treatment of

chronic hepatitis B. We present the preclinical characterization

of NVR 3-778 and its antiviral properties in cells expressing

HBV. Methods: HBV core assembly was examined using fluorescence

quenching and electron microscopy. HBV replication

inhibition was measured as reduction of secreted HBV DNA in

HBV producing HepG2.2.15 cells. The production, encapsidation

and secretion of HBV RNA was determined using Northern

blot and Quantigene assays. Antiviral activity of NVR 3-778

against nucleoside resistant HBV variants was determined by

cell based phenotyping. HBV broad spectrum antiviral activity

was examined by testing NVR 3-778 across all HBV genotypes

(A-H). Results: NVR 3-778 inhibited HBV replication in

HepG2.2.15 cells and could fully block the production of HBV

DNA.. The compound could effectively induce mis-assembly of

recombinant HBV core protein and electron microscopy indicated

the formation of capsid-like particles. In HBV expressing

cells, NVR 3-778 blocked the encapsidation of HBV pgRNA,

HBV DNA formation, and the release of HBV DNA and RNA

containing particles. NVR 3-778 was similarly active against

wild-type and HBV nucleos(t)ide resistant variants with defined

amino acid changes within the reverse transcriptase protein:

rtL180M/M204V, rtN236T, rtA181V, rtA181V/N236T, and

rtL180M/M204V/N236T. Furthermore, in transfection assays

NVR 3-778 inhibited HBV replication across representative

HBV strains from genotypes A to H (EC 50

values from 0.20 to

0.58 mM). Conclusions: Preclinical antiviral profiling studies

showed that the antiviral HBV replication inhibitor NVR 3-778

could induce mis-assembly of core protein into capsid-like particles

in vitro. Treatment of HBV producing cells with NVR 3-778

was associated with inhibition of pgRNA encapsidation. Consequently,

the downstream processes of HBV DNA production

and secretion of infectious HBV DNA-containing virions (Dane

particles) were inhibited. In addition, NVR 3-778 also blocked

the secretion of HBV RNA-containing particles. NVR 3-778 was

active against HBV variants resistant to nucleos(t)ide analogs

and was broadly active across all HBV genotypes. These results

support the clinical development of NVR 3-778 as a potential

new therapy for chronic hepatitis B patients alone or in combination

with nucleoside analogs or other antiviral agents.


Angela Lam - Employment: Novira Therapeutics, Merck & Co

Christine Espiritu - Employment: Novira Therapeutics

Mollie Kelly - Independent Contractor: Novira Therapeutics

George D. Hartman - Management Position: Novira Therapeutics

Klaus Klumpp - Board Membership: Riboscience LLC; Employment: Novira Therapeutics


The following authors have nothing to disclose: Suping Ren, Robert Vogel, Vincent

Lau, Lalo Flores


Dual-gRNAs and gRNA-microRNA (miRNA)-gRNA ternary

cassette combined CRISPR/Cas9 system and RNAi

approach promotes the clearance of HBV cccDNA

Jie Wang, Fengmin Lu; Peking University Health Science Center,

Beijing, China

Purpose: To investigate the potential use of CRISPR/Cas9

approach for eradicating hepatitis B virus (HBV) infection, we

designed and screened for the most effective gRNAs against

HBV of genotypes A-D, and developed a novel gRNA-microRNA

(miRNA)-gRNA ternary cassette. Methods: A total of

15 gRNAs against HBV of genotypes A-D were designed. 11

combinations of two above gRNAs (dual-gRNAs) covering the

different regions of HBV genome were chosen. Based on the

efficiency of dual-gRNAs in suppressing HBV replication, a

novel gRNA-miRNA-gRNA ternary cassette driven by a single

U6 promoter was designed by integrating an anti-HBV primiR31

mimic between two HBV-specific gRNAs, in which two

gRNAs could be released through Drosha/DGCR8 processing.

The HBV replication was examined by the measurement

of HBV surface antigen (HBsAg) and e antigen (HBeAg) in

the culture supernatant. The destruction of HBV-expressing vec-


tor was examined by polymerase chain reaction (PCR) and

sequencing method, and the destruction of cccDNA was examined

by KCl precipitation, plasmid-safe ATP-dependent DNase

digestion, rolling circle amplification and quantitative PCR combined

method. Results: All of gRNAs could significantly reduce

the HBsAg or HBeAg production in the culture supernatant,

which was dependent on the region in which gRNA against.

All of dual-gRNAs could efficiently suppress the HBsAg and

HBeAg production for HBV of genotypes A-D, and the efficacy

of dual-gRNAs in suppressing HBsAg and HBeAg production

was significantly increased when compared to the single gRNA

used alone. Furthermore, with PCR direct sequencing we confirmed

that these dual-gRNAs could specifically destroy HBV

expressing template by removing the fragment between the

cleavage sites of the two used gRNAs. The gRNA-miR-HBVgRNA

ternary cassette exerted a synergistic effect in efficiently

inhibiting HBV replication and destroying HBV-expressing template

in vitro and in vivo. We determined that the optimal stem

extended length of pri-miRNA was 38 base pairs in our ternary

cassette. Most importantly, together with RNAi approach

the gRNA-miR-HBV-gRNA ternary cassette showed the potent

activity on the destruction of HBV cccDNA. Conclusions: These

results suggested that dual-gRNAs guided CRISPR/Cas9 system

could efficiently destroy HBV expressing templates (genotypes

A-D). Together with RNAi approach, the gRNA-miR-HBV-gRNA

ternary cassette showed the potent activity on the destruction

of HBV cccDNA. Since HBV cccDNA was an obstacle for the

elimination of chronic HBV infection, thus the CRISPR/Cas9 system

may be a potential tool for the clearance of HBV cccDNA.


The following authors have nothing to disclose: Jie Wang, Fengmin Lu

both GSEA and IPA. Strikingly, there was a comparable transcriptional

response to TLR7-CM treatment in HBV-infected and

uninfected PHH (p0.80), including a comparable

induction of IFN-stimulated gene (ISG) signature. Notably,

APOBEC3B (A3B) mRNA levels were only weakly induced

by TLR7-CM (mean = 2.6-fold), and A3A expression was not

significantly stimulated. Consistent with the lack of viral interference

in the hepatocyte response to TLR7-CM, HBV infection

did not result in prominent global transcriptional response in

Mock-CM treated PHH. Knock-down of IFNAR1 mRNA (~90%

reduction) abrogated the antiviral activity of TLR7-CM, suggesting

that one or more of the ISGs strongly induced by treatment

(e.g. MX1, IFI16, IFIT1 and IFITM1) may be HBV restriction

factors. Conclusion: These data demonstrate that type I IFN is a

key antiviral cytokine induced by GS-9620 in PBMCs, and that

HBV does not interfere with IFN-signaling in human hepatocytes.

In addition, transcriptional profiling identified candidate

HBV restriction factors, but indicated that APOBEC3A likely

does not mediate the antiviral activity of TLR7-CM in HBV-infected



Li Li - Employment: Gilead Sciences

Congrong Niu - Employment: Gilead Science

Stephane Daffis - Employment: Gilead Sciences

Hilario Ramos - Employment: Gilead Sciences

Eduardo Salas - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

Christian Voitenleitner - Employment: Gilead Sciences

William E. Delaney - Employment: Gilead Sciences; Patent Held/Filed: Gilead

Sciences; Stock Shareholder: Gilead Sciences

Simon P. Fletcher - Employment: Gilead Sciences; Stock Shareholder: Gilead



HBV Does Not Modulate the Transcriptional Response

to TLR7-Induced Cytokines in Highly Infected Primary

Human Hepatocytes

Li Li, Congrong Niu, Stephane Daffis, Hilario Ramos, Eduardo

Salas, Christian Voitenleitner, William E. Delaney, Simon P.

Fletcher; Gilead Sciences, Foster City, CA

Background & Aims: GS-9620, an oral agonist of toll-like

receptor 7 (TLR7), is in phase 2 trials for the treatment of

chronic hepatitis B (CHB). We previously demonstrated that

prolonged exposure to antiviral cytokines directly induced by

TLR7 activation, such as interferon-α (IFN-α), potently inhibited

HBV in primary human hepatocytes (PHH) in vitro. Here we

characterized the transcriptional response of PHH to TLR7-induced

cytokines in order to identify potential HBV restriction

factors and to determine whether HBV infection modulates

signaling of these cytokines in hepatocytes. Methods: PHH

from two different donors were infected with HBV or mock

infected, and 13 days later were treated with media from

healthy human PBMCs stimulated with either GS-9620 (TLR7

conditioned media; TLR7-CM) or DMSO (control; Mock-CM).

High level infection (≥75% PHH infected) was confirmed by

HBV core staining. Whole transcriptome analysis of PHH

± HBV at 8, 24 and 48 hours post-treatment with TLR7-CM

and Mock-CM was performed by RNA-Seq. Transcriptional

responses were characterized by gene set enrichment analysis

(GSEA) and Ingenuity Pathway Analysis (IPA). Knock-down of

IFN-α/β receptor 1 (IFNAR1) mRNA was performed by siRNA,

and included a negative (scramble) control. Results: TLR7-CM

induced a comparable transcriptional signature in PHH from

two independent donors. In both donors, TLR7-CM induced the

greatest transcriptional response at 8 hours post-treatment, with

IFN signaling pathways being the top induced pathways by


GalNAc-siRNA conjugate ALN-HBV targets a highly

conserved, pan-genotypic X-orf viral site and mediates

profound and durable HBsAg silencing in vitro and in


Laura Sepp-Lorenzino, Andrew G. Sprague, Tara Mayo, Tuyen

M. Nguyen, Svetlana Shulga Morskaya, Huilei Xu, Stuart Milstein,

Greg Hinkle, Pia Kasperkovitz, Richard G. Duncan, Natalie Keirstead,

Brenda Carito, Lauren Moran, Prasoon Chaturvedi, Krishna

C. Aluri, Husain Attarwala, Renta M. Hutabarat, Ju Liu, Chris Tran,

Qianfan Wang, Benjamin S. Brigham, Akin Akinc, Klaus B. Charisse,

Vasant Jadhav, Satya Kuchimanchi, Martin A. Maier, Muthiah

Manoharan, Rachel Meyers, Tadeusz Wyrzykiewicz, Haroon

Hashmi, Julie Donovan, Tim Mooney, Daniel Freedman, Tanya

P. Sengupta, Karin Galil, Eoin Coakley, Patrick Haslett; Alnylam

Pharmaceuticals, Cambridge, MA

Background: Despite the prevalence of chronic HBV infection

(CHB), there are no highly effective therapies allowing sustained

off-treatment viral control and/or cure of HBV infection.

An RNAi therapeutic targeting the HBV genome has the potential

to achieve a “functional cure” by effectively decreasing

expression of all viral gene products, including tolerogenic viral

antigens, e.g., HBsAg. Methods and Results: ALN-HBV is a

hepatocyte-targeted GalNAc-siRNA conjugate with Enhanced

Stabilization Chemistry (ESC) for subcutaneous (SC) administration.

It targets a site in the HBV X protein open reading

frame (ORF), thereby targeting all four HBV RNA transcripts

for degradation by RNA interference. Bioinformatic analyses

indicate a >95% perfect sequence homology with a database

of 3,950 full length HBV viral genomes incorporating genotypes

A through J, and a low potential for off-target activity

against host genes. Pan-genotypic activity was confirmed in


vitro, using reporter constructs representative of genotypes A-J.

In HepG2.2.15 cells, ALN-HBV mediates viral transcript silencing

with ED 50

s of 12-19 pM, as determined for amplicons in

the P and S ORFs. In vivo, in an AAV-HBV mouse model, a

single 3 mg/kg SC dose of ANL-HBV resulted in mean 1.6

log 10

reduction in plasma HBsAg (3.6 log 10

max reduction) at

10-15 days post dosing. After 3 weekly doses of 3 mg/kg SC,

mean HBsAg silencing >2.9 log 10

was observed, with several

animals being below the level of quantitation (100 days after the last dose.

In vitro metabolic stability, and mouse and rat pharmacokinetics

and liver exposure were consistent with ESC chemistry

which enables the long-lived pharmacologic response. Exploratory

toxicology and histopathology were performed in rats

sacrificed 24 hr after 3 weekly doses of 30 and 100 mg/kg

SC, and revealed excellent tolerability. Additional pharmacology

studies are in progress, including combination studies

with ALN-PDL, an RNAi drug targeting PD-L1 in hepatocytes.

ALN-PDL is expected to result in augmentation of HBV-specific

cellular immunity in the liver, thereby improving immunological

control of HBV infection locally, while avoiding the immunotoxicity

of systemic immune checkpoint blockade. Conclusion:

ALN-HBV is a single, hepatocyte-targeted siRNA conjugate

targeting a highly conserved region in the HBV genome. It

mediates specific, potent and durable silencing of HBV viral

transcripts and HBsAg expression. ALN-HBV is being developed

as finite treatment in combination with standard-of-care

nucleos(t)ide analogs as a means for inducing a functional cure

in CHB patients. A CTA filing is planned for late 2015.


Laura Sepp-Lorenzino - Employment: Alnylam

Andrew G. Sprague - Employment: Alnylam Pharmaceuticals; Stock Shareholder:

Alnylam Pharmaceuticals

Tuyen M. Nguyen - Employment: Alnylam Pharmaceutical; Stock Shareholder:

Alnylam Pharmaceutical

Svetlana Shulga Morskaya - Employment: Alnylam Pharmaceuticals; Stock Shareholder:

Alnylam Pharmaceuticals

Stuart Milstein - Employment: Alnylam

Greg Hinkle - Employment: Alnylam Pharmaceuticals

Richard G. Duncan - Employment: Alnylam Pharmaceuticals; Stock Shareholder:

Alnylam Pharmaceuticals

Krishna C. Aluri - Employment: Alnylam Pharmaceuticals

Husain Attarwala - Employment: Alnylam Pharmaceuticals

Renta M. Hutabarat - Employment: Alnylam Pharmaceuticals

Ju Liu - Employment: Alnylam

Benjamin S. Brigham - Employment: Alnylam Pharmaceuticals

Akin Akinc - Employment: Alnylam Pharmaceuticals

Klaus B. Charisse - Employment: Alnylam Pharmaceuticals

Vasant Jadhav - Employment: Alnylam Pharmaceuticals

Satya Kuchimanchi - Employment: Alnylam Pharmaceuticals

Martin A. Maier - Employment: Alnylam Pharmaceuticals; Stock Shareholder:

Alnylam Pharmaceuticals

Muthiah Manoharan - Employment: Alnylam

Rachel Meyers - Employment: Alnylam Pharmaceuticals

Haroon Hashmi - Employment: Alnylam Pharmaceuticals; Stock Shareholder:

Alnylam Pharmaceuticals

Daniel Freedman - Employment: Alnylam Pharmaceuticals

Tanya P. Sengupta - Employment: Alnylam Pharmaceuticals

Karin Galil - Consulting: Alnylam Pharmaceuticals

Eoin Coakley - Employment: AbbVie; Stock Shareholder: AbbVie

Patrick Haslett - Employment: Alnylam Pharmaceuticals

The following authors have nothing to disclose: Tara Mayo, Huilei Xu, Pia

Kasperkovitz, Natalie Keirstead, Brenda Carito, Lauren Moran, Prasoon

Chaturvedi, Chris Tran, Qianfan Wang, Tadeusz Wyrzykiewicz, Julie Donovan,

Tim Mooney


Safety and efficacy of daclatasvir plus sofosbuvir with

or without ribavirin for the treatment of chronic HCV

genotype 3 infection: Interim results of a multicenter

European compassionate use program

Tania M. Welzel 1 , Joerg Petersen 2 , Peter Ferenci 3 , Michael

Gschwantler 4 , Kerstin Herzer 5 , Markus Cornberg 6 , Eckart Schott 7 ,

Thomas Berg 8 , Ulrich Spengler 9 , Ola Weiland 10 , Marc van der

Valk 11 , Andreas Geier 12 , Jürgen K. Rockstroh 9 , Markus Peck-Radosavljevic

3 , Yue Zhao 13 , Maria Jesus Jimenez Exposito 14 , Stefan

Zeuzem 1 ; 1 Universitätsklinikum der Johann Wolfgang Goethe

Universität, Frankfurt, Germany; 2 IFI Institut für Interdisziplinäre

Medizin, Hamburg, Germany; 3 Medizinische Universität Wien,

Vienna, Austria; 4 Wilhelminenspital, Vienna, Austria; 5 Universitätsklinikum

Essen (AöR), Essen, Germany; 6 Medizinische

Hochschule Hannover, Hannover, Germany; 7 Charité Universitätmedizin

Berlin, Berlin, Germany; 8 Universitätsklinikum Leipzig,

Leipzig, Germany; 9 Universitätsklinikum Bonn, Bonn, Germany;

10 Karolinska Institutet, Solna, Sweden; 11 Academic Medical Center,

Amsterdam, Netherlands; 12 Universitätsklinikum Würzburg,

Würzburg, Germany; 13 Bristol-Myers Squibb, Hopewell, NJ;

14 Bristol-Myers Squibb, Princeton, NJ

BACKGROUND: Despite progress in the development of well

tolerated, all-oral, interferon-free therapies, the treatment of

HCV genotype (GT) 3 infection remains challenging. Current

treatment options are limited, and debate continues regarding

the optimal regimen and duration of treatment, especially for

treatment-experienced patients with liver cirrhosis. Here we

report interim results on the combination daclatasvir (DCV) plus

sofosbuvir (SOF) with or without ribavirin (RBV) for the treatment

of GT3-infected patients from a European compassionate use

program (CUP). METHODS: This CUP enrolled adult patients

with chronic HCV infection who were at a high risk of hepatic

decompensation or death within 12 months if left untreated,

and who had no available treatment options. Patients received

DCV 60mg + SOF 400mg QD, with RBV added at the physician’s

discretion. Recommended duration of treatment was

24 weeks. This interim analysis includes 101 GT 3-infected

patients enrolled in the CUP with available data on March 16,

2015, of whom 24 patients have available SVR12 data to

date. RESULTS: Most patients were male (68%), white (90%);

median age: 54 years (31–75). Fifty-nine (58%) were treatment

experienced. Most patients had cirrhosis (81%); 46 of them

(56%) were Child-Pugh class B/C; and 13 (16%) had MELD

scores ≥15. Seven patients were liver transplant recipients; 15

were HIV/HCV coinfected. Median baseline HCV RNA was

5.6 log 10

IU/mL. Overall, SVR12 rates among GT3-infected

patients with HCV RNA available data were 92% (22/24).

One patient meets criteria for relapse. SVR12 rates among

sub-groups are presented in the Table. Updated SVR12 data

will be presented. Twenty-two patients experienced at least one

serious adverse event (AE); 5 patients discontinued therapy due

to AEs (pneumonia/multi-organ failure, physical deterioration,

hepatic encephalopathy, syncope, undetermined); 2 patients

died (multi-organ failure, liver related). CONCLUSION: In this

preliminary analysis, DCV in combination with SOF±RBV led

to high SVR12 rates in HCV GT3-infected patients, regardless

of cirrhosis status, and was well tolerated. The use of RBV did

not affect efficacy outcomes.


†All liver transplant patients had cirrhosis, ‡Includes 3 liver transplant



Tania M. Welzel - Advisory Committees or Review Panels: Novartis, Janssen,

Gilead, Abbvie, Boehringer-Ingelheim+, BMS

Joerg Petersen - Advisory Committees or Review Panels: Bristol-Myers Squibb,

Gilead, Novartis, Merck, Bristol-Myers Squibb, Gilead, Novartis, Merck; Grant/

Research Support: Roche, GlaxoSmithKline, Roche, GlaxoSmithKline; Speaking

and Teaching: Abbott, Tibotec, Merck, Abbott, Tibotec, Merck

Peter Ferenci - Advisory Committees or Review Panels: Idenix, Gilead, MSD,

Janssen, Salix, AbbVie, BMS; Patent Held/Filed: Madaus Rottapharm; Speaking

and Teaching: Gilead, Roche

Michael Gschwantler - Advisory Committees or Review Panels: Janssen, BMS,

Gilead, AbbVie; Speaking and Teaching: Janssen, BMS, Gilead, AbbVie

Markus Cornberg - Advisory Committees or Review Panels: Merck (MSD Germamny),

Roche, Gilead, Novartis, Abbvie, Janssen Cilag, BMS; Grant/Research

Support: Merck (MSD Germamny), Roche; Speaking and Teaching: Merck (MSD

Germamny), Roche, Gilead, BMS, Novartis, Falk, Abbvie

Eckart Schott - Advisory Committees or Review Panels: Gilead, Roche, Bayer,

BMS, Abbvie; Speaking and Teaching: Gilead, Novartis, Roche, MSD, Bayer,

Falk, BMS, Janssen, Abbvie

Thomas Berg - Advisory Committees or Review Panels: Gilead, BMS, Roche,

Tibotec, Vertex, Jannsen, Novartis, Abbott, Merck, Abbvie; Consulting: Gilead,

BMS, Roche, Tibotec; Vertex, Janssen; Grant/Research Support: Gilead, BMS,

Roche, Tibotec; Vertex, Jannssen, Merck/MSD, Boehringer Ingelheim, Novartis,

Abbvie; Speaking and Teaching: Gilead, BMS, Roche, Tibotec; Vertex, Janssen,

Merck/MSD, Novartis, Merck, Bayer, Abbvie

Ola Weiland - Advisory Committees or Review Panels: Merk, BMS, Medivir, Gilead,

AbbVie; Grant/Research Support; Speaking and Teaching: Merk, Roche,

BMS, Novartis, Janssen, Medivir, Gilead, AbbVie

Marc van der Valk - Advisory Committees or Review Panels: gilead, msd, bms,

abbvie, janssen cilag, viiV, roche

Andreas Geier - Advisory Committees or Review Panels: AbbVie, Janssen; Speaking

and Teaching: BMS, Gilead, Sequana, Falk, Novartis

Jürgen K. Rockstroh - Advisory Committees or Review Panels: Abbvie, BI, BMS,

Merck, Roche, Tibotec, Abbvie, Bionor, Tobira, ViiV, Gilead, Janssen; Consulting:

Novartis; Grant/Research Support: Merck; Speaking and Teaching: Abbott,

BI, BMS, Merck, Roche, Tibotec, Gilead, Janssen, ViiV

Markus Peck-Radosavljevic - Advisory Committees or Review Panels: Bayer, Gilead,

Janssen, BMS, AbbVie; Consulting: Bayer, Boehringer-Ingelheim, Jennerex,

Eli Lilly, AbbVie; Grant/Research Support: Bayer, Roche, Gilead, MSD, AbbVie;

Speaking and Teaching: Bayer, Roche, Gilead, MSD, Eli Lilly, AbbVie, Bayer

Maria Jesus Jimenez Exposito - Employment: Bristol-Myers Squibb

Stefan Zeuzem - Consulting: Abbvie, Bristol-Myers Squibb Co., Gilead, Merck

& Co., Janssen

The following authors have nothing to disclose: Kerstin Herzer, Ulrich Spengler,

Yue Zhao


Sofosbuvir/GS-5816+GS-9857 for 6 or 8 Weeks in

Genotype 1 or 3 HCV-infected Patients

Edward J. Gane 1 , Robert H. Hyland 2 , Yin Yang 2 , Luisa M. Stamm 2 ,

Diana M. Brainard 2 , John G. McHutchison 2 , Catherine A. Stedman

3 ; 1 Auckland Clinical Studies, Auckland, New Zealand; 2 Gilead

Sciences, Inc, Foster City, CA; 3 Christchurch Clinical Studies

Trust, Christchurch, New Zealand

Background: Sofosbuvir (SOF), GS-5816, and GS-9857 target

3 distinct viral proteins, NS5B, NS5A, and NS3, respectively.

The combination of these pangenotypic agents has the potential

to improve efficacy and may reduce treatment duration

across different patient populations. We previously reported

safety and efficacy of SOF/GS-5816 + GS-9857 administered

for 4 or 6 weeks in patients with HCV genotype 1 (GT1) infection,

including treatment-naïve (TN) GT1 patients without cirrhosis;

TN GT1 patients with cirrhosis; and GT1 patients who had

failed a regimen containing at least 2 direct antiviral agents.

We now evaluate the safety and efficacy of this regimen

administered for 6 or 8 weeks in more difficult-to-treat populations,

including treatment experienced (TE) GT 1 patients with

cirrhosis who had failed PEG/RBV; TE GT 1 patients who had

failed protease inhibitor (PI) + PEG/RBV; TN GT 3 patients with

cirrhosis; and TE GT 3 patients with cirrhosis who had failed

PEG/RBV. Methods: All patients received once daily SOF/

GS-5816 400mg/100mg in a fixed dose combination, plus

GS-9857 100mg. Treatment duration of each group differed

according to the patient’s baseline characteristics: 8 weeks

for TE GT 1 patients who had failed prior PEG/RBV, 8 weeks

for TE GT 1 patients who had failed PI + PEG/RBV, 6 weeks

for TN GT 3 patients and 8 weeks for TE GT 3 patients. The

primary efficacy endpoint was SVR12, defined as HCV RNA



SVR12 results from the Phase II, open-label IMPACT

study of simeprevir (SMV) in combination with daclatasvir

(DCV) and sofosbuvir (SOF) in treatment-naïve and

-experienced patients with chronic HCV genotype 1/4

infection and decompensated liver disease

Eric Lawitz 1 , Fred Poordad 1 , Julio A. Gutierrez 1 , Thomas Kakuda 2 ,

Gaston Picchio 3 , Greet Beets 4 , Ann Vandevoorde 4 , Peter Van

Remoortere 2 , Bert Jacquemyn 4 , Gemma Quinn 4 , Donghan Luo 2 ,

Sivi Ouwerkerk-Mahadevan 5 , Leen Vijgen 4 , Veerle Van Eygen 4 ,

Maria Beumont 5 ; 1 Texas Liver Institute, University of Texas Health

Science Center, San Antonio, TX; 2 Janssen Research & Development,

LLC, Titusville, NJ; 3 Infectious Diseases, Janssen Research

& Development, San Francisco, CA; 4 Janssen Infectious Diseases

BVBA, Beerse, Belgium; 5 Janssen Research & Development,

Beerse, Belgium

Purpose: The ongoing IMPACT study (NCT02262728)

assessed SMV+DCV+SOF in HCV genotype (GT)1/4-infected

patients (pts) with decompensated liver disease. We present

sustained virologic response 12 weeks (w) after end of treatment

(SVR12) from an interim analysis (IA). Methods: Treatment-naïve

or PegIFN±RBV treatment-experienced cirrhotic

adults with Child-Pugh (CP) score


patients ± cirrhosis ± HIV co-infection. Injection drug use is the

major risk factor for HCV transmission in many settings; however,

data on interferon-free treatment outcomes among PWID

are very limited. C-EDGE COSTAR is a Phase 3 trial evaluating

efficacy and safety of GZR/EBR among treatment naïve

HCV GT1/4/6-infected patients ± cirrhosis who are receiving

opioid agonist therapy (OAT) (methadone or buprenorphine).

METHODS: This double-blind, placebo-controlled study randomized

patients 2:1 to an immediate treatment group (ITG) or

a deferred treatment group (DTG). The ITG received GZR/

EBR for 12 weeks; the DTG received placebo for 12 weeks,

followed by 12 weeks of open label GZR/EBR. Use of non-prescribed

drugs was monitored by urine drug screening, but

was not exclusionary to study participation. HCV RNA levels

were measured using COBAS TaqMan v2.0 (lower limit of

quantitation 5x

ULN after normalization while on therapy. SVR12 results will

be presented. CONCLUSION: Preliminary data indicate that

GZR/EBR is safe and highly effective in patients with chronic

HCV GT1, 4, or 6 receiving OAT, and supports enhanced

efforts to address barriers to HCV treatment access for PWID.


Gregory Dore - Board Membership: Gilead, Merck, Abbvie, Bristol-Myers

Squibb; Grant/Research Support: Gilead, Merck, Abbvie, Bristol-Myers Squibb;

Speaking and Teaching: Gilead, Merck, Abbvie, Bristol-Myers Squibb

Frederick Altice - Grant/Research Support: Gileaad, NIH, NIDA, SAMHSA,

HRSA; Speaking and Teaching: Merck, Bristol Myers Squibb, Gilead, Rush Practice

Point Communications

Alain H. Litwin - Advisory Committees or Review Panels: Merck, Gilead; Grant/

Research Support: Merck, Gilead

Olav Dalgard - Advisory Committees or Review Panels: MSD, Janssen Cilag,

Medivir, Gilead, Abbvie; Grant/Research Support: MSD, Medivir, Gilead

Edward J. Gane - Advisory Committees or Review Panels: Novira, AbbVie, Janssen,

Gilead Sciences, Janssen Cilag, Achillion, Merck, Tekmira; Speaking and

Teaching: AbbVie, Gilead Sciences, Merck

Anne Luetkemeyer - Grant/Research Support: Gilead, Abbvie, Pfizer, Bristol

Myers Squibb, Merck

Ronald Nahass - Advisory Committees or Review Panels: Gilead, MErck, Janssen,

BMS; Grant/Research Support: Gilead, Merck, Janssen, BMS; Speaking and

Teaching: Gilead, Merck, Janssen

Cheng-Yuan Peng - Advisory Committees or Review Panels: AbbVie, BMS, Gilead,

MSD, Roche

Brian Conway - Advisory Committees or Review Panels: Vertex Pharmaceuticals,

Merck, Boehringer Ingelheim, Jannsen Pharmaceuticals; Grant/Research Support:

Vertex Pharmaceuticals, Merck, Boehringer Ingelheim, Jannsen Pharmaceuticals,

AbbVie, Gilead Sciences, Gilead Sciences

Jason Grebely - Advisory Committees or Review Panels: Merck, Gilead; Grant/

Research Support: Merck, Gilead, Abbvie, BMS

Anita Y. Howe - Employment: Merck Research Laboratory

Bach-Yen T. Nguyen - Employment: Merck

Janice Wahl - Employment: Merck & Co,

Eliav Barr - Employment: Merck

Michael Robertson - Employment: Merck; Stock Shareholder: Merck

The following authors have nothing to disclose: Oren Shibolet, Heather L. Platt


98%–100% SVR4 in HCV Genotype 1 Non-Cirrhotic

Treatment-Naïve or Pegylated Interferon/Ribavirin Null

Responders With the Combination of the Next Generation

NS3/4A Protease Inhibitor ABT-493 and NS5A

Inhibitor ABT-530 (SURVEYOR-1)

Fred Poordad 2 , Franco Felizarta 3 , Armen Asatryan 1 , Tarek I. Hassanein

4 , Humberto I. Aguilar 5 , Jacob P. Lalezari 6 , J. Scott Overcash

7 , Teresa Ng 1 , Ran Liu 1 , Chih-Wei Lin 1 , Federico J. Mensa 1 ,

Jens Kort 1 ; 1 AbbVie, Inc., North Chicago, IL; 2 Texas Liver Institute,

University of Texas Health Science Center, San Antonio, TX; 3 Private

practice, Bakersfield, CA; 4 Southern California GI and Liver

Centers and Southern California Education and Research Center,

Coronado, CA; 5 Louisiana Research Center, Shreveport, LA;

6 Quest Clinical Research, San Francisco, CA; 7 eStudySite, San

Diego, CA

Background: The next-generation HCV direct-acting antivirals

(DAAs) ABT-493, an NS3/4A protease inhibitor identified by

AbbVie and Enanta, and ABT-530, an NS5A inhibitor, are

characterized by potent pangenotypic in vitro antiviral activity

against major HCV genotypes (GTs), including activity against

key known resistance-associated variants and a high barrier

to resistance selection. Monotherapy with ABT-493 or ABT-

530 resulted in a mean 4 log 10

IU/mL decline from baseline

in HCV plasma viral load in GT1-infected subjects with and

without compensated cirrhosis. Purpose: In this phase 2 study,

treatment with ABT-493 and ABT-530 for 12 weeks is evaluated

in HCV GT1-infected subjects without cirrhosis. Efficacy

and safety results are reported here. Methods: Non-cirrhotic

GT1-infected treatment-naïve (TN) or pegylated interferon/ribavirin

(pegIFN/RBV) null responder subjects received once-daily

ABT-493 200 mg + ABT-530 120 or 40 mg for 12 weeks,

and subsequently were followed for 24 weeks. The primary

efficacy endpoint was sustained virologic response 12 wks

after the last dose of study drug (SVR12). Safety was evaluated

by adverse event (AE) monitoring, laboratory testing,

and other standard assessments. Results: 79 subjects (male,

52%; median [range] age, 54.0 [26.0–70.0] years; GT1a,

81%; GT1b, 19%; TN, 63%; pegIFN/RBV null responders,

37%; fibrosis >F2, 25%; median [range] HCV RNA log 10


mL, 6.8 [4.4–7.5]) were enrolled, 40 received ABT-493 200

mg+ABT-530 120 mg and 39 received ABT-493 200 mg and

ABT-530 40 mg. SVR 4 weeks after the last dose of study drug

(SVR4) is available in all 79 subjects. SVR4 was achieved in

29 of 29 (100%) pegIFN/RBV null responders and 49 of 50

(98%) TN subjects. One TN subject relapsed at post-treatment

week 4; further characterization of this relapse and complete

post-treatment week 12 data (SVR12) will be available. There

were no treatment-related serious AEs or clinically relevant

laboratory findings. The most common AEs (reported in >5%

of subjects) were fatigue, headache, nausea, diarrhea, and

anxiety. Conclusions: Once-daily 12-week treatment with the

next-generation HCV DAAs ABT-493 and ABT-530 of GT1

infection in non-cirrhotic TN and pegIFN/RBV null responders

resulted in high SVR4 (98%–100%) rates. One treatment

relapse was observed. Based on these encouraging results,

the SURVEYOR-1 study with once-daily ABT-493 and ABT-530

has been expanded to include GT1 subjects with compensated

cirrhosis and evaluate a shorter, 8 week treatment duration in

non-cirrhotic subjects.



Fred Poordad - Advisory Committees or Review Panels: Abbott/Abbvie, Achillion,

BMS, Inhibitex, Boeheringer Ingelheim, Pfizer, Genentech, Idenix, Gilead,

Merck, Vertex, Salix, Janssen, Novartis; Grant/Research Support: Abbvie,

Anadys, Achillion, BMS, Boehringer Ingelheim, Genentech, Idenix, Gilead,

Merck, Pharmassett, Vertex, Salix, Tibotec/Janssen, Novartis

Franco Felizarta - Grant/Research Support: AbbVie, Gilead, Janssen, Merck,

BMS, Boehringer-Ingelheim, Vertex, Roche; Speaking and Teaching: AbbVie,

Gilead, Janssen, Merck

Armen Asatryan - Employment: AbbVie

Tarek I. Hassanein - Advisory Committees or Review Panels: AbbVie, Bristol-Myers

Squibb; Grant/Research Support: AbbVie Pharmaceuticals, Obalon, Bristol-Myers

Squibb, Eiasi Pharmaceuticals, Gilead Sciences, Janssen R&D, Idenix

Pharmaceuticals, Ikaria Therapeutics, Merck Sharp & Dohme, NGM BioPharmaceuticals,

Ocera Therapeutics, Salix Pharmaceuticals, Sundise, TaiGen Biotechnology,

Takeda Pharmaceuticals, Vital Therapies, Tobria; Speaking and

Teaching: Baxter, Bristol-Myers Squibb, Gilead, Salix, AbbVie

Humberto I. Aguilar - Speaking and Teaching: abbvie, gilead

Teresa Ng - Employment: AbbVie; Patent Held/Filed: AbbVie; Stock Shareholder:


Ran Liu - Employment: Abbvie

Chih-Wei Lin - Employment: Abbvie

Federico J. Mensa - Employment: Abbvie Inc.; Stock Shareholder: Abbvie Inc.

Jens Kort - Employment: AbbVie Inc.; Stock Shareholder: AbbVie Inc.

The following authors have nothing to disclose: Jacob P. Lalezari, J. Scott Overcash


An Integrated Analysis of 402 Compensated Cirrhotic

Patients With HCV Genotype (GT) 1, 4 or 6 Infection

Treated With Grazoprevir/Elbasvir

Ira M. Jacobson 1 , Eric Lawitz 2 , Paul Y. Kwo 3 , Christophe Hezode 4 ,

Cheng-Yuan Peng 5 , Anita Y. Howe 6 , Peggy Hwang 6 , Janice

Wahl 6 , Michael Robertson 6 , Eliav Barr 6 , Barbara A. Haber 6 ;

1 Medicine, Mt. Sinai Beth Israel, New York, NY; 2 Texas Liver Institute,

University of Texas Health Science Center, San Antonio, TX;

3 Indiana University School of Medicine, Indianapolis, IN; 4 Henri

Mondor Hospital, Creteil, France; 5 China Medical University Hospital,

Taichung, Taiwan; 6 Merck & Co., Inc., Kenilworth, NJ

Purpose: Treatment of cirrhotic HCV-infected patients (pts) often

requires ribavirin (RBV) and long durations of therapy. In the

C-EDGE trials, 12-16 weeks’ therapy with grazoprevir 100

mg/elbasvir 50 mg (GZR/EBR) ± RBV was highly efficacious

in a diverse population of pts. An analysis of the efficacy/

safety of GZR/EBR ± RBV among compensated cirrhotics in

the phase 2/3 GZR/EBR program was conducted. Methods:

Treatment-naive (TN) and treatment-experienced (TE) pts with

compensated cirrhosis received GZR/EBR ±RBV for 12 (TN) or

12-18 weeks (TE) in 6 studies. The primary end point was HCV



could predict the presence of various cancer entities in patients

with hepatocellular, colorectal and non-small cell lung carcinoma

(HCC, CRC, NSCLC, resp.), as a tool for cancer screening

and therapy response monitoring. Methods: MPs were

isolated from serum of patients with HCC, CRC, NSCLC and

from healthy donors. MP profiling was done after differential

ultracentrifugation using FACS analysis for the presence and

quantity of AnnexinV + /EpCAM + and AnnexinV + /EpCAM + /

CD147 + taMPs. Results: AnnexinV + /EpCAM + and AnnexinV

+ /EpCAM + /CD147 + taMPs highly significantly separated

patients with carcinomas from healthy controls. The calculated

cut-off values for AnnexinV + /EpCAM + taMPs were 36.6

/1AnnexinV + MPs in NSCLC, 35.8/1kAnnexinV + MPs in CRC

and 31.4/1kAnnexinV + taMPs in HCC. Additionally, AnnexinV

+ /EpCAM + /CD147 + taMPs indicated the presence of cancer

as well as AnnexinV + /EpCAM + taMPs alone. AnnexinV + /

EpCAM + taMPs and AnnexinV + /EpCAM + /CD147 + taMPs

were increased 2.4-fold (p


respectively. The median (IQR) time intervals were 46.5 (62)

and 47.5 (87) days between MRE and biopsy and between

ARFI and biopsy, respectively. For diagnosing any fibrosis,

MRE had an area under ROC curve (AUROC) of 0.799 (95%

CI, 0.723-0.875), which was significantly (p=0.012) higher

than ARFI, which had an AUROC of 0.664 (95% CI, 0.568-

0.760). The AUROCs of MRE for diagnosing ≥ stages 2, 3,

and 4 fibrosis were 0.885 (95% CI, 0.816-0.953), 0.934

(95% CI, 0.863-1.000), and 0.882 (95% CI, 0.729-1.000),

and those for ARFI were 0.848 (95% CI, 0.776-0.921), 0.896

(95% CI, 0.824-0.968), and 0.862 (95% CI, 0.721-1.000).

MRE had a higher AUROC than ARFI for discriminating dichotomized

fibrosis stages at every dichotomization cutpoint, but

the difference in AUROC was smaller as the dichotomization

cutpoint increased. Conclusion: In a head-to-head comparison

and using contemporaneous liver biopsy as the gold standard,

MRE provides significantly higher diagnostic accuracy than

ARFI for diagnosing any fibrosis in NAFLD patients.


Claude B. Sirlin - Grant/Research Support: GE, Pfizer, Bayer, Guerbet, Siemens

Rohit Loomba - Advisory Committees or Review Panels: Galmed Inc, Tobira Inc,

Arrowhead Research Inc; Consulting: Gilead Inc, Corgenix Inc, Janssen and

Janssen Inc, Zafgen Inc, Celgene Inc, Alnylam Inc, Inanta Inc, Deutrx Inc; Grant/

Research Support: Daiichi Sankyo Inc, AGA, Merck Inc, Promedior Inc, Kinemed

Inc, Immuron Inc, Adheron Inc

The following authors have nothing to disclose: Jeffrey Y. Cui, Elhamy Heba,

Carolyn Hernandez, William Haufe, Jonathan Hooker


The predictive value of 13 C-Methacetin breath test (MBT)

for liver transplantation (LT) and death: a seven year

follow-up study of 206 chronic hepatitis C patients

Oliver Goetze 2,1 , Monika Breuer 1 , Andreas Geier 2,1 , Michael

Fried 1 , Achim Weber 4 , Yaron Ilan 3 , Beat Mullhaupt 1 ; 1 Department

of Gastroenterology & Hepatology, University Hospital Zurich,

Zurich, Switzerland; 2 Division of Hepatology, University Hospital

Würzburg, Würzburg, Germany; 3 Department of Medicine,

Hebrew University-Hadassah Medical Center, Jerusalem, Israel;

4 Institute of Surgical Pathology, University Hospital Zurich, Zurich,


In patients with chronic liver disease, currently used biomarkers

demonstrate limited mid- to long-term predictive values to

forecast deterioration of liver function leading to liver transplantation

(LT) or liver related death. Aim: To determine the

predictive value of the 13 C-Methacetin Breath Test (MBT)

(BreathID ® ; Exalenz) in forseeing deterioration of liver function

leading to LT and/or death in patients with chronic HCV infection.

Methods: A total of 265 chronic HCV infected patients

were enrolled, out of which 206 patients (71 females) without

co-infections and/or liver comorbidities were considered for

this analysis. The average BMI was 24.8 kg/m 2 (SD 4.0 kg/

m 2 ), 183 patients underwent liver biopsy and ISHAK scoring

and 196 patients underwent MBT. 13 C-Methacetin peak percent

dose recovery rate (PDR-Peak) was used as determinant

parameter for this analysis. Patients were followed for up to

7 years according to standard of care. During the course of

follow-up some patients underwent antiviral treatment as part of

routine patient management. Primary analysis was performed

on all patients up to one of the following: liver-transplantation,

death or response to antiviral treatment, resulting in 161

and 158 patients with biopsy and/or MBT results respectively.

Predictive value was assessed using ROC curve and survival

analyses. Results: In seven years follow up, PDR-Peak, using

cut-off of 20%/h, predicted LT and/or death with HR=12.07

(95%CI 3.48;41.84),p


black tea. The mean ALT noted in 657 individuals within 3

months of TE was 60.6 U/L. The average BMI was 25.7 kg/m 2

and weight was 71.7 kg. Average daily alcohol consumption

was 5 g/day. The average liver stiffness for the 1155 individuals

was 8.4 kPa. CAP was available from 2013 in 719 individuals

with average steatosis measures of 214 dB/m. After

MLR taking into account age, alcohol, smoking and weight,

individuals with HCV benefited from coffee intake with LSM

on average 2 kPa less with intake of 2 or more cups of coffee

daily (p = 0.026). This was not seen in those with HBV or

NAFLD. In NAFLD a decrease in CAP of 23 dB/m (9%) was

noted in individuals who drank ≥2 cups of coffee (p = 0.032).

There was no effect of coffee consumption on CAP in other

groups. Conclusions: Individuals with self-reported daily coffee

intake ≥2 cups of coffee/ day had a significant decrease in

liver stiffness, which was not seen in those with HBV or NAFLD.

Similar daily coffee intake resulted in significant decrease in

liver steatosis in NAFLD. These findings are consistent with the

literature to date and add to the growing body of evidence

suggesting coffee may be a beneficial supplement in some

liver diseases.


The following authors have nothing to disclose: Alexander Hodge, Sarah P. Lim,

Evan Goh, Ophelia H. Wong, Philip Marsh, Virginia Knight, Yong Song


Serum Lipidomic Profiling for Screening Potential Biomarkers

of Liver Cirrhosis among Patients with Chronic

Hepatitis C

Ana Maria Passos-Castilho 1 , Maria Lucia Ferraz 2 , Valdemir M.

Carvalho 3 , Karina H. Cardozo 3 , Luciana Kikuchi 4 , Aline Chagas 4 ,

João Renato R. Pinho 4 , Michele S. Gomes-Gouvêa 4 , Fernanda

Malta 4 , Flair J. Carrilho 4 , Celso Granato 1,3 ; 1 Division of Infectious

Diseases, Federal University of Sao Paulo, Sao Paulo, Brazil;

2 Department of Gastroenterology, Federal University of Sao Paulo,

Sao Paulo, Brazil; 3 Fleury SA Group, Sao Paulo, Brazil; 4 Department

of Gastroenterology, University of Sao Paulo School of Medicine,

Sao Paulo, Brazil

Background: Liver cirrhosis (LC) is the 14th most common cause

of death in adults worldwide, resulting in 1.03 million deaths

per year. Chronic hepatitis C (CHC) is one of the main causes

of LC. Non-invasive markers of fibrosis have been increasingly

studied as they may represent a more informative, safe and

accessible diagnostic and/or screening tool for at-risk patients.

Aim: To explore the serum lipidome profiles of LC to identify

potential biomarkers. Methods: A total of 182 subjects were

enrolled from 2011 to 2014. An ultraperformance liquid chromatography–mass

spectrometry (UPLC-MS) lipidomic method

was used to characterize serum profiles from LC (n=59), CHC

(n=65) and healthy subjects (n=58). Patients were diagnosed

by clinical, laboratory and imaging evidence of LC while

healthy controls had normal liver function and no infectious diseases.

Reversed-phased analysis was performed on a Waters

ACQUITY IClass UPLC system coupled to a Waters Synapt-MS

hidrid quadrupole-time of flight mass spectrometer operating

in the positive ion electrospray mode with a mass scan range

200–1200 m/z for data acquisition in continuous mode. All

data were processed with Progenesis software (Waters). The

resulting multivariate data set was analyzed with MetaboAnalyst

(TMIC) using supervised partial least-squares discriminate

analysis (PLS-DA). Potential biomarkers were selected according

to the variable importance in the projection (VIP) and statistical

significance was evaluated using Mann-Whitney test. The

receiver operating characteristic (ROC) curve was performed

to assess the accuracy of selected biomarkers in LC diagnosis.

To identify the potential biomarkers, the HMDB and Lipid

Maps databases were queried with the exact mass. Results:

The UPLC–MS-based serum lipidomic profile detected 51 of

59 LC cases, performing with a diagnostic accuracy of 87%.

The 3 potential biomarkers differentiated LC from CHC individually

with 76 to 88% sensitivity and 72 to 74% specificity.

They all presented with fold change higher than 2 and p <

0.0001 in univariate analyses. The combination of the 3 potential

biomarkers resulted in an area under the curve of 0.95,

86% sensitivity and 88% specificity. The model was validated

with 1000 permutation tests (p < 0.001) to prevent overfitting.

3-Hydroxy-cis-5-tetradecenocylcarnitine, a hydroxy fatty acid

involved in many metabolic and proinflammatory pathways,

exhibited a significant increase in LC and a decrease in CHC

and was proposed as an important indicator of LC. Conclusions:

UPLC-MS lipid profiling proved to be an efficient and

convenient tool for diagnosis and screening of LC in an at-risk



João Renato R. Pinho - Employment: Hospital Israelita Albert Einstein

The following authors have nothing to disclose: Ana Maria Passos-Castilho,

Maria Lucia Ferraz, Valdemir M. Carvalho, Karina H. Cardozo, Luciana Kikuchi,

Aline Chagas, Michele S. Gomes-Gouvêa, Fernanda Malta, Flair J. Carrilho,

Celso Granato


Comparison of Liver Transplant-related Survival Benefit

in Patients With vs Without Hepatocellular Carcinoma in

the United States.

George N. Ioannou 1,2 , Kristin Berry 1 ; 1 Veterans Affairs Puget

Sound Healthcare System, Seattle, WA; 2 University of Washington,

Seattle, WA

Background and Aims Patients with T2 hepatocellular carcinoma

(HCC) can obtain an exception allowing them to undergo

liver transplantation at much lower actual Model for End Stage

Liver Disease (MELD) scores than patients without HCC. We

aimed to compare patients transplanted with and without HCC

with regards to transplantation-related survival benefit. Methods

We modeled the post-transplant survival of adult, first-time

liver transplant recipients with (n=9135) or without (n=25,890)

HCC from 2002-2013 using Cox proportional hazards regression.

We modeled waitlist survival of patients listed for transplantation

with (n=15,605) or without (n=85,229) HCC using

competing risks analysis and a combined outcome of death

or liver failure, defined as MELD score ≥30. We used these

survival models to calculate monthly transition probabilities and

5-year life expectancies. Survival benefit was calculated as the

difference between post-transplant and waitlist life expectancy.

Results Five-year survival benefit increased with actual MELD

score for both patients with and without HCC from just a few

months in patients with low MELD scores (6-8) to 4 years in

patients with the highest MELD scores (36-40). The survival

benefit of patients with HCC was similar to that of patients

without HCC who had the same actual MELD score, irrespective

of tumor burden or serum alpha fetoprotein level. Because

patients with HCC were transplanted at a lower mean MELD

score (13.3±6.2) than patients without HCC (21.8±8.0), a

dramatically lower mean 5-year survival benefit was achieved

by transplanting patients with HCC (0.12 years/patient) than

patients without HCC (1.47 years/patient). Conclusions The

HCC MELD exception policy unintentionally resulted in a dramatic

reduction in transplant-related survival benefit.


Five-year transplant-related survival benefit as a function of MELD

score shown separately in patients with or without HCC

considered, wait time 18 months (HR 1.6, 95% CI

1.01-2.5, p=0.043) and AFP >400 ng/mL at HCC diagnosis

(HR 3.0, 95% CI 1.7-5.5, p100 ng/mL at LT

(HR 1.6, 95% CI 1.04-2.6, p


LT. Therefore, MHE, i) should not to be ignored and ii) deserves

to be treated in order to reduce the risk of neurological complications

occurring post-LT.


The following authors have nothing to disclose: Marc-André Clément, Cristina R.

Bosoi, Mélanie Tremblay, Chantal Bemeur, Christopher F. Rose


The Functional Basis for Cognitive Improvement after

Liver Transplant: A Prospective Brain MRI Study

Vishwadeep Ahluwalia, James Wade, HoChong Gilles, Melanie

White, Ariel Unser, Edith A. Gavis, Mohammad S. Siddiqui, Velimir

A. Luketic, Arun J. Sanyal, Puneet Puri, Douglas M. Heuman,

Scott C. Matherly, R. Todd Stravitz, Richard K. Sterling, Michael

Fuchs, Jasmohan S. Bajaj; VCU and McGuire VAMC, Richmond,


Cirrhotics have varying degrees of cognitive recovery post-liver

transplant (LT) depending on their prior hepatic encephalopathy(HE)

status but the mechanism is unclear. Aim: Define the

functional basis of cognitive change in LT recipients. Method:

Cirrhotics on the transplant list underwent standard cognitive

testing & questionnaires regarding depression (Beck Depression,

BDI), QOL (Sickness impact profile, SIP has psych &

physical components) pre/6 months post-LT. Cognitive tests

were number connection A/B (NC), digit symbol (DS) & block

design (BD) which test for psychomotor speed, set-shifting and

visuo-motor coordination. We also performed brain functional

MRI while subjects underwent the inhibitory control test (ICT)

within the scanner. ICT tests for response inhibition, working

memory & psychomotor speed. The outcomes are lures (that

need to be inhibited) & targets (need to be responded to).

Changes in brain activation on correct lure inhibition & target

response were compared before & after LT. Results: We

enrolled 67 cirrhotics of which 11 died prior, 12 still await &

5 pts were


may help refine post-LT HCC surveillance strategies and identify

patients who would derive the most benefits from future post-LT

adjuvant therapies.


0 points for: AFP


from BM macrophages and Kupffer cells in a dose-dependent

manner; 5-10nM MCC950 abrogated such IL-1β release. Conclusions:

These data demonstrate that MCC950, a potent and

selective NLRP3 inflammasome inhibitor, prevents or reverses

inflammation, injury and fibrosis in two different murine models

of NASH. These effects could be explained, at least in part, by

the effective abrogation of cholesterol crystal-induced NLRP3

activation and IL-1β release in BM macrophages and Kupffer

cells. Targeting NLRP3 is a logical new direction in NASH



The following authors have nothing to disclose: Auvro R. Mridha, Alexander

Wree, Avril A. Robertson, Narci C. Teoh, Matthew A. Cooper, Ariel E. Feldstein,

Geoffrey C. Farrell


Role of Fatty Acid Desaturase 1 (FADS1) in Nonalcoholic

Fatty Liver Disease (NAFLD)

Shaminie Athinarayanan 1 , Yang-Yi Fan 2 , Robert Chapkin 2 , Wanqing

Liu 1 ; 1 Medicinal Chemistry and Molecular Pharmacology,

Purdue University, West Lafayette, IN; 2 Center for Translational

Environmental Health Research, Texas A&M, College Station, TX

Background: NAFLD and nonalcoholic steatohepatitis (NASH)

are common chronic liver diseases. Previous studies have

demonstrated that decreased hepatic and blood levels of

n-3 polyunsaturated fatty acids (PUFA) are associated with

NAFLD/NASH. The FADS1 gene encodes delta-5 desaturase

(D5D), one of the rate-limiting enzymes involved in n-3 PUFA

metabolism. We hypothesized that FADS1 is involved in the

pathogenesis of NAFLD/NASH via modulating hepatic lipid

homeostasis and other pathways. Methods: In order to corroborate

our hypothesis, we used both the HepG2 cell line

and a Fads1 knockout (null) mouse model. The FADS1 gene

was knocked down using siRNA in HepG2 cells. Palmitic acid

(PA)-induced lipid accumulation was compared between the KD

cells and the control siRNA group. In addition, RNA-seq based

hepatic transcriptome data were compared between the null

(n=4) and wild-type (n=4) mice. Differential gene expression

data were analyzed using EdgeR from Bioconductor. Detailed

pathway analyses were performed by combining both a targeted

approach to examine well-established lipid homeostasis

pathways as well as a global, unsupervised enrichment analysis

using Ingenuity Pathway Analysis (IPA). Results: FADS1

knock down in HepG2 cells significantly increased PA-induced

intracellular lipid accumulation by 60% (p < 0.001). Liver samples

from Fads1 null mice exhibited a significantly (p



Hepatocytes Release Ceramide-rich Proinflammatory

Extracellular Vesicles in an IRE1alpha-dependent manner

Eiji Kakazu 1,2 , Amy S. Mauer 1 , Harmeet Malhi 1 ; 1 Gastroenterology

and Hepatology, Mayo Clinic, Rochester, MN; 2 Department of

Community Medical Supports, Tohoku University, Aobaku, Japan

Palmitate (PA), a lipotoxic free fatty acid, is implicated in

hepatocyte apoptosis, macrophage-mediated liver inflammation,

and activation of the IRE1alpha branch of the endoplasmic

reticulum (ER) stress response - all key features of

progressive nonalcoholic steatohepatitis (NASH). Recently,

extracellular vesicles (EVs) have been implicated in NASH.

However, the exact pathways of hepatocyte EV generation

under lipotoxic conditions and their downstream effects on

macrophages are undefined. Therefore, we hypothesized that

hepatocytes release proinflammatory PA-induced EVs via an

ER stress response. To test this hypothesis we employed immortalized

mouse hepatocytes (IMH) from wild-type (WT) or IRE1a

knockout (KO) mice, Huh 7 and primary mouse hepatocytes

(PMH). Cells were treated with 400μM PA or thapsigargin

(Tg) to induce ER stress. EVs were isolated from cell culture

supernatants by ultracentrifugation and characterized by immunofluorescence

and western blotting for EV markers, electron

microscopy (EM) and by nanoparticle tracking analysis (NTA)

for morphology and size. Ceramides were measured by mass

spectrometry. CRISPR/Cas9 technology was used to delete

XBP1. Results: PA and Tg significantly increased EV release

in all three hepatocyte cells tested (IMH, Huh7 and PMH). The

released EVs were 100nm in size, decorated with EV markers

CD63, TSG101 and LAMP1, and demonstrated characteristic

cup-shaped morphology on EM. As PA and Tg activate an ER

stress response, we next tested cells lacking in each of the three

canonical ER stress sensors, IRE1alpha, ATF6alpha and PERK.

Both PA and Tg induced a significant EV release in cells lacking

ATF6 alpha or PERK; however, PA and Tg-induced EV release

was significantly suppressed in IRE1alpha KO cells. We tested

the involvement of XBP1 signaling in this phenomenon and

found a reduction in EV response in cells lacking XBP1. Next

we measured ceramides in PA-induced EVs as they are implicated

in EV biogenesis. PA-induced EVs were enriched in C16

ceramide; this enrichment occurred in an IRE1alpha-dependent

manner. Inhibition of de novo ceramide synthesis by myriocin

ameliorated PA-induced EV release, and exogenous C16 ceramide

resulted in EV release in IRE1alpha KO and WT cells.

Lastly, PA-stimulated EVs were chemotactic to macrophages.

Conclusions: PA induces C16 ceramide-enriched EV release

in an IRE1alpha-dependent manner. PA-induced EVs stimulate

macrophage chemotaxis and this may be a mechanism for the

recruitment of macrophages to the liver under lipotoxic conditions.

We hypothesize that interference with this macrophage

recruitment response may be a therapeutic avenue in NASH.


The following authors have nothing to disclose: Eiji Kakazu, Amy S. Mauer,

Harmeet Malhi


Deficiency of intestinal mucin-2 protects mice from

diet-induced fatty liver disease and obesity

Phillipp Hartmann, Caroline T. Seebauer, Magdalena Mazagova,

Angela Horvath, Lirui Wang, Cristina Llorente-Izquierdo, Katharina

Brandl, Samuel B. Ho, David A. Brenner, Bernd Schnabl; University

of California San Diego, La Jolla, CA

Background: Non-alcoholic fatty liver disease (NAFLD) and

obesity are characterized by altered gut microbiota, inflammation,

and gut barrier dysfunction. The aim of this study was to

investigate the role of mucin-2 (Muc2) as the major component

of the intestinal mucus layer in the development of fatty liver

disease and obesity. Methods and Results: We studied experimental

fatty liver disease and obesity induced by high-fat diet

(HFD) feeding for 16 weeks in wild-type (WT) littermate and

mucin-2 knockout mice. Muc2 deficiency protected mice from

HFD-induced obesity and fatty liver disease as evidenced by a

significantly reduced body weight and lower hepatic triglyceride

concentrations, respectively. Furthermore, Muc2 -/- mice displayed

significantly decreased weights of subcutaneous and

epididymal white adipose tissue and brown adipose tissue

relative to WT mice on HFD. There was no difference in food

intake and fecal triglyceride content between the HFD groups

that would explain differences in phenotype. HFD-fed Muc2 -/-

mice exhibited improved glucose tolerance and responsiveness

to insulin, as evidenced by intraperitoneal glucose tolerance

and insulin tolerance tests, as well as reduced inflammation

in white adipose tissue than WT littermates. Furthermore, they

showed an upregulated expression of key genes involved in

lipolysis, such as adipose triglyceride lipase (ATGL) and hormone-sensitive

lipase (HSL), and in fatty acid β-oxidation, e.g.

palmitoyl acyl-CoA oxidase 1 (ACOX1), in white adipose tissue

compared with WT mice after 16 weeks of HFD feeding.

Interleukin-22 (IL-22) is not only crucial for maintaining intestinal

homeostasis, but administration of IL-22 to obese mice has

been shown to improve insulin sensitivity, decrease chronic

inflammation and reduce body weight. Most interestingly,

Muc2 -/- mice showed increased intestinal gene expression

of IL-22 and IL-22 target genes Reg3b and Reg3g relative to

WT mice fed HFD. Plasma levels of IL-22 were significantly

higher in HFD-fed Muc2 -/- mice as compared with WT mice.

After bypassing the mucus layer by intraperitoneal injections

of bacteria-derived flagellin, differences in IL-22 plasma levels

between HFD-fed WT and Muc2 -/- mice vanished, suggesting

that the mucosal immune system produces increased amounts

of IL-22 in the absence of a protective mucus barrier. Conclusion:

An impaired gut barrier elicits a strong intestinal immune

response in Muc2 deficient mice, which is characterized by the

induction of IL-22. Increased systemic IL-22 mediates beneficial

metabolic and anti-inflammatory effects, and protects against

obesity and fatty liver disease.


Angela Horvath - Grant/Research Support: Instutut Allergosan

Samuel B. Ho - Grant/Research Support: Gilead, Genentech; Speaking and

Teaching: Prime Education, Inc

The following authors have nothing to disclose: Phillipp Hartmann, Caroline T.

Seebauer, Magdalena Mazagova, Lirui Wang, Cristina Llorente-Izquierdo, Katharina

Brandl, David A. Brenner, Bernd Schnabl



FoxO3 increases miR-34a to cause palmitate-induced

cholangiocytes lipoapoptosis

Sathish Kumar Natarajan, Cody J. Wehrkamp, Ashley M. Mohr,

Mary A. Smith, Sizhao Lu, Duygu Dee Harrison-Findik, Justin L.



Abstract: Non-alcoholic fatty liver disease (NAFLD) patients

have elevated plasma saturated free fatty acid (FFA) levels.

We had recently demonstrated the critical role of FoxO3 and

its downstream target, PUMA in FFA-induced cholangiocyte

lipoapoptosis and provided insights for biliary damage in a

subset of NAFLD patients. Here, we explored whether miR-34a

have a role in cholangiocyte lipoapoptosis. Methods: Apoptosis

was assessed by TUNEL, characteristic nuclear morphology

staining with DAPI, and caspase 3/7 activity assay. miR-34a

levels were measured using quantitative real time-PCR. FoxO3,

cleaved PARP, cleaved caspase 3 and miR-34a target expression

was examined by Western blot. Results: Treatment of cholangiocytes

with palmitate (PA) showed 7-20 fold increase in

caspase3/7 activity in normal immortalized cholangiocytes,

H69 and cell lines derived from cholangiocarcinoma, KMCH,

Mz-ChA-1 and HuCCT cells. PA treatment also resulted in a dramatic

increase in the levels of caspase cleaved products such

as cleaved PARP and cleaved caspase 3 suggesting cholangiocyte

lipoapoptosis. Interestingly, treatment with PA significantly

increased levels of miR-34a in cholangiocytes and hepatocyte

cell line, Huh7. PA-induced increase in miR-34a levels were

abolished in cholangiocytes transduced with FoxO3 shRNA,

implicating that miR-34a is a transcriptional target of FoxO3.

Further, cholangiocytes transfected with anti-miR-34a were protected

from PA-induced lipoapoptosis suggesting a major role

for miR-34a. Further, direct and indirect targets of miR34a such

as Sirt1, receptor tyrosine kinase (cMET), Kruppel like factor 4

(KLF4), fibroblast growth factor receptor (FGFR1 and 4) were

all decreased in PA-treated cholangiocytes. In addition, our

data also shows that cholangiocyte apoptosis and miR-34a are

dramatically increased in the liver from mice fed with high-fat

high-sucrose for 3 months compared to control fed animals.In

conclusion, PA induces the expression of proapoptotic miR-34a

via FoxO3 and increased miR-34a levels results in targeting

protein deacetylase Sirt1 and anti-apoptotic proteins like cMET

and KLF4 resulting in cholangiocyte lipoapoptosis.


The following authors have nothing to disclose: Sathish Kumar Natarajan, Cody

J. Wehrkamp, Ashley M. Mohr, Mary A. Smith, Sizhao Lu, Duygu Dee Harrison-Findik,

Justin L. Mott


Prior Blockade of TNF-α Signaling Promotes Liver

Regeneration and Offers Strategies for Improving Outcomes

after Liver Resection Surgery



The presumed beneficial role in liver regeneration (LR) of

pro-inflammatory cytokines, such as TNF-α or IL6, contrasts

with deleterious effects of these cytokines in other settings,

e.g., after ischemia/reperfusion, inflammation, etc. Recently,

cell transplantation-induced liver injury was shown to activate

multiple chemokines/cytokines/receptors, which were essentially

normalized after prior blockade of TNF-α by the drug,

Etanercept (ETN), with superior transplanted cell engraftment

and proliferation during liver repopulation in retrorsine (Ret)/

partial hepatectomy (PH) rat model. To resolve what role TNF-α

may serve in LR, we hypothesized that administering ETN to

block TNF-α before PH will allow determination of whether LR

will be impaired or if it would still proceed. Groups of F344

rats were established for 2/3 PH alone or ETN followed by

2/3 PH along with untreated controls. Time-course analysis

at 6, 32 or 72 h and 7 d after these procedures showed ETN

was well tolerated and liver/body weight ratios were actually

superior rather than inferior in ETN-pretreated rats with PH,



experience a fibrotic phase dominated by inflammatory macrophages,

followed by a phase of fibrosis remodelling characterised

by restorative macrophages. Gpnmb- mice show a

higher level of circulating hepatic enzymes in the phase of

fibrosis remodelling, associated with higher proliferation of

parenchymal cells and higher levels of inflammatory cytokines

in the tissue. Moreover those mice show a lower number of

macrophages and T cells in the phase of fibrosis resolution,

determined by immunohistochemistry. Flow cytometry analysis

unveiled a possible skew to a pro-inflammatory phenotype in

the recovering liver. Key cytokines have been investigated as

a possible cross-regulator of phagocytosis and tissue proliferation.

The role of IL6 as a pro-proliferative agent in the liver

is well known. No information is available on its role as a

modulator of phagocytosis. Both in vivo and in vitro IL6 shows

a pro-phagocytic effect possibly through the activation of the

STAT3 pathway downstream the IL6 receptor. Collectively

our data suggest that phagocytosis could be the gatekeeper

between the necessary phase of initial inflammation and the

following phase of tissue remodelling.


The following authors have nothing to disclose: Lara Campana, Antonella Pellicoro,

Rebecca Aucott, Stephen Greenhalgh, Katherine L. Hull, Stuart J. Forbes,

John P. Iredale


Dynamic shift of microbiota and its relationship with

hepatic gene expression during liver regeneration

Hui-Xin Liu 1 , Clarissa S. Rocha 2 , Satya Dandekar 2 , Yu-Jui Yvonne

Wan 1 ; 1 Medical Pathology and Laboratory Medicine, UC DAVIS,

Sacramento, CA; 2 Medical Microbiology and Immunology, UC

DAVIS, Davis, CA

ABSTRACT: The pathways that regulate hepatocyte proliferation

and liver regeneration have been extensively studied within

the liver. However, the signaling contribution specifically from

the gut microbiota involved in liver regeneration is poorly

understood. We have temporally profiled the microbiota, bile

acids (BA), and hepatic transcriptomes in regenerating livers

obtained from mice 0, 1 hour, 1, 2, 3, 5, 7, and 9 days post

2/3 partial hepatectomy (Phx) and established their interactive

relationships. Our data showed that liver resection led

to rapid changes in the gut microbiota that was reflected in

increased abundance of Bacteroidetes including S24-7 and

Rikenellaceae and decreased abundance of Firmicutes including

Clostridiales, Lachnospiraceae, and Ruminococcaceae.

These changes persisted throughout the course of liver regeneration.

Short Time-series Expression Miner analysis of RNA-Sequencing

data of 6,125 genes, which had greater than 2 folds

changes at their mRNA levels compared day 0 controls, generated

six unique patterns: peaked at day 1 (691), 2 (818),

and 3 (275), dipped at day 2 (175), continuously increasing

(340) or decreasing (558) during the course of regeneration.

Correlation analyses revealed close associations between the

expression of genes involved in BA homeostasis, metabolism,

and immune function with the altered abundance of Ruminococcacea,

Lachnospiraceae, and S24-7. Quantification of BA

demonstrated a rapid increase of free and conjugated BAs 1

hour after liver resection due to a sudden loss of liver mass. The

hepatic conjugated BA overload was correlated with increased

abundance of Bacteroidetes, which mediate BA conjugation

by expressing 7α-dehydroxylase. The possibility of shifts in the

microbiota profile as a consequence of changes in BA composition

following liver resection remains to be determined. Conclusion:

This is the first report to show that microbiota profile is

altered during liver regeneration. Moreover, there appears to

be a close relationship between microbiota composition and

hepatic gene expression pattern. The data suggested a significant

role of microbiota in contributing to the liver regeneration.


The following authors have nothing to disclose: Hui-Xin Liu, Clarissa S. Rocha,

Satya Dandekar, Yu-Jui Yvonne Wan


Annexin A6 is necessary for liver regeneration and glucose

homeostasis in mice

Carles Rentero Alfonso 1,2 , Anna Alvarez-Guaita 1 , Stephen E.

Moss 3 , Thomas Grewal 4 , Carlos Enrich 1,2 ; 1 Biologia Cel.lular,

Immunologia i Neurociences, Universitat de Barcelona, Barcelona,

Spain; 2 Centre de Recerca Biomèdica CELLEX, Institut d’Investigacions

Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain;

3 Department of Cell Biology, UCL Institute of Ophthalmology, London,

United Kingdom; 4 Faculty of Pharmacy, University of Sydney,

Sydney, NSW, Australia

Annexin A6 (AnxA6) belongs to a conserved family of Ca 2+ -

and phospholipid-binding proteins which interact with membranes

upon increase of intracellular Ca 2+ concentration. We

have previously shown the relevance of AnxA6 in the regulation

of endocytic and exocytic pathways, intracellular cholesterol

homeostasis, in the EGFR/PKCα/Ras/MAPK signaling

pathway and in membrane remodeling. However, although

AnxA6 is one of the major proteins in the liver (0.25% total

protein), its function in the physiology of this organ remains

unknown. After partial hepatectomy (PHx), an acute proliferative

and metabolic stress, quiescent hepatocytes are triggered

to progress through the cell cycle, showing a synchronous onset

of DNA synthesis with a cellular response that involves cell

activation and tissue remodeling. During this period of liver

regeneration, the liver has to retain the major physiological

tasks such as the synthesis and secretion of plasma proteins,

lipid homeostasis and its metabolic function to ensure viability

of the organism. The aim of the present study was to investigate

the in vivo function of AnxA6 during liver regeneration using

an AnxA6 knock-out (AnxA6 -/- ) mouse model. To this objective,

2/3 PHx was performed in wild-type C57BL/6 control and

AnxA6 -/- mice. Histological, electron microscopy, biochemical

and metabolic studies after PHx were performed. Primary

hepatocytes were also isolated and cultured to confirm the

liver-specificity. After PHx, AnxA6 -/- mice exhibit a dramatic

reduction of survival rate (87.5% at 72h post-PHx). However,

the exogenous administration of glucose before and during

PHx restored AnxA6 -/- survival rate after PHx, suggesting a link

between AnxA6 and hepatic energetic metabolism. A comprehensive

analysis of glucose metabolism experiments pointed to

an impairment of liver gluconeogenesis in AnxA6 -/- mice, also

observed in starving mice. A biochemical approach allowed us

to elucidate a role for AnxA6 in the intracellular trafficking of

SLC38A2, the major liver L-Alanine transporter, which is essential

for gluconeogenic hepatic substrate incorporation during

liver regeneration and mice starvation. We conclude that

AnxA6 is a new regulator of hepatic gluconeogenesis essential

for the trafficking of the alanine transporter SLC38A2 into

the hepatocyte sinusoidal plasma membrane and subsequent

alanine uptake, the major gluconeogenic substrate during both

liver regeneration and fasting.


The following authors have nothing to disclose: Carles Rentero Alfonso, Anna

Alvarez-Guaita, Stephen E. Moss, Thomas Grewal, Carlos Enrich



Akt-mediated FoxO1 inhibition is required for liver

regeneration after partial hepatectomy in mice

Montserrat Pauta 1,3 , Noemi Rotllan 4,5 , Ana Fernandez-Hernando 6 ,

Cedric Langhi 7 , Jordi Ribera 1,2 , Loreto Boix 8,2 , Jordi Bruix 8,2 ,

Wladimiro Jiménez 1,9 , Yajaira Suarez 4,5 , David A. Ford 7 , Angel

Baldan 7 , Morris J. Birnbaum 10 , Manuel Morales-Ruiz 1,2 , Carlos

Fernandez-Hernando 4,5 ; 1 Biochemistry and Molecular Genetics,

Hospital Clinic, Barcelona, Spain; 2 IDIBAPS, CIBERehd, Barcelona,

Spain; 3 IDIBAPS, Barcelona, Spain; 4 Vascular Biology and Therapeutics

Program, Yale University School of Medicine, New Haven,

CT; 5 Integrative Cell Signaling and Neurobiology of Metabolism

Program, Section of Comparative Medicine and Department of

Pathology, Yale University School of Medicine, New Haven, CT;

6 Departments of Medicine and Cell Biology, Leon H. Charney

Division of Cardiology, New York University School of Medicine,

New York, NY; 7 Edward A. Doisy Department of Biochemistry &

Molecular Biology, and Center for Cardiovascular Research, Saint

Louis University, Saint Louis, MO; 8 Barcelona Clinic Liver Cancer

(BCLC) Group, Liver Unit, Hospital Clinic of Barcelona, University

of Barcelona, Barcelona, Spain; 9 Department of Physiological

Sciences I, IDIBAPS, CIBERehd, University of Barcelona School of

Medicine, Barcelona, Spain; 10 Institute for Diabetes, Obesity, and

Metabolism, Perelman School of Medicine, University of Pennsylvania,

Philadelphia, PA

Introduction and aim: Understanding the hepatic regenerative

process has clinical interest since the effectiveness of many

treatments for chronic liver diseases is conditioned by an efficient

liver regeneration. Experimental evidence points to the

need of a temporal coordination between cytokines, growth

factors and metabolic signaling pathways to enable successful

liver regeneration. One intracellular mediator that acts as a

signal integration node for these processes is the serine-threonine

kinase Akt, being Akt1 and Akt2 the most abundant

isoforms expressed in the liver. Therefore, the aim of this study

was to demonstrate whether Akt, globally or through any of

its isoforms, has a significant role in liver regeneration. Methods:

Two-thirds partial hepatectomy (PH) was performed in

eight-old-week male Akt1 -/- , Akt2 -/- , Akt1 loxP/loxP , Akt2 loxP/loxP ,

FoxO1 loxP/loxP and wild-type (WT) mice. In addition, we generated

the Akt1 loxP/loxP ;Akt2 loxP/loxP ;FoxO1 loxP/loxP mice (TLKO)

by crossing the Akt1 loxP/loxP ;Akt2 loxP/loxP mice (DLKO) with the

FoxO1 loxP/loxP mice. The liver specific DLKO and TLKO deficient

mice were generated by treating the floxed mice with adeno-associated

virus encoding for the Cre-recombinase driven

by the Alpha-Fetoprotein promoter. Mice from each group

were sacrificed at various times post-hepatectomy (0 days, 2d,

4d and 7d; n = 5-10) for evaluating biochemical and liver

function parameters. Results: The hepatic deficiency of single

Akt1 or Akt2 does not influence liver regeneration after PH.

However, DLKO mice show impaired liver regeneration and

a significant increase mortality (compared with WT, Akt1 -/-

and Akt2 -/- mice; P10-fold;

p50%) by 15min,

and only approached basal levels by 96h post-PH. TMX-DTG

mice (with reduced Hh signaling) accumulated 50% fewer

Gli2+ cells, and 2-3 fold less Gli1 and Gli2 mRNA (p


approaches that inhibit Hh signaling down-regulate YAP, profibrogenic

YAP targets and aSMA, showing that Hh regulates

YAP and suggesting that YAP may be a downstream effector of

pro-EMT Hh signaling.


The following authors have nothing to disclose: Marzena Swiderska-Syn, Wing-

Kin Syn, Guanhua Xie, Mark Jewell, Richard T. Premont, Gregory A. Michelotti,

Anna Mae Diehl


Region-Based Projections of Deceased Liver Donors from

2015-2025: An Analysis of Proposed UNOS Liver Allocation


Neehar D. Parikh 1 , Wesley J. Marrero Colon 2 , Yongcai Xu 2 , Anna

S. Lok 1 , David W. Hutton 3 , Mariel S. Lavieri 2 ; 1 Division of Gastroenterology,

University of Michigan, Ann Arbor, MI; 2 Industrial and

Operations Engineering, University of Michigan, Ann Arbor, MI;

3 School of Public Health, University of Michigan, Ann Arbor, MI

Background: To help remedy the geographic disparity in liver

transplant (LT) allocation the United Network of Organ Sharing

(UNOS) has recently proposed regional redistricting plans.

While current geographic inequity will improve, it is unclear to

what extent demographic trends in the US will impact redistricting.

We aimed to determine the impact of population demographic

trends on liver redistricting proposals. Method: We

performed a secondary analysis of the Organ Procurement

and Transplantation Network database of all adult donors from

2000 to 2012 and calculated the total number of donors available

and transplanted donor livers stratified by age, race, and

body mass index (BMI) group per year. We used National

Health and Nutrition Examination Survey and Centers for Disease

Control and Prevention historical data to stratify the US

population by age, sex, race, and BMI. We then used US population

age and race projections provided by the US Census

Bureau and the Weldon Cooper Center for Public Service and

made regional projections of available donors from 2015 to

2025, incorporating the proposed 8- and 4-region redistricting

plans proposed by UNOS. Given the uncertainty in LT demand,

we used donors/100,000 population age 18-84 (D/100K) as

a measure of equity. We calculated the mean and variation

between regions, by calculating the percentage difference

between the highest and lowest D/100K per allocation model.

Results: The overall projected D/100K will decrease from 2.72

to 2.65 over the next decade. The projected percentage variation

in 2015 and 2025 in each model is shown in the Table.

In the current 11-region allocation system, the 2015 range in

D/100K is 2.45-2.89, and is projected to be 2.42-2.83 in

2025. In the proposed 8-region allocation system the 2015

range is 2.49-2.89, and is projected to be 2.42-2.83 in 2025.

In the 4-region allocation system, the 2015 range is 2.49-2.79

and is projected to be 2.42-2.71 in 2025. Conclusions: Of

the current UNOS allocation schemes, the 4-region allocation

model reduces the geographic variation to the greatest extent.

This is projected to be maintained as the US population ages

and undergoes demographic shifts over the next decade. However,

this should be balanced by consequences of redistricting

such as increased cold ischemia time and costs associated with

organ transport.


Liver transplantation waiting list mortality in PSC

patients is low as compared to non-PSC patients and

consistent across laboratory MELD and MELD exception

candidates: a nationwide study in the Netherlands

Annemarie C. de Vries 1 , Madelon Tieleman 1 , Bart van Hoek 2 ,

Aad P. van den Berg 3 , Wojciech G. Polak 4 , Jan Ringers 5 , Robert J.

Porte 6 , Cynthia Konijn 7 , Robert A. de Man 1 , Henk R. van Buuren 1 ,

Bettina E. Hansen 1 , Herold J. Metselaar 1 ; 1 Gastroenterology and

Hepatology, Erasmus MC University Medical Center, Rotterdam,

Netherlands; 2 Gastroenterology and Hepatology, Leiden University

Medical Center, Leiden, Netherlands; 3 Gastroenterology and

Hepatology, University Medical Center Groningen, Groningen,

Netherlands; 4 Surgery, Erasmus MC University Medical Center,

Rotterdam, Netherlands; 5 Surgery, Leiden University Medical

Center, Leiden, Netherlands; 6 Surgery, University Medical Center

Groningen, Groningen, Netherlands; 7 Dutch Transplantation Foundation,

Leiden, Netherlands

Background: PSC patients with end-stage disease form a heterogeneous

group due to their varying complications. This

hinders laboratory MELD score prioritization on the liver transplantation

(LTx) waiting list, resulting in both lab MELD (LM)

and MELD exception (ME) PSC candidates. Aim: To assess LTx

waiting list mortality of LM and ME PSC candidates as compared

to non-PSC patients. Methods: Patients aged ≥18 years

who were listed for LTx in the Netherlands after introduction of

the MELD score prioritization to December 31th 2013 were

included. Data were recorded until November 2014. Exclusion

criteria were reLTx, HU status or combined organ transplantation.

A competing risk analysis was performed. Results: During

the study period 852 candidates (M 579/ F 273; median age

54.0 yrs) were listed for LTx. Median lab MELD score at listing

was not significantly different between PSC patients (n=146)

and non-PSC patients (n=706) (13.5 vs. 13.0; p=0.51).

ME points were granted in resp. 22 PSC and 227 non-PSC

patients (HR 0.34; p



Bart van Hoek - Advisory Committees or Review Panels: Janssen-Cilag, Bristol

Meyers Squib, Gilead, Merck, Abbvie

Robert A. de Man - Advisory Committees or Review Panels: Norgine; Grant/

Research Support: Biotest

Henk R. van Buuren - Grant/Research Support: Intercept, Zambon Nederland BV

The following authors have nothing to disclose: Annemarie C. de Vries, Madelon

Tieleman, Aad P. van den Berg, Wojciech G. Polak, Jan Ringers, Robert J. Porte,

Cynthia Konijn, Bettina E. Hansen, Herold J. Metselaar


Liver Transplant Outcomes and Survival Benefit for

Obese Patients in the United States: Are We Disadvantaging

the Morbidly Obese?

Barry Schlansky 1 , Willscott E. Naugler 1 , Susan L. Orloff 2 , C. Kristian

Enestvedt 2 ; 1 Gastroenterology/Hepatology, Oregon Health &

Science University, Portland, OR; 2 Abdominal Organ Transplantation,

Oregon Health & Science University, Portland, OR

Purpose. Over 85% of U.S. centers adhere to national practice

guidelines that consider morbid obesity to be a contraindication

to liver transplantation (LT) based on inferior post-LT

survival compared to the non-obese. In the present era, LT outcomes

and survival benefit for obese patients have not been

well studied. Methods. We evaluated the association of body

mass index (BMI) with wait list and post-LT outcomes in patients

wait listed for LT from 2005 to 2014, using the United Network

for Organ Sharing database. We categorized BMI by the

World Health Organization classification, with 18.5-29.9 kg/

m 2 defining normal weight, 30-34.9 obesity, 35-39.9 severe

obesity, and ≥40 morbid obesity. We followed patients from

LT to death or graft loss, from wait listing to death before LT

or receipt of LT, and from wait listing to death before or after

LT (intention-to-treat, ITT). We used Cox regression to evaluate

post-LT and ITT mortality and LT survival benefit, and competing

risk regression to evaluate wait list mortality versus receipt

of LT. We also explored temporal trends of these outcomes

in an expanded cohort from 2002. Results. 3.9% of 80,221

waitlisted patients and 3.5% of 38,177 transplanted patients

were morbidly obese. ITT survival was lower for morbidly

obese compared to lower BMI patients (log-rank p


sis of complications during the donor hospitalization. Baseline

data (PRE) 2/08-7/10 (29 months) were compared to donors

receiving the pain protocol (POST) 3/13-4/15 (26 months).

An external monitor validated the findings. Each center was IRB

approved. RESULTS: 97 donors received SOC (PRE) and 75

donors received the pain protocol (POST). Complications due

to opioids were nausea (82%), hypoxia



Incidence and Impact of Decompensating Events in Primary

Biliary Cirrhosis - Results of an International Follow

Up Study of 3030 Patients.

Maren H. Harms 1 , Willem J. Lammers 1 , Harry L. Janssen 2 , Christophe

Corpechot 3 , Pietro Invernizzi 4 , Marlyn J. Mayo 5 , Pier Maria

Battezzati 6 , Annarosa Floreani 7 , Albert Pares 8 , Frederik Nevens 9 ,

Andrew Mason 10 , Kris V. Kowdley 11 , Cyriel Y. Ponsioen 12 , Tony

Bruns 13 , George N. Dalekos 14 , Douglas Thorburn 15 , Gideon

Hirschfield 16 , Nicholas F. LaRusso 17 , Ana Lleo 4 , Nora Cazzagon

7 , Irene Franceschet 7 , Llorenc Caballeria 8 , Kalliopi Zachou 14 ,

Raoul Poupon 3 , Angela C. Cheung 2 , Palak J. Trivedi 16 , Marco

Carbone 4 , Keith D. Lindor 18 , Henk R. van Buuren 1 , Bettina E.

Hansen 1 ; 1 Gastroenterology and Hepatology, Erasmus University

Medical Center, Rotterdam, Netherlands; 2 Liver Clinic, Toronto

Western & General Hospital, University Health Network, Toronto,

ON, Canada; 3 Centre de Référence des Maladies Inflammatoires

des VoiesBiliaires, Hôpital Saint-Antoine, Paris, France; 4 Liver Unit

and Center for Autoimmune Liver Diseases, Humanitas Clinical

and Research Center, Rozzano, Italy; 5 Digestive and Liver diseases,

UT Southwestern Medical Center, Dallas, TX; 6 Department

of Health Sciences, Università degli Studi di Milano, Milan, Italy;

7 Department of Surgery, Oncology and Gastroenterology, University

of Padua, Padua, Italy; 8 Liver Unit, Hospital Clínic, CIBERehd,

IDIBAPS, University of Barcelona, Barcelona, Spain; 9 Dept of

Hepatology, University Hospitals Leuven, KULeuven, Leuven, Belgium;

10 Divison of Gastroenterology and Hepatology, University of

Alberta, Edmonton, AB, Canada; 11 Liver Care Network, Swedish

Medical Center, Seattle, WA; 12 Dept of Gastroenterology and

Hepatology, Academic Medical Center, Amsterdam, Netherlands;

13 Department of Gastroenterology and Hepatology, University of

Jena, Jena, Germany; 14 Institute of Internal Medicine and Hepatology,

University of Thessaly, Larissa, Greece; 15 The Sheila Sherlock

Liver Centre, The Royal Free Hospital, London, United Kingdom;

16 NIHR Biomedical Research Unit and Centre for Liver Research,

University of Birmingham, Birmingham, United Kingdom; 17 Dept

Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN;

18 Arizona State University, Phoenix, AZ

Background Clinical events of decompensation are considered

indicators of poor prognosis in primary biliary cirrhosis (PBC).

Few studies have assessed the incidence of decompensating

events and the related outcome of patients. Methods Long-term

follow-up (FU) data of ursodeoxycholic acid (UDCA) treated

patients were derived from 17 North-American and European

centers. Decompensation was defined as a first event of ascites,

variceal bleeding, or encephalopathy, whichever came

first. Patients with events prior to baseline or in first year of FU

were excluded. Risk factor analysis was performed using Cox

proportional hazard models. Results Of 3030 UDCA-treated

PBC patients, 92 patients (3.0%) were excluded. Median FU

was 8.4 yrs (IQR 4.7-12.9). A decompensating event occurred

in 275 patients: ascites:167 (61%), variceal bleeding:76

(27%), encephalopathy:24 (9%), multiple:8 (3%). 1-, 3- and

5- year transplantation-free survival with or without event was

59% vs 99%, 35% vs 97% and 19% vs 94% respectively (time

dependent HR40.6; 95%CI 29.6-55.7). Survival did not significantly

differ between different types of events. Multivariable

analysis showed that at time of first event, the following

factors are predictive of survival: age at time of event (per

10 yrs) (HR 1.39; 95%CI 1.09-1.63), calendar year of event

(HR0.97; 95%CI 0.94-0.99), bilirubin>2x upper limit of normal

(ULN) (HR2.98; 95%CI 1.99-4.45) and normal albumin

(HR1.68; 95%CI 1.12-2.53). Survival of patients with normal

albumin and bilirubin

2xULN (p


LTx. However, prognostic models based on population-based

cohorts are lacking. Various biomarkers of disease progression

have been assessed for PSC, and the potential prognostic

value of serum alkaline phosphatase (ALP) over time has been

discussed. The aim of this study was to create a prognostic

model for PSC consisting of disease characteristics and biochemical

variables, and to validate this model in an external

cohort. Methods 692 PSC patients were identified in a large,

population-based PSC cohort from the Netherlands. Disease

characteristics and biochemical variables during long term

follow-up (FU) were retrieved from patient records. Clinical

endpoints were: development of cholangiocarcinoma, LTx,

or PSC-related death. Biochemical tests were transformed by

log transformation, missing values were imputed by multiple

imputation. All variables were assessed as potential predictors

of survival by univariate analysis. To calculate the prognostic

index (PI), a Cox proportional hazards model was developed

and internally validated with bootstrap. The model was

then validated in an external cohort of 259 PSC patients from

UK. Results The median FU was 85 months (range 0-468

months). All disease characteristics and biochemical variables

were considered for the model. After variable selection by

LASSO, multivariate Cox models were fitted, and parameters

estimated from 20 imputation datasets were averaged. The

following formula was created: PI=1.409*PSC type (0/1) 1

+ 0.021*Age_PSC diagnosis - 2.420*log_AlbuminxULN 2 +

2.073*abs(log_TrombocytesxULN-0.5) 2 + 0.469*log_AspartateAminotransferase(AST)xULN

2 + 0.565*log_ALPxULN 2 +

0.528*log_TotalBilirubinxULN 2 1: PSC type: Large duct=1;

Small duct=0 2: xULN= times upper limit of normal A higher

PI indicated a worse prognosis, corresponding to a shorter

endpoint-free survival. The PI yielded a c-statistic of 0.715 in

the development dataset, 0.705 in internal validation (adjusted

for optimism with 1000 times bootstrap), and 0.683 in the

external validation dataset. Conclusion A novel prognostic PSC

model based on PSC type, age at PSC diagnosis, albumin,

thrombocytes, AST, ALP and bilirubin which shows adequate

performance in internal and external validation cohorts, allows

to determine complication-free survival probability for PSC

patients over time.


Ulrich Beuers - Consulting: Intercept via University of Amsterdam, Novartis via

University of Amsterdam; Grant/Research Support: Falk, Zambon; Speaking and

Teaching: Falk Foundation, Gilead, Roche, Shire

Cyriel Y. Ponsioen - Advisory Committees or Review Panels: Takeda; Consulting:

AbbVIE; Grant/Research Support: AbbVIE, Schering Plough, Dr. Falk Pharma,

Tramedico Netherlands, Takeda

The following authors have nothing to disclose: Elisabeth M. de Vries, Junfeng

Wang, Kate D. Williamson, Mariska M. Leeflang, Kirsten Boonstra, Roger W.

Chapman, Ronald Geskus


Clinical Epidemiology of Primary Biliary Cirrhosis based

on a Large US Laboratory Database: Incidence and

Trends in Serum Alkaline Phosphatase

W. Ray Kim 1 , Tracy J. Mayne 2 , Tonya Marmon 3 , David Shapiro 4 ,

Keith D. Lindor 5 ; 1 Division of Gastroenterology and Hepatology,

Stanford University School of Medicine, Stanford, CA; 2 Outcomes

Research, Intercept Pharmaceuticals, New York, NY; 3 Biostatistics,

Intercept Pharmaceuticals, San diego, CA; 4 Office of the Chief

Medical Officer, Intercept Pharmaceuticals, San Diego, CA; 5 College

of Health Solutions, Arizona State University, Tempe, AZ

Backround/Aims: Large scale epidemiological data on primary

biliary cirrhosis (PBC) are scarce. Using a nationwide database

covering approximately 30% of US adults, we estimate

PBC incidence and describe trends in serum alkaline phosphatase

(ALP) activities following the diagnosis. Methods: Electronic

data from a large commercial laboratory were queried

to extract all AMA and ALP results between Jan 2010 and Dec

2013. Following AASLD guidelines, we defined probable PBC

by positive anti-mitochondrial antibodies (AMA) and elevated

serum ALP. AMA positivity was defined by a titer > 1:20 or M2

antibody > 25.0 U, and elevated ALP (> upper limit of normal

(ULN), 120 U/L) by the peak ALP before and up to 30-days

after the first positive AMA. In subjects with PBC, serum ALP

was followed for 24 months after the first positive AMA (baseline)

and the highest ALP was identified per 6-month interval.

Results: Out of over 576,000 persons receiving AMA testing,

16,492 unique individuals (2.9%) tested positive. In 5,380

(33%), serum ALP was elevated at baseline, while another 727

AMA-positive patients developed elevated ALP subsequent to

a positive AMA result. Thus, a total of 6,107 met the criteria

for probable PBC, giving rise to a crude incidence rate of

5.8 per 100,000 over 4 years. In the figure, of the 5,380

patients, 47% had ALP > x2ULN, including 25% with ALP >

3xULN. Following the diagnosis, the proportion of elevated

ULN decreased, presumably as a result of therapy. By 3-6

months, 61% of patients had ALP < 1.5xULN. Subsequent

trends did not show further improvement in ALP – as of 24

months post diagnosis, 69% had elevated ALP, including 35%

with ALP > 1.5xULN. Conclusion: This largest epidemiological

survey for PBC in US to date suggests that annually more than

5000 Americans meet the diagnosis of probable PBC. Two

years after the diagnosis, more than a third continue to have

significantly elevated ALP.


W. Ray Kim - Advisory Committees or Review Panels: Bristol Myers Squibb,

Gilead Sciences, Abbvie, Merck

Tracy J. Mayne - Employment: Intercept Pharmaceuticals

Tonya Marmon - Employment: Intercept Pharmaceuticals, Inc; Stock Shareholder:

Intercept Pharmaceuticals, Inc

David Shapiro - Employment: Inttercept Pharmaceuticals; Management Position:

Intercept Pharmaceuticals; Stock Shareholder: Intercept Pharmaceuticals

The following authors have nothing to disclose: Keith D. Lindor



Gender and IBD phenotype are independent predictors

of death or transplantation and of malignancy in Primary

Sclerosing Cholangitis – a multicenter retrospective

study of the International PSC Study Group (IPSCSG)

Tobias J. Weismuller 1 , Bettina E. Hansen 2 , Palak J. Trivedi 3 , Mohamad

Imam 6 , Henrike Lenzen 5 , Cyriel Y. Ponsioen 6 , Kristian Holm 7 ,

Ulrich Beuers 6 , Annika Bergquist 8 , Daniel Gotthardt 9 , Hanns-Ulrich

Marschall 10 , Douglas Thorburn 11 , Rinse K. Weersma 12 , Johan

Fevery 13 , Tobias Mueller 14 , Olivier Chazouillères 15 , Christoph

Schramm 16 , Konstantinos Lazaridis 4 , Brian D. Juran 4 , Martti A.

Färkkilä 19 , Stephen P. Pereira 18 , Sven Almer 17,8 , Cynthia Levy 20 ,

Andrew Mason 21 , Christopher L. Bowlus 22 , Annarosa Floreani 23 ,

Emina Halilbasic 24 , Michael Trauner 24 , Kidist K. Yimam 25 , Piotr

Milkiewicz 26,27 , Dep K. Huynh 28 , Albert Pares 29 , Christine N.

Manser 30 , George N. Dalekos 31 , Bertus Eksteen 32 , Gabi I. Kirchner

38 , Christoph Sarrazin 37 , Vincent Zimmer 36 , Luca Fabris 35 ,

Pietro Invernizzi 34 , Felix Braun 33 , Marco Marzioni 39 , Christoph

P. Berg 40 , Ansgar W. Lohse 16 , Gideon Hirschfield 3 , Christian

P. Strassburg 1 , Michael P. Manns 5 , Keith D. Lindor 4 , Tom H.

Karlsen 7 , Kirsten M. Boberg 7 ; 1 Department of Internal Medicine I,

University of Bonn, Bonn, Germany; 2 Department of Gastroenterology

and Hepatology, Erasmus University Medical Center, Rotterdam,

Netherlands; 3 National Institute for Health Research (NIHR)

Birmingham Liver Biomedical Research Unit (BRU) and Centre for

Liver Research, University of Birmingham, Birmingham, United

Kingdom; 4 Department of Gastroenterology and Hepatology,

Mayo Clinic, Rochester, MN; 5 Department of Gastroenterology,

Hepatology and Endocrinology, Hannover Medical School, Hannover,

Germany; 6 Department of Gastroenterology & Hepatology,

Academic Medical Center, Amsterdam, Netherlands; 7 Norwegian

PSC Research Center and Section for Gastroenterology, Department

of Transplantation Medicine, Division of Cancer Medicine,

Surgery and Transplantation, Oslo University Hospital, Rikshospitalet,

Oslo, Norway; 8 Center for Digestive Diseases, Division of

Hepatology, Karolinska University Hospital and Karolinska Institutet,

Stockholm, Sweden; 9 Dept. of Gastroenterology, Infectious

Diseases and Intoxications, University Hospital Heidelberg, Heidelberg,

Germany; 10 Department of Molecular and Clinical Medicine,

Sahlgrenska Academy, University of Gothenburg, Gothenburg,

Sweden; 11 The Sheila Sherlock Liver Centre and UCL Institute for

Liver and Digestive Health, Royal Free Hospital, London, United

Kingdom; 12 Department of Gastroenterology and Hepatology, University

of Groningen and University Medical Center Groningen,

Groningen, Netherlands; 13 Department of Hepatology, University

Hospital Gasthuisberg, Leuven, Belgium; 14 Department of Internal

Medicine, Hepatology and Gastroenterology, Charité Universitätsmedizin,

Berlin, Germany; 15 Service d’Hépatologie, Hôpital Saint

Antoine, Assistance Publique-Hôpitaux de Paris,Faculté de Médecine

Pierre et Marie Curie, Paris, France; 16 1st Department of Medicine,

University Medical Center Hamburg Eppendorf, Hamburg,

Germany; 17 Gastroenterology & Hepatology, Linköping University,

Linköping, Sweden; 18 Institute for Liver and Digestive Health, UCL,

London, United Kingdom; 19 Division of Gastroenterology, Department

of Medicine, Helsinki University Central Hospital, Helsinki,

Finland; 20 University of Miami, Miami, FL; 21 Division of Gastroenterology

and Hepatology, University of Alberta, Edmonton, AB,

Canada; 22 Division of Gastroenterology and Hepatology, University

of California Davis, Davis, CA; 23 Department of Surgery,

Oncology and Gastroenterology, University of Padova, Padova,

Italy; 24 Division of Gastroenterology and Hepatology, Department

of Internal Medicine III, Medical University of Vienna, Vienna,

Austria; 25 Department of Hepatology and Liver Transplantation,

California Pacific Medical Center, San Francisco, CA; 26 Department

of Clinical and Molecular Biochemistry, Pomeranian Medical

University, Szczecin, Poland; 27 Liver and Internal Medicine Unit,

Department of General, Transplant and Liver Surgery, Medical

University of Warsaw, Warsaw, Poland; 28 Department of Gastroenterology

and Hepatology, Royal Adelaide Hospital, Adelaide,

SA, Australia; 29 Liver Unit, Hospital Clinic, IDIBAPS, CIBERehd,

University of Barcelona, Barcelona, Spain; 30 Division for Gastroenterology

and Hepatology, University Hospital Zurich (USZ), Zurich,

Switzerland; 31 Department of Medicine and Research Laboratory

of Internal Medicine, School of Medicine, University of Thessaly,

Larissa, Greece; 32 University of Calgary, Snyder Institute for

Chronic Diseases, Alberta, AB, Canada; 33 Campus Kiel, UKSH,

Kiel, Germany; 34 Humanitas Clinical and Research Center, Center

for Autoimmune Liver Diseases, Rozzano (MI), Italy; 35 Department

of Molecular Medicine, University of Padua School of Medicine,

Padua, Italy; 36 Department of Medicine II, Saarland University

Medical Center, Homburg, Germany; 37 Department of Internal

Medicine 1, Johann Wolfgang Goethe-University Hospital, Frankfurt,

Germany; 38 Department of Internal Medicine 1, University

Hospital of Regensburg, Regensburg, Germany; 39 Department of

Gastroenterology, Università Politecnica delle Marche, Ancona,

Italy; 40 Department of Gastroenterology, Hepatology, and Infectiology,

Medical Clinic, University of Tübingen, Tübingen, Germany

Primary sclerosing cholangitis (PSC) is a male predominant

disease bearing a strong association with inflammatory bowel

disease (IBD). The aims of this study were to address impact

of gender and IBD phenotype on liver-related outcomes across

a large, internationally representative multi-centre registry.

All patients diagnosed with PSC between 1/1/1980 and

12/31/2010 in 37 institutions from 17 countries were included

in the study. Data on clinical presentation, survival, liver transplantation

(LT), IBD, colorectal neoplasia and hepatobiliary

malignancy (HBM) were collected. Cox regression analyses,

stratified by region and adjusted for age, year of diagnosis,

diagnose (PSC/small duct PSC/ with AIH overlap) with IBD

as a time dependent covariate were used to study the effect of

gender on the risk of HBM, colorectal neoplasia and the risk

of LT and death. 7119 patients were included (65.5% male,

mean age at diagnosis 37.4 years in males and 40.4 years

in females (p



Palak J. Trivedi - Grant/Research Support: Wellcome Trust

Cyriel Y. Ponsioen - Advisory Committees or Review Panels: Takeda; Consulting:

AbbVIE; Grant/Research Support: AbbVIE, Schering Plough, Dr. Falk Pharma,

Tramedico Netherlands, Takeda

Ulrich Beuers - Consulting: Intercept via University of Amsterdam, Novartis via

University of Amsterdam; Grant/Research Support: Falk, Zambon; Speaking and

Teaching: Falk Foundation, Gilead, Roche, Shire

Hanns-Ulrich Marschall - Consulting: Albireo

Olivier Chazouillères - Consulting: APTALIS, MAYOLY-SPINDLER

Andrew Mason - Advisory Committees or Review Panels: AbbVie, Novartis,

Intercept; Grant/Research Support: Abbvie, Gilead, Astellas

Christopher L. Bowlus - Advisory Committees or Review Panels: Gilead Sciences,

Inc; Grant/Research Support: Gilead Sciences, Inc, Intercept Pharmaceuticals,

Bristol Meyers Squibb, Takeda, Lumena, Merck; Speaking and Teaching: Gilead

Sciences, Inc

Michael Trauner - Advisory Committees or Review Panels: MSD, Janssen, Gilead,

Abbvie; Consulting: Phenex; Grant/Research Support: Intercept, Falk Pharma,

Albireo; Patent Held/Filed: Med Uni Graz (norUDCA); Speaking and Teaching:

Falk Foundation, Roche, Gilead

Christoph Sarrazin - Advisory Committees or Review Panels: Bristol-Myers

Squibb, Janssen, Merck/MSD, Gilead, Roche, Abbvie, Janssen, Merck/MSD;

Consulting: Merck/MSD, Merck/MSD; Grant/Research Support: Abbott, Roche,

Merck/MSD, Gilead, Janssen, Abbott, Roche, Merck/MSD, Qiagen; Speaking

and Teaching: Gilead, Novartis, Abbott, Roche, Merck/MSD, Janssen, Siemens,

Falk, Abbvie, Bristol-Myers Squibb, Achillion, Abbott, Roche, Merck/MSD, Janssen

Gideon Hirschfield - Advisory Committees or Review Panels: Intercept Pharma;

Consulting: Dignity Sciences, GSK, NGM Bio, Lumena, J & J; Grant/Research

Support: BioTie; Speaking and Teaching: Falk Pharma

Christian P. Strassburg - Advisory Committees or Review Panels: Novartis, Roche;

Speaking and Teaching: Novartis, Merz, MSD, Falk Pharma, BMS, Abbvie

Michael P. Manns - Consulting: Roche, BMS, Gilead, Boehringer Ingelheim,

Novartis, Idenix, Achillion, GSK, Merck/MSD, Janssen, Medgenics; Grant/

Research Support: Merck/MSD, Roche, Gilead, Novartis, Boehringer Ingelheim,

BMS; Speaking and Teaching: Merck/MSD, Roche, BMS, Gilead, Janssen, GSK,


The following authors have nothing to disclose: Tobias J. Weismuller, Bettina

E. Hansen, Mohamad Imam, Henrike Lenzen, Kristian Holm, Annika Bergquist,

Daniel Gotthardt, Douglas Thorburn, Rinse K. Weersma, Johan Fevery,

Tobias Mueller, Christoph Schramm, Konstantinos Lazaridis, Brian D. Juran,

Martti A. Färkkilä, Stephen P. Pereira, Sven Almer, Cynthia Levy, Annarosa

Floreani, Emina Halilbasic, Kidist K. Yimam, Piotr Milkiewicz, Dep K. Huynh,

Albert Pares, Christine N. Manser, George N. Dalekos, Bertus Eksteen, Gabi

I. Kirchner, Vincent Zimmer, Luca Fabris, Pietro Invernizzi, Felix Braun, Marco

Marzioni, Christoph P. Berg, Ansgar W. Lohse, Keith D. Lindor, Tom H. Karlsen,

Kirsten M. Boberg


Ig Repertoire of Autoantigen-Specific B cells in Primary

Biliary Cirrhosis

Weici Zhang 1 , Patrick S. Leung 1 , Ying Sun 1,2 , Thomas P. Kenny 1 ,

Guo-Xiang Yang 1 , Xiao-Song He 1 , Christopher L. Bowlus 3 , Ross

L. Coppel 4 , Ignacio Sanz 6 , Aftab A. Ansari 5 , M. Eric Gershwin 1 ;

1 Internal Medicine, University of California, Davis, CA; 2 Hepatology,

Center for Non-Infectious Liver Diseases, Beijing 302 Hospital,

Beijing, China; 3 Division of Gastroenterology and Hepatology,

University of California at Davis, Sacramento, CA; 4 Department

of Microbiology, Monash University, Melbourne, VIC, Australia;

5 Department of Pathology, Emory University School of Medicine,

Atlanta, GA; 6 Department of Medicine, Division of Rheumatology

and Lowance Center for Human Immunology, Emory University,

Atlanta, GA

Background: Autoantibodies to the E2 subunit of pyruvate

dehydrogenase (PDC-E2) are the serological hallmark of PBC.

Interestingly, a subpopulation of anti-PDC-E2 autoantibodies

are cross-reactive to chemical xenobiotics including 2-octynoic

acid (2OA) and 6, 8-bis (acetylthio) octanoic acid (SAc), both

putative etiological agents of PBC. Recent studies suggest that

anti-PDC-E2 is derived from de novo activated autoantigen-specific

B cells, or from B cells previously primed by xenobiotics

when self-tolerance was originally compromised. Examination

of antigen specific plasmablasts from PBC demonstrate an

extraordinary frequency of circulating plasmablasts specific

for PDC-E2 without significant detection of reactivity to xenobiotic

antigens; this is in striking contrast to comparable sera

analysis. We propose that circulating PDC-E2 plasmablasts

result from positive selection to xenobiotics, i.e. the “original

sin” was the creation of a neoantigen between a chemical

xenobiotic and a self peptide. Our aim herein was to identify

Ig sequences from plasmablasts directed to PDC-E2. Methods:

1,680 single plasmablast cells (CD3 – CD19 + CD20 lo/– CD27 hi C-

D38 hi ) were obtained from PBC with high PDC-E2-specific

plasmablasts (>30%). Thereafter, Ig heavy chain (IgH) and Igk

or Igl light chain (IgL) gene transcripts were amplified using a

IgG, IgA and IgM H and L chain gene specific primer cocktail

and sequenced. A total of 420 functional paired IgH and

Igk or Igl gene sequences were analyzed. CDR3 length and

frequency of positively and negatively charged residues in

various V gene libraries were analyzed. Differential analysis

was performed comparing IgM versus IgG or IgA gene libraries.

Results: Our data revealed a decreased diversity of IgH

and IgL usage with preferential usage of IGHV3-23, IGHV3-

30, IGHV3-7, IGHV4-39, IGHV3-15, IGHV1-18, IGHV3-74

and IGKV3-20, IGKV2-28. Moreover, the usage of IGHV3-

23 and IGHV3-30 increased by two-fold in PBC compared to

controls. Thirty-two clonal V region sequences in the repertoire

were identified and shared among PBC patients. Interestingly,

the unique rearrangements accounted for 0.84% of the total

sequenced IgH rearrangements and the majority of sequences

are highly mutated with an average of 17 replacement mutations

per sequence. Conclusion: We suggest that the original

breach of tolerance was secondary to molecular mimicry in

a genetically susceptible host to a neoantigen that includes a

chemical xenobiotic of PDC-E2; subsequently, through determinant

spreading and autoantibody maturation, the predominant

hallmark of PBC arises, autoantibodies directed at the inner

lipoyl domain of PDC-E2.


Christopher L. Bowlus - Advisory Committees or Review Panels: Gilead Sciences,

Inc; Grant/Research Support: Gilead Sciences, Inc, Intercept Pharmaceuticals,

Bristol Meyers Squibb, Takeda, Lumena, Merck; Speaking and Teaching: Gilead

Sciences, Inc

Ignacio Sanz - Advisory Committees or Review Panels: Pfizer; Consulting: Genentech,

Sanofi; Grant/Research Support: MedImmune

The following authors have nothing to disclose: Weici Zhang, Patrick S. Leung,

Ying Sun, Thomas P. Kenny, Guo-Xiang Yang, Xiao-Song He, Ross L. Coppel,

Aftab A. Ansari, M. Eric Gershwin



Intrahepatic Cholestasis of Pregnancy (ICP) in U.S. Latinas

and Chileans: Ancestry Analysis, Admixture Mapping,

and Biochemical Features

Laura Bull 1,2 , Donglei Hu 2,3 , Sohela Shah 1,2 , Luisa Temple 1,2 ,

Karla Silva 4,5 , Scott Huntsman 2,3 , Jennifer Melgar 1,2 , Mary

Geiser 1,2 , Ukina Sanford 1,2 , Juan Ortiz 5,6 , Richard Lee 7 , Juan P.

Kusanovic 4,5 , Elad Ziv 3,2 , Juan Vargas 8,9 ; 1 Medicine, San Francisco

General Hospital, UCSF, San Francisco, CA; 2 Institute for

Human Genetics, UCSF, San Francisco, CA; 3 Medicine, UCSF,

San Francisco, CA; 4 Center for Research and Innovation in Maternal-Fetal

Medicine, Hospital Dr. Sótero del Río, Puente Alto, Chile;

5 Obstetrics and Gynecology, Hospital Dr. Sótero del Río, Puente

Alto, Chile; 6 Obstetrics and Gynecology, Clínica Santa María,

Santiago, Chile; 7 Obstetrics and Gynecology, Los Angeles County

+ University of Southern California Medical Center, Los Angeles,

CA; 8 Obstetrics, Gynecology and Reproductive Sciences, UCSF,

San Francisco, CA; 9 Radiology, UCSF, San Francisco, CA

Purpose: In the Americas, women with Indigenous American

ancestry are at increased risk of intrahepatic cholestasis of

pregnancy (ICP). We hypothesized that ancestry-related

genetic factors contribute to this increased risk, and that the

statistically powerful genetic admixture mapping approach

could allow mapping of an ICP susceptibility locus. We considered

that diagnosis, features, and management of ICP might

differ between U.S. and Chilean clinical sites. Methods: We

collected patient data and performed biochemical assays and

SNP genotyping on samples from U.S. Latinas and Chilean

women, with and without ICP. The study sample included 198

women with ICP (90 from the U.S. and 108 from Chile) and

174 pregnant control women (69 from the U.S. and 105 from

Chile). We compared overall genetic ancestry between cases

and controls, and performed genome-wide admixture mapping,

taking country of enrollment into account, to screen for

ICP susceptibility loci. We characterized features of ICP at the

U.S. and Chilean clinical sites. Results: Cases had a greater

proportion of Indigenous American ancestry than did controls

(p=0.034); when U.S. and Chilean study samples were analyzed

separately, this difference was apparent in the U.S., but

not the Chilean, study sample, potentially due to differences in

population history. Admixture mapping allowed identification

of one locus for which Native American ancestry was associated

with increased risk of ICP at a genome-wide level of

significance (P = 3.1 x 10 -5 , P corrected

= 0.035). This locus, on

chromosome 2, has an odds ratio of 4.48 (95% CI: 2.21-9.06)

for 2 versus zero Indigenous American chromosomes; the 10

Mb 95% confidence interval does not contain any previously

identified hereditary ‘cholestasis genes.’ ICP appears biochemically

more severe in the U.S., than Chilean, study sample.

Differences in management include 1) ursodeoxycholate was

prescribed to a greater proportion of U.S. than Chilean patients

(48% vs 1%); 2) most U.S. cases were delivered by 37 weeks

gestation (or at time of diagnosis if later), while induction of

labor is not usually performed until 40 weeks at the Chilean

site; and 3) cesarean birth is more common in Chilean cases

than controls, while rates are similar between U.S. cases and

controls. Conclusions: Our results indicate that genetic factors

contribute to risk of developing ICP in the Americas, and support

the utility of clinical and genetic studies of ethnically mixed

populations for increasing our understanding of ICP. Greater

characterization of diagnosis, management and outcomes of

ICP in different countries may improve our understanding of

ICP, and its potential subtypes.


Sohela Shah - Employment: Qiagen Bioinformatics

The following authors have nothing to disclose: Laura Bull, Donglei Hu, Luisa Temple,

Karla Silva, Scott Huntsman, Jennifer Melgar, Mary Geiser, Ukina Sanford,

Juan Ortiz, Richard Lee, Juan P. Kusanovic, Elad Ziv, Juan Vargas


The effects of apoptosis inhibition on pulmonary alveolar

type 2 epithelial cell alterations in experimental

hepatopulmonary syndrome after common bile duct


Wenli Yang, Bingqian Hu, Michael B. Fallon, Junlan Zhang; The

university of Texas Health Science Center, Houston, TX

Introduction: Abnormal oxygenation due to lung ventilation-perfusion

mismatch drives the development of hepatopulmonary

syndrome (HPS). In addition to alterations in microvascular

perfusion, dysfunction in type II alveolar epithelial cells (AT2),

which play a critical role in maintaining alveolar ventilation

through surfactant protein (SPs) production, develops in experimental

HPS. Specifically, AT2 cell apoptosis and decreased

SP production associated with increased circulating bile acid

levels and bile acid nuclear receptor (FXRα) activation, reduce

alveolar airspace and impair ventilation. However, the relative

effects of FXRα activation on AT2 cell apoptosis and SP

expression and whether apoptosis inhibition influences AT2

cells and the development of HPS are unknown. Aim: To evaluate

the effects of apoptosis inhibition on FXRα activation in

AT2 cells and in experimental HPS. Methods: A murine AT2

cell line (MLE-12) was treated with a FXRα agonist (GW4064,

1-10mM) in the presence or absence of a pan caspase inhibitor

(Z-VAD-FMK). SD rats underwent common bile duct ligation

(CBDL) or sham operation and were treated with Z-VAD-FMK

(1.0mg/kg/day, IP, for 3 weeks). Lung and cell apoptosis was

determined by TUNEL staining or cleaved caspase-3 levels.

Co-staining of TUNEL and SP-C was performed to assess lung

AT2 cell apoptosis. SP levels were assessed by westerns or qRT-

PCR. Lung morphology (alveolar mean chord length, Lm) and

gas exchange (PO 2

) were measured to evaluate HPS. Results:

GW4064 treatment in MLE-12 cells caused a dose dependent

increase in cleaved caspase-3 protein expression and reduction

of SP-C and SP-B mRNAs. Z-VAD-FMK pretreatment attenuated

GW4064 induced cleaved caspase-3 protein increases

(76.0±6.1% reduction, p



Statins activate the canonical hedgehog-signaling and

aggravate non-cirrhotic portal hypertension, but inhibit

the non-canonical hedgehog signaling and cirrhotic portal


Frank E. Uschner 1 , Ganesh Ranabhat 1 , Michaela Granzow 1 , Steve

S. Choi 2 , Sabine Klein 1 , Robert Schierwagen 1 , Esther Raskopf 1 ,

Sebastian Gautsch 1 , Peter F. Van der Ven 3 , Christian P. Strassburg

1 , Dieter O. Fürst 3 , Kanishka Hiththetiya 4 , Tilman Sauerbruch 1 ,

Anna Mae Diehl 2 , Jonel Trebicka 1 ; 1 Medical Clinic I, University of

Bonn, Bonn, Germany; 2 Department of Medicine, Duke University,

Durham, NC; 3 Department of Molecular Cell Biology, University of

Bonn, Bonn, Germany; 4 Institute of Pathology, University of Bonn,

Bonn, Germany

Background: Portal hypertension is a source of complications in

patients with liver cirrhosis, as well as in patients with portal vein

obstruction. In both conditions, increased outflow resistance

and hyperperfusion of splanchnic vessels trigger angiogenesis.

Extrahepatic angiogenesis exacerbates hyperperfusion of

the splanchnic vessels and aggravates portal hypertension. In

cirrhosis, intrahepatic angiogenesis is involved in progression

of fibrosis since activated hepatic stellate cells (HSC) produce

collagen and serve as pericytes. HSC activation and activity

are promoted by Hedgehog (Hh) pathway, which further regulates

vascular integrity and angiogenesis in different tissues.

Statins not only blunt liver fibrosis and decreases portal pressure,

but also interacts with the Hh. This study investigated the

differences of angiogenesis in cirrhotic and non-cirrhotic portal

hypertension with special emphasis on the canonical (Shh/Gli)

and non-canonical (Shh/RhoA) pathway. Methods: Cirrhotic

(BCL; CCl 4

) and non-cirrhotic (partial portal vein ligation/

PPVL) rats received either atorvastatin (15mg/kg, 7d) or control

chow. Invasive hemodynamics were assessed by coloured

microspheres. Implantation of matrigel (s.c. and i.p.) assessed

angiogenesis in vivo. In vitro angiogenesis was analyzed in

primary rat HSC and LX-2 cells, after atorvastatin incubation

and transfection with RhoA plasmids. Expression was analyzed

using RT-PCR and Western blot. Results: Atorvastatin decreased

portal pressure, shunt flow and angiogenesis in cirrhotic rats,

whereas atorvastatin increased these parameters in PPVL rats.

Hh was upregulated in experimental and human liver cirrhosis,

but was blunted by atorvastatin. The transfection of LX-2 cells

with a RhoA plasmid increased Hh protein levels and inhibition

of the Hh-pathway reduced profibrotic markers, which clearly

indicate a non-canonical activation of Hh-pathway in HSC.

Moreover, atorvastatin blocked this non-canonical Hh-pathway

RhoA-dependently in activated HSCs. Interestingly, hepatic and

extrahepatic Hh-pathway was enhanced in PPVL rats, which

resulted in increased angiogenesis. Discussion: In summary,

statins caused contrary effects in cirrhotic and non-cirrhotic

portal hypertension. Atorvastatin inhibited the non-canonical

Hh-pathway and angiogenesis in cirrhosis. In portal vein

obstruction, statins enhanced the canonical Hh-pathway and

aggravated PHT and angiogenesis. In conclusion, the broad

use of statins with its beneficial effects in liver cirrhosis still

requires caution in therapy of portal hypertension without cirrhosis.


Christian P. Strassburg - Advisory Committees or Review Panels: Novartis, Roche;

Speaking and Teaching: Novartis, Merz, MSD, Falk Pharma, BMS, Abbvie

The following authors have nothing to disclose: Frank E. Uschner, Ganesh

Ranabhat, Michaela Granzow, Steve S. Choi, Sabine Klein, Robert Schierwagen,

Esther Raskopf, Sebastian Gautsch, Peter F. Van der Ven, Dieter O. Fürst,

Kanishka Hiththetiya, Tilman Sauerbruch, Anna Mae Diehl, Jonel Trebicka


Hepatocyte Tissue Factor Contributes to the Hypercoagulable

State in a Mouse Model of Chronic Liver Injury

Pierre-Emmanuel Rautou 1,2 , Kohei Tatsumi 3 , Silvio Antoniak 3 ,

Albert P. Owens III 3 , Erica Sparkenbaugh 3 , Anna K. Kopec 4 , Rafal

Pawlinski 3 , James P. Luyendyk 4 , Nigel Mackman 3 ; 1 Hepatology,

Hôpital Beaujon - APHP, Clichy, France; 2 Inserm U970; Paris cardiovascular

Research center @ HEGP, Paris, France; 3 Department

of Medicine, Division of Hematology and Oncology, McAllister

Heart Institute, University of North Carolina at Chapel Hill, Chapel

Hill, NC; 4 Department of Pathobiology and Diagnostic Investigation,

Michigan State University, East Lansing, MI

Background & Aims: Patients with chronic liver disease and cirrhosis

have a dysregulated coagulation system and are prone

to thrombosis. The basis for this so-called procoagulant imbalance

is not completely understood. Tissue factor (TF) is the

primary initiator of coagulation in vivo. Patients with cirrhosis

have increased TF activity in white blood cells and circulating

microparticles. The aim of our study was to determine the role

of TF and the contribution of different cellular sources of TF to

the hypercoagulable state in a mouse model of chronic liver

injury. Methods: We measured levels of TF activity in the liver,

white blood cells and microparticles, and a marker of activation

of coagulation [thrombin-antithrombin complexes (TATc)]

in the plasma of mice subjected to bile duct ligation for 12

days. We used wild-type mice, mice with a global TF deficiency

(low TF mice), and mice deficient for TF in either myeloid

cells (TF flox/flox ,LysM Cre mice) or in hepatocytes (TF flox/flox /Albumin

Cre ). Data are presented as median (interquartile range).

Results: Wild-type mice with liver injury had increased levels

of TATc [9.7 (7.6-10.5) vs. 6.0 (5.7-7.0) ng/mL; p



The association of non-selective beta-blocker use and

acute kidney injury in patients with decompensated

cirrhosis admitted into hospital - A Study from the North

American Consortium for the Study of End Stage Liver

Diseae (NACSELD).

Florence Wong 1 , Jacqueline G. O’Leary 2 , K. Rajender Reddy 3 ,

Guadalupe Garcia-Tsao 4 , Scott W. Biggins 5 , Michael B. Fallon 6 ,

Puneeta Tandon 7 , Ram M. Subramanian 8 , Benedict Maliakkal 9 ,

Paul J. Thuluvath 10 , Hugo E. Vargas 11 , Patrick S. Kamath 12 , Leroy

Thacker 13 , Jasmohan S. Bajaj 14,15 ; 1 Medicine, University of

Toronto, Toronto, ON, Canada; 2 Medicine, Hepatology, Baylor

University Medical Center, Dallas, TX; 3 Gastroenterology & Hepatology,

University of Pennsylvania, Philadelphia, PA; 4 Digestive

Diseases, Yale University School of Medicine, New Haven, CT;

5 Gastroenterology, University of Colorado, Denver, CO; 6 Gastroenterology,

Hepatology & Nutrition, University of Texas Health Science

Center, Houston, TX; 7 Medicine, Gastroenterology, University

of Alberta, Edmonton, AB, Canada; 8 Gastroenterology, Emory

University Medical Centre, Atlanta, GA; 9 Gastroenterology, University

of Rochester, Rochester, NY; 10 Institute of Digestive Health

and Liver Disease, Mercy Hospital, Baltimore, MD; 11 Gastroenterology

& Hepatology, Mayo Clinic, Scottsdale, AZ; 12 Internal

Medicine, Gastroenterology & Hepatology, Mayo Clinic, Rochester,

MN; 13 Biostatistics, Virginia Commonwealth University, Richmond,

VA; 14 Gastroenterology, Hepatology & Nutrition, Virginia

Commonwealth University, Richmond, VA; 15 Gastroenterology,

McQuire VA Medical Center, Richmond, VA

Non-selective beta-blocker (NSBB) use may be detrimental

in patients with cirrhosis & refractory ascites, although this

remains controversial. Theoretically, NSBB use in decompensated

cirrhosis (DC) may decrease renal perfusion and predispose

patients to renal impairment. Aim: To determine the

impact of NSBB use on the risk i) for acute kidney injury (AKI)

in hospitalized patients with DC, and ii) of infection to potentially

accentuate this risk. Methods: NACSELD is a 16-center

consortium that prospectively enrols non-elective cirrhotic inpatients.

The presence of AKI (defined by Angeli et al, Gut 2015)

at admission or its development during admission was evaluated

and compared between those taking NSBB (NSBB+)

vs those not (NSBB-) at admission. Further comparison was

made between NSBB+ patients ± an infection during admission.

Results: 981 cirrhotics (57±10 years, men: 64.6%,

MELD: 19.5±7.3) with mostly alcoholic (43%) and hepatitis

C (22.4%) cirrhosis were enrolled. Significantly more NSBB+

patients had a history of variceal bleeding and diabetes



of AKI. Peak AKIN stage reached was stage III:II:I in 80(66%):

31(25%):11(9%) patients, respectively. Presence of any E-OF

was strongly associated with both development of new AKI

(p=0.004, OR 6.7, 95% CI 1.86-24.1) and progression of

AKI (p=0.04, OR 2.28, 95%CI 1.01-5.1). This risk further

increased with increase in the number of E-OFs (p=0.001, OR

1.41, 95%CI 1.2-1.7). Further, AKI at admission alone was not

associated with mortality, but predicted mortality together with

any E-OF (p150000) and liver stiffness

(LS) ( 60% and an IQR/

Median < 30% were considered. SS was US guided. Results:

99 patients were included: 63% women; age 59 (± 6.6) years;

BMI 28 (± 4.8); MELD 9.7 (± 3.4); 80% Child-Pugh A. LS was

invalid in 2 patients and SS in 19. Platelets was 136.000 (±

54.000), LS was 24 kPa (± 12) and SS was 53 kPa (± 18).

Esophageal varices were detected in 54% of patients, 40%

small and 14% large size. Gastric varices were present in 5%

of patients, and GEV in 55%. Platelets (120.000 ± 45.000

vs 155.000 ± 57.000), LS (28.4 ± 13.1 vs 18.4 ± 7.6 kPa)

and SS (59.5 ± 17.0 vs 43.3 ± 17.9 kPA) were significantly

different among patients with or without GEV (p

6 months, n=83); and G4, past HCV infection (positive anti-

HCV and negative HCV RNA PCR, n=15). The serum samples

were tested using our HCV-Ags EIA before and after denaturation.

RESULTS. All 38 patients in G1 tested negative for

HCV-Ags, indicating 100% specificity of our HCV-Ags EIA. All

85 patients with acute (G2, n=2) or chronic (G3, n=83) HCV

infection (genotypes 1-6), and serum HCV RNA ranged from

1,124 to 14,400,000 IU/mL quantified using PCR, tested positive

for HCV-Ags with or without serum sample denaturation,

indicating 100% sensitivity of the HCV-Ags EIA. The mean

coefficient of variation (CVs) was 9.8% and 6.4%, respectively,

for intra- precision assay and inter-assay reproducibility.

In 2 cases with acute HCV infection, HCV-Ags were detectable

59-65 days before anti-HCV test became positive. To further

assess sensitivity, serum samples with low HCV RNA levels

(214-1160 IU/mL) were serially diluted and tested. The lower

limits of detection of the HCV-Ags assay using denatured or

undenatured sera were equivalent to serum HCV RNA levels

of 150 IU/mL and 250 IU/mL, respectively. Using denatured

serum samples, 6/15 patients in G4 tested positive; however,

none tested positive for HCV-Ags using undenatured serum

samples. Additional studies showed that denaturation of these

serum samples released HCV-Ags sequestered in HCV-ICs in

these patients that may result in false positivity for active HCV

infection. CONCLUSIONS. The highly specific and sensitive

HCV-Ags EIA has a lower limit of detection equivalent to serum


HCV RNA levels of 150-250 IU/mL. Testing undenatured, but

not denatured serum samples reliably differentiated active from

past HCV infection and accomplished screening and diagnosis

of HCV infection in one step.


The following authors have nothing to disclose: Ke-Qin Hu, Wei Cui


Hepatitis C Virus (HCV) Mortality Patterns in the British

Columbia Hepatitis Testers Cohort (BC-HTC)

Mel Krajden 1,2 , Amanda Yu 1 , Darrel Cook 1 , Margot E. Kuo 1 ,

Maria Alvarez 1 , Mark Tyndall 3,2 , Naveed Z. Janjua 1,2 ; 1 Clinical

Prevention Services, BC Centre for Disease Control, Vancouver,

BC, Canada; 2 University of British Columbia, Vancouver, BC, Canada;

3 BC Centre for Disease Control, Vancouver, BC, Canada

Objective: To describe the population-level impacts of HCV

acquisition risks vs. chronic infection on mortality for HCV

negative and positive testers. Methods: The British Columbia

Hepatitis Testers Cohort (BC-HTC) includes 1,135,947 individuals

tested for HCV or reported to public health as a HCV

case from 1990-2013, linked to their corresponding healthcare

administrative data. Cohort entry was delayed one year

for each individual and deaths within one year of HCV diagnosis

were excluded, i.e., one year lagging. We computed

age-adjusted annual all-cause, liver- and drug-related mortality

rates/100,000 population and age-, sex- and year-adjusted

standardized mortality ratios (SMRs) for: 1) seroconverters,

those with known HCV acquisition timeframes and risk activities;

2) anti-HCV positive on initial testing, the majority of whom

were chronically infected and most of whom no longer engage

in acquisition risk activities; and 3) HCV negatives. Results:

Of 1,028,227 individuals included in this analysis, 64,876

(6.3%) were HCV positive. Overall, 16.3% (10,572/64,876)

of HCV positive vs. 6.4% (61,623/963,351) of HCV negative

individuals died. For HCV positives, age-adjusted allcause

mortality increased from 0.11/100,000 in 1993 to

15.0 in 2005 and stayed in this range thereafter. Liver-related

mortality increased from 0.03/100,000 in 1993 to 2.5 in

2005 to 4.7 in 2013. Drug-related mortality increased from

0.03/100,000 in 1993, peaked at 3.2 in 2005 and declined

to 1.3 by 2013. All-cause, liver- and drug-related mortality

rates were consistently higher for males vs. females. All-cause

mortality for seroconverters was significantly higher than those

with chronic HCV and HCV negatives (SMRs: 4.8, 3.1 and

1.0). Liver-related mortality was significantly lower for seroconverters

than for chronic HCV and was lowest for HCV negatives

(SMRs: 10.4, 17.0 and 1.4). Similarly, liver cancer mortality

was significantly lower for seroconverters than for chronic HCV

and was lowest for HCV negatives (SMRs: 3.8, 17.5 and 1.3).

However, drug-related mortality was highest for seroconverters

compared to chronic HCV and HCV negatives (SMRs: 17.0,

11.4 and 0.9). Conclusions: The BC-HTC enables comprehensive

assessment of the mortality impacts of HCV infection and

creates an opportunity to differentiate acquisition risk mortality

from the impacts of chronic infection. These data illustrate that

the excess mortality related to HCV acquisition risks is distinct

from viral sequelae for a significant proportion of the HCV

infected population. Therefore antiviral treatment on its own

will not optimize mortality reductions in the affected populations.


Mel Krajden - Grant/Research Support: Roche, Merck, Siemens, Boerhinger

Ingelheim, Hologic

The following authors have nothing to disclose: Amanda Yu, Darrel Cook, Margot

E. Kuo, Maria Alvarez, Mark Tyndall, Naveed Z. Janjua


The Association of Sustained Virological Response and

All-cause Mortality After Interferon-based Therapy for

Chronic Hepatitis C (HCV) in a Large U.S. Community-based

Health Care Delivery System

Lisa M. Nyberg 1 , Xia Li 1 , Su-Jau Yang 1 , Kevin Chiang 1 , T. Craig

Cheetham 1 , Susan Caparosa 1 , Jose R. Pio 1 , Zobair M. Younossi 2 ,

Anders H. Nyberg 1 ; 1 Kaiser Permanente, San Diego, CA; 2 Department

of Medicine, Inova Fairfax Medical Campus, Fairfax, VA

Background: Previous studies performed at tertiary care centers

have shown that sustained virological response (SVR) to interferon-based

therapy for HCV is associated with a reduction

of liver-related and all-cause mortality (1-2). The aim of this

study was to determine if SVR to interferon-based therapy in

a community-based health care setting is associated with a

reduction in all-cause mortality and to examine for differences

in those with and without cirrhosis. Methods: A retrospective

cohort study at Kaiser Permanente, Southern California (KPSC),

a large community-based integrated health care system including

3.5 million members. Inclusion criteria: a diagnosis code

and/or positive lab test for HCV RNA (index date) 1/1/02-

12/31/13; age ≥ 18 years at index date, ≥ 12 months continuous

membership before and after index date. Exclusion

criteria: HCV diagnosis after 1/1/13 and liver transplant or

HCC on or before index date. SVR was determined for patients

treated for HCV with interferon-based therapy and stratified for

cirrhosis vs non-cirrhosis. Mortality data were obtained from the

California Department of Public Health Vital Statistics of California

Report. Results: Total study cohort: 24,968 with chronic

HCV; of those 10,449 (42%) had cirrhosis. Overall mortality

during the study period was 18.5% (4,608/24,968). Mortality

among those with cirrhosis was 33.2% (3,470/10,449) vs

7.8% (1,138/14,519) among those without cirrhosis. Among

patients treated for HCV, 45.1% (2348/5203) achieved SVR;

mortality for those who achieved SVR was 5.4% (127/2348)

vs 18.9% (540/2855) for those without SVR. Mortality in

treated patients, with and without cirrhosis, is shown in Table

1. Conclusions: An approximate 3-fold reduction in all-cause

mortality is seen in patients with HCV who are treated and

achieve SVR compared to those without SVR. Cirrhotics who

are treated and achieve SVR have a > 3-fold reduction in mortality

compared to the mortality in treated cirrhotics who do not

achieve SVR. New, more potent, HCV treatment regimens with

higher SVR have the potential of significantly reducing future

mortality due to HCV. Further study is ongoing with matching

for severity of liver disease to more specifically examine the

effect of SVR on mortality compared to that due to the natural

history of HCV/cirrhosis. 1. van der Meer, et al. JAMA 2012

Dec26;308(24):2584-93. 2. Berenguer, et al. Hepatology

2009 Aug;50(2):407-13

Mortality in HCV patients treated with interferon-based therapy.

Cirrhosis: N=2603; No Cirrhosis: N=2600


Lisa M. Nyberg - Grant/Research Support: Merck, Gilead, Abbvie

T. Craig Cheetham - Grant/Research Support: Gilead, BMS

Zobair M. Younossi - Advisory Committees or Review Panels: Salix, Janssen,

Vertex; Consulting: Gilead, Enterome, Coneatus

The following authors have nothing to disclose: Xia Li, Su-Jau Yang, Kevin Chiang,

Susan Caparosa, Jose R. Pio, Anders H. Nyberg



Increasing Prevalence of Cirrhosis among US Adults

with Chronic Hepatitis C Virus Infection: Results from

NHANES 1988-1994 and 1999-2012

Prowpanga Udompap, Ajitha Mannalithara, Nae-Yun Heo, Donghee

Kim, W. Ray Kim; Division of Gastroenterology and Hepatology,

Stanford University School of Medicine, Stanford, CA

Background & Aims: Hepatitis C virus(HCV) is a major cause

of end stage liver disease and hepatocellular carcinoma worldwide.

While cirrhosis underlies these complications, the prevalence

of HCV cirrhosis in the US remains unknown. We aim

to determine the prevalence of advanced fibrosis/cirrhosis and

to identify factors associated with advanced fibrosis/cirrhosis

in people with chronic hepatitis C in the US population. Methods:

Using the National Health and Nutrition Examination Survey(NHANES)

data, we identified participants with detectable

serum HCV RNA and assessed liver fibrosis using validated

surrogate indicators, including APRI and FIB-4 scores. The prevalence

of cirrhosis was determined for survey participants for

Era 1 (1988-94), Era 2 (1999-2006) and Era 3 (2007-12).

Results: Out of 52,644 participants with age ≥20, 736 (1.4%)

had HCV. Based on APRI score, 6.6% (95%CI:2.2-11.0) of

US adults with HCV infection in Era 1, 7.6% (95%CI:3.4-

11.8) in Era 2 and 17.0% (95%CI:8.0-26.0) in Era 3 were

estimated to have cirrhosis (Ishak stage 5-6). The prevalence

of advanced fibrosis (Ishak stage 4-6) based on FIB-4 was

8.6%, 10.1%, and 16.0% for Eras 1, 2, and 3, respectively.

These data project to 370,500 Americans with cirrhosis and

347,800 with advanced fibrosis in the most recent era. The

higher prevalence of cirrhosis (determined by APRI) in the

recent era was associated with increasing age (OR=1.04,

95%CI:1.02-1.07), diabetes (OR=2.33, 95%CI:1.01-5.40)

and obesity (OR=2.96, 95%CI:1.15-7.57). When FIB-4 score

was used, advanced fibrosis was associated with increasing

age (OR=1.08, 95%CI:1.05-1.11) and diabetes (OR=3.37,

95%CI:1.24-9.5). Conclusion: Nearly one in five US adults

with HCV infection have cirrhosis. The prevalence of cirrhosis

is rising over time, which is in part attributable to the increasing

age of the birth cohort with HCV infection. In addition, comorbidities

such as diabetes and obesity accelerate liver fibrosis.

These data further underscore the current recommendations

for HCV screening in asymptomatic individuals and highlights

the need for systematic assessment for liver fibrosis and comprehensive

medical management in those with HCV infection.

Characteristics of participants


Rectal Shedding of HCV in HCV/HIV Co-infected Men

Andrew L. Foster 1,2 , Michael Gaisa 1 , Rosanne M. Hijdra 1,3 , Karen

B. Jacobson 1 , Samuel Turner 1,2 , Tristan Morey 1,2 , Daniel S. Fierer 1 ;

1 Infectious Diseases, Mount Sinai School of Medicine, New York,

NY; 2 James Cook University, Cairns, QLD, Australia; 3 Amsterdam

Medical Center, Amsterdam, Netherlands

Introduction An epidemic of hepatitis C virus (HCV) infection

is occurring among HIV-infected men who have sex with men

(MSM). Epidemiological studies suggest that sexual transmission

is fueling the epidemic. Blood and semen have been considered

as potential mechanisms of transmission, but there are

still transmission circumstances that remain unexplained. We

hypothesized that HCV may be shed into rectal fluid of HCV/

HIV co-infected MSM. Methods Written informed consent was

obtained from 45 HIV-infected MSM with HCV infection and

paired blood and rectal fluid specimens were obtained. Rectal

fluid was collected using a moistened polyester-tipped swab

that was gently inserted into the rectum. The swab was placed

into transport medium, vortexed, and the supernatant analysed

for HCV using COBAS AMPLICOR (Roche Diagnostics), lower

limit of quantification 43 IU/mL, lower limit of detection 7 IU/

mL. The experimentally-determined efficiency of HCV absorption

to and elution from the swabs was used to calculate the

rectal HCV viral load (VL). Rectal sexually-transmitted infection

(STI) and blood syphilis testing were also performed. Results

Visual inspection of the swabs showed no visible blood on

any. HCV was detected in 20 (47%) of 43 specimens. Among

those samples with HCV detected, the median rectal VL was

2.92 log 10

IU/mL (IQR 2.92, 4.27). Rectal HCV detection was

associated with serum HCV VL >5 log 10

IU/mL (p=0.011),

and there was a high correlation between the magnitude of

blood VL and rectal HCV VL (correlation coefficient 0.688,



patients in care with hepatitis C virus infection (HCV) will

achieve this status in the near future. However, the long term

prognosis in terms of risk or predictors of developing hepatocellular

carcinoma (HCC) among patients with SVR remains

unclear. Methods: A retrospective cohort study using data from

the national VA HCV Clinical Case Registry in patients with

HCV diagnosed (positive HCV RNA) between 10/1999 and

1/2009 who had at-least one year of follow up in the VA.

HCV treatment (pegylated interferon with or without ribavirin)

and SVR status (RNA test negative at least 12 weeks after

the end of treatment) were ascertained. Cox proportional hazards

models were used to examine the effect of demographic,

virological and clinical factors on time to HCC among those

who achieved SVR through end of 2011 while adjusting for

medical comorbidity. We excluded those with HCC diagnostic

codes before SVR date. Results: We had 33,005 HCV-infected

individuals who received HCV treatment during the study timeframe.

Of these, 10,817 patients achieved SVR and 10765

had no HCC before SVR. Their mean age was 53.1 (sd, 6.3),

12.4% were 60 years or older, and 12.9% were Black, 64.4%

non-Hispanic white, and 3.4% Hispanic. Majority (95.3%)

were males; and 53.6% had genotype 1, 25.0% genotype

2, 13.5% genotype 3, 0.7% genotype 4, and 7.2% had no

genotype test available. A total of 124 patients developed

HCC during a total follow of 38,394 person year follow up

(median duration of follow up after SVR was 2.7 years) at

an incidence rate of 0.32% per year. In the cohort with SVR,

patients 60 years and older (HR=6.59 c/w younger age),

Hispanics (HR=2.16, 1.03-4.50 c/w non-Hispanic whites) and

those with cirrhosis (HR=5.58, 3.21 - 9.68) or diabetes (1.82

(0.92 - 3.61) were at an increased risk of developing HCC

compared to their counterparts. Conclusions: The risk of HCC

after HCV cure remains elevated (0.3% per year). Older age

at the time of SVR, Hispanic ethnicity, and presence of cirrhosis

and diabetes are associated with an increased HCC risk

among those who achieve SVR.


Hashem B. El-Serag - Grant/Research Support: WAKO, Gilead

The following authors have nothing to disclose: Peter Richardson, Fasiha Kanwal


Prevalence of Pre-Treatment NS5A Resistance Associated

Variants in Genotype 1 Patients Across Different

Regions Using Deep Sequencing and Effect on Treatment

Outcome with LDV/SOF

Stefan Zeuzem 2 , Masashi Mizokami 3 , Stephen Pianko 4 , Alessandra

Mangia 5 , Kwang-Hyub Han 6 , Ross Martin 1 , Evguenia

S. Svarovskaia 1 , Hadas Dvory-Sobol 1 , Brian Doehle 1 , Phillip S.

Pang 1 , Steven J. Knox 1 , John G. McHutchison 1 , Diana M. Brainard

1 , Michael D. Miller 1 , Hongmei Mo 1 , Wan-Long Chuang 7 ,

Ira M. Jacobson 8 , Gregory Dore 9 , Mark S. Sulkowski 10 ; 1 Gilead

Sciences Inc, Foster City, CA; 2 Department of Internal Medicine,

J.W. Goethe University Hospital, Frankfurt am Main, Germany;

3 National Center for Global Health and Medicine, Tokyo, Japan;

4 Department of Gastroenterology, Monash Medical Centre, Victoria,

VIC, Australia; 5 Liver Unit, IRCCS-Ospedale Casa Sollievo

della Sofferenza, San Giovanni Rotondo, Italy; 6 Department of

Internal Medicine, Yonsei University College of Medicine, Seoul,

Korea (the Republic of); 7 Internal Medicine, Kaohsiung Medical

University Hospital, Kaohsiung, Taiwan; 8 Medicine, Mt. Sinai Beth

Israel, New York, NY; 9 Kirby Institute, UNSW Australia, Sydney,

NSW, Australia; 10 School of Medicine, Johns Hopkins University,

Baltimore, MD

Background: The efficacy of some HCV regimens is influenced

by the presence of certain NS5A resistance associated variants

(RAVs). To investigate if the pretreatment prevalence of

NS5A RAVs in genotypes (GT) 1a and 1b varied by country,

race or ethnicity, comprehensive analyses were performed

using deep sequencing of NS5A from more than 5000 patients

from 17 countries. Methods: NS5A deep sequencing analysis

with a 1% cut-off was performed. GT1a RAVs were defined

as K24G/N/R, M28A/G/T/V, Q30H/G/R/E/K, L31any,

P32L, S38F, H58D, A92K/T, Y93any, and GT1b RAVs were

L31any, P32L, P58D, A92K, Y93any. Results: Pretreatment

samples from 5046 patients were analyzed. Prevalence of

NS5A RAVs was similar between the different regions for both

GT1a and GT1b HCV infected patients (Table). In addition, no

significant differences were observed in NS5A RAV prevalence

between patients with different race or ethnicity. In the subset of

patients who were treated with LDV/SOF for 12 weeks, SVR12

rates were similar in GT1b patients with and without pretreatment

NS5A RAVs across all regions. In GT1a patients with

pretreatment RAVs, SVR12 rates in North America were 91%

(75/82) compared to 98% without NS5A RAVs. All seven

GT1a patients who relapsed had pretreatment NS5A RAVs

conferring >1000-fold reduced susceptibility to LDV (H58D,

Y93H/N/F or multiple RAV combinations). However, 18 GT1a

patients with similar RAVs conferring >1000-fold reduced

susceptibility achieved SVR12 after LDV/SOF for 12 weeks.

Conclusions: No significant difference in prevalence of NS5A

pretreatment RAVs was observed between different regions,

races or ethnicities. Overall, high SVR12 rates (91-100%) were

observed in patients with or without NS5A RAVs with LDV/

SOF. In GT1a patients, lower SVR rate (72%) was observed

in patients with pretreatment NS5A RAVs conferring high level

(>1000-fold) resistance to NS5A inhibitors.

NA: Too few patients treated with LDV/SOF


Stefan Zeuzem - Consulting: Abbvie, Bristol-Myers Squibb Co., Gilead, Merck

& Co., Janssen

Stephen Pianko - Advisory Committees or Review Panels: Roche, Novartis, GIL-

EAD, Roche, Novartis; Consulting: GILEAD; Speaking and Teaching: JANSSEN

Alessandra Mangia - Advisory Committees or Review Panels: ROCHE, Janssen,

MSD, ROCHE, Janssen, MSD, Boheringer ; Consulting: Gilead; Grant/Research

Support: Shering-Plough, Shering-Plough

Ross Martin - Employment: Gilead Sciences

Evguenia S. Svarovskaia - Employment: Gilead Sciences Inc; Stock Shareholder:

Gilead Sciences Inc

Hadas Dvory-Sobol - Employment: Gilead Sciences; Stock Shareholder: Gilead


Brian Doehle - Employment: Gilead Sciences

Phillip S. Pang - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

Steven J. Knox - Employment: Gilead Sciences

John G. McHutchison - Employment: Gilead Sciences; Stock Shareholder: Gilead


Diana M. Brainard - Employment: Gilead Sciences; Stock Shareholder: Gilead


Michael D. Miller - Employment: Gilead Sciences, Inc.; Stock Shareholder: Gilead

Sciences, Inc.

Hongmei Mo - Employment: Gilead Science Inc


Wan-Long Chuang - Advisory Committees or Review Panels: Gilead, Abbvie;

Speaking and Teaching: BMS, Roche, MSD

Ira M. Jacobson - Consulting: AbbVie, Achillion, Alnylam, Bristol Myers Squibb,

Enanta, Gilead, Janssen, Merck; Grant/Research Support: AbbVie, Bristol Myers

Squibb, Gilead, Janssen, Merck, Tobira; Speaking and Teaching: AbbVie, Bristol

Myers Squibb, Gilead, Janssen

Gregory Dore - Board Membership: Gilead, Merck, Abbvie, Bristol-Myers

Squibb; Grant/Research Support: Gilead, Merck, Abbvie, Bristol-Myers Squibb;

Speaking and Teaching: Gilead, Merck, Abbvie, Bristol-Myers Squibb

Mark S. Sulkowski - Advisory Committees or Review Panels: Merck, AbbVie,

Janssen, Gilead, BMS; Grant/Research Support: Merck, AbbVie, Janssen, Gilead,


The following authors have nothing to disclose: Masashi Mizokami, Kwang-Hyub



Highly Successful Retreatment with Ledipasvir (LDV) and

Sofosbuvir (SOF) in HCV GT-1 Patients Who Failed Initial

Short Course Therapy with Combination DAA Regimens


Eleanor Wilson 1,2 , Sarah Kattakuzhy 1 , Zayani Sims 2 , Mary

McLaughlin 3 , Angie Price 1 , Hongmei Mo 4 , Anu Osinusi 4 , Henry

Masur 2 , Anita Kohli 5 , Shyam Kottilil 1 ; 1 Infectious Disease, Institute

for Human Virology/University of Maryland School of Medicine,

Rockville, MD; 2 Critical Care Medicine Department, National Institutes

of Health, Bethesda, MD; 3 National Institute of Allergy and

Infectious Diseases, National Institutes of Health, Bethesda, MD;

4 Gilead Sciences, Foster City, CA; 5 Hepatology, St. Joseph’s Hospital

and Medical Center, Phoenix, AZ

Introduction: Directly-acting antivirals (DAAs) dramatically

improved HCV therapy but retreatment options for patients

who have failed therapy with combination DAAs have not

been studied. Our study aim is to determine if HCV genotype-1

patients who failed short course combination therapy with 3

or 4 DAAs can be successfully treated with 12 weeks of LDV/

SOF. Material and Methods: In this single-center, open-label,

phase 2a trial, HCV mono-infected persons with early stage

(F0-F2) liver fibrosis and previous exposure to combination

DAA therapy only (LDV/SOF with GS-9451 +/- GS-9669)

were eligible to enroll and receive 12 weeks of LDV/SOF.

HCV RNA was measured with the Abbott assay, lower level

of quantitation (LLOQ) 12 IU/ml. The primary endpoint was

defined as HCV viral load (VL) < LLOQ 12 weeks after end

of therapy (SVR12). Deep sequencing of NS5B and NS5A

regions was performed at baseline by Illumina next generation

sequencing technology. Results: The study enrolled 34 persons,

and 32 (94%) completed therapy with 12 weeks of LDV/

SOF. Two patients withdrew consent after Day 0. Participants

were predominantly male (82%) and black (85%), median

age 60.5 years (IQR 57.0 – 63.8) and BMI 26.8 kg/m 2 (IQR

25.3 – 29.3). Baseline HCV VL was 1.3 x 10^6 IU/mL (IQR

5.8x10^5 – 3.9x10^6), 73.5% (25/34) were infected with

HCV genotype 1a, and median Metavir fibrosis stage was

1. Time from relapse to retreatment was 22 weeks (IQR 18 –

23). SVR12 rates were 91% (31/34; ITT) and 97% (31/32,

per protocol). For SVR rates by original treatment group (per

protocol), see Figure. At baseline, 28/33 patients (85%) had

resistance-associated variants (RAVs) consistent with >25 fold

resistance in NS5A. Of all patients completing therapy, 1

patient with NS5A RAVs relapsed. Conclusions: For the first

time, we demonstrate a high SVR rate following retreatment

with DAAs in patients who have previously failed DAA-only



Hongmei Mo - Employment: Gilead Science Inc

Anu Osinusi - Employment: gilead sciences

The following authors have nothing to disclose: Eleanor Wilson, Sarah Kattakuzhy,

Zayani Sims, Mary McLaughlin, Angie Price, Henry Masur, Anita Kohli,

Shyam Kottilil


Effectiveness of Ledipasvir/Sofosbuvir in Treatment

Naïve Genotype 1 Patients Treated in Routine Medical


Lisa I. Backus 1,2 , Pamela S. Belperio 1 , Troy Shahoumian 1 , Timothy

P. Loomis 1 , Larry A. Mole 2 ; 1 Office of Public Health/Population

Health, Veterans Affairs Palo Alto Health Care System, Palo Alto,

CA; 2 Department of Medicine, VA Palo Alto Healthcare System,

Palo Alto, CA

Aim: Assess the effectiveness of ledipasvir/sofosbuvir±ribavirin

(LDV/SOF±RBV) in treatment naïve genotype 1 (GT1) hepatitis

C virus (HCV)-infected veterans treated in routine medical practice.

Methods: This observational, intent-to-treat cohort analysis

used the Veterans Affairs’ Clinical Case Registry to identify all

treatment naïve GT1 HCV-infected veterans initiating 8 or 12

weeks of LDV/SOF±RBV by 31 December 2014. Patients were

excluded for liver transplantation or baseline HCV RNA


ics with baseline HCV RNA



Improving liver function and delisting of patients awaiting

liver transplantation for HCV cirrhosis: do we ask

too much to DAA?

Audrey Coilly 1,2 , Georges-Philippe Pageaux 22 , Pauline Houssel-Debry

7 , Christophe Duvoux 3 , Sylvie Radenne 6 , Victor de

Ledinghen 15 , Danielle Botta-Fridlund 11 , Anaïs Vallet-Pichard 13 ,

Rodolphe Anty 9 , Vincent Di Martino 5 , Filomena Conti 10 , Marie Line

Debette-Gratien 20 , Laurent Alric 16 , Armando Abergel 17 , Camille

Besch 19 , Helene Montialoux 18 , Pascal Lebray 10 , Sebastien Dharancy

4 , Francois Durand 12 , Louis d’Alteroche 21 , Florian Charier 8 ,

Olivier Chazouillères 14 , Jérôme Dumortier 24 , Vincent Leroy 23 , Jean-

Charles Duclos-Vallee 1,2 ; 1 Centre Hepato-Biliaire, AP-HP Hopital

Paul-Brousse, Villejuif, France; 2 Unit 1193, INSERM, Villejuif,

France; 3 AP-HP Hopital Henri Mondor, Creteil, France; 4 CHRU

de Lille, Lille, France; 5 CHU Jean Minjoz, Besançon, France; 6 HCL

- Hopital Croix Rousse, Lyon, France; 7 CHU de Rennes - Hopital

Pontchaillou, Renes, France; 8 CHU de Poitiers, Poitiers, France;

9 CHU de Nice, Nice, France; 10 AP-HP Hopital Pitie-Salpetrière,

Paris, France; 11 AP-HM Hopital La Conception, Marseille, France;

12 AP-HP Hopital Beaujon, Clichy, France; 13 AP-HP Hopital Cochin,

Paris, France; 14 AP-HP Hopital Saint-Antoine, Paris, France; 15 CHU

de Bordeaux - Hopital Haut-Leveque, Pessac, France; 16 CHU de

Toulouse - Hopital Purpan, Toulouse, France; 17 CHU de Clermont-Ferrand,

Clermont-Ferrand, France; 18 CHU de Rouen, Rouen,

France; 19 CHU de Strasbourg - Hopital Hautepierre, Strasbourg,

France; 20 CHU de Limoges, Limoges, France; 21 CHU de Tours

- Hopital Trousseau, Tours, France; 22 CHU de Montpellier - Hopital

Saint-Eloi, Montpellier, France; 23 CHU de Grenoble - Hopital

Michallon, Grenoble, France; 24 HCL - Hopital Edouard Herriot,

Lyon, France

Background: Combinations of DAA have shown excellent

results to treat HCV-infection in cirrhotic patients (pts). But some

issues remain unresolved regarding efficacy in pts awaiting

liver transplantation (LT) and impact on access to LT. Methods:

This cohort study enrolled 151 pts registered in 23 centers

on French LT list (male: 79%, age 56±7 years), treated with

sofosbuvir ± ribavirin (n=84) ± daclatasvir (n=90) or simeprevir

(n=9) or ledipasvir (n=17). Meantime between listing and

starting therapy was 23±57 weeks (wks). All pts were cirrhotic.

LT indication was HCC in 85(56%) pts. 112(74%) pts were

treatment experienced including 46% of non-responders, failures

to 1rst generation protease inhibitors in 32 or sofosbuvir

in 16 pts. Majority of pts were genotype 1 (56%) and 3 (24%).

Results: Mean follow-up was 44±19 wks [12-79]. Mean baseline

MELD score (MELD), platelet count, bilirubin and albumin

levels were 10±5 [6-32], 89±50 G/L[23-347], 39±42μmol/L

[5-405], 33±7 g/L [15-47]. 73 pts (48%) were decompensated

(bilirubin > 50μmol/L in 34(23%) ± ascites in 53(35%)

± hepatic encephalopathy (HE) in 29(19%)). Indetectability

of HCV RNA was obtained at 7±4 wks [1-24]. Among 117

pts, 103(88%) achieved SVR12. Among 73 decompensated

pts, a complete response (normal bilirubin level, no ascites,

no HE) was observed in 14(19%) after 12 wks and 31(42%)

at 12 wks post-treatment. When achieving SVR12, ascites or

HE persisted in 12/44 (27%) and 7/14 (50%). Variations of

MELD are shown in Table 1. Among patients with baseline

MELD score≥20, 83% still an indication of LT at the end of treatment

(MELD≥15). 45(30%) pts were transplanted. Ten (6%) pts

were delisted because of liver function improvement. A serious

adverse event occurred in 24(19%) pts, mainly liver events

(37%), anemia (25%) and sepsis (25%). Conclusion: Among

151 cirrhotic pts awaiting LT treated with IFN-free regimens,

88% achieved SVR12. Only 6% of pts were delisted due to

liver function improvement. A complete clinical and biological

response was observed in 42%, raising hopes that more pts

could be delisted. However, major clinical improvement was

observed in a minority of patients especially when MELD score

was ≥20, suggesting that antiviral treatment might be postponed

after LT in this population. Final results will be provided

in a larger population.

Variations of MELD score in 66 pts with decompensated condition

from baseline to the end of treatment


Audrey Coilly - Consulting: Novartis, Astellas, Janssen, Bristol-Myers-Squibb,

Merck Sharp & Dohme, Gilead, Roche

Georges-Philippe Pageaux - Advisory Committees or Review Panels: Roche,

Roche, Roche, Roche; Board Membership: Astellas, Astellas, Astellas, Astellas

Pauline Houssel-Debry - Speaking and Teaching: NOVARTIS, ASTELLAS, GILEAD

Christophe Duvoux - Advisory Committees or Review Panels: Novartis, Roche,

Novartis, Roche, Novartis, Roche, Novartis, Roche; Speaking and Teaching:

Astellas, Astellas, Astellas, Astellas

Victor de Ledinghen - Board Membership: Janssen, Gilead, BMS, Abbvie; Speaking

and Teaching: AbbVie, Merck, BMS, Gilead

Danielle Botta-Fridlund - Consulting: GILEAD, ABBVIE, BMS, MSD

Anaïs Vallet-Pichard - Independent Contractor: Schering Plough, Gilead, BMS,


Vincent Di Martino - Advisory Committees or Review Panels: Gilead, France,

Abbvie, BMS France; Board Membership: MSD France; Consulting: Gilead,

France; Speaking and Teaching: Janssen, BMS France, Gilead France

Laurent Alric - Board Membership: Schering Plough, Schering Plough, Schering

Plough, Schering Plough; Consulting: MSD; Speaking and Teaching: Roches,

BMS, Gilead, Roches, BMS, Gilead, Roches, BMS, Gilead, Roches, BMS, Gilead,

MSD, Abbvie

Armando Abergel - Consulting: gilead, msd, bms; Speaking and Teaching: abbvie

Pascal Lebray - Grant/Research Support: Schering-Plough, Schering-Plough, Schering-Plough,

Schering-Plough; Speaking and Teaching: Gilead, Gilead, Gilead,


Sebastien Dharancy - Advisory Committees or Review Panels: CHIESI; Board

Membership: NOVARTIS; Speaking and Teaching: ASTELLAS

Francois Durand - Advisory Committees or Review Panels: Astellas, Novartis,

BMS; Speaking and Teaching: Gilead

Olivier Chazouillères - Consulting: APTALIS, MAYOLY-SPINDLER

Jérôme Dumortier - Board Membership: Novartis, Astellas, Roche; Consulting:

Novartis; Grant/Research Support: Novartis, Astellas, Roche, MSD, GSK

Vincent Leroy - Board Membership: Abbvie, BMS, Gilead; Consulting: Janssen,

MSD; Speaking and Teaching: Abbvie, BMS, Gilead, Janssen, MSD

The following authors have nothing to disclose: Sylvie Radenne, Rodolphe Anty,

Filomena Conti, Marie Line Debette-Gratien, Camille Besch, Helene Montialoux,

Louis d’Alteroche, Florian Charier, Jean-Charles Duclos-Vallee


Detecting Drug-Induced Liver Injury in Patients with

Decompensated Chronic Hepatitis C: A Review of the

SOLAR-1 and SOLAR-2 Studies

Andrew J. Muir 2 , Michael R. Charlton 5 , Phillip S. Pang 1 , Luisa

M. Stamm 1 , Diana M. Brainard 1 , John G. McHutchison 1 , Nezam

H. Afdhal 3 , Paul B. Watkins 4 ; 1 Gilead Sciences, Foster City, CA;

2 Duke University Medical Center, Durham, NC; 3 Division of Gastroenterology,

BIDMC, Boston, MA; 4 The Hamner-UNC Institute for

Drug Safety Sciences, Chapel Hill, NC; 5 Intermountain Medical

Center, Murray, UT

Background: The identification of drug-induced liver injury

(DILI) is challenging in patients with underlying liver disease,

particularly in the setting of decompensated cirrhosis which has

recently become a target population for novel therapies. Traditional

laboratory criteria (e.g., elevation in serum ALT > 5x

upper limit of normal) may not be appropriate for patients with


advanced liver disease. The aim of this analysis was to develop

new criteria for identifying DILI associated with direct-acting

antivirals in end-stage liver disease patients. Methods: The

SOLAR-1 and SOLAR-2 studies assessed the safety and efficacy

of ledipasvir/sofosbuvir plus ribavirin (LDV/SOF+RBV) for 12

or 24 weeks in patients with advanced liver disease pre- or

post-liver transplant. Laboratory data, including total and direct

bilirubin and ALT and AST relative to baseline, from patients

with CPT B and C cirrhosis (N=328) were used to determine

criteria to screen for possible cases of DILI. Cases of interest,

including clinical outcome data, were further evaluated for DILI

by an expert panel. DILI was classified as possible or unlikely,

where unlikely required the identification of a clear non-drug

related etiology of the decompensation or laboratory abnormalities.

Results: Overall, on-treatment elevations above the

upper limit of normal were common for total bilirubin (90%,

n=295), ALT (48%, n=157) and AST (90%, n=294). Further

analysis of total bilirubin identified only 11 of 295 patients in

whom the direct bilirubin had a treatment-emergent elevation of

greater than 1 mg/dL. Adjudication of these 11 cases revealed

1 possible case of DILI. The use of ALT and AST criteria, including

a cutoff of 5x nadir, did not identify further cases of interest

beyond those identified by the >1 mg/dL increase in direct

bilirubin. Median ALT and AST at baseline were 62 IU/ml and

100 IU/ml, respectively; only 3 subjects reached a maximum

ALT or AST > 2x baseline. ALT and AST improved (on-treatment

ALT maximum < baseline) in 91% and 92% of patients,

respectively. Conclusions: In patients with CPT B or C cirrhosis

and HCV treated with LDV/SOF+RBV, elevation from baseline

in direct bilirubin of > 1mg/dL was a clinically sensitive and

conservative cutoff that identified patients who should be further

evaluated for the possibility of DILI. No additional possible

cases of DILI were identified by using ALT or AST criteria. An

adjudication of all biochemical and clinical events of interest

(including deaths and liver transplantations) of all patients in

the SOLAR-1 and SOLAR-2 studies will be presented.


Andrew J. Muir - Advisory Committees or Review Panels: BMS, Gilead, Janssen,

Merck; Consulting: Theravance; Grant/Research Support: Abbvie, Abbvie, BMS,

Gilead, Janssen, Merck, Achillion, Lumena

Michael R. Charlton - Grant/Research Support: GIlead Sciences, Merck, Janssen,

AbbVie, Novartis

Phillip S. Pang - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences

Luisa M. Stamm - Employment: Gilead Sciences

Diana M. Brainard - Employment: Gilead Sciences; Stock Shareholder: Gilead


John G. McHutchison - Employment: Gilead Sciences; Stock Shareholder: Gilead


Nezam H. Afdhal - Advisory Committees or Review Panels: Trio Helath Care;

Board Membership: Journal Viral hepatitis; Consulting: Merck, EchoSens, BMS,

Achillion, GlaxoSmithKline, Springbank, Gilead, AbbVie; Grant/Research Support:

Gilead; Stock Shareholder: Springbank

Paul B. Watkins - Consulting: Abbott, Actelion, Boerringer-Ingelheim, Cempra,

Alecra, Roche, Merck, Reservlogix, Intercept, Janssen, Novartis, Otsuka, Pfizer,

Sanolfi, Takeda, UCB, Bristol-Myers Squibb, GSK

the underlying mechanism driving apoptosis remains unclear.

In this study we investigated the role of autophagy in regulating

apoptosis in NAFLD. Methods and Results: Administration of

palmitic acid (PA) to HepG2 cells increased Annexin V-positive/7-AAD-negative

cells and induced apoptosis from 8 hours,

which was found to be mediated by activation of IRE1-JNK and

PERK-CHOP pathways. PA increased the expression level of

LC3-II and decreased the autophagic flux index, which can be

measured by monitoring LC3-II turnover using bafilomycin A1,

from 3 hours. These findings suggest that PA inhibits the autophagic

process after autophagosome formation. In PA-treated

HepG2 cells, mTOR signaling, a negative regulator of autophagy,

was inhibited and the expressions levels of Beclin1,

Atg7 and Atg5 were unaltered. In contrast, the expression

level of Rubicon, a negative regulator of autophagosome-lysosome

fusion, was clearly upregulated by PA treatment from

2 hours. siRNA-mediated Rubicon knockdown clearly abolished

PA-induced inhibition of autophagic flux, reduced ER

stress and ameliorated PA-induced apoptosis. While PA treatment

did not change the levels of Rubicon mRNA expression,

a pulse chase assay revealed that Rubicon protein stability

was enhanced by PA treatment. Consistent with in vitro findings,

mice on high fat diet (HFD) showed increased Rubicon

expression from 1 month, autophagy inhibition from 2 months

and increased apoptosis from 3 months in the liver. Electron

microscopy revealed that isolation membranes and autophagosomes

in the liver of mice fed HFD were increased, suggesting

the inhibition of autophagy after autophagosome maturation.

Hepatocyte-specific Rubicon knockout (KO) mice on HFD for 4

months improved autophagy inhibition, ER stress and hepatocyte

apoptosis compared with wild-type littermates. They also

showed the significant reduction of liver volume to approximately

normal level, which was accompanied by significant

and substantial reduction of triglycerides in their livers. Finally,

we investigated Rubicon expression levels in human liver samples

by western blotting under approval of institutional ethics

committee. Rubicon expression levels in steatotic livers were

higher than those in non-steatotic livers. Conclusion: Increased

expression of Rubicon induced by PA and HFD suppresses

autophagic flux and promotes hepatocyte apoptosis by increasing

ER stress. Enhancement of autophagy by inhibiting Rubicon

may provide new approaches for treatment of NAFLD.


Hayato Hikita - Grant/Research Support: Bristol-Myers Squibb

Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD

K.K., Bristol-Meyer Squibb, Mitsubishi Tanabe Pharma Corparation, Toray Industories

Inc. ; Speaking and Teaching: MSD K.K., Bristol-Meyer Squibb, Janssen

Pharmaceutical Companies

The following authors have nothing to disclose: Satoshi Tanaka, Sadatsugu

Sakane, Yasutoshi Nozaki, Yugo Kai, Tasuku Nakabori, Yoshinobu Saito,

Ryotaro Sakamori, Tomohide Tatsumi


Enhanced expression of Rubicon inhibits autophagy and

promotes apoptosis by increasing endoplasmic reticulum

stress in nonalcoholic fatty liver disease

Satoshi Tanaka, Hayato Hikita, Sadatsugu Sakane, Yasutoshi

Nozaki, Yugo Kai, Tasuku Nakabori, Yoshinobu Saito, Ryotaro

Sakamori, Tomohide Tatsumi, Tetsuo Takehara; Gastroenterology

& Hepatology, Osaka University, Suita, Japan

Background and Aim: Hepatocyte apoptosis is a characteristic

feature of nonalcoholic fatty liver disease (NAFLD). However,



In vivo reprogramming of myofibroblasts into hepatocytes

as a therapy for liver fibrosis

Milad Rezvani 1 , Regina Español-Suñer 1 , Yann Malato 1 , Laure

Dumont 1 , Andrew A. Grimm 2 , Eike Kienle 3 , Julia Bindmann 1 , Ellen

Wiedtke 3 , Syed J. Naqvi 1 , S. C. Derderian 4 , Robert F. Schwabe 5 ,

Dirk Grimm 3 , Holger Willenbring 1,6 ; 1 Institute of Regeneration

Medicine, University of California San Francisco, San Francisco,

CA; 2 Department of Pediatrics, Division of Gastroenterology,

Hepatology, and Nutrition, University of California San Francisco,

San Francisco, CA; 3 Department of Infectious Diseases, Cluster of

Excellence CellNetworks, BioQuant BQ0030, Heidelberg University

Hospital, Heidelberg, Germany; 4 4Department of Surgery,

Division of Pediatric Surgery, University of California San Francisco,

San Francisco, CA; 5 Department of Medicine,, Columbia

University, New York, NC; 6 Department of Surgery, Division of

Transplantation, University of California San Francisco, San Francisco,


Repeated hepatocyte loss in chronic liver injury can exceed

the regenerative capabilities of hepatocytes and lead to liver

fibrosis, a form of scarring characterized by replacement of

hepatocytes by collagen produced by myofibroblasts (MFs).

The structural and molecular changes associated with liver

fibrosis further impair liver function, leading to liver failure.

The only cure for advanced liver fibrosis is liver transplantation,

but donor organs are scarce. As a strategy to replenish

the hepatocyte mass and limit collagen deposition in the

chronically injured liver, we developed in vivo reprogramming

of MFs into hepatocytes by overexpression of hepatic transcription

factors. To facilitate clinical translation, we delivered

these genes to MFs using adeno-associated viral (AAV) vectors,

which are not toxic and do not integrate into the genome. We

first investigated the feasibility of generating induced hepatocytes

from primary MFs (MF-iHeps) using AAV vectors expressing

FOXA1, FOXA2, FOXA3, GATA4, HNF1α and HNF4α in

vitro. We found that AAV vectors were effective in generating

expandable MF-iHeps resembling previously reported iHeps

generated from fibroblasts with integrating retroviral or lentiviral

vectors. Specifically, MF-iHeps lost most of their original

cell identity and acquired hepatocyte gene and protein expression

and functions like low-density lipoprotein uptake, glycogen

storage and cytochrome P450 activity. Next, we established

hepatic reprogramming of MFs in vivo. For this we used a

lineage-tracing mouse model in which MFs and their progeny

are constitutively labeled. To induce liver fibrosis, we treated

these mice with carbon tetrachloride. After intravenous injection

of the AAV vectors we observed the formation of MF-iHeps

and reduced liver fibrosis. Like primary hepatocytes MF-iHeps

proliferated in response to liver injury in both CCl4-treated

and fumarylacetoacetate hydrolase-deficient mice. To assess

hepatocyte differentiation of MF-iHeps, we isolated them by

laser-capture microscopy and analyzed their gene expression

with microarrays. MF-iHeps closely resembled primary hepatocytes,

with the exception of minimal residual MF identity. We

confirmed these results using functional assays, including analysis

of hepatic glucose metabolism, and ascertained stability

of MF-iHep differentiation in mice followed for 6 months. Our

results establish repurposing of MFs as a potential new therapy

for liver fibrosis that not only reduces fibrosis but also increases

the functional hepatocyte mass. By using AAV vectors, which

proved effective and safe in liver-directed human gene therapy,

our strategy lends itself well to clinical translation.


Robert F. Schwabe - Consulting: Merck

The following authors have nothing to disclose: Milad Rezvani, Regina Español-

Suñer, Yann Malato, Laure Dumont, Andrew A. Grimm, Eike Kienle, Julia Bindmann,

Ellen Wiedtke, Syed J. Naqvi, S. C. Derderian, Dirk Grimm, Holger



Protective role of miR-122 against acetaminophen toxicity

is due to suppression of Cyp2e1 and Cyp1a2

Vivek K. Chowdhary 3 , Huban Kutay 3 , Laura James 2 , William M.

Lee 1 , Kalpana Ghoshal 3 ; 1 UT Southwestern Medical Center, Dallas,

TX; 2 University of Arkansas for Medical Science, Little Rock,

AR; 3 Pathology, Comprehensive Cancer Center, The Ohio State

University College of Medicine, Columbus, OH

Approximately 2000 cases of acute liver failure occur annually

in the United States and acetaminophen (APAP) accounts for

nearly 50% of cases. miR-122 is the most abundant, conserved

liver-specific microRNA that maintains metabolic homeostasis

and functions as a tumor suppressor. Although circulating

miR-122 is a sensitive biomarker of APAP toxicity in humans

and rodents, its role in this injury has not been elucidated. To

investigate whether miR-122 has any protective role against

APAP toxicity, we gave APAP (500mg/Kg) intraperitoneally

to the Mir122 fl/fl (WT) and liver-specific miR-122 knockout

(Mir122 fl/fl ; Alb-Cre) (aka LKO) mice generated in our lab

(PMID: 22820288), and monitored their survival. Mortality

rate of LKO mice was significantly higher than that of WT mice




CX3CR1 is a gatekeeper for intestinal barrier integrity:

Limiting steatohepatitis by maintaining intestinal


Kai M. Schneider 1 , Veerle Bieghs 1 , Felix Heymann 1 , Wei Hu 1 ,

Daniela Dreymueller 2 , Lijun Liao 1 , Mick Frissen 1 , Andreas Ludwig 2 ,

Nikolaus Gassler 3 , Oliver Pabst 4 , Eicke Latz 5,6 , Gernot Sellge 1 ,

John Penders 7 , Frank Tacke 1 , Christian Trautwein 1 ; 1 Department

of Internal Medicine III, RWTH Aachen University, Aachen, Germany;

2 Institute of Pharmacology and Toxicology, Medical Faculty,

University Hospital RWTH Aachen, Aachen, Germany; 3 Institute

of Pathology, University Hospital RWTH Aachen, Aachen, Germany;

4 Institute of Molecular Medicine, University Hospital RWTH

Aachen, Aachen, Germany; 5 Institute of Innate Immunity, University

Hospital, University of Bonn, Bonn, Germany; 6 Department of

Medicine, University of Massachusetts Medical School, Worcester,

MA; 7 Department of Medical Microbiology, School of Nutrition

and Translational Research in Metabolism, Maastricht University

Medical Center, Maastricht, Netherlands

Background: Non-alcoholic fatty liver disease (NAFLD) represents

the most common liver disease in Western societies

and is regarded as the hepatic manifestation of the metabolic

syndrome. The G-Protein-coupled chemokine receptor Cx3cr1

has been shown to play a central role in many metabolic diseases

including type-2 diabetes, atherosclerosis and obesity.

However, the role of Cx3cr1 for NAFLD progression is unclear.

Recent data indicate that intestinal dysbiosis drives NASH

development and Cx3cr1 is essential for intestinal homeostasis.

Based on these findings, we hypothesized that Cx3cr1

plays a role in regulating the gut-liver axis and therefore has

implications for NASH progression. Methods: Male wildtype

(WT) and Cx3cr1 -/- mice were fed either a normal chow diet

(NCD), high-fat diet (HFD) or methionine-choline-deficient diet

(MCD) to induce steatohepatitis. For eradicating the intestinal

microbiota, the drinking water of the mice was supplemented

with broad-spectrum non-resorbable antibiotics. Cx3cr1-signaling

was studied using Bone-marrow-derived macrophages

(BMDMs). Results: On HFD or MCD Cx3cr1 -/- mice showed

more severe hepatic steatosis and inflammation, as well as systemic

glucose intolerance compared to WT controls. Mechanistically,

in-vitro experiments with BMDMs identified Cx3cr1 as

a regulator of macrophage homeostasis by modulating inflammasome

activation. Accordingly, Cx3cr1 deficiency in mice

was associated with significantly altered intestinal microbiota

composition, which was linked to an impaired intestinal barrier

including thinner colonic mucus layers, reduction of tight junction

expression and a decrease in colonic resident phagocytic

macrophages. Concomitantly, these intestinal changes led to

an increased translocation of endotoxin and stronger activation

of pathogen recognition receptors (PRRs) in livers of Cx3cr1 -/-

mice, thereby triggering an enhanced inflammatory response

in the liver. Strikingly, depletion of the intestinal microbiota

by administration of broad-spectrum antibiotics (AB) did not

only suppress the number of infiltrating macrophages, but also

promoted a restorative phenotype of liver macrophages. Consequently,

AB-treated mice demonstrated a marked improvement

of steatohepatitis and glucose tolerance. Conclusion: Our

data demonstrate that microbiota-mediated activation of the

innate immune responses via Cx3cr1 is crucial for controlling

steatohepatitis progression, thereby recognizing CX3CR1 as

an essential gatekeeper in this scenario.


Frank Tacke - Advisory Committees or Review Panels: Tobira; Grant/Research

Support: Novartis, Noxxon; Speaking and Teaching: BMS, Gilead, Falk, MSD,

Janssen, Abbvie

Christian Trautwein - Grant/Research Support: BMS, Novartis, BMS, Novartis;

Speaking and Teaching: Roche, BMS, Roche, BMS

The following authors have nothing to disclose: Kai M. Schneider, Veerle Bieghs,

Felix Heymann, Wei Hu, Daniela Dreymueller, Lijun Liao, Mick Frissen, Andreas

Ludwig, Nikolaus Gassler, Oliver Pabst, Eicke Latz, Gernot Sellge, John Penders


Clonal analysis of the human liver reveals adaptable

stem cell dynamics

Malcolm Alison, Biancastella Cereser, Hemant Kocher, Stuart A.

McDonald; Centre for Tumour Biology, Barts Cancer Institute, London,

United Kingdom

The study of cell lineages through heritable genetic lineage

tracing is well established in experimental animals. However,

using such techniques it is still unclear whether the liver conforms

to a stem cell and lineage system, as seen for example

in the bone marrow and gut. We have sought to resolve

this question in the human liver using a variety of investigative

techniques. The mitochondrial genome is highly prone

to non-pathogenic mutations resulting in a deficiency in cytochrome

c oxidase (CCO). Such mutations can be used as markers

of clonal expansion and understanding the phylogenetics

of these mutations will help in identifying stem cell derived

clonal populations and their dynamics. Analysis of many hundreds

of periportal and hepatic venous regions revealed that

the majority of CCO-deficient cell patches abutted portal tracts,

and when adjacent to hepatic veins the CCO-deficient patch

typically extended from the portal tract. Furthermore, CCO-deficient

patches were associated with trifurcating terminal portal

tracts, but not with conducting portal tracts. Secondly we analyzed

the methylation patterns of CpG islands in the promoter

regions of non-expressed genes in CCO-deficient cell patches

with respect to the portal tract-hepatic vein axis. Patterns from

microdissected areas within CCO-deficient patches became

diverse a short distance away from portal tracts, suggesting

that most periportal and centrilobular hepatocytes are of similar

mitotic age and only hepatocytes in the immediate periportal

zone revealed patterns that were proportional to the size

of the clone. Next generation sequencing (NGS) of human

mtDNA was also employed to determine the clonal dynamics in

CCO-deficient patches. Some mutations were common (clonal)

to all cells within a patch, while some were confined (private)

to cells at different points through the patch. Overall the methylation

and NGS data suggest that CCO patches developed

initially as a rapid expansion (perhaps through acute damage)

which then ceased and allowed cell by cell diversity to accrue.

Interestingly, the centrilobular regions revealed a higher mutation

burden, indicative of these being older cells having had

more time for subclonal expansion of mutations. Our studies

employing CCO activity, promoter methylation and NGS are

consistent with the human liver being a lineage system, presumably

emanating from periportal hepatic progenitor cells,

the so-called streaming liver. However, this process appears to

be very slow in normal homeostasis and rapid perhaps during

periods of acute damage and suggests an adaptable stem cell



The following authors have nothing to disclose: Malcolm Alison, Biancastella

Cereser, Hemant Kocher, Stuart A. McDonald



MicroRNA-21 is a Potential Link between Non-alcoholic

Fatty Liver Disease and Hepatocellular Carcinoma via

Modulation of the HBP1-p53-Srebp1c Pathway

Heng Wu, Guisheng Song; Medicine, University of Minnesota,

Minneapolis, MN

Background: Nonalcoholic fatty liver disease (NAFLD) is a

major risk factor for hepatocellular carcinoma (HCC). However,

the mechanistic pathways that link both disorders are

essentially unknown. Our study was designed to investigate

the role of microRNA-21 in the pathogenesis of NAFLD and its

potential involvement in HCC. Methods: Wild-type mice maintained

on a high fat diet (HFD) received tail-vein injections of

microRNA-21-ASO (anti-sense oligonucleotide) or miR-21 mismatched

ASO for 4 or 8 weeks. Livers were collected after that

time period for lipid content and gene expression analysis. The

role of microRNA-21 in carcinogenesis was analyzed by softagar

colony formation, cell cycle analysis, and xenograft tumor

assay. Results: The expression of microRNA-21 was increased

in the livers of HFD-treated mice and human HepG2 cells incubated

with fatty acid. MicroRNA-21 knockdown in those mice

and HepG2 cells impaired lipid accumulation and growth of

xenograft tumor. Further studies revealed that Hbp1 was a

novel target of microRNA-21 and a transcriptional activator of

p53. It is well-established that p53 is a tumor suppressor and

an inhibitor of lipogenesis by inhibiting Srebp1c. As expected,

microRNA-21 knockdown led to increased HBP1 and p53 and

subsequently reduced lipogenesis and delayed G1/S transition,

and the additional treatment of HBP1-siRNA antagonized

the effect of microRNA-21-ASO, suggesting that HBP1

mediated the inhibitory effects of microRNA-21-ASO on both

hepatic lipid accumulation and hepatocarcinogenesis. Mechanistically,

microRNA-21 knockdown induced p53 transcription,

which subsequently reduced expression of genes controlling

lipogenesis and cell cycle transition. In contrast, the opposite

result was observed with overexpression of microRNA-21,

which prevented p53 transcription. Conclusion: Our findings

reveal a novel mechanism by which microRNA-21, in part,

promotes hepatic lipid accumulation and cancer progression

by interacting with the Hbp1-p53-Srebp1c pathway, and suggest

the potential therapeutic value of microRNA-21-ASO for

both disorders.


The following authors have nothing to disclose: Heng Wu, Guisheng Song


Comparison And Outcomes Of 5% Albumin Vs 0.9%

Normal Saline Fluid Resuscitation In Cirrhotics Presenting

With Sepsis Induced Hypotension – A Randomized

Controlled Trial - Fluid Resuscitation In Septic Shock In

Cirrhosis (FRISC Protocol)

Cyriac A. Philips 1 , Ashok K. Choudhury 1 , Amrish Sahney 1 , Rakhi

Maiwall 1 , Lalita G. Mitra 2 , Shiv K. Sarin 1 ; 1 Hepatology and Transplant

Medicine, Institute of Liver and Biliary Sciences, New Delhi,

India; 2 Anesthesia and Critical Care, Institute of Liver and Biliary

Sciences, New Delhi, India

Background&Aims:Fluid resuscitation and choice of fluid for

use in sepsis induced hypotension (SIH)is generally done as per

Surviving Sepsis Guidelines (SSG). There is however, no data

on fluid resuscitation protocols in cirrhosis patients. We investigated

the efficacy, hemodynamic and perfusion effects of

human albumin (HA) versus 0.9% normal saline(NS)in cirrhotics

presenting with SIH. Patients&Methods: 308 patients of cirrhosis

with SIH were randomized to receive either HA(5%,250ml

bolus over 15 mins,n=154) or NS(30mL/kg over 30 mins);primary

end-point (PE) being increase in mean arterial pressure

(MAP)>65 mm Hg at 3 hr; secondary end-points of effects

on heart rate(HR),lactate [delta lactate (dLAC), lactate clearance

(cLAC)], urine output (UO) from baseline, at 1, 2 and

3 hr and survival at 1 wk. Results:154 patients each, in HA

(males,76%,mean age 49.7 yr) or NS group (79.2%,47 yr);(-

MAP-53.8±6.2/54.4±6 mmHg,HR–93.6±22.6/93.4±16.8/

bpm,LAC–6.94±2.2/6.83±2.6 mmol/L) and severity scores

(CTP11.8±1.9/12.2±1.9,MELD 32±8.6/30±8.3,SOFA-

9.99±2.5/10.4±2.8) had comparable baseline parameters.

MAP>65mmHg at 1hr and sustenance of MAP at 3hr were in



idly. Our objective was to project the number of HCV patients

needing treatment in 2015 and beyond, and the long-term

health outcomes under different treatment penetration rates.

Method: We used our previously published Hepatitis C Disease

Burden Simulation model (HEP-SIM), which projected the

changing prevalence of HCV in the United States. The HEP-SIM

model was validated with NHANES studies and other published

data. We simulated the current clinical management of

HCV from 2001 onwards, which included risk-based screening

until 2013 and addition of birth-cohort screening afterwards.

We modeled antiviral treatment in different waves starting with

peginterferon-ribavirin (PEG-RBV) until 2011, followed by the

launch of boceprevir/telaprevir in 2012, sofosbuvir/simeprevir

in 2014, and finally oral DAAs in 2015. We also implemented

changes in insurance status because of the Affordable Care

Act. We projected the number of patients needing treatment

in 2015 and beyond under varying treatment capacity/penetration

scenarios. We also projected patients’ fibrosis score,

HCV awareness status, and access to insurance. Results: We

estimated that in 2015, 2 million noninstitutionalized patients

would be chronically infected and viremic. Among these

patients, 1.1 million would be potential DAA candidates, i.e.,

aware of their HCV status and insured. At the current annual

treatment capacity of 200,000 patients, it will take at least 10

years to treat the majority of the patients. By 2025, the number

of treatment candidates would decline to fewer than 50,000

patients. Even in the DAA era, 320,000 patients would die

because of HCV, 32,000 patients would get liver transplants,

202,000 would develop decompensated cirrhosis (DC) and

156,000 would progress to hepatocellular carcinoma (HCC)

by 2050. Doubling the annual treatment capacity could avoid

8,000 deaths, 700 transplants, 7,000 DC and 4,000 HCC

cases by 2050. Conclusions: Though the majority of patients

aware of their HCV will be treated in the next 10 years, the

HCV burden would still remain substantial unless aggressive

screening and treatment policies are implemented. Increasing

HCV treatment capacity is essential to decrease disease burden

and improve health outcomes of HCV patients in the US, and

decrease health resource utilization.


Jagpreet Chhatwal - Consulting: Merck & Co., Inc., Gilead, Complete HEOR

Solutions; Grant/Research Support: NIH/National Center for Advancing Translational


The following authors have nothing to disclose: Xiaojie Wang, Fasiha Kanwal,

Mina Kabiri, Turgay Ayer, Julie M. Donohue, Mark S. Roberts


An international, phase 2 randomized controlled trial of

the dual PPAR α-δ agonist GFT505 in adult patients with


Vlad Ratziu 1 , Stephen A. Harrison 2 , Sven M. Francque 3 , Pierre

Bedossa 4 , Lawrence Serfaty 5 , Manuel Romero-Gomez 6 , Paul

Cales 7 , Manal F. Abdelmalek 8 , Stephen H. Caldwell 9 , Joost

Drenth 10 , Quentin M. Anstee 11 , Dean W. Hum 12 , Rémy Hanf 12 ,

Alice Roudot 12 , Sophie Megnien 12 , Bart Staels 13 , Arun J. Sanyal 14 ;

1 Hepatology, Hopital Pitie Salpetriere, Paris, France; 2 Department

of Medicine, Gastroenterology & Hepatology Service,, Brooke

Army Medical Center, Fort Sam Houston, TX; 3 Department of Gastroenterology

& Hepatology, Antwerp University Hospital, University

of Antwerp, Antwerp, Belgium; 4 Department of Pathology,

Hôpital Beaujon, University Paris-Denis Diderot, Clichy, France;

5 Service d’Hépatologie,, Hôpital Saint-Antoine, APHP, UPMC Paris

6, Paris, France; 6 Hospital Universitario de Valme, Unit for the

Clinical Management of Digestive Diseases and CIBERehd, Sevilla,

Spain; 7 Hepatology Department, University Hospital & LUNAM

University, Angers, France., Angers, France; 8 Duke University,

Duke university, Durham, NC; 9 University of Virginia, Gastroenterology

& hepatology Division, Charlottesville, VA; 10 Radboud

University Medical Center, Department of Gastroenterology and

Hepatology, Nijmegen, Netherlands; 11 Institute of Cellular Medicine,,

Faculty of Medical Sciences, Newcastle University, Newcastle

upon Tyne, United Kingdom; 12 Genfit SA, Loos, France;

13 Université Lille 2, INSERM U1011, European Genomic Institute

for Diabetes (EGID), Institut Pasteur de Lille, Lille, France, Lille,

France; 14 Virginia Commonwealth University, Richmond, VA

Peroxisome proliferator-activated receptor α-δ dual agonists,

such as GFT505, are a promising therapy for NASH as they

improve hepatic insulin sensitivity, glucose homeostasis, lipid

metabolism, and inflammation. Methods. In this randomized

controlled trial (56 European and US centers) 274 patients (pts)

(full analysis set, FAS) with histologically-defined non-cirrhotic

NASH received GFT505 80 mg or 120 mg QD vs placebo (PLB)

for one year. The primary outcome was resolution of NASH

without worsening of fibrosis. Data were analyzed according

to baseline severity (histological NAS score) and center effect.

Biopsies were read by a single pathologist. Results. 237 pts

had entry and end-of-treatment biopsies (ITT population). While

the a priori primary endpoint did not meet significance, after

controlling for baseline severity and center effect, pts in the

120 mg arm had a 1.94 (CI 1.08-3.48, p=0.027) higher relative

risk (RR) of achieving the primary end-point compared to

PLB, while the RR was 1.68 (0.92-3.05, p=0.091) for the 80

mg arm. Results were similar in the FAS where pts missing the

second biopsy were counted as failures. In pts with moderate

activity (NAS 4 or 5) the response rate was 27.5% in the 120

mg arm vs. 19.5% for the PLB arm. In those with severe activity

(NAS>5) it was 14.8% vs. 0%, respectively. In the 120 pts

with NAS>4 from centers that recruited >1 patient/arm, the

response rate was 29% and 5% in the 120 mg and PLB arms,

respectively, p=0.01. A >2 point NAS reduction was obtained

in 48% and 21% of patients respectively, p=0.01. Compared

to PLB, the 120 mg arm improved ballooning (45% vs. 23%,

p=0.02), inflammation (55% vs. 33%, p=0.05) and steatosis

(35.5% vs. 18%, NS). In the 120 mg arm, resolution of NASH,

resulted in a significant improvement in fibrosis (mean change

-0.67 vs. +0.09 in non-responders, p


as HbA1c and FFA in diabetic pts, all on top of standard of

care therapies. Tolerability was excellent without weight gain,

cardiac events or safety signal. Conclusion. In NASH patients

120 mg daily of GFT505 induced histological improvement

and resolution of NASH, significantly more often than PLB.

The excellent safety and tolerability and the improvement in

cardiometabolic risk profile makes GFT505 an ideal drug candidate

to be tested in phase 3 trials


Vlad Ratziu - Advisory Committees or Review Panels: GalMed, Abbott, Genfit,

Enterome, Gilead; Consulting: Tobira, Intercept, Exalenz, Sanofi-Synthelabo,


Stephen A. Harrison - Advisory Committees or Review Panels: Merck, Nimbus

Discovery, Fibrogen, RuiYi, CLDF; Consulting: NGM Biopharmaceuticals; Speaking

and Teaching: Gilead, Abbvie, Janssen, CLDF

Lawrence Serfaty - Board Membership: BMS, Gilead; Consulting: Merck; Speaking

and Teaching: Roche, Janssen, Merck, Janssen, BMS, Gilead

Manuel Romero-Gomez - Advisory Committees or Review Panels: Roche Farma,SA.,

MSD, S.A., Janssen, S.A., Abbott, S.A.; Grant/Research Support: Ferrer,


Paul Cales - Consulting: BioLiveScale

Manal F. Abdelmalek - Consulting: Islet Sciences; Grant/Research Support:

Tobira, Gilead Sciences, NIH/NIDDK, Synageva, Genfit Pharmaceuticals,

Immuron, Galmed, TaiwanJ Pharma, Intercept, NGM Pharmaceuticals

Stephen H. Caldwell - Advisory Committees or Review Panels: Vital Therapy;

Grant/Research Support: Genfit, Gilead Sciences, Immuron, Hyperion, Immuron,


Quentin M. Anstee - Advisory Committees or Review Panels: Genfit, Intercept,

Raptor; Grant/Research Support: GSK; Speaking and Teaching: Abbott Laboratories

Dean W. Hum - Management Position: Genfit

Rémy Hanf - Management Position: GENFIT

Alice Roudot - Employment: GENFIT

Sophie Megnien - Employment: GENFIT

Bart Staels - Advisory Committees or Review Panels: MSD; Consulting: Genfit

Arun J. Sanyal - Advisory Committees or Review Panels: Bristol Myers, Gilead,

Genfit, Abbott, Ikaria, Exhalenz; Consulting: Salix, Immuron, Exhalenz, Nimbus,

Genentech, Echosens, Takeda, Merck, Enanta, Zafgen, JD Pharma, Islet

Sciences; Grant/Research Support: Salix, Genentech, Intercept, Ikaria, Takeda,

GalMed, Novartis, Gilead, Tobira; Independent Contractor: UpToDate, Elsevier

The following authors have nothing to disclose: Sven M. Francque, Pierre

Bedossa, Joost Drenth


NGM282, A Novel Variant of FGF-19, Demonstrates

Biologic Activity in Primary Biliary Cirrhosis Patients

with an Incomplete Response to Ursodeoxycholic Acid:

Results of a Phase 2 Multicenter, Randomized, Double

Blinded, Placebo Controlled Trial

Marlyn J. Mayo 3 , Alan J. Wigg 4 , Stuart K. Roberts 5 , Hays

Arnold 19 , Tarek I. Hassanein 7 , Barbara A. Leggett 8 , John P. Bate 9 ,

Martin Weltman 10 , Elizabeth J. Carey 11 , Andrew J. Muir 12 , Geoff

McCaughan 14 , Steven J. Bollipo 15 , Stuart C. Gordon 16 , Peter W.

Angus 13 , Stephen Riordan 17 , Mitchell L. Shiffman 18 , Elisa Young 6 ,

Lei Ling 1 , Jian Luo 1 , Michael Elliott 1 , Stephen Rossi 1 , Alex M.

DePaoli 1 , Alex J. Thompson 2 ; 1 NGM Biopharmaceuticals, Inc.,

South San Francisco, CA; 2 St. Vincent’s Hospital, Melbourne, VIC,

Australia; 3 UT Southwestern, Dallas, TX; 4 Flinders Medical Center,

Adelaide, SA, Australia; 5 Alfred Hospital, Melbourne, VIC,

Australia; 6 Novotech, Sydney, NSW, Australia; 7 Southern California

Liver Research Center, Coronado, CA; 8 Royal Brisbane and

Women’s Hospital, Brisbane, QLD, Australia; 9 Royal Adelaide

Hospital, Adelaide, SA, Australia; 10 Nepean Hospital, Sydney,

NSW, Australia; 11 Mayo Clinic - Arizona, Scottsdale, AZ; 12 Duke

University, Durham, NC; 13 Austin Hospital, Heidelberg, VIC, Australia;

14 University of Sydney, Sydney, NSW, Australia; 15 John

Hunter Hospital, New Lambton, NSW, Australia; 16 Henry Ford

Hospital, Detroit, MI; 17 Prince of Wales Hospital, Sydney, NSW,

Australia; 18 Liver Institute of Virginia, Richmond, VA; 19 Digestive

Research Center, San Antonio, TX

Background: PBC patients with an incomplete biochemical

response to ursodeoxycholic acid (UDCA) are at increased

risk for disease progression and with limited treatment options.

NGM282 is a novel engineered variant of FGF-19 that inhibits

the CYP7A1-mediated bile acid (BA) synthesis in animals and

healthy volunteers. The biologic activity was therefore evaluated

in PBC patients. Methods: 45 subjects with a baseline

(BL) alkaline phosphatase (ALP) >1.67xULN after 1yr of UDCA

were randomized to NGM282 0.3 or 3mg vs placebo (PBO)

as a daily SC injection for 28d. Change from BL ALP was the

primary endpoint, with key secondary efficacy endpoints of

liver chemistries and BA synthesis (7a-hydroxy-4-cholesten-3-

one or C4). Pruritus was measured at all study visits with the

5D Itch and Visual Analogue Score (VAS). Post-hoc sub-analyses

evaluated ALP response by BL ALP< or >3xULN. Results:

All study arms were balanced for typical PBC patient characteristics

(female=91%, mean age=56y, mean BL ALP=297

IU/L, mean UDCA dose=15mg/kg/d). Pruritus at BL was 54%,

64% and 67% of PBO, 0.3mg and 3mg arms, respectively.

NGM282-treated subjects had a significant, dose-dependent

reduction in ALP vs PBO (p3xULN (Table 1). Significant reductions were also

seen in liver chemistries (p90% supporting a potent biologic effect. NGM282

was safe and well tolerated with no observed safety signals.

Adverse events occurring >10% of both NGM282 treatment

arms were diarrhea (PBO=6.7%, 0.3mg=21.4%, 3mg=25%),

headache (PBO=6.7%, 0.3mg=14.3%, 3mg=25%) and nausea

(PBO=6.7%, 0.3mg=14.3%, 3 mg=12.5%), the majority

of which were mild. There was no clinically significant evidence

of drug-induced pruritus by either VAS or 5D itch. Conclusions:

These data demonstrate the biologic activity of NGM282 in

PBC and support a therapeutic potential in other BA-mediated

diseases. Studies are ongoing of longer duration, increased

dose and BL predictors of response to identify optimal PBC

patient populations for potential treatment with NGM282.


Table 1 Mean change in liver and BA Parameters


Marlyn J. Mayo - Grant/Research Support: Intercept, Salix, NGM, Lumena,


Stuart K. Roberts - Board Membership: AbbVie, Gilead

Tarek I. Hassanein - Advisory Committees or Review Panels: AbbVie, Bristol-Myers

Squibb; Grant/Research Support: AbbVie Pharmaceuticals, Obalon, Bristol-Myers

Squibb, Eiasi Pharmaceuticals, Gilead Sciences, Janssen R&D, Idenix

Pharmaceuticals, Ikaria Therapeutics, Merck Sharp & Dohme, NGM BioPharmaceuticals,

Ocera Therapeutics, Salix Pharmaceuticals, Sundise, TaiGen Biotechnology,

Takeda Pharmaceuticals, Vital Therapies, Tobria; Speaking and

Teaching: Baxter, Bristol-Myers Squibb, Gilead, Salix, AbbVie

Barbara A. Leggett - Advisory Committees or Review Panels: MSD, MSD, MSD,

MSD; Speaking and Teaching: Roche, Roche, Roche, Roche, Gilead

Andrew J. Muir - Advisory Committees or Review Panels: BMS, Gilead, Janssen,

Merck; Consulting: Theravance; Grant/Research Support: Abbvie, Abbvie, BMS,

Gilead, Janssen, Merck, Achillion, Lumena

Geoff McCaughan - Advisory Committees or Review Panels: Gilead

Stuart C. Gordon - Advisory Committees or Review Panels: Janssen; Consulting:

Merck, Gilead, BMS, CVS Caremark, Amgen, AbbVie; Grant/Research Support:

Merck, Gilead, AbbVie, Intercept Pharmaceuticals, Exalenz Sciences, Inc., BMS

Peter W. Angus - Advisory Committees or Review Panels: Gilead Sciences, BMS;

Grant/Research Support: Gilead sciences

Mitchell L. Shiffman - Advisory Committees or Review Panels: Merck, Gilead,

Boehringer-Ingelheim, Bristol-Myers-Squibb, Abbvie, Janssen, Acchillion; Consulting:

Roche/Genentech; Grant/Research Support: Merck, Gilead, Boehringer-Ingelheim,

Bristol-Myers-Squibb, Abbvie, Beckman-Coulter, Achillion, Lumena,

Intercept, Novartis, Gen-Probe; Speaking and Teaching: Roche/Genentech,

Merck, Gilead, Abbvie, Janssen, Bayer

Lei Ling - Employment: NGM Biopharmaceuticals, Inc.

Jian Luo - Employment: NGM Biopharmaceuticals

Michael Elliott - Employment: NGM; Stock Shareholder: NGM

Stephen Rossi - Employment: NGM Biopharmaceuticals, Inc; Stock Shareholder:

NGM Biopharmaceuticals, Gilead Sciences

Alex M. DePaoli - Employment: NGM Biopharmaceuticals

Alex J. Thompson - Advisory Committees or Review Panels: Gilead, Abbvie,

BMS, Merck, Spring Bank Pharmaceuticals, Arrowhead, Roche; Grant/Research

Support: Gilead, Abbvie, BMS, Merck; Speaking and Teaching: Roche, Gilead,

Abbvie, BMS

The following authors have nothing to disclose: Alan J. Wigg, Hays Arnold, John

P. Bate, Martin Weltman, Elizabeth J. Carey, Steven J. Bollipo, Stephen Riordan,

Elisa Young

in non-diabetic NASH derived from the PIVENS vitamin E and

placebo groups and from non-diabetics in the FLINT placebo

group. Methods: Two efficacy measures from FLINT were

applied to our pooled data: histologic improvement, defined

as ≥ 2 point improvement in NAS with no worsening of fibrosis

or NASH resolution. Safety estimates paralleled those used in

FLINT and included incidence of cardiac events and changes in

lipid levels. Logistic regression models were used to summarize

the odds ratio (OR) effects, confidence limits, and p-values on

the pooled vitamin E treatment vs no vitamin E treatment efficacy

estimates; Fisher’s exact test was used to assess cardiac

events. Results: A total of 250 patients were randomized to

vitamin E (n=80) or placebo (n=72) in PIVENS or to placebo

(n=98) in FLINT and had both baseline and end-treatment liver

biopsies; 53 had diabetes (21%) and 197 were non-diabetic

(79%); 105 (42%) received vitamin E and 145 (58%) did not

in the PIVENS or FLINT trials. Vitamin E use was associated

with histologic improvement in diabetic (OR 4.4, 95% CI 1.1,

18.0, p=0.04) and non-diabetic patients (OR 3.1, 95% CI

1.7, 5.8, p


decompensation, reduced risk for hepatocellular carcinoma,

and reduced liver-related mortality. We propose that cure of

CHC (defined as SVR of at least 12 weeks) can also lead to

regression of advanced fibrosis and/or cirrhosis and that Fibroscan

elastography may be used to confirm reversal of fibrosis.

METHODS: We conducted a retrospective chart review to

identify patients treated for CHC with concomitant advanced

fibrosis or cirrhosis based on clinical features, Fibroscan scores

and/or biopsy. Advanced fibrosis was defined as FibroScan

score 11-13.9Kpa and cirrhosis was defined as Fibroscan

score > 14KPa. This was followed by prospective and retrospective

Fibroscan and clinical data collection at 6 month intervals,

up to 14 years. RESULTS: 201 subjects were enrolled, 58

are currently eligible for analysis with at least 2 measurements

of fibrosis. Mean age was 60.7 years, 70.7% were male, and

most were Non-Hispanic (81%) white (91.4%). At baseline

25.9% had hypertension, 12.1 % had diabetes, 12.1% had

hyperlipidemia and the average BMI was 27.7 (SD 5). Of

the 34 subjects who had cirrhosis at baseline, 18 (52.9%)

demonstrated improvement by Fibroscan, with a median time

to improvement of 2.8 years (IQR 1.0-3.5). Improvement was

defined as a change in stage of fibrosis rather than a change in

Fibroscan score. Of the 17 subjects who had advanced fibrosis,

16 (66.7%) demonstrated improvement, with a median time of

2.0 years (IQR 1.2-2.5). Gender, BMI, age, individual medical

problems and HCV genotype were not found to be associated

with improvement or worsening of baseline liver disease. ALT,

albumin and INR changes did not correlate with fibrosis regression.

However, platelet count change (increased counts in subjects

who demonstrated improved liver disease) approached

statistical significance with a p=0.06 in subjects with cirrhosis.

CONCLUSION: The majority of our subjects, 34/58 (58.6%),

with advanced fibrosis or cirrhosis demonstrated improvement

by Fibroscan scores after a median follow-up (after treatment

of CHC) of 2.5 years (range: 0.5-14 years, IQR: 1- 3.5 years).

We have yet been not able to establish factors that may predict

improvement of cirrhosis or advanced fibrosis. Most enrolled

patients are still awaiting follow-up Fibroscan test results anticipated

to be completed in 2015.


Catherine T. Frenette - Speaking and Teaching: Bayer, Salix, Gilead; Stock

Shareholder: Gilead

Paul J. Pockros - Advisory Committees or Review Panels: Janssen, Merck, BMS,

Gilead, AbbVie; Consulting: Lumena, Beckman Coulter; Grant/Research Support:

Intercept, Janssen, BMS, Gilead, Lumena, Beckman Coulter, AbbVie, RMS,

Merck; Speaking and Teaching: AbbVie, Janssen, Gilead

The following authors have nothing to disclose: Ana Maria Crissien, William B.

Minteer, Jason J. Pan


Circulating extracellular vesicles and their microRNA

cargos are potential novel biomarkers with a functional

role of miR-122 in monocyte activation in alcoholic hepatitis

Banishree Saha, Fatemah Momen-Heravi, Shashi Bala, Karen

Kodys, Gyongyi Szabo; Medicine, UMASS Med School, Worcester,


Purpose: Alcohol and its metabolites induce hepatocyte damage

and recruitment of inflammatory monocytes/macrophages

and neutrophils leading to alcoholic hepatitis. Currently there

is no reliable biomarker of alcoholic hepatitis. Extracellular

vesicles (EVs) found in the circulation carry mRNA, microRNA

(miRs) and proteins. EVs can serve as biomarkers and mediate

intercellular communication. miR-122 is abundantly expressed

in hepatocytes, not in immune cells and increased levels of circulating

miR-122 were found in liver injury. We hypothesized

that EV-associated miRs can serve as biomarkers and modulate

intercellular signaling between hepatocytes and immune

cells in alcoholic hepatitis. Methods: EVs were isolated from

chronic alcohol-fed (5 weeks of Lieber DeCarli diet) or pair-fed

mice sera and from serum of patients with alcoholic hepatitis.

EVs were characterized by transmission electron microscopy,

western blot, nanoparticle tracking analysis system and

miRNA analysis. Results: The total number of circulating EVs

was significantly increased in alcohol-fed mice as compared

to control mice. Exosomes (40-150nm) represented most of

the EVs (~80%). MicroRNA array of circulating EVs revealed

a significant increase of 7 inflammatory miRs including: miR-

192, 122, 30a, 744, 1246, 30b and miR-130a in alcohol-fed

mice compared to controls. The ROC analyses indicated excellent

diagnostic value of miR-192, 122, and 30a to identify

alcohol-induced liver injury. In patients with acute alcoholic

hepatitis, we found a significant increase in the number of

circulating EVs compared to normal controls and miR-192 and

miR-30a were significantly increased in the EVs from alcoholic

hepatitis patients. Serum miR-122 was increased after alcohol

binge drinking. In the liver, miR-122 is abundantly expressed

in hepatocytes and monocytes/macrophages have low levels.

In vitro experiments revealed that exosomes derived from

ethanol-treated human hepatocytes were taken up by monocytes

and transferred mature miR-122 into monocytes. This horizontally

transferred miR-122 inhibited the hemeoxygenase-1

expression, a target of miR-122 and sensitized monocytes to

LPS stimulation to increase production of pro-inflammatory cytokines,

TNF-α and IL-1β; all of these effects were inhibited by

exosome-mediated delivery of a miR-122 inhibitor in monocytes.

Conclusion: Elevated levels of EVs and their miR signature

could serve as biomarkers of alcoholic hepatitis. This study

reveals a novel EV-mediated mechanism of alcohol-induced

communication between hepatocytes and monocytes by transferring

hepatocyte-derived miR-122 that reprograms monocytes

promoting inflammation in alcoholic hepatitis.


The following authors have nothing to disclose: Banishree Saha, Fatemah

Momen-Heravi, Shashi Bala, Karen Kodys, Gyongyi Szabo


Fat-specific Protein 27/CIDEC Promotes Alcoholic Steatohepatitis

in Mice and Humans

Ming-Jiang Xu 1 , Yan Cai 1 , Hua Wang 1 , José T. Altamirano 2 ,

Gemma Odena 3 , Frank J. Gonzalez 4 , Ramon Bataller 3 , Bin Gao 1 ;

1 NIAAA, NIH, Rockville, MD; 2 Vall d’Hebron Institut de Recerca,

Barcelona, Spain; 3 University of North Carolina, Chapel Hill, NC;

4 National Institutes of Health, Bethesda, MD

Objectives: Alcoholic steatohepatitis (ASH) is the progressive

form of alcoholic liver disease that leads to cirrhosis and

hepatocellular carcinoma. There is an urgent need to identify

molecular drivers in order to develop targeted therapies.

Here the functions of mouse fat-specific protein 27 (Fsp27)/

human cell death activator CIDEC (the human homologue of

Fsp27) were investigated. Methods and results: We developed

a mouse model with chronic (8 weeks)-plus-binge ethanol feeding,

which mimics the drinking patterns of alcoholic hepatitis

(AH) patients, produced severe ASH and mild fibrosis. Microarray

analyses revealed that mouse Fsp27/human CIDEC gene

was increased in this animal model and human ASH samples.

Fsp27 is expressed at high levels in adipose tissues but at

very low levels in normal liver. Chronic-plus-binge ethanol feeding

markedly upregulated hepatic Fsp27 mRNA and protein

expression. Silencing the Fsp27 gene by shRNA or genetic

disruption ameliorated chronic-plus-binge ethanol-induced


ASH. Inhibition of peroxisome proliferator-activated receptor

g (PPARg) or cyclic-AMP-responsive-element binding protein H

(CREBH) attenuated chronic-plus-binge ethanol-induced elevation

of Fsp27a and FSP27b mRNA, respectively, and subsequently

ameliorated liver injury. Overexpression of Fsp27 and

ethanol exposure synergistically induced mitochondrial reactive

oxygen species production and hepatocyte injury in vivo and

in vitro, which is likely owing to the mitochondrial location of

FSP27 leading to the decreased mitochondrial complex I activity.

Finally, hepatic expression of CIDEC mRNA was elevated

and positively correlated with hepatic steatosis, disease severity,

and mortality in AH patients. Conclusion: FSP27/CIDEC

gene product promotes ASH in chronic-plus-binge ethanol-fed

mice and in human AH. Targeting CIDEC gene is likely to be a

novel therapeutic targets for the treatment of ASH.

overgrowth was similar to WT mice, alcohol-fed Reg3b and

Reg3g deficient mice showed a significantly higher number of

mucosa-associated bacteria in the small intestine as compared

with their respective WT littermates. This was accompanied

by a significantly increased number of bacteria translocating

through intestinal epithelial cells and more positive mesenteric

lymph node cultures in alcohol-fed Reg3b -/- and Reg3g -/- mice.

Interestingly, absence of Reg3b or Reg3g did not affect the

intestinal paracellular barrier function as determined by systemic

LPS level and fecal albumin. Most importantly, Reg3b

and Reg3g deficient mice showed more alcoholic liver disease

as assessed by higher plasma ALT levels, increased hepatic

triglycerides and inflammation. To corroborate our data, we

generated transgenic mice overexpressing Reg3g under the

control of the intestine specific villin promoter. Consistent with

our results, Reg3g transgenic mice showed significantly less

mucosa-associated bacteria than WT littermates after alcohol

feeding. Overexpression of intestinal Reg3g suppressed

translocation of bacteria through intestinal epithelial cells to

mesenteric lymph nodes, while paracellular permeability and

systemic LPS levels were not changed after alcohol feeding.

Reg3g transgenic mice were protected from alcohol-induced

liver injury, steatosis and inflammation. Conclusion: Intestinal

Reg3b and Reg3g prevent bacterial colonization of epithelial

surfaces, which reduces translocation of viable bacteria and

prevents alcoholic liver disease. Reg3 proteins are novel and

promising targets in the treatment of alcohol-induced liver disease.


The following authors have nothing to disclose: Lirui Wang, Peng Chen, Lora V.

Hooper, Bernd Schnabl


Ramon Bataller - Advisory Committees or Review Panels: Sandhill; Consulting:

VTI, Oncozyme Pharma

The following authors have nothing to disclose: Ming-Jiang Xu, Yan Cai, Hua

Wang, José T. Altamirano, Gemma Odena, Frank J. Gonzalez, Bin Gao


Antimicrobial proteins Reg3b and Reg3g protect mice

from alcoholic liver disease by preventing bacterial


Lirui Wang 1 , Peng Chen 1 , Lora V. Hooper 2 , Bernd Schnabl 1 ;

1 University of California San Diego, La Jolla, CA; 2 Department of

Immunology, The University of Texas Southwestern Medical Center,

Dallas, TX

Background: Antimicrobial proteins are secreted by intestinal

epithelial cells and Paneth cells. They represent the first

line of defense against pathogens and maintain homeostasis

with commensal bacteria. We have previously shown that the

expression of the C-type lectin regenerating islet derived-3

(Reg3) is suppressed in the small intestine after chronic alcohol

use in mice and humans, yet functional consequences of

lower Reg3 expression on the intestinal microbiota, bacterial

translocation and alcoholic liver disease are unknown. The

aim of our study was to investigate the role of Reg3b and

Reg3g in chronic alcoholic liver disease. Methods and Results:

A Lieber-DeCarli model was used to induce intestinal dysbiosis,

bacterial translocation and liver disease in mice. After alcohol

feeding for 8 weeks, wild-type (WT) littermate mice showed

bacterial overgrowth of the luminal and the mucosa-associated

microbiota in the small intestine. While luminal bacterial


Hepatocytes from mice on intragastric feeding model of

alcoholic steatohepatitis release extracellular vesicles

with specific microRNA cargo that modulate hepatic

stellate cell and macrophage phenotype

Akiko Eguchi 1 , Jihoon Kim 1 , Lucila Ohno-Machado 1 , Hidekazu

Tsukamoto 2 , Ariel E. Feldstein 1 ; 1 UCSD, La Jolla, CA; 2 USC, Los

Angeles, CA

Liver inflammation and fibrosis are key histological features

associated with prognosis in patients with alcoholic steatohepatitis

(ASH). Extracellular vesicles (EVs) are released during cell

stress or demise, can contain a barcode of the cell of origin

including specific microRNAs and are growingly recognized

as key cell-to-cell communicators. Here we tested the hypothesis

that during ASH development, hepatocyte damage release EVs

with a microRNA signature that can fuse with hepatic stellate

cells (HSC) and Kupffer cells (KC) to regulate their phenotype.

Methods: C57/B6 mice were placed on intragastric feeding

model of continuous ethanol infusion or control diet for 4 weeks

to reproduce a physiologically relevant model of ASH. The

extent of steatosis, inflammation, and fibrosis was assessed

by histological and molecular analyses on liver specimens.

Isolated hepatocytes (HC) or KC from ASH or control mice

were incubated in medium-EV free serum and HC- or KC-derived

EVs were isolated by ultracentrifugation from culture

medium. A complete characterization of EVs was performed

by FACS, electron microscopy, dynamic light scattering. HC-EV

biological function was investigated in isolated primary HSCs

or KCs derived from C57/B6 mice by cell morphology or

qPCR. Comprehensive encapsulated miRNA in HC-EVs were

assessed via transcriptome using illumina miRNA-seq. Results:

We observed highly significant differences in the levels of HCor

KC-EVs between control group and ASH (1.3x10 6 HC-EV/


ml of medium in ASH versus 1.4x10 5 HC-EV/ml of medium

in control, p


ing significant reduction in certain long- (hexadecanoic and

heptadecanoic), medium (hexanoic and octanoic), and short

(butanoic) free fatty acids. Remarkably, the levels of octanoic

acid (known to have antimicrobial properties) were dramatically

reduced (~48 fold) in mice fed USF+EtOH compared to

SF+EtOH fed animals. A decline in certain fecal amino acids

(e.g. serine and glycine) was observed in USF+EtOH fed animals.

Conclusions: These data support an important role of

dietary lipids in ALD pathogenesis, and provide insight into

mechanisms of ALD development. A diet enriched in USF not

only enhanced alcohol-induced liver injury, but also caused

major fecal metagenomic and metabolomic changes that may

play an etiologic role in observed liver injury. Characterization

of both microbiota composition and function is an important

approach to investigate host–microbial interaction. Our data

suggest that dietary lipids can potentially serve as interventions

for the prevention/treatment of ALD.


Shirish Barve - Speaking and Teaching: Abbott

Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco,

Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline;

Speaking and Teaching: Roche

The following authors have nothing to disclose: Irina Kirpich, Wenke Feng, Xinmin

Yin, Xiaoli Wei, Xiang Zhang


PEGylated TRAIL treatment ameliorates liver cirrhosis in

rats by targeting activated hepatic stellate cells

Ogyi Park 1,2 , Yumin Oh 1,2 , Magdalena Swierczewska 1,2 , Jong

Sung Park 1,2 , Justin Hanes 2 , Martin Pomper 1 , Bin Gao 3 , Seulki

Lee 1,2 ; 1 The Russell H. Morgan Department of Radiology and

Radiological Sciences, Johns Hopkins University, Baltimore, MD;

2 The Center for Nanomedicine at the Wilmer Eye Institute, Johns

Hopkins University School of Medicine, Baltimore, MD; 3 Laboratory

of Liver Diseases, National Institute of Alcohol Abuse and

Alcoholism, National Institutes of Health, Bethesda, MD

Background: Liver fibrosis is a common outcome of chronic

liver disease and leads to liver failure and cancer. Still, no targeted

anti-fibrotic therapy exists at this time. Activated hepatic

stellate cells (aHSCs) are the originators of liver fibrosis; therefore,

eradication of aHSCs is a logical strategy to stop and/

or reverse liver fibrosis. However, there are no effective strategies

to specifically deplete apoptosis-resistant aHSCs during

fibrosis without systemic toxicity. TRAIL is known to selectively

induce apoptosis in cancer cells or transformed cells by binding

to its death receptors (DRs) while sparing normal tissue.

But because recombinant TRAIL suffers from a short half-life

and low potency, it failed to show efficacy in cancer clinical

trials. Here we introduce a longer-acting, more stable form

of TRAIL that shows striking anti-fibrotic activity in rat fibrosis

and cirrhosis models with no detected hepatotoxicity. Methods:

We developed PEGylated TRAIL (TRAIL PEG

) by stabilizing a

potent homotrimer TRAIL of iLZ-TRAIL with a 5kDa PEG. In in

vivo studies, SD rats were induced liver fibrosis and cirrhosis

by CCl 4

and treated with 4 mg/kg of TRAIL PEG

daily for 10

days for fibrosis and two weeks for cirrhosis. In in vitro studies,

primary human HSCs were culture-activated and TRAIL PEG

effects were investigated on quiescent and activated HSCs.

Primary human hepatocytes were used to determine hepatotoxicity


. Results: Intravenously injected TRAIL PEG

has a markedly extended half-life in non-human primates and

no toxicity in primary human hepatocytes. We found DRs are

upregulated in human cirrhotic livers because of aHSCs. In

vitro primary human aHSCs, but not quiescent HSCs, become

sensitive to TRAIL PEG

-induced apoptosis via DR- and DISC-activated

caspase-8-dependent mechanisms. Intravenous TRAIL PEG

directly induced apoptosis of aHSCs and ameliorated CCl 4


fibrosis/cirrhosis in rats by simultaneously down-regulating

multiple key fibrogenic molecules in vivo. TRAIL PEG


caused selective toxicity to hepatocellular carcinoma (HCC)

cells. Conclusions: TRAIL-based therapies could serve as firstin-class

therapeutics for liver fibrosis/cirrhosis, cirrhosis-associated

HCC and possibly other fibrotic diseases. Our results not

only introduce the unique activity that TRAIL-based compounds

have in liver fibrosis but also warrant clinical translation of


for severe liver fibrosis therapy.


The following authors have nothing to disclose: Ogyi Park, Yumin Oh, Magdalena

Swierczewska, Jong Sung Park, Justin Hanes, Martin Pomper, Bin Gao,

Seulki Lee


Statins, ACE inhibitors and ARB use is associated with

reduced mortality and morbidity in chronic liver diseases

– a nationwide cohort study in Sweden

Knut Stokkeland 1,2 , Christine Takami Lageborn 3 , Anders Ekbom 4 ,

Jonas Höijer 5 , Matteo Bottai 5 , Per Stål 6 , Karin Söderberg Löfdal 7 ;

1 Department of Medicine, Visby Hospital, Visby, Sweden; 2 Karolinska

Institutet, Department of Medicine, Gastroenterology and

Hepatology, Stockholm, Sweden; 3 Karolinska Institutet, Stockholm,

Sweden; 4 Department of Medical Epidemiology and Biostatistics,

Karolinska Institutet, Stockholm, Sweden; 5 Unit of Biostatistics,

IMM, Karolinska Institutet, Stockholm, Sweden; 6 Division of Hepatology,

Karolinska Hospital, Stockholm, Sweden; 7 Division of Clinical

Pharmacology, Department of Laboratory Medicine, Karolinska

Institutet, Stockholm, Sweden

Aim: To explore whether current medication in patients with

chronic liver diseases affect overall mortality, liver-related mortality

and liver-related morbidity. Methods: We performed a

register-based cohort study in Sweden with patients with a

first-time diagnosis of chronic liver disease between 2005 and

2012. We studied the use of statins, angiotensin converting

enzyme inhibitors (ACEi), angiotensin receptor blockers (ARB),

acetylsalicylic acid (ASA), non-steroidal anti-inflammatory

drugs (NSAID), selective seretonin re-uptake inhibitors (SSRI)

and antibiotics. We measured total mortality, liver-specific

mortality and liver-specific morbidity. We used register data

from the Patient Register, the Prescribed Drug Register and the

Death Certificate Register. Liver-specific morbidity and mortality

included all liver diseases, esophageal varices and liver

cancer. Results: We analyzed 70 546 patients who had been

seen in hospital out-patient services or hospitalized with chronic

liver disease for the first time between 2005 and 2012. We

found a reduction in mortality risk for statin users (n= 8 688)

with a hazard ratio range between 0.42 (95% CI: 0.28-0.63)

for patients with autoimmune hepatitis and 0.91 (0.63-1.32)

for primary biliary cirrhosis with a similar risk reduction for

liver-specific mortality and liver-related morbidity. There was

a significant mortality reduction for patients exposed to ARB

(n= 6204) between 0.62 (0.53-0.72) in alcoholic liver disease

and 0.91 (0.64-1.32) in primary biliary cirrhosis and a similar

reduction for patients exposed to ACEi (n=9 714). There was

a significant reduction of liver morbidity in patients exposed to

ASA (n= 9 768) for all chronic liver diseases, even among users

of NSAID (n= 14 119). An increased risk for all-cause mortality

was seen for almost all liver diseases in patients exposed to

SSRI (n= 11 048), with a range between 1.25 (1.16-1.35) and

1.61 (1.30-1.98), and a reduction in risk of liver-specific morbidity

for almost all diagnoses. There was a reduction in risk for

liver morbidity for patients exposed to antibiotics (n= 27 756)

between 0.55 (0.44-0.70) and 0.77 (0.73-0.80). Conclusion:


Several frequently used drug classes in patients with chronic

liver disease are associated with reduced all-cause mortality,

and liver-specific morbidity and mortality. Statin, ACEi and

ARB use was associated with lower risk of total mortality, liver-specific

mortality and liver-specific morbidity. ASA use was

associated with reduced liver-specific mortality, and NSAID,

SSRI and antibiotics exposure was associated with a reduction

in risk for liver morbidity.


Anders Ekbom - Advisory Committees or Review Panels: Centocor, Schering-Plough,

Centocor, Schering-Plough, Centocor, Schering-Plough, Centocor,

Schering-Plough; Speaking and Teaching: Astra Zeneca, Astra Zeneca, Astra

Zeneca, Astra Zeneca

The following authors have nothing to disclose: Knut Stokkeland, Christine

Takami Lageborn, Jonas Höijer, Matteo Bottai, Per Stål, Karin Söderberg Löfdal

that becomes significantly more accurate than its 2 composite

tests, and solves discrepancies between these tests. The

combined test that associates elastography and a blood test,

used with its detailed fibrosis classification, should become the



Victor de Ledinghen - Board Membership: Janssen, Gilead, BMS, Abbvie; Speaking

and Teaching: AbbVie, Merck, BMS, Gilead

Vincent Leroy - Board Membership: Abbvie, BMS, Gilead; Consulting: Janssen,

MSD; Speaking and Teaching: Abbvie, BMS, Gilead, Janssen, MSD

Isabelle Fouchard-Hubert - Speaking and Teaching: JANSSEN, BMS, GILEAD,


Paul Cales - Consulting: BioLiveScale

The following authors have nothing to disclose: Jerome Boursier, Oberti Frederic


Evaluation of EASL recommendation for the non-invasive

diagnosis of liver fibrosis in chronic hepatitis C

Jerome Boursier 1,3 , Victor de Ledinghen 2 , Vincent Leroy 4 , Oberti

Frederic 1,3 , Isabelle Fouchard-Hubert 1,3 , Paul Cales 1,3 ; 1 Hepato-gastroenterology

Department, University Hospital, Angers,

France; 2 Hepatology Department, University Hospital, Bordeaux,

France; 3 HIFIH Laboratory EA3859, University, Angers, France;

4 Hepato-Gastroenterology Department, University Hospital, Grenoble,


Introduction. The EASL has proposed the first recommendations

about non-invasive testing for the evaluation of liver disease

severity. For chronic hepatitis C (CHC), it is proposed to associate

transient elastography (TE) with a blood test (BT), and to

accept the diagnosis if both are in agreement. We aimed to

validate this rule and to improve it by using a combined fibrosis

test that simultaneously associates both BT and TE (BET). Methods.

679 CHC patients with liver biopsy, TE, and a patented BT

were included. The primary outcome was severe fibrosis (Metavir

F≥3, prevalence: 31.9%). Diagnostic cut-off for this target

was evaluated according to the maximum Youden index or the

threshold between the 2 classes F2±1 and F3±1 of a detailed

fibrosis classification including 6 fibrosis classes. Results.

AUROC was: BT: 0.829, TE: 0.846, BET: 0.882 (p


Hans Huber - Consulting: MiNA Therapeutics; Stock Shareholder: Merck & Co

Robert Habib - Board Membership: MiNA Therapeutics Ltd; Management Position:

MiNA Therapeutics Ltd; Stock Shareholder: MiNA Therapeutics Ltd

Pål Sætrom - Patent Held/Filed: MiNA Therapeutics, Ltd; Stock Shareholder:

MiNA Therapeutics, Ltd

Nagy Habib - Board Membership: EMcision Limited, Omnicyte Limited, Apterna

Limited, MiNA Therapeutics Ltd; Patent Held/Filed: EMcision Limited, Omnicyte

Limited; Stock Shareholder: EMcision Limited, Omnicyte Limited, Apterna Limited,

MiNA Therapeutics Ltd

The following authors have nothing to disclose: Kai-Wen Huang, Anjaneyulu

Muragundla, Aravindakshan Jayaprakash, Prashant Vadnal, John Rossi


Fibrosis is not just fibrosis - Extracellular matrix turnover

is markedly different in two types of viral hepatitis, suggesting

different fibrotic representations

Mette J. Nielsen 1,2 , Morten A. Karsdal 1 , Konstantin Kazankov 3 ,

Aleksander Krag 2 , Diana J. Leeming 1 , Jacob George 4 , Henning

Gronbaek 3 , Detlef Schuppan 5,6 ; 1 Nordic Bioscience A/S, Herlev,

Denmark; 2 Department of Gastroenterology and Hepatology,

Odense University Hospital, University of Southern Denmark,

Odense, Denmark; 3 Department of Medicine V, Aarhus University

Hospital, Aarhus, Denmark; 4 Storr Liver Unit, Westmead Millennium

Institute, Westmead Hospital and University of Sydney,

Sydney, NSW, Australia; 5 Institute of Translational Immunology,

University Medical Center, Mainz, Germany; 6 Division of Gastroenterology,

Beth Israel Deaconess Medical Center, Harvard Medical

School, Boston, MA

Background and aim: Fibrosis is the result of a dysregulated

tissue remodelling leading to excessive and abnormal accumulation

of extracellular matrix (ECM). The developmental pattern

of fibrosis depends on the underlying aetiology causing the

fibrosis such as; portal-portal, portal-central, or central-central

septa, albeit the molecular composition and turnover remain

to be investigated. In this study we investigated the level of

ECM remodelling in two seemingly similar types of viral hepatitis,

namely hepatitis B and C. Methods: In a cross-sectional

study design, specific protein fragments of matrix metalloprotease

degraded type I, III, IV and VI collagen (C1M, C3M,

C4M, C6M) and type III and IV collagen formation (Pro-C3

and P4NP7S) were assessed in plasma from 403 chronic

hepatitis C patients and 197 chronic hepatitis B patients by

specific ELISAs. The grade of inflammation and fibrosis was

scored according to the Metavir Activity and Fibrosis Scoring

systems based on liver biopsy. Results: Plasma levels of

P4NP7S, C3M, C4M, and C6M were significantly elevated

in HBV compared to HCV (p



Objective Determination of Hepatitis B Phenotype using

Biochemical and Serological Markers: Immune Tolerant,

Immune Active, Inactive Carrier and Indeterminant Status

Adrian M. Di Bisceglie 1 , Manuel Lombardero 2 , Jeffrey Teckman 1 ,

Lewis R. Roberts 3 , Harry L. Janssen 4 , Steven H. Belle 2 , Jay H. Hoofnagle

5 ; 1 Saint Louis University, St. Louis, MO; 2 University of Pittsburgh,

Pittsburgh, PA; 3 Mayo Clinic, Rochester, MN; 4 University of

Toronto, Toronto, ON, Canada; 5 NIH, Bethesda, MD

Background: HBV infection is frequent world-wide and may

result in progressive liver disease or HCC. Effective therapies

are available but treatment is usually reserved for patients (pts)

with selected biochemical and serological profiles (phenotypes).

Currently, criteria for defining HBV phenotype are not

standardized and are based upon expert opinion rather than

medical evidence. Aim: To determine the distribution of phenotypes

in a large cohort of North American pts with chronic

HBV infection using standardized definitions and calculation.

Methods: Epidemiologic, demographic, biochemical and virologic

features of pts enrolled into the HBRN Cohort Study at 19

US and 1 Canadian site were analyzed, assigning pts into 1

of 4 phenotypes: immune tolerant (IT), chronic hepatitis B with

(CHB e+) or without HBeAg (CHB e-) or inactive carrier (IC)

based upon baseline HBV DNA and ALTs. Cut-off HBV DNA

values used to define phenotypes were 10 4 IU/ml for e-negative

and 10 5 for e-positive pts. ALT cut-offs were analyzed using

either fixed values (30 U/L for men, 20 U/L for women) or

using each local lab ULN. Pts not fitting into a phenotype were

designated “Indeterminant”. Independently, local investigators

assigned a phenotype at baseline based on available laboratory

values and clinical impression. Results: 1398 adults (51%

male, 71% Asian) had data needed to determine phenotype.

The table shows the distribution of phenotypes calculated by

two methods of assessing ALT and physician assessment. Only

moderate agreement was found between clinician-determined

and calculated phenotype using fixed ALTs (Kappa 0.39, 95%

CI 0.36-0.42). Of note, only 13% of pts were designated Indeterminant

by clinicians compared to 20-40% by computer calculation.

Using the Fixed ALT groups for comparison, IT was

most distinct, being generally younger, more frequently Asian

and female. The most frequent clinical phenotype identified

was Indeterminant, the majority of whom (83%) had elevated

ALT values despite low levels of HBV DNA, not apparently

associated with higher BMI, alcohol consumption or HBV genotype.

Clinician estimates often differed from the calculated

phenotype. Conclusions: The clinical significance of HBV phenotypes

using these standardized definitions requires further

study based on long term follow up of these patient cohorts but

there is clearly a need to re-examine categorization of pts with

chronic HBV infection to more accurately predict their outcomes

and need for therapy.


Adrian M. Di Bisceglie - Advisory Committees or Review Panels: Gilead, AbbVie,

Novartis, Bayer, BTG; Grant/Research Support: Gilead, AbbVie

Jeffrey Teckman - Consulting: Dicerna, Isis Pharmaceuticals, Vertex, Proteostasis,

Genkyotex, The Alpha-1 Project, Retrophin, RxCelerate, Velgene; Grant/

Research Support: Alnylam, Arrowhead, Alpha-1 Foundation, Gilead

Lewis R. Roberts - Grant/Research Support: Bristol Myers Squibb, ARIAD Pharmaceuticals,

BTG, Wako Diagnostics, Inova Diagnostics, Gilead Sciences, Five

Prime Therapeutics

Harry L. Janssen - Consulting: AbbVie, Bristol Myers Squibb, GSK, Gilead Sciences,

Innogenetics, Merck, Medtronic, Novartis, Roche, Janssen, Medimmune,

ISIS Pharmaceuticals, Tekmira; Grant/Research Support: AbbVie, Bristol Myers

Squibb, Gilead Sciences, Innogenetics, Merck, Medtronic, Novartis, Roche,

Janssen, Medimmune

The following authors have nothing to disclose: Manuel Lombardero, Steven H.

Belle, Jay H. Hoofnagle


Length of antiviral therapy and cirrhosis are key factors

in the development of hepatocellular carcinoma among

U.S. veterans with chronic hepatitis B

Gina Choi, Marina Serper, David E. Kaplan, Kimberly A. Forde;

Gastroenterology, University of Pennsylvania, Philadelphia, PA

Background: The risk of hepatocellular carcinoma (HCC) and

the effect of anti-viral therapy in U.S. populations with chronic

hepatitis B (HBV) is poorly defined. Aim: To examine the incidence

and impact of anti-viral therapy on the development of

HCC among a national cohort of veterans with chronic HBV.

Methods: A retrospective cohort study using the VA Corporate

Data Warehouse was performed between 1999-2013. The

primary exposure variable was antiviral therapy, defined as

≥6 months of therapy with oral nucleosides. Additional covariates

such as demographics and clinical variables such as cirrhosis,

alcohol abuse, dyslipidemia, hepatitis C (HCV), and

HIV were obtained. Multivariable Cox proportional hazard

models were used to evaluate associations between exposures

and the primary outcome of HCC. Results: A total of 26,704

veterans had a HBsAg+ result; N=9001 were confirmed to

have chronic HBV. The median follow-up time was 7.6 years

(IQR 4.2–10.9). Patients were predominantly male (96%),

white (45%) and middle aged (M=51, SD 11). A total of 20%

had HCV, 7% had HIV, and 23% had alcohol abuse. The

prevalence of dyslipidemia was 52%; 13% had cirrhosis. The

overall incidence of HCC was 5.6 per 1000 person-years;

26 per 1000 person-years with cirrhosis and 1.9 per 1000

person-years without cirrhosis. The median exposure time of

antiviral therapy was 3.6 years (IQR 1.4-7.1). A total of 38%

(N=3333) received antiviral therapy for ≥6 months; 23%

received ≥3 years and 16% received therapy for ≥5 years.

In multivariable models adjusted for age, race, cirrhosis, alcohol

abuse, and dyslipidemia, antiviral therapy ≥5 years was

associated with decreased incidence of HCC (HR 0.77, 95%

CI 0.60-0.98, p=0.03), whereas ≥4 years of antiviral therapy

was not protective (HR 1.04, CI 1.03-1.05). Antiviral therapy

for ≥5years was associated with a decreased incidence of

HCC among cirrhotics (HR 0.56, CI 0.41-0.78, p



Serum Alanine Aminotransferase (ALT) and Hepatitis

B Virus (HBV) DNA Flares In Pregnant and Postpartum

Women With Chronic Hepatitis B (CHB)

Christine Y. Chang 1 , Natali Aziz 2 , Huy N. Trinh 3 , Mindie H.

Nguyen 1 ; 1 Division of Gastroenterology and Hepatology, Stanford

University Medical Center, Palo Alto, CA; 2 Obsterics and Gynecology,

Stanford University School of Medicine, Stanford, CA; 3 San

Jose Gastroenterology, San Jose, CA

Background: During pregnancy, alterations in the immune system

allow the mother to tolerate the semiallogenic fetus, but

their effects on CHB activity are poorly understood. Methods:

To examine flares in HBV DNA (≥1 log increase from baseline)

and/or ALT (≥2xULN or baseline) during pregnancy and 6

months postpartum, we performed a retrospective study of 88

pregnancies in 74 CHB expectant mothers who were not on

anti-HBV therapy for ≥1 year prior to pregnancy at 2 U.S.

centers. Results: Most were Asian (93%), with a mean age

of 32±4, median gravida of 2 (IQR: 1-3), and median parity

of 0 (IQR: 0-1). Mean log HBV DNA was 2.0±0.8 IU/mL at

baseline in low HBV DNA mothers (≤2000 IU/mL, n=33), and

6.1±2.0 in high HBV DNA mothers (>2000 IU/mL, n=55). At

baseline, low HBV DNA mothers had lower ALT (≤x2ULN: 82%

vs. 47%, p=0.001) and were more likely to be HBeAg- (90%

vs. 44%, p



The following authors have nothing to disclose: Prabhu P. Gounder, Lisa Bulkow,

Mary Snowball, Susan Negus, Philip R. Spradling, Brian J. McMahon


Relationship between hepatocellular carcinoma development

and serum viral markers in chronic hepatitis B

patients who achieved undetectable serum HBV DNA

while on long-term nucleoside analogue therapy

Ka-Shing Cheung 1 , Wai-Kay Seto 1 , Danny Wong 2 , Ching-Lung

Lai 1 , Man-Fung Yuen 1 ; 1 Medicine, Queen Mary Hospital, The University

of Hong Kong, Hong Kong, Hong Kong; 2 Queen Mary

Hospital, Hong Kong, Hong Kong

Background: Hepatitis B surface antigen (HBsAg) and hepatitis

B core-related antigen (HBcrAg) are risk factors for the

development of hepatocellular carcinoma (HCC). Linearized

HBsAg (HQ-HBsAg) is a more sensitive assay to measure

HBsAg level. However, their roles in predicting HCC development

are unknown when there is profound viral suppression

by nucleos(t)ide analogues (NA). Methods: Seventy-six chronic

hepatitis B (CHB) patients who developed HCC in 2007-2014

despite undetectable serum HBV DNA level after at least oneyear

NA therapy were recruited. 152 CHB patients without

HCC were recruited as controls. They were matched by age,

gender, HBeAg status, cirrhosis status, undetectable serum HBV

DNA and duration of NA therapy with the 76 HCC patients in

a 2:1 ratio. Serum HBsAg, HQ-HBsAg and HBcrAg levels were

analysed and compared between the two groups. Results: A

statistically significant difference in the HBcrAg level was noted

between the HCC group and non-HCC groups (10.2 and 1.7

kU/mL respectively, p=0.005), but was not observed in HBsAg

or HQ-HBsAg levels. The area under receiver operating characteristic

curve (AUROC) was 0.61 (95% CI: 0.54-0.69) with a

negative predictive value (NPV) of 77.0% when a cutoff value

of HBcrAg level ≥7.8 kU/mL was used. The odds ratio of HCC

development was 3.27 (95% CI: 1.84-5.80). In the subgroup

analysis for non-cirrhotic patients, a statistically significant

difference in the HBcrAg level was noted between the HCC

group and non-HCC groups (10.2 and 1.0 kU/mL respectively,

p=0.001). The AUROC was 0.70 (95% CI: 0.58-0.81) with a

NPV of 80.6% when a cutoff value of HBcrAg level ≥7.9 kU/

mL was used. The odds ratio of HCC development was 5.95

(95% CI: 2.35-15.07). Conclusion: HBcrAg (but not HBsAg or

HQ-HBsAg) can predict HCC risk in CHB patients who achieve

undetectable serum HBV DNA while receiving NA therapy.

Future prospective studies are warranted to further define the

role of HBcrAg level in this group of patients.


Wai-Kay Seto - Advisory Committees or Review Panels: Gilead Science; Speaking

and Teaching: Bristol-Myers Squibb

Ching-Lung Lai - Advisory Committees or Review Panels: Bristol-Myers Squibb,

Gilead Sciences Inc; Consulting: Bristol-Myers Squibb, Gilead Sciences, Inc;

Speaking and Teaching: Bristol-Myers Squibb, Gilead Sciences, Inc

Man-Fung Yuen - Advisory Committees or Review Panels: GlaxoSmithKline,

Bristol-Myers Squibb, Pfizer, GlaxoSmithKline, Bristol-Myers Squibb, Pfizer,

GlaxoSmithKline, Bristol-Myers Squibb, Pfizer, GlaxoSmithKline, Bristol-Myers

Squibb, Pfizer; Grant/Research Support: Roche, Bristol-Myers Squibb,

GlaxoSmithKline, Gilead Science, Roche, Bristol-Myers Squibb, GlaxoSmith-

Kline, Gilead Science, Roche, Bristol-Myers Squibb, GlaxoSmithKline, Gilead

Science, Roche, Bristol-Myers Squibb, GlaxoSmithKline, Gilead Science

The following authors have nothing to disclose: Ka-Shing Cheung, Danny Wong


A New Clinically-Based Staging System for Distal Cholangiocarcinoma

Maria E. Lozada 1 , Jonggi Choi 1 , Roongruedee Chaiteerakij 1,2 ,

Ju Dong Yang 1 , Nasra H. Giama 1 , Steven Alberts 3 , Gregory J.

Gores 1 , Lewis R. Roberts 1 ; 1 Division of Gastroenterology and

Hepatology, Mayo Clinic College of Medicine, and Mayo Clinic

Cancer Center, Rochester, MN; 2 Department of Medicine, Faculty

of Medicine, Chulalongkorn University and King Chulalongkorn

Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand;

3 Division of Medical Oncology, Mayo Clinic College of Medicine,

and Mayo Clinic Cancer Center, Rochester, MN

Background: Clinical predictors of survival for each subtype of

cholangiocarcinoma, intrahepatic, perihilar and distal (dCCA)

are limited. Currently available staging systems can be used to

predict prognosis for resectable dCCA but are not relevant to

patients with unresectable disease. Based on these limitations,

we aimed to construct a clinical staging system that stratifies

dCCA patients. Methods: We identified 170 treatment-naïve

dCCA patients seen at Mayo Clinic, Rochester from January

1, 2000 through May 31, 2014. Data were retrospectively

abstracted from the electronic medical record. Overall median

survival (MS), the primary endpoint, was estimated using the

Kaplan-Meier method and compared using the log-rank test.

Cox-proportional hazards analysis was used to identify predictors

of survival. Performance of the staging system was assessed

using concordance index. Results: Of the 170 dCCA patients,

93 (58%) were male, the mean age was 67 years, and 19

(12%) had primary sclerosing cholangitis. The MS of the entire

cohort was 17.0 months (95% confidence interval [CI]: 14.0-

20.5). Baseline ECOG performance status, post-stenting CA

19-9 and disease extent were significantly associated with survival

and further incorporated into a 4-tier staging system. A

total of 143 patients were classified into the new staging system.

Overall MS for patients in Stage I, Stage II, Stage III and

Stage IV were 46.8, 14.3, 8.1, and 3.4 months, with hazard

ratios (95% CI) of 1.0 (ref.), 1.9 (1.0-3.4), 4.1 (2.3-7.0), and

9.7 (5.1-18.5), respectively. Concordance index for the new

staging system was 0.73. Conclusion: We have developed a

new staging system based on non-operative clinical variables

that classifies dCCA patients into 4 distinct stages with homogeneous

survival. Given the small cohort, validation to confirm

these findings is crucial. However, this staging system could

potentially be a key prognostic tool for better stratification of

patients enrolled in clinical trials of novel therapies for dCCA.


Gregory J. Gores - Advisory Committees or Review Panels: Conatus

Lewis R. Roberts - Grant/Research Support: Bristol Myers Squibb, ARIAD Pharmaceuticals,

BTG, Wako Diagnostics, Inova Diagnostics, Gilead Sciences, Five

Prime Therapeutics


The following authors have nothing to disclose: Maria E. Lozada, Jonggi Choi,

Roongruedee Chaiteerakij, Ju Dong Yang, Nasra H. Giama, Steven Alberts


Trends in the burden of cirrhosis and hepatocellular carcinoma

by underlying liver disease in US Veterans from


Lauren A. Beste 2 , Steven Leipertz 2 , Pamela Green 2 , Jason A. Dominitz

2,1 , David Ross 3 , George N. Ioannou 2,1 ; 1 University of Washington,

Seattle, WA; 2 Veterans Affairs Puget Sound Healthcare

System, Seattle, WA; 3 Office of Public Health, Department of Veterans

Affairs, Washington, D.C., DC

Background & Aims Cirrhosis and hepatocellular carcinoma

(HCC) are predicted to increase in the U.S. but the accuracy

of prior forecasts and the contributions from various etiologies

remain unclear. We aimed to determine the burden of

cirrhosis and HCC according to underlying etiology from

2001-2013. Methods We developed a national retrospective

cohort of Veterans Affairs (VA) patients diagnosed with cirrhosis

(n=129,998) or HCC (n=21,326) from 2001-2013.

We used laboratory results, ICD-9 codes, and biometrics to

identify underlying etiologies. Results In 2013, VA provided

care to 5,720,614 individuals, of whom 60,553 (1.06%) had

cirrhosis and 7,670 (0.13%) had HCC. Hepatitis C virus (HCV)

was present in an increasing proportion of cirrhosis and HCC

between 2001-2013, reaching 48% of cirrhosis cases and

deaths and 67% of HCC cases and deaths by 2013. Cirrhosis

prevalence nearly doubled from 2001-2013 (664 to 1058

per 100,000 enrollees), driven by HCV and nonalcoholic fatty

liver disease (NAFLD). Cirrhosis incidence ranged from 159

to 193 per 100,000 patient-years. Deaths in patients with

cirrhosis increased from 83 to 126 per 100,000 patient-years,

largely driven by HCV. HCC incidence increased 2.5-fold from

17 to 45 per 100,000 patient-years. HCC mortality tripled

from 13 to 37 per 100,000 patient-years, driven overwhelmingly

by HCV with much smaller contributions from NAFLD and

alcoholic liver disease. Conclusions Cirrhosis prevalence and

mortality and HCC incidence and mortality increased from

2001-2013 driven by HCV with a smaller contribution from

NAFLD. If current trends continue, cirrhosis prevalence will

peak in 2021. Healthcare systems will need to accommodate

rising numbers of patients with cirrhosis and HCC.




Jason A. Dominitz - Employment: Department of Veterans Affairs; Grant/Research

Support: Gilead Pharmaceuticals

The following authors have nothing to disclose: Lauren A. Beste, Steven Leipertz,

Pamela Green, David Ross, George N. Ioannou


Multi-platform analysis of Telomerase Reverse Transcriptase

(TERT) gene alterations in Hepatocellular Carcinoma


Renumathy Dhanasekaran 1 , Kyle Covington 2 , Jennifer Watt 2 ,

Andrew D. Cherniack 3 , Daniel R. O’Brien 4 , Ju-Seog Lee 5 , Chad

Creighton 2 , Donna M. Munzy 2 , Lewis R. Roberts 1 , David A.

Wheeler 2 ; 1 Gastroenterology and Hepatology, Mayo Clinic, Rochester,

MN; 2 Baylor College of Medicine, Houston, TX; 3 Broad

Institute of MIT and Harvard, Cambridge, MA; 4 Mayo Clinic, Rochester,

MN; 5 The University of Texas MD Anderson Cancer Center,

Houston, TX

Background: Telomerases are enzymatic protein complexes

which maintain telomere length and play an important role in

cancer cell immortalization. Mechanisms of TERT gene activation

and clinical associations of TERT gene alterations in

HCC are not completely understood. Methods: We used data

from TCGA analysis of HCC primary tumor from 192 patients.

Germline DNA from blood or benign surrounding liver was

used as reference. Mutational, copy number, mRNAseq and

viral genomic integration analyses were performed. Results:

The TERT gene was found to be significantly overexpressed

in HCC tumor tissue when compared to benign surrounding

liver (pA (C228T) and 1,295,250 G>A (C250T)) were found.

The majority of tumors had the C288T mutation (92.1%) and

six (7.9%) tumors had the C250T mutation. Patients with TERT

promoter mutation were older (p=0.03) and predominantly

male (p=0.02). Patients with TERT promoter mutation were

more likely to have hepatitis C than no mutation (33% vs 10%;

p=0.02). In contrast, patients with TERT promoter mutation

were less likely to have hepatitis B than no mutation (14% vs.

27%; p=0.05). Also, patients with TERT promoter mutation

had worse survival than patients without the mutation (3 year

survival 59% vs. 39%; p=0.02). Further, we report here the first

case of a germline mutation in the TERT promoter in HCC in a

30 year old male who did not have any risk factors for HCC.

Copy number analyses showed TERT gene amplification in

7% of tumor samples; TERT was the fourth most common gene

found to be amplified in HCC. There was significant correlation

between TERT gene amplification and TERT mRNA overexpression.

In the subset of patients with HBV infection, the integration

of HBV viral genome into TERT promoter region was identified

in 13.6% (6/44) of patients and this was the most common site

of recurrent HBV integrations. Conclusion: This is the first comprehensive

multi-platform analysis of the various mechanisms of

TERT gene alterations in HCC and their association with patient

outcomes. TERT promoter mutations were found to be the most

common somatic mutation in HCC and were associated with

poor survival. We identify TERT promoter somatic mutations,

TERT gene amplification and HBV integrations into the TERT

promoter region as various mechanisms of TERT gene alterations.

Also, we report here the first case of germline mutation

of the TERT promoter gene in HCC, possibly resulting in an

inherited predisposition to HCC.



Andrew D. Cherniack - Grant/Research Support: Bayer AG

Lewis R. Roberts - Grant/Research Support: Bristol Myers Squibb, ARIAD Pharmaceuticals,

BTG, Wako Diagnostics, Inova Diagnostics, Gilead Sciences, Five

Prime Therapeutics

The following authors have nothing to disclose: Renumathy Dhanasekaran, Kyle

Covington, Jennifer Watt, Daniel R. O’Brien, Ju-Seog Lee, Chad Creighton,

Donna M. Munzy, David A. Wheeler


Targeting the TGF-β Pathway Increases Patient Survival

for Hepatocellular Cancer

Jian Chen 1 , Jiun-Sheng Chen 1 , Wilma Jogunoori 1 , Young Jin Gi 1 ,

Yun Seong Jeong 1 , Xiaoping Su 1 , Asif Rashid 1 , Bibhuti Mishra 2 ,

Jon White 2 , Milind Javle 1 , Marta L. Davila 1 , John R. Stroehlein 1 ,

Xuemei Wang 1 , Jeffrey S. Morris 1 , Rehan Akbani 1 , Lopa Mishra 1 ;

1 The University of Texas MD Anderson Cancer Center, Houston,

TX; 2 Institute of Clinical Research, Veterans Affairs Medical Center,

Washington DC, DC

Liver cancers that include hepatocellular cancer (HCC) and

intrahepatic cholangiocarcinoma are among the most challenging

and lethal malignancies with a degree of complexity seldom

seen in other cancers. Patients with HCC have a poor survival

of 9-11 months. However, key driver pathways and identifying

specific populations responsive to targeted therapeutics have

remained elusive. Recent clinical studies show that targeting

TGF-β in patients with high TGF-β level improves survival up

to 21 months. Therefore, we hypothesized that genomic, transcriptomic

profiles coupled with mouse mutants and functional

analyses may identify specific patients with HCC with a high

response rate to targeting TGF-β and present an attractive new

treatment strategy. Methods and Results: (1) We analyzed the

transcriptome of 488 hepatocellular cancers and screened for

mutations in the TGF-β pathway in 202 HCCs from The Cancer

Genome Atlas (TCGA). We found aberrant TGF-β superfamily

in 72% of HCC with mutations in 38%, the greatest number

being for the Smad adaptor β2SP (6%). (2) Increased levels of

TGF-β genes were designated as an “activated” signature that

is associated with hepatic fibrosis. Decreased levels of TGF-β

genes were defined as an “inactivated” signature, which was

associated with the loss of TGF-β tumor suppressor function.

HCCs characterized by the “inactivated” TGF-β signature were

associated with a significantly poorer survival particularly in

early stage HCCs, compared to HCCs with the “activated”

TGF-β signature (p=0.0027). (3) The TGF-β pathway is statistically

significantly associated with DNA repair genes. (4)

We performed (80x) whole-genome sequence analysis of eight

additional HCCs to define the role of TGF-β in their development

and characterize a potential novel “driver mutations” in

HCV- and alcohol-associated HCC. (5) Further functional analyses

were performed in mouse mutants in the TGF-β pathway

that spontaneously develop HCC, patient derived xenografts,

and human cell lines. Increased levels of TGF-β associated-E3

ligases, including KEAP1, and PJA1 were found in 20% of

HCCs. Targeting these E3 ligases revealed a strong response

using patient derived xenografts. Conclusions: The molecular

signatures of the TGF-β pathway we characterize here appear

to have prognostic significance. The additional association

with the DNA repair pathway supports new approaches to

developing biomarkers. TGF-β and TGF-β-associated-E3 ligases

are activated in distinct groups of liver cancer patients. It may

be possible to identify subsets of patients, defined by the activation

or inactivation of TGF-β signaling, who may respond more

effectively to specific treatments.


The following authors have nothing to disclose: Jian Chen, Jiun-Sheng Chen,

Wilma Jogunoori, Young Jin Gi, Yun Seong Jeong, Xiaoping Su, Asif Rashid,

Bibhuti Mishra, Jon White, Milind Javle, Marta L. Davila, John R. Stroehlein,

Xuemei Wang, Jeffrey S. Morris, Rehan Akbani, Lopa Mishra


A Study for the Risk Factors of Hepatocellular Carcinoma

in Cirrhotic Patients with Chronic Hepatitis B

Yuanqing Zhang 1 , Lijun Peng 1,2 , Yirong Cao 1 , Zhiping Zeng 1 ,

Yujing Wu 1 , Hong Shi 1 , Shiyao Chen 1 , Jiyao Wang 1 , Scott L.

Friedman 1,3 , John J. Sninsky 1,4 , Jinsheng Guo 1 ; 1 Division of Digestive

Diseases, Zhong Shan Hospital, Fu Dan University, Shanghai,

China; 2 Department of Gastroenterology, Linyi People’s Hospital,

Linyi, China; 3 Division of Liver Diseases,, Mount Sinai Hospital,

Icahn School of Medicine at Mount Sinai, New York, NY; 4 Celera/Quest

Diagnostics, Alameda, CA

Background and Aims: Chronic hepatitis B virus infection and

cirrhosis are important risk factors for hepatocellular carcinoma

(HCC), however the HCC risk can vary widely among individuals.

The aim of this study was to identify the clinical factors

and host genetic single nucleotide polymorphisms (SNPs) that

are associated with HCC risk in cirrhotic patients with CHB.

Methods: Data from 949 Chinese Han patients with chronic

HBV infection were analyzed by single and multivariate logistic

regression analysis in cirrhotic patients without (n = 281) and

with HCC (n = 434) to identify the clinical factors associated

with HCC risk. In a parallel study, DNA was extracted from

879 chronic HBV patients including non-cirrhotic HBV carriers

and CHB patients (n = 234), cirrhotic patients without (n =

257) and with HCC (n = 388) for genotyping of ten candidate

SNPs using polymerase chain reaction-ligase detection

reaction method. The correlations between the candidate SNPs

and HCC risk were analyzed by chi-square test followed by

logistic regression analysis. Results: 1. Ineffective antiviral treatment

(OR=5.2), fatty liver (OR=3.4), family history of HCC

(OR=2.3), drinking history (OR=2.2), and age≥50 (OR=1.8)

were independent risk factors for HCC in cirrhotic patients with

CHB. Longer HBV infection increased the HCC risk whereas

sustained virologic suppression significantly lowered HCC

risk compared to those without enough response or untreated

patients, especially in patients with decompensated cirrhosis.

Even after achieving viral suppression, 36.5% (62/170) of cirrhotic

patients with CHB still developed HCC. Fatty liver, family

history of HCC and HBV infection were significantly associated

with the HCC development in these patients. 2. After adjusted

for influencing factors, TLR4 rs11536889 was found to be a

protective factor (OR for CC vs. GG+GC or GG =0.4 and

0.6, respectively) whereas SPP1 rs2853744 (OR for TT vs. GG

and GT+TT vs. GG =1.9 and 3.1, respectively) and AP3S2

rs2290351 (OR for GA+AA or GA vs. GG =2.5 and 2.9,

respectively) were risk factors of HCC in cirrhotic patients with

CHB. In cirrhotic patients with CHB and drinking history, MLEC

rs7976497 (OR for TT vs. CC=2.8, CT+TT vs. CC=3.6) and

SOCS3 rs4969168 (OR for GG vs. AA=2.4) were found to be

risk factors of HCC. Conclusion: Ineffective antiviral treatment,

fatty liver, family history of HCC, drinking history and age≥50

are risk factors for HCC. Sustained suppression of HBV does

not remove the risk of HCC. Specific host genetic factors may

impact on HCC development in cirrhotic patients with CHB

including a protective SNP in TLR4, and risk SNPs in SPP1,

AP3S2, MLEC, and SOCS3.



Scott L. Friedman - Advisory Committees or Review Panels: Pfizer Pharmaceutical;

Consulting: Conatus Pharm, Exalenz, Genfit, Exalenz Biosciences, Eli Lilly PHarmaceuticals,

Fibrogen, Boehringer Ingelheim, Nitto Corp., Immune Therapeutics,

Synageva, Roche/Genentech Pharmaceuticals, DeuteRx, Abbvie, Novartis,

RuiYi, Kinemed, Sanofi Aventis, Takeda Pharmaceuticals, Nimbus Therapeutics,

Bristol Myers Squibb, Astra Zeneca, Sandhill Medical Devices, Galmed, Northern

Biologics, Enanta Pharmaceuticals, Regado Bioscience, Raptor Pharmaceuticals,

Teva Pharmaceuticals, Zafgen Pharmaceuticals, Merck Pharmaceuticals,

Debio Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira

Pharm; Stock Shareholder: Angion Biomedica, Intercept Pharma

John J. Sninsky - Consulting: Roche Diagnostics; Employment: CareDx

The following authors have nothing to disclose: Yuanqing Zhang, Lijun Peng,

Yirong Cao, Zhiping Zeng, Yujing Wu, Hong Shi, Shiyao Chen, Jiyao Wang,

Jinsheng Guo


Diabetes, HBV infection and smoking are independent

risk factors for developing hepatocellular carcinoma

on non-fibrotic liver in the NoFLIC French multicenter

case-control study.

Jean-Frédéric Blanc 1 , Adelaide Doussau 2 , Marie-Quitterie Picat 2 ,

Aline Maillard 2 , Caroline Bouyssou 1 , Charlotte E. Costentin 3 , Olivier

Rosmorduc 4 , Isabelle Rosa 5 , Luc Letenneur 2 , Karen Leffondre 6 ,

Jessica Zucman-Rossi 6 , Marianne Ziol 7 ; 1 Hepatology and Digestive

Oncology Unit, CHU bordeaux, Bordeaux, France; 2 ISPED,

Bordeaux, France; 3 Hepatology, Hopital Henri Mondor, Creteil,

France; 4 Heptology, Hopital saint-Antoine, Paris, France; 5 Hepatology,

CHI Créteil, Créteil, France; 6 INSERM U1162, Paris, France;

7 Pathologie, Hopital Jean Verdier, Bondy, France

risk factors for developing nfHCC. Conclusion NoFLIC is the

first case-control study dedicated to the study of factors associated

with the occurrence of HCC in non-fibrotic liver (F0/F1).

The first analysis shows a significant association between metabolic

syndrome, diabetes, HBV infection and smoking with the

occurrence of HCC in non-fibrotic liver suggesting an important

role of these factors in liver carcinogenesis.


Jean-Frédéric Blanc - Advisory Committees or Review Panels: Lilly / Imclone,

Merck Serono, Sanofi; Speaking and Teaching: Bayer, BMS, Roche, Abbvie

Olivier Rosmorduc - Advisory Committees or Review Panels: Syrtex, IPSEN;

Speaking and Teaching: Bayer

Jessica Zucman-Rossi - Advisory Committees or Review Panels: Astellas, Celgene;

Consulting: Pfizer; Grant/Research Support: Integragen; Speaking and Teaching:

Bayer, Lilly

Marianne Ziol - Grant/Research Support: Echosens

The following authors have nothing to disclose: Adelaide Doussau, Marie-Quitterie

Picat, Aline Maillard, Caroline Bouyssou, Charlotte E. Costentin, Isabelle

Rosa, Luc Letenneur, Karen Leffondre

Introduction The occurrence of HCC in non-fibrotic liver (nfHCC)

is a rare event (5% of HCC patients) and factors contributing to

their onset remain poorly understood. The aim of the case-control

exploratory study NoFLIC was to identify factors involved

in hepatocarcinogenesis in non-fibrotic liver. Patients and

methods NoFLIC is a French multicenter case-control study that

included consecutive patients who developed HCC in a non-fibrotic

liver, F0 or F1, proven by histological analysis. Controls

were matched for age and gender, they were recruited in

people with gastro-intestinal screening and normal endoscopy,

or in rheumatology departments. Clinical data were collected

including the parameters of the metabolic syndrome defined

according to the NCEP-ATP3 criteria, viral serology, quantification

of alcohol and tobacco use. Data were taken from the

patient’s medical record, the results of blood test performed for

this study and a specific epidemiologic survey. Logistic regressions

adjusted for age, sex and geographical origin, were performed

to determine the association of each of the risk factors

with the status of the patient (case / control). A multivariate

analysis was performed to study the effect of significant risk

factors. Results A total of 109 nfHCC (cases) and 163 controls

were included in 12 hospitals between 2010 and 2013.

Mean age of the nfHCC patients was 64.4 years, with 68.8%

male. Active HBV infection was detected in 6 cases (OR 11.4,

95% 1.18-110.84, p=0.035). History of alcohol abuse was

more frequent in nfHCC (OR 2.13, 95% 1.09-4.16 p=0.026).

A metabolic syndrome was identified in 28% of the patients

developing nfHCC (OR 3.3; 95% CI 1.62-6.82 p=0.001).

Among the components of metabolic syndrome, treated diabetes

or fasting glucose ≥ 6.1mmol/l was significantly associated

with nfHCC occurrence (OR 3.08; 95% CI 1.67-5.69

p=0.0002), while there was no significant association with

BMI, hypertension and dyslipidemia. Smoking (defined by at

least 100 smoked cigarettes and at least with a weekly smoking

habit) was also associated with nfHCC (OR 2.18 adjusted

for age and sex; 95% CI 1.23-3.83 p = 0.0067). In multivariate

analysis, HBV infection (p=0.024), metabolic syndrome

(P=0.0023) and tobacco use (p=0.0062) were independent



Neurodevelopmental Outcomes in Patients with Biliary

Atresia and Native Liver at Ages 1 and 2 Years: Results

from ChiLDReN

Vicky L. Ng 1 , Lisa G. Sorensen 16 , Estella M. Alonso 2 , Emily M.

Fredericks 3 , Wen Ye 21 , Saul J. Karpen 17 , Benjamin Shneider 18 ,

Jorge A. Bezerra 4 , Jean P. Molleston 5 , Karen F. Murray 6 , Philip

Rosenthal 7 , Kasper S. Wang 19 , Kathleen M. Loomes 8 , Paula M.

Hertel 9 , Nanda Kerkar 20,22 , Kathleen B. Schwarz 10 , Yumirle P.

Turmelle 11 , Barbara A. Haber 12 , Averell H. Sherker 15 , John C.

Magee 13 , Ronald J. Sokol 14 ; 1 Division of Pediatric Gastroenterology,

Hepatology and Nutrition, The Hospital for Sick Children,

University of Toronto, Toronto, ON, Canada; 2 Division of Pediatric

Gastroenterology, Hepatology and Nutrition, Ann & Robert H.

Lurie Children’s Hospital, Chicago, IL; 3 Division of Child Behavioral

Health, University of Michigan and C.S. Mott Children’s

Hospital, Ann Arbor, MI; 4 Division of Pediatric Gastroenterology,

Hepatology and Nutrition, Cincinnat Children’s Hospital Medical,

Cincinnati, OH; 5 Pediatric Gastroenterology, Hepatology

and Nutrition, Indiana Universtiy School of Medicine/Riley Hospital

for Children, Indianapolis, IN; 6 Division of Gastroenterology

and Hepatology, University of Washington Medical Center,

Seattle Children’s, Seattle, WA; 7 Division of Gastroenterology,

Hepatology and Nutrition, Department of Pediatrics, University

of California, San Francisco, San Francisco, CA; 8 Pediatric Gastroenterology,

Hepatology and Nutrition, Children’s Hospital of

Philadelphia, Philadelphia, PA; 9 Baylor College of Medicine,

Houston, TX; 10 Johns Hopkins School of Medicine, Baltimore, MD;

11 Pediatric Gastroenterology, Hepatology and Nutrition, Washington

University School of Medicine, St. Louis, MO; 12 Infectious

Disease, Clinical Research, Merck, North Wales, PA; 13 University

of Michigan Medical School, Ann Arbor, MI; 14 Section of Pediatric

Gastroenterology, Hepatology and Nutrition, Department of Pediatrics,

University of Colorado School of Medicine, Denver, CO;

15 Liver Diseases Research Branch, National Institute of Diabetes

and Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, MD; 16 Ann & Robert H. Lurie Children’s Hospital of

Chicago, Northwestern University Feinberg School of Medicine,

Chicago, IL; 17 Pediatric Gastroenterology, Hepatology and Nutrition,

Emory University School of Medicine/Children’s Healthcare

of Atlanta, Atlanta, GA; 18 Pediatric Gastroenterology, Hepatology

and Nutrition, Baylor College of Medicine, Houston, TX; 19 Division

of Pediatric Surgery, Children’s Hospital Los Angeles, Los Angeles,

CA; 20 Children’s Hospital of Los Angeles, Los Angeles, CA;

21 Department of Biostatistics, University of Michigan, Ann Arbor,

MI; 22 Mount Sinai, New York, NY

Background: One of the most serious consequences of liver

disease and malnutrition early in life is neurodevelopmental

(ND) delay for which prompt identification and intervention

may improve long-term outcomes. We hypothesized i) biliary

atresia (BA) patients with their native liver at ages 1 and 2

yrs will have significant ND delays and ii) that specific demographic

and clinical variables will predict worse ND outcomes.

Methods: Infants with BA, birth weight >2 kg, who received

hepatoportoenterostomy (HPE) and were enrolled between

2004-2012 in the multi-center, prospective NIDDK-funded

ChiLDReN study (PROBE-NCT00061828) underwent ND testing

at ages 12±2 mos (T1) and 24±2mos (T2) using either

Bayley Scales of Infant Development-2(B2) or Bayley-3(B3),

based on era. A subset of these infants was also enrolled in a

blinded randomized placebo-controlled study of corticosteroids

as adjunctive therapy to HPE (START–NCT00294684). Scores

(normative mean=100±15) were categorized as ≥100, 85-99,



were included. Clinical and biochemical data were collected

and compared with subsequent endoscopies to compare the

efficacy and safety of prophylactic therapy versus non-treatment.

Results: 273 endoscopies from 70 patients (42M) mean

age 8.8 +/- 4.7y, were included. Diagnosis was portal vein

thrombosis, biliary atresia, and other liver diseases, such as

Wilson’s disease, autoimmune hepatitis, cystic fibrosis and

congenital hepatic fibrosis in 23 (33%), 25 (35%) and 22

(32%), respectively. Subjects were in the surveillance program

for mean time of 4.8 years +/- 2.7 years (range, 9 months-8

years). Variceal grading was as per UK national guidelines.

Prophylactic treatment was administered in 177 (65%) endoscopies

versus 96 (35%). Treatment group showed improvement

of varices in 127 (72%) endoscopies, no change in 39

(22%) and worsening of varices in 11 (6%). The non-treatment

group showed improvement of varices in 16 (16%) endoscopies,

no change in 38 (40%) and worsening of varices in 42

(43%). Mean values for serum bilirubin, platelet count (PLT),

haemoglobin (Hb) and INR at the time of endoscopy in the

prophylaxis group and non-prophylaxis treatment group were

18.65mg/dL and 21.11mg/dL [n.v. 3-20], 109.1x10 9 /L and

103.95x10 9 /L [n.v. 180-400], 118.91g/L and 110.5g/L

[n.v. 115-155], 1.28 and 1.25 [n.v. 0.9-1.2], respectively.

No significant difference was found between treatment and

non-treatment groups in bilirubin (p=0.4), PLT (p=0.4), Hb

(p=0.38) or INR (p=0.5). Blood products prior to the procedure

were required in 33 endoscopies (12%), as per protocol.

Seven (10%) patients suffered a single GI bleed, all but one of

which were in the no prophylactic treatment group. No other

complications were recorded in either group. Conclusion: Prophylactic

treatment of GI varices results in an overall reduction

of varices and GI bleeds. Children with PTH could benefit from

surveillance endoscopy programs with prophylactic treatment

in the form of EVL or EST as it has been shown to be both safe

and effective.


The following authors have nothing to disclose: Harry Sutton, Mark Davenport,

Alastair J. Baker, Anil Dhawan, Tassos Grammatikopoulos


Molecular genetic dissection of 101 neonatal/infantile

intrahepatic cholestasis using targeted next-generation


Takao Togawa, Tokio Sugiura, Koichi Ito, Takeshi Endo, Shinji

Saitoh; Department of Pediatrics and Neonatology, Nagoya City

University Graduate School of Medical Sciences, Nagoya, Japan

Neonatal/infantile intrahepatic cholestasis (NIIC) is a heterogeneous

disorder and caused by mutations in a number of

genes, making molecular diagnosis challenging. Our aim is

to clarify the molecular diagnosis for subjects with NIIC using

next-generation sequencing (NGS) and bioinformatics pipeline.

We further sought genotype-phenotype correlation to

help comprehensive understanding of NIIC. Neonatal/infantile

cholestasis was defined by the level of serum direct bilirubin;

D.Bil≥1 mg/dL, if T.Bil8.6 kPa to detect METAVIR F3-F4 fibrosis.

Several studies in children have used different LSM cut-points to

identify fibrosis. However, validation of these LSM cut-points in

children has not been reported. METHODS: Patients at Boston

Children’s Hospital who underwent LSM from October 2006-

May 2015 were eligible for this prospective two-phase study.

All patients must have had a liver biopsy within 12 months

prior to enrollment. The METAVIR system was used to stage

fibrosis by pathologists blinded to LSM. Patients enrolled from

October 2006- March 2011 were in the test group used to

develop cut-points for discriminating F3-F4 and F4 fibrosis.

Patients enrolled from April 2011-May 2015 were the validation

group to test the accuracy of these cut-points. Diagnostic

performance of TE was assessed by sensitivity (Se), specificity

(Sp), accuracy (Ac) and area under the receiver operating

characteristic (AUC). RESULTS: 263 subjects were enrolled; 97

in the test group and 166 in the validation group. Clinical/

demographic data of the two groups were similar. Overall,

the subjects were 46% female, aged 2 mo–24 yrs (median


13y), with indications for biopsy of autoimmune LD (19%),

viral infection (18%), cholestatic LD (9%), PSC (12%), non-alcoholic

fatty LD (11%), and post-transplantation (2%). METAVIR

stages were: F0 (20%), F1 (29%), F2 (20%), F3 (17%), F4

(14%). Median time from LSM to biopsy was 50 days (IQR

21-144). Fasting prior to TE was known for 23%. In the test

group, the cut-point to discriminate F3-F4 was >8.6 kPa with

Se=79%, Sp=83%, Ac=81% and AUC=0.83. The cut-point for

F4 was >11.5 kPa (Se=74%, Sp=82%, Ac=80%, AUC=0.81).

Applied to the validation group, the cut-point of >8.6 kPa to

detect F3-F4 had Se=67%, Sp=69% and Ac=69%. For subjects

who fasted prior to TE (N=45), the performance for this

cut-point improved (Se=92%, Sp=70%, Ac=76%). To detect

F4, the cut-point of >11.5 kPa had Se=78%, Sp=80% and

Ac=80%. In fasting subjects (N=45), the accuracy improved

(Se=86%, Sp=84%, Ac=84%). CONCLUSIONS: This is the first

study in children and young adults in which LSM cut-points for

F3-F4 and F4 fibrosis are defined and evaluated in separate

test and validation samples. This study supports recent findings

that accuracy of TE improves with fasting. TE can be an important

tool for identifying advanced fibrosis in children.


Christine K. Lee - Grant/Research Support: Echosens

Maureen M. Jonas - Advisory Committees or Review Panels: Gilead Sciences;

Grant/Research Support: Gilead, Bristol Myers Squibb, Roche, Echosens

The following authors have nothing to disclose: Paul D. Mitchell, Roshan Raza,

Sarah Harney, Shanna M. Wiggins


Hospitalizations for Respiratory Syncytial Virus, Influenza,

and Other Vaccine Preventable Illnesses in Liver

Transplant Recipients at Freestanding US Children’s


Amy G. Feldman 1,4 , Brenda Beaty 2 , Shikha Sundaram 1,4 , Allison

Kempe 3 ; 1 Pediatric Gastroenterology, Hepatology and Nutrition,

Children’s Hospital Colorado, Aurora, CO; 2 University of Colorado,

Aurora, CO; 3 Pediatrics, Children’s Hospital Colorado,

Aurora, CO; 4 University of Colorado School of Medicine, Aurora,


Objectives: Influenza and respiratory syncytial virus (RSV) are

common infections requiring pediatric hospitalizations with

annual rates of 0.05% and 0.5% respectively. Rates of these

same illnesses in the pediatric liver transplant population are

unknown. The goals of this study were to examine among liver

transplant recipients at centers participating in the Pediatric

Health Information System (PHIS) dataset: (1) number of hospitalizations

for RSV, influenza, and other vaccine preventable

illnesses (VPIs); (2) morbidity and mortality associated

with these hospitalizations; and (3) costs associated with these

hospitalizations. Methods: Retrospective cohort study of subjects

< 18 years of age who underwent liver transplantation at

a PHIS center between January 1, 2004, and December 31,

2013. Subsequent hospitalizations during this time period for

RSV, influenza, and 10 other VPIs were ascertained using ICD-

9-CM diagnosis codes. Data were collected on clinical care,

outcomes, and costs during these hospitalizations. Results:

2,554 pediatric liver transplant recipients were identified;

511 (20.0%) of the patients had a total of 799 cases of RSV,

influenza, or other VPI. Overall, RSV, influenza, and rotavirus

were the most common infections (7.2%, 6.6%, and 5.8% of

cohort respectively). The mean time from transplant to infection

was 1.7 years (SD 1.7 years). Ninety-two patients (3.6%)

had infection during their transplant hospitalization, 8 of these

had multiple infections. In the first year post-transplant, 4.0%

of all patients had a hospitalization for RSV and 3.3% had

a hospitalization for influenza. The death rate during these

hospitalizations was 4.9% for RSV and 2.4% for influenza.

Excluding those infections that occurred during initial transplant

hospitalization (when all patients receive mechanical ventilation

and ICU care), rejection occurred in 15.9% of patients

with RSV and 13.4% of patients with influenza, ventilation in

9.3% (RSV) and 5.9% (influenza), and ICU level care in 20.9%

(RSV) and 12.8% (influenza). The median inflation adjusted

costs for hospitalization were $ 15,934 (RSV) and $7806

(influenza). Conclusions: VPIs occur in 1 of every 5 pediatric

liver transplant recipients at a rate of up to 66 times higher than

in the general pediatric population. RSV and influenza were

the most common infections and most cases occurred in the

first two years following transplant, demonstrating the importance

of ensuring vaccination in all patients prior to liver transplant.

Further research on clinical utility and cost-effectiveness

of Palivizumab to prevent RSV in select transplant candidates/

recipients is needed.


The following authors have nothing to disclose: Amy G. Feldman, Brenda Beaty,

Shikha Sundaram, Allison Kempe


Liver Fibrosis is Present in One-Third of Adults with

Alpha-1 Antitrypsin Deficiency Without Overt Liver Disease

Virginia C. Clark 1 , George W. Marek 2 , Tracie L. Kurtz 3 , Chen

Liu 4 , Farshid Rouhani 3 , Mark Brantly 3 ; 1 Medicine, Division of Gastroenterology,

Hepatology, and Nutrition, University of Florida,

Gainesville, FL; 2 Medicine, University of Florida, Gainesville, FL;

3 Medicine, Pulmonary Critical Care, University of Florida, Gainesville,

FL; 4 Pathology, University of Florida, Gainesville, FL

Rationale: Alpha-1 antitrypsin deficiency (AATD) is a risk factor

for the development of cirrhosis and liver related mortality in

affected individuals. We hypothesize individuals with AATD

have subclinical liver injury and fibrosis not detected by routine

testing. The primary aim of this study is to define the prevalence

and histologic spectrum of liver disease in adults with

AATD. Methods: Adults with confirmed AATD Pi*ZZ and other

rare AATD alleles participated in the IRB approved study after

informed consent was obtained. A percutaneous liver biopsy

was performed on eligible subjects. A single liver pathologist

staged the biopsy for fibrosis (Ishak stage 1-6). The findings of

AATD in PAS-D globules were scored qualitatively to describe

the total observed (rare 0-1; moderate 2-3; severe 4-5). Individuals

with a clinical history of decompensated liver disease were

excluded. Results: The predominant AATD genotype is Pi*ZZ

(n=73), and two patients with rare alleles are included Pi*ZM-


and Pi*null. The mean age was 57 years, range 25-71

and 61% (n=46) were female. The mean age of AATD diagnosis

is 45.3 years, range 8 months-68 years. Pulmonary symptoms

were present at AATD diagnosis in 64% (n=48) and 28%

(n=21) were diagnosed because of family member. Only 5%

(n=4) were identified by abnormal liver test. Pulmonary function

characteristics included a baseline FEV1 1.95L (0.37-5.69L)

and % predicted FEV1 of 61.5 (14-117). The prevalence of

clinically significant liver fibrosis defined by Ishak fibrosis score

≥2 is 34.7% (n=25). Men were more likely to have advanced

fibrosis (Ishak ≥3, 22.2%, n=6) compared to women (2.2%

n=1). ALT (22.5 vs 29.3 IU/L, p=0.03) and GGT (26.3 vs

51.7, p=0.0006) were higher in those with Ishak ≥2, but mean

values were within normal range. PAS-D globules correlated

with fibrosis stage (spearman r= 0.5224, p


and risk for liver disease in AATD is needed to direct screening

and novel therapies toward patients who could benefit most.

In this cross section of older AATD subjects, over one-third had

clinically significant liver fibrosis confirmed by biopsy. This is

much higher than our previously reported prevalence of 7.9%

using patient self-reported data. We also confirmed our previous

finding that ALT and GGT for affected subjects fall within

normal range, which limits clinical utility. The histologic spectrum

was varied both in fibrosis and qualitative presence of

AATD globules. Future studies will evaluate clinical risk factors

and predictors for advanced liver disease.


The following authors have nothing to disclose: Virginia C. Clark, George W.

Marek, Tracie L. Kurtz, Chen Liu, Farshid Rouhani, Mark Brantly


Bile Acid Sequestrant Prevents NAFLD and NASH in

Western Diet Fed Mice Independent of FXR

Yuhuan Luo 1 , Xiaoxin Wang 1 , David J. Orlicky 2 , Moshe Levi 1 ;

1 Medicine, Univ Colorado, Aurora, CO; 2 Pathology, University of

Colorado, Aurora, CO

Bile acid sequestrants (BAS) are orally administered nonabsorbable

polymers that decrease serum cholesterol and serum glucose

in patients and animal models of type 2 diabetes mellitus.

We evaluated the effect of the BAS sevelamer in mice with diet

induced obesity and insulin resistance that develop NAFLD and

NASH. C57BL/6J male mice were fed with 1) control low fat

(LF) diet or 2) western (WD) diet (high fat, high sucrose, cholesterol)

for 5 months. The diets contained a) no addition or b) 2%

sevelamer. Treatment with sevelamer prevented the increases

in body weight, liver weight, serum triglycerides and serum

cholesterol. Histopathological analysis revealed diet induced

NAFLD and NASH mouse model resulted in panlobular macrosteatosis

with some microvesicular steatosis, and increases in

peri-portal inflammation and peri-portal and zone 1 sinusoidal

fibrosis extending into zones 2 & 3 in places. Sevelamer treatment

significantly decreased steatosis, inflammation and fibrosis

scores. There was decreased FXR signaling in the intestine

and the liver (FGF15 and SHP in the intestine as well as SHP

in the liver). There was also increased liver bile acid synthesis

(Cyp7a1) and expression of bile acid transporters. In addition,

there were significant decreases in SREBP-1c, ACC and FAS,

resulting in decreased neutral lipid accumulation. Sevelamer

also prevented or reversed WD-induced upregulation of profibrotic

(COL1A1, α-SMA, TGF-β) and proinflammatory (CCL2,

IL-1β, and TNF-α) genes in the liver. These effects of sevelamer

were independent of FXR as in FXR KO mice fed a western diet

sevelamer had similar beneficial effects in improving NAFLD/

NASH as in wild type mice. BAS treatment therefore prevents

diet induced NAFLD and NASH. This beneficial effect is largely

independent of FXR but may be mediated through activation of

the TGR5 signaling pathway.


Moshe Levi - Grant/Research Support: Intercept, Genzyme-Sanofi, Daiichi Sankyo,

Merck, Novartis

The following authors have nothing to disclose: Yuhuan Luo, Xiaoxin Wang,

David J. Orlicky


Targeting enterohepatic bile acid signaling as a novel

approach to modulate hepatic autophagic activity in

maintaining cholesterol homeostasis

Yifeng Wang, Yifeng Ding, Hong-Min Ni, Wen-Xing Ding, Tiangang

Li; Pharmacolgy, KUMC, Kansas City, KS

Liver plays a key role in whole body cholesterol homeostasis.

In addition, hepatic free cholesterol accumulation results in

hepatocyte injury in non-alcoholic steatohepatitis (NASH). Lysosomes

play an important role in maintaining cellular cholesterol

homeostasis by regulating cholesterol ester hydrolysis that

affects pathways such as cholesterol uptake, secretion and bile

acid synthesis. New studies revealed that autophagy mediates

the cellular transport of cholesterol ester from lipid droplets to

lysosomes. Goal: this study investigated the role and regulation

of hepatic autophagy in cholesterol metabolism. Methods: the

effect of cholesterol accumulation on autophagy flux and lysosome

function was investigated in hepatocytes and in mice.

The impact of autophagy impairment on hepatic and plasma

cholesterol metabolism was studied in liver-specific ATG5

knockout mice. The effect of cholestyramine on hepatic autophagy

was studied in mice. Results: Feeding mice a cholesterol-containing

diet or an atherogenic diet for 6 days increased

liver LC3-II and p62 levels in response to hepatic cholesterol

accumulation. Both free cholesterol loading and treatment with

low density lipoprotein (LDL) increased LC3B-II and p62 levels

in human hepatocytes and HepG2 cells, however, only free

cholesterol loading, but not LDL treatment, increased lysosome

size and decreased cathepsin B activities in HepG2 cells, suggesting

free cholesterol accumulation can also impair lysosome

function. Consistently, inhibition of cholesterol esterification

with an ACAT inhibitor to increase cellular free cholesterol

levels blocked autophagosome/lysosome fusion and caused

CASPASE-3 activation in HepG2 cells. In autophagy-defective

liver-specific ATG5 knockout mice, hepatic cholesterol was

unchanged, but FPLC analysis showed that plasma LDL-cholesterol

was significantly elevated. As previous studies suggested

that autophagy was impaired in NASH, we found that cholestyramine

feeding increased hepatic autophagy in both lean

and ob/ob mice, which was possibly due to the removal of

bile acid-mediated inhibition of autophagosome/lysosome

fusion. Conclusion: this study suggests that hepatic autophagy

is required for maintaining whole body cholesterol homeostasis.

Hepatic free cholesterol accumulation impairs hepatic

autophagy, which exacerbates hypercholesterolemia and

hepatocyte-injury in NASH. As we recently showed that bile

acids inhibit hepatic autophagy, targeting enterohepatic bile

acid signaling by bile acid binding resin may be an effective

approach to induce hepatic autophagy and attenuate cholesterol-induced

liver injury.


The following authors have nothing to disclose: Yifeng Wang, Yifeng Ding, Hong-

Min Ni, Wen-Xing Ding, Tiangang Li


Anti-Fibrotic Effect of Autotaxin and LPA1R Antagonists

in a Rodent Model of NASH.

Malavi Madireddi 1 , Gretchen Bain 3 , James Swaney 2 , Brian J.

Murphy 1 , Simeon Taylor 1 ; 1 Metabolic and Fibrotic Diseases, Bristol

Myers Squibb, West Windsor, NJ; 2 Inception Sciences, San

Diego, CA; 3 PharmAkea, Inc., San Diego, CA

Nonalcoholic steatohepatitis (NASH) is a condition where

increased fat accumulation concomitant with inflammation

results in liver cell damage. Patients with NASH have elevated


serum lipids and diabetes. Early disease is clinically asymptomatic,

therefore often presenting only when critical hepatic

function is compromised. A wound healing response in general

occurs in injured liver tissue, and the persistence of this

response may result in liver fibrosis. At this time, there is no

treatment for NASH, and left untreated leads to hepatocellular

carcimona and liver cirrhosis. Current management focuses on

treating underlying cause of injury, as there are no targeted

therapeutics to interrupt fibrotic response; liver transplant is

the only option for late-stage cirrhosis. The enzyme Autotaxin

(ATX) hydrolyzes lysophosphatidylcholine to produce lysophosphatidic

acid (LPA), a multi-functional bioactive lipid mediator.

ATX is a major determinant of LPA levels in circulation, and the

pathophysiological function of ATX is attributed to its ability

to produce LPA. There is mounting literature supporting serum

ATX as an indicator of the severity of liver disease. Here we

investigated two small molecule inhibitors of ATX and LPA1

receptor in the STAM ® rodent model of liver fibrosis to pharmacologically

interrogate these targets in NASH. AM063 and

BMT-053011 are low nanomolar inhibitor of ATX and LPA1

receptor respectively. The STAM ® mouse model of NASH is

induced by a single subcutaneous injection of streptozotocin

two days after birth and by feeding a high fat diet. Starting at

6 weeks after disease initiation, BMT-053011 was administered

twice daily, while AM063 and Telmisartan were administered

once daily. Treatment was continued for the following 3

weeks after initiation. AM063 showed significant decreases in

plasma TG, fibrosis area and TNF-a expression levels, with a

decreasing trend in blood glucose and macrophage activation

compared to the Vehicle group. Treatment with BMT-053011

showed significant decreases in liver weight, NAS, the fibrosis

area and inflammation. The results, together with the decrease

in ALT and inflammatory gene expression levels, demonstrate

anti-inflammatory and hepatoprotective effects. Importantly, as

NAS is one of the clinical endpoints for assessing the activity

of NASH (Sanyal AJ. et al., Hepatology, 2011; 54:344), the

observed changes in the treatment groups suggest potential

translatability of BMT-053011 as an anti-NASH therapeutic

for humans.


Malavi Madireddi - Employment: Bristol Myers Squibb

Gretchen Bain - Employment: PharmAkea Therapeutics

Simeon Taylor - Consulting: Isis Pharmaceuticals, Aegerion, Yabao; Stock Shareholder:

Bristol-Myers Squibb, Gilead, Celgene

The following authors have nothing to disclose: James Swaney, Brian J. Murphy


Treatment with the FXR-TGR5 dual agonist INT-767

arrests and reverses progression of NASH in mice fed a

Western diet

Xiaoxin Wang 1 , Yuhuan Luo 1 , David J. Orlicky 2 , Luciano Adorini 3 ,

Moshe Levi 1 ; 1 Medicine, Univ Colorado, Aurora, CO; 2 Pathology,

University of Colorado, Aurora, CO; 3 Intercept Pharmaceuticals,

New York, NY

Obesity and insulin resistance have become leading causes

of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic

steatohepatitis (NASH). In the present study, we determined in

mice with established NASH if treatment with INT-767, a dual

agonist of the nuclear receptor farnesoid X receptor (FXR) and

the G protein coupled receptor TGR5, can decrease disease

progression. In these studies, C57BL/6Jmice were first fed a

low fat (LF) or a Western diet (WD: high fat, high sucrose, and

high cholesterol) for 3 months and then treated orally for an

additional 2 months with vehicle or INT-767, 30 mg/kg body

weight/d. Compared to mice fed a LF diet, feeding a WD

for 5 months resulted in panlobular macrosteatosis with some

microvesicular steatosis, increased peri-portal inflammation and

lipogranuloma formation, and peri-portal and zone 1 sinusoidal

fibrosis extending in places into zones 2 & 3. When mice

on WD were treated with INT-767, there were marked and significant

decreases in the percentage of mice showing hepatic

macrosteatosis (83%), microsteatosis (100%), inflammation

(100%) and fibrosis (47%) compared to non-treated WD fed

mice. These histopathological changes were accompanied by

significant decreases in liver cholesterol content (52%), as well

as decreases in the lipogenic transcription factor SREBP-1c,

pro-inflammatory mediator MCP-1, and profibrotic matrix protein

Col1a1. INT-767 treatment also increased liver expression

of proteins involved in mitochondrial biogenesis and energy

expenditure including AMPK, Sirtuin 1, PGC-1α, Sirtuin 3, and

ERRα. Using FXR knockout mice, the FXR selective agonist INT-

747 (obeticholic acid), and the TGR5 specific agonist INT-777,

we further determined that FXR plays a major role in mediating

the effects of INT-767 in the liver, including the increases in

PGC-1α and Sirtuin 3. The dual FXR-TGR5 agonist INT-767 is

therefore able to markedly and significantly arrest and reverse

progression of liver disease even when treatment is started in

the presence of obesity, insulin resistance, and NASH.


Luciano Adorini - Employment: Intercept Pharmaceuticals

Moshe Levi - Grant/Research Support: Intercept, Genzyme-Sanofi, Daiichi Sankyo,

Merck, Novartis

The following authors have nothing to disclose: Xiaoxin Wang, Yuhuan Luo,

David J. Orlicky


DRX-065, the stabilized R-enantiomer of pioglitazone is

without PPARg agonist activity and exhibits the beneficial

in vivo pharmacodynamic effects for the treatment


Sheila H. DeWitt, Vincent Jacques, Lex H. Van der Ploeg; DeuteRx,

Andover, MA

Background: Pioglitazone is approved for the treatment of

type 2 diabetes mellitus (T2DM) and is currently the only drug

recommended off-label to treat patients with biopsy-proven

nonalcoholic steatohepatitis (NASH). However, the use of

pioglitazone for NASH has been limited due the side effect

of weight gain. Pioglitazone is a mixture of two mirror-image

compounds (the R- and S-enantiomers) that rapidly interconvert

in vivo. The pharmacokinetic (PK) and pharmacodynamic

(PD) properties of the individual enantiomers has never been

reported in humans. Methods: We replaced the exchangeable

hydrogen at the chiral center of pioglitazone by deuterium,

a stable, naturally occurring isotope of hydrogen. Deuterium

at the chiral center stabilizes the enantiomers and reduces

their rate of interconversion. Stability studies were completed

in aqueous buffer and in mouse and human plasma. In vitro

PPARg agonist activity was determined in a TRAP220 coactivator

recruitment assay. In vitro mitochondrial respiration activity

was assessed in intact C2C12 cells. PK and weight gain studies

of the enantiomers were completed in male C57BL/6 mice.

Efficacy was evaluated in a db/db mouse model of T2DM,

where effects on metabolic and inflammatory markers were

assessed. Results: Preparation of the deuterium-stabilized enantiomers

of pioglitazone, d-R-pioglitazone (DRX-065) and d-S-pioglitazone

(d-S-pio) enabled the evaluation of the properties of

the individual enantiomers. DRX-065 is not a PPARg agonist

at concentrations up to 100 mM while d-S-pio accounts for

all of the PPARg agonist activity observed with racemic pioglitazone.

DRX-065 is exclusively responsible for the effects of


pioglitazone on mitochondrial respiration. In the db/db mouse

model of T2DM, DRX-065 significantly decreases blood glucose,

serum triglycerides, and fatty acids, as well as markers of

inflammation (cytokines, serum amyloid A). At a therapeutically

effective dose in mice, DRX-065 also does not induce weight

gain, unlike pioglitazone or d-S-pio. The effects of d-S-pio on

weight gain are expected based on its strong PPARg agonist

activity. Conclusion: We demonstrated in preclinical experiments

that the stabilized deuterated R-enantiomer of pioglitazone,

DRX-065, has pharmacological properties desirable

for the treatment of NASH (mitochondrial function modulation,

non-steroidal anti-inflammatory effects, and glucose lowering

effects) without the undesired PPARg-related weight gain side

effects. DRX-065 represents a potentially significant therapeutic

improvement over pioglitazone, a drug already recommended

off-label for the treatment of NASH.


The following authors have nothing to disclose: Sheila H. DeWitt, Vincent

Jacques, Lex H. Van der Ploeg


Peretinoin, an acyclic retinoid, suppresses steatohepatitis

and development of tumorigenesis by activated

Atg16L1-dependent autophagy in mice fed an atherogenic

high-fat diet.

Hikari Okada 1 , Masao Honda 1 , Kai Takegoshi 1 , Taro Yamashita 1 ,

Naoto Nagata 2 , Toshinari Takamura 2 , Takuji Tanaka 3 , Shuichi

Kaneko 1 ; 1 Gastroenterology, Kanazawa University Graduate

School of Medical Science, Kanazawa, Japan; 2 Department of

Disease Control and Homeostasis, Kanazawa University Graduate

School of Medical Sciences, Kanazawa, Japan; 3 Cancer Research

and Prevention, The Tohkai Cytopathology Institute, Gifu, Japan

Objective Peretinoin, acyclic retinoid, is under phase III clinical

trials in Japan to prevent the recurrence and development of

HCC after surgical removal of the primary tumors. The mechanism

of peretinoin for preventing HCC remains unclear. Mice

fed an atherogenic high-fat diet (Ath HFD) developed steatohepatitis

followed by hepatic fibrosis and HCC progression

(Hepatology 2007). Here we investigated the suppressive

effects of peretinoin on steatohepatitis and tumorigenesis in

Ath HFD mice. Materials and Methods Three groups of 8-weekold

mice (n=15-20/group) were fed a control diet or Ath HFD

containing 0.01% or 0.03% peretinoin for 12, 30, and 60

weeks. Then, 0.01% peretinoin was added to the Ath HFD

at 40 weeks to examine the reversible effect of peretinoin on

established fibrosis and steatosis in the liver. The degree of liver

steatosis, hepatic fibrosis, tumor incidence, and liver weight

was calculated. Expression of IL6, IL1β TNFα, CEBPα, collagen

I/IV, pSTAT3, pNFkβ, ATG5, ATG7, ATG16L1, LC3b,

and Lamp2 was evaluated by immunohistochemical staining,

real-time PCR, and western blotting. The promoter analysis of

ATG16L1 was performed by reporter gene assay and ChIP

assay. Primary hepatocytes were isolated from male C57BL/6

mice treated Ath HFD for 12 weeks and the effect of peretinoin

on primary hepatocytes was evaluated in vitro. Results Mice

fed an Ath HFD developed liver steatosis and liver fibrosis after

12 and 30 weeks, and developed liver tumors after 60 weeks

at 70% frequency. Peretinoin markedly reduced steatosis and

fibrosis at 12 and 30 weeks and reduced tumor incidence to

approximately 30%. Expression of IL6, IL1β, TNFα, collagen

I/IV, pSTAT3, and pNFkβ was suppressed to approximately

60% in mice fed an Ath HFD containing peretinoin compared

with mice fed only an Ath HFD. We found peretinoin induced

autophagy that was shown by the increased autophagosome

formation by electron microscopy. We found increased LC3-II

and co-localization of autophagosome (LC3-II) and lysosome

(Lamp2) by immune fluorescent staining. Among various autophagy

related factors, peretinoin increased the expression of

Atg16L1 and promoted Atg16L1-dependent autophagy. In primary

hepatocyte, palmitic acid and IL6 suppressed the expression

of Atg16L1, while peretinoin rescued the Atg16L1 and

suppressed pSTAT3 and pNFkβ. We found peretinoin induced

the expression of CEBPα and CEBPα activated the promoter

activity of Atg16L1. Conclusion Peretinoin suppresses the development

of liver steatosis, inflammation, and tumorigenesis by

activating Atg16L1-dependent autophagy. These results support

the clinical efficacy of peretinoin for preventing HCC.


Hikari Okada - Employment: Kanazawa University

Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,

Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,

Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,

Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer

Japan, Eli lilly Japan

The following authors have nothing to disclose: Masao Honda, Kai Takegoshi,

Taro Yamashita, Naoto Nagata, Toshinari Takamura, Takuji Tanaka


Effects of norfloxacin therapy on survival in patients

with Child-Pugh class C cirrhosis: Results of a randomized,

double-blind, placebo-controlled, multicenter trial

Richard Moreau 1,2 , Laure Elkrief 2 , Christophe Bureau 3 , Jean-

Marc Perarnau 4 , Thierry Thevenot 5 , Faouzi Saliba 6 , Isabelle Ollivier-Hourmand

15 , Alexandre Louvet 7 , Pierre Nahon 8 , Frédéric

Oberti 9 , Rodolphe Anty 10 , Sophie Hillaire 11 , Blandine Pasquet 12 ,

Violaine Ozenne 13 , Marika Rudler 14 , Marie Angele Robic 3 , Louis

d’Alteroche 4 , Vincent Di Martino 5 , Pierre-Emmanuel Rautou 2 , Nathalie

Gault 16 , Didier Lebrec 1,2 ; 1 UMR S1149, Center for Research

on Inflammation (CRI), Inserm and Paris Diderot University, Clichy,

France; 2 DHU UNITY, Service d’hépatologie, Hôpital Beaujon,

APHP, Clichy, France; 3 Service d’hépato-gastroentérologie,, Hôpital

Purpan CHU Toulouse et Université Paul Sabatier, Toulouse,

France; 4 Unité d’Hépatologie, Hépatogastroentérologie, CHRU

Tours, Tours, France; 5 Service d’Hépatologie et de Soins Intensifs

Digestifs, Hôpital Jean Minjoz, Besançon, France; 6 Centre

Hépato-Biliaire, Hôpital Paul Brousse, APHP, Villejuif, France;

7 Service des maladies de l’appareil digestif, Hôpital Huriez, Lille,

France; 8 Service d’Hépatologie, Hôpital Jean Verdier, APHP,

Bondy, France; 9 Service d’hépato-gastroentérologie, CHU d’Angers,

Angers, France; 10 Pôle Digestif and Inserm 1065, CHU

de Nice, Nice, France; 11 Service de Médecine Interne, Hôpital

Foch, Suresnes, France; 12 Unité de recherche clinique Paris Nord,

Hôpital Bichat, APHP, Paris, France; 13 Hépato-gastroentérologie,

Hôpital Lariboisière, APHP, Paris, France; 14 Hépatologie, GH

Pitié-Salpêtrière, APHP, Paris, France; 15 Service d’hépato-gastro-entérologie

et nutrition and Unité 1930, CHU Côte de Nacre,

Caen, France; 16 Unité de recherche clinique Paris Nord, Hôpital

Beaujon, APHP, Clichy, France

Background & Aims: Whether long-term oral antibiotic therapy

using a fluoroquinolone should be used to improve prognosis in

patients with advanced cirrhosis is debated. Thus we addressed

this question by investigating the effects of the administration

of norfloxacin (a fluoroquinolone) in patients with advanced

cirrhosis. Methods: We performed a multicenter, double-blind

trial in which we randomly assigned patients with Child-Pugh

class C cirrhosis to once-daily 400 mg oral norfloxacin (144

patients) or placebo (147 patients) for 6 months. Surviving

patients were followed-up for further 6 months after treatment

cessation. The primary end point was overall survival at 6

months; secondary end points included overall survival at 3

and 12 months, and liver-related complications (bacterial infec-


tion, kidney failure, encephalopathy, variceal hemorrhage).

Patients were censored at the time of liver transplantation or

spontaneous bacterial peritonitis. Results: 291 patients were

enrolled rather than the projected 392 because of slow recruitment

and termination of funding. Excessive alcohol consumption

was the cause of cirrhosis in 115 patients (79.9%) of the

norfloxacin group and 108 patients (73.5%) of the placebo

group. By 6 months, 19 deaths (13.2%) occurred in the norfloxacin

group as compared with 27 deaths (18.4%) in the

placebo group, with a hazard ratio (HR) for death at 6 months

of 0.69 (95% confidence interval [CI], 0.38 to 1.24; P=0.21).

The HR for death at 3 months and 12 months were 0.53 (95%

CI, 0.28 to 1.02; P=0.05) and 0.76 (0.48 to 1.22; P=0.26),

respectively. There were no significant differences in terms of

6-month rates of infections (20.8% vs. 22.4%), kidney failure

(6.9% vs. 7.5%), encephalopathy (16.7% vs. 19.0%), variceal

hemorrhage (3.5% vs. 4.1%). Post-hoc analysis showed that at

enrollment, there were 56 patients (39.2%) in the norfloxacin

group and 63 (42.9%) in the placebo group who had histological

features of alcoholic hepatitis (P=0.52). In this subgroup

of patients, the HR for death at 3 months, 6 months and 12

months were 0.32 (95% CI, 0.12 to 0.86; P=0.01), 0.49

(95% CI, 0.21 to 1.15; P=0.09) and 0.47 (95% CI, 0.23 to

0.97; P=0.03), respectively. Conclusions: In this study, 6-month

oral norfloxacin therapy did not improve overall survival and

did not affect the development of liver-related complications

in patients with Child-Pugh class C cirrhosis. In the subgroup

of patients with alcoholic hepatitis, norfloxacin therapy may

increase 3-month overall survival suggesting early beneficial

effects of the antibiotic in this subgroup of patients.


Christophe Bureau - Speaking and Teaching: Gore

Jean-Marc Perarnau - Consulting: Gore and Associates

Faouzi Saliba - Advisory Committees or Review Panels: Novartis, Roche, Genzyme,

Vital therapies; Grant/Research Support: Astellas; Speaking and Teaching:

Gambro, MSD, Gilead

Isabelle Ollivier-Hourmand - Speaking and Teaching: gilead, jansen, bayer

Vincent Di Martino - Advisory Committees or Review Panels: Gilead, France,

Abbvie, BMS France; Board Membership: MSD France; Consulting: Gilead,

France; Speaking and Teaching: Janssen, BMS France, Gilead France

Pierre-Emmanuel Rautou - Speaking and Teaching: Gilead

The following authors have nothing to disclose: Richard Moreau, Laure Elkrief,

Thierry Thevenot, Alexandre Louvet, Pierre Nahon, Frédéric Oberti, Rodolphe

Anty, Sophie Hillaire, Blandine Pasquet, Violaine Ozenne, Marika Rudler, Marie

Angele Robic, Louis d’Alteroche, Nathalie Gault, Didier Lebrec


Ischemic hepatitis following acute variceal bleed is

ameliorated by N- acetyl cysteine (NAC) in cirrhotics: A

prospective randomized controlled trial

Avinash Kumar, Ankur Jindal, Rakhi Maiwall, Lovkesh Anand, Amrish

Sahney, Shiv K. Sarin; Institute Of Liver and Biliary Sciences,

New Delhi, India

Background & aims. Ischemic hepatitis (IH) following acute

variceal bleed (AVB) in cirrhotics carries ominous prognosis.

Consequent hypotension & hepatic ischemia may result in centrilobular

necrosis . N-acetylcysteine (NAC), a potent anti-oxidant

may prevent cell injury & improve tissue oxygen delivery.

We investigated the efficacy & safety of NAC in the prevention

of IH & survival. Methods: Of 530 consecutive patients

admitted with UGI bleed (Aug 2013-Apr 2015), cirrhotics with

acute variceal bleed (n=214) were randomized in a prospective

open label study (NCT02015403) to receive either standard

of care (SOC; iv fluids, terlipressin, endoscopic variceal

ligation, blood/blood products & antibiotics) plus NAC (150

mg/kg/hr for 1 h followed by 12.5mg/kg/hr for 4 h, followed

by 6.25 mg/kg for 67 hrs) [Group A(GrA), n=107) or

SOC alone [Group B(GrB),n=107]. Patients were monitored

for presence & severity of IH (increase in AST of ≥5 X ULN &

bilirubin ≥1.5 X ULN in


patients, 50(4.9%) were found to have definite HP; 4 were

excluded (2 on L-dopa and 2 on bromocriptine).The patients

in Gr A and B were comparable in age (54.8± 6.7 vs., 54.3

±7.4 yr, p=0.7) gender(Males 86%vs. 83%, p= 0.5),etiology

of cirrhosis, MELD (17.9 ± 3.8vs. 18.2 ± 4.2,p=0.3),

baseline arterial ammonia(131 ± 7.4vs. 132 ± 7.9mmol/l,

p=.4), number of patients with recurrent HE: 13/22(59%)

vs. 14/24(58%), p=0.7,persistent HE: 8/22 (36%) vs. 9/24

(37.5%), p=0.6, and porto-systemic shunts: 17/21(80) vs.

19/24(79), p= 0.6. Complete &partial response was seen in

(none vs. 7 (29%), p


on the underlying mechanisms of action of taurine and further

long-term evaluation in cirrhosis and portal hypertension are



Mattias Mandorfer - Consulting: Janssen; Speaking and Teaching: AbbVie, Gilead,

Janssen, Boehringer Ingelheim, Bristol-Myers Squibb, Roche

Thomas Reiberger - Consulting: Xtuit; Grant/Research Support: Roche, Gilead,

MSD, Phenex; Speaking and Teaching: Roche, Gilead, MSD

Michael Trauner - Advisory Committees or Review Panels: MSD, Janssen, Gilead,

Abbvie; Consulting: Phenex; Grant/Research Support: Intercept, Falk Pharma,

Albireo; Patent Held/Filed: Med Uni Graz (norUDCA); Speaking and Teaching:

Falk Foundation, Roche, Gilead

Markus Peck-Radosavljevic - Advisory Committees or Review Panels: Bayer, Gilead,

Janssen, BMS, AbbVie; Consulting: Bayer, Boehringer-Ingelheim, Jennerex,

Eli Lilly, AbbVie; Grant/Research Support: Bayer, Roche, Gilead, MSD, AbbVie;

Speaking and Teaching: Bayer, Roche, Gilead, MSD, Eli Lilly, AbbVie, Bayer

The following authors have nothing to disclose: Remy Schwarzer, Rafael Paternostro,

Birgit B. Heinisch, Philipp Schwabl, Arnulf Ferlitsch


Anticoagulation in patients with cirrhosis and portal

vein thrombosis is associated with increased portal vein

recanalization and better prognosis.

Carlos Noronha Ferreira 1 , Teresa Rodrigues 2 , Ana Júlia Pedro 3 ,

Paula Ferreira 1 , Margarida Sobral Dias 1 , Afonso Gonçalves 4 ,

Leonor Xavier Brito 1 , Fatima Serejo 1 , Rui T. Marinho 1 , Cilénia

B. Costa 1 , Narcisa Fatela 1 , Helena Cortez-Pinto 1 , Fernando

Ramalho 1 , Paula Alexandrino 1 , José F. Velosa 1 ; 1 Serviço de Gastrenterologia

e Hepatologia, Hospital de Santa Maria, Centro

Hospitalar Lisboa Norte, Lisboa, Portugal; 2 Laboratório de Biomatemática,

Faculdade de Medicina de Lisboa, Lisboa, Portugal;

3 Serviço de Medicina 2, Hospital de Santa Maria - Centro Hospitalar

Lisboa Norte, Lisboa, Portugal; 4 Serviço de Imagiologia,

Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, Lisboa,


Introduction: Cirrhosis is recognized as a prothrombotic state.

A recent study showed that prophylactic anticoagulation prevented

portal vein thrombosis (PVT) and decreased episodes

of decompensation of cirrhosis. Aims: To analyze the effect

of anticoagulation on recanalization of non-tumoral PVT in

patients with cirrhosis and its effect on prognosis. Methods: 69

consecutive patients with cirrhosis diagnosed with non-tumoral

PVT were studied. The clinical features at diagnosis of PVT and

factors associated with anticoagulation use were studied. Decision

to start anticoagulation was taken at the discretion of the

clinician managing the patient. The effect of anticoagulation

on PVT recanalization and mortality was analyzed. Results:

The average age was 58.6±11.8 years and 44(64%) were

males. Severity of cirrhosis: Median(Range) Child–Pugh(CP)

score: 8(5-15), MELD score:13(6-35). CP class: A-15(22%),

B-32(46%), C-22(32%). At diagnosis of PVT, 55(80%) were

symptomatic. Variceal bleeding(VB) in 30(46%) and abdominal

pain in 19(29%) were the main clinical presentations.

Anticoagulation (LMWH–9, warfarin–16) was administered in

25(36%) patients one of whom with cavernoma. Patients with

VB were less likely to be given anticoagulation (p=0.037).

There were no differences in age, gender, etiology, severity of

cirrhosis and extent of PVT in patients receiving, or not, anticoagulation.

Recanalization of PVT was assessed by at least one

imaging study in 60 patients and recanalization (Total–13, partial

– 9) of the portal vein was documented in 22(37%) patients.

Median (Range) follow–up was 21(0-376) months. At the end

of follow-up, 29(42%) patients died, of which sixteen deaths

were related to infectious complications with no deaths due to

anticoagulation related bleeding. By Cox regression analysis,

factors associated with mortality at the end of follow-up were:

Age (HR 1.040, 95% C.I. 1.002–1.078, p=0.037), CP score

(HR 1.35, 95% C.I. 1.18–1.55, p


Additional resources from government for HCV could aid in the

elimination of HCV from the United States.

Cost of HCV in the era of DAAs

($1.5 billion USD), shielding x-ray components ($13.2 billion

USD), and reducing greenhouse gas emissions ($413 billion

USD). Conclusions: The scope of benefit conferred by new

HCV therapies is large due to the high mortality risk of HCV,

the curative effect of therapy and the size of the population

infected. However, at current prices the incremental cost of

therapy represents a 3478-fold increase in current nationwide

spending on hepatitis C, more than 11% of all national health

expenditures, and 11 times the annual NIH budget. Concurrent

efforts to increase access to treatment as well as reduce drug

costs should be prioritized.


The following authors have nothing to disclose: Mai T. Pho, Andrew I. Aronsohn,

Benjamin P. Linas, Daniel C. Johnson, Jake R. Morgan, William V. Padula, David

O. Meltzer


Jagpreet Chhatwal - Consulting: Merck & Co., Inc., Gilead, Complete HEOR

Solutions; Grant/Research Support: NIH/National Center for Advancing Translational


The following authors have nothing to disclose: Xiaojie Wang, Mark S. Roberts,

Mina Kabiri, Turgay Ayer, Julie M. Donohue, Fasiha Kanwal


Treating Hepatitis C in the US: measuring impact and

value in the context of other major health interventions

Mai T. Pho 1 , Andrew I. Aronsohn 1 , Benjamin P. Linas 2 , Daniel

C. Johnson 1 , Jake R. Morgan 2 , William V. Padula 1 , David O.

Meltzer 1 ; 1 Medicine, University of Chicago, Chicago, IL; 2 Boston

Medical Center, Boston, MA

Background: Hepatitis C (HCV) results in significant morbidity

and mortality in the US. All-oral therapies, while curative, are

costly. We estimate the economic value of providing treatment

for individuals with HCV relative to other major interventions

such as decreasing greenhouse gas emissions. Methods: We

calculate the net monetary benefit (NMB) of therapy provided

for all individuals estimated to be infected with HCV in the US

using a value of statistical life (VSL) and value of statistical life

year (VSLY) framework. A VSL of 5 million US dollars (USD) is

used, and we calculate the VSLY as the quotient of the VSL and

the discounted expected remaining life-years for the general

population. We estimate the total population of HCV based

on prevalence, demographic and genotype data from the

National Health and Nutrition Evaluation Survey from 2003-

2010. The NMB of therapy is the product of VSLY and the

remaining discounted life-years associated with treatment and

no treatment of the total population, subtracted by the incremental

cost. Regimens examined included 12 weeks of sofosbuvir

(SOF) and ledipasvir (LDV) for genotype (GT)1, 12-16

weeks of SOF and ribavirin (RBV) for GT2, and 24 weeks of

SOF and RBV for GT3 infections. Remaining life expectancy

and costs are projected using the Hepatitis C Virus Cost-Effectiveness

(HEP-CE) simulation model. Prices for HCV drugs are

varied from 100% to 55% to reflect offsets due to rebates and

other discounts. Benefits and costs are reported in the context

of federally regulated health interventions. Results: Compared

to no treatment, treating 2.7 million individuals with HCV will

result in 9.5 million life-years gained at an incremental cost of

$347 billion USD. The NMB of HCV therapy is $2.2 trillion

USD. When the price of medications is discounted by 45%,

the NMB of therapy is $2.4 trillion USD, with an incremental

cost of therapy of $187 billion USD. The NMB of HCV

treatment exceeds influenza vaccinations at long-term facilities


Hepatitis C (HCV) Infection in Baby Boomers Are Independently

Associated with Mortality and Resource Utilization

Mehmet Sayiner 2 , Mark Wymer 2 , Pegah Golabi 2 , Joel S. Ford 3 ,

Indie Srishord 2 , Zobair M. Younossi 2,1 ; 1 Center For Liver Disease,

Department of Medicine, Inova Fairfax Hospital, Falls Church,

VA; 2 Betty and Guy Beatty Center for Integrated Research, Inova

Health System, Falls Church, VA; 3 Department of Medicine, Inova

Health System, Falls Church, VA

Background and aim: HCV is more common among Baby

boomers (BB) (born 1946-1965). As this cohort age, they will

increasingly become Medicare eligible. Our aim was to evaluate

resource utilization and short term mortality of BB-Medicare

recipients with HCV. Method: We used inpatient and outpatient

Medicare databases (2005-2010) with clinical data,

admission and discharge information, ICD-9 codes, procedure

codes, and billing information. Medicare patients who were

considered BB were identified using all HCV-related ICD-9

codes. Charlson Index was calculated as a measure of comorbidity.

The outcome measures included resource utilizations

[payment per case and inpatient length of stay (LOS)] and short

term mortality (90 days). Results: We included 1,153,862

BB who received Medicare services from 2005 to 2010. Of

these, 3.2% (N=37,365) were HCV (+). During this period,

Medicare-BB in the inpatient setting increased from 39,793 to

55,235, and their claims increased from 78,924 to 106,232.

During the study period, both overall mortality [8.94% to

10.25% (p


category of 45-49 (4.21%, [3.14-5.28]), ESRD (966.31%,

[954.86-977.88]), disabled status (43.22%, [41.67-44.80]),

Charlson score (46.78%, [46.31-47.26]), and year study

(2.72%, (2.58-2.85]) were all independently associated with

an increase in total payments. Conclusions: Prevalence of HCV

is higher in Baby boomers Medicare recipients. For the entire

BB cohort, HCV positivity is independently associated with

higher mortality and resource utilization.


Zobair M. Younossi - Advisory Committees or Review Panels: Salix, Janssen,

Vertex; Consulting: Gilead, Enterome, Coneatus

The following authors have nothing to disclose: Mehmet Sayiner, Mark Wymer,

Pegah Golabi, Joel S. Ford, Indie Srishord


Bariatric surgery for the treatment of nonalcoholic steatohepatitis:

Results of a Markov Model

Matthew Klebanoff 1,2 , Kathleen E. Corey 2,3 , Lee M. Kaplan 2,3 ,

Raymond T. Chung 2,3 , Chin Hur 2,3 ; 1 Institute for Technology

Assessment, Massachusetts General Hospital, Boston, MA; 2 Gastrointestinal

Unit, Massachusetts General Hospital, Boston, MA;

3 Harvard Medical School, Boston, MA

Obese individuals are at heightened risk of developing nonalcoholic

steatohepatitis (NASH), which can give rise to cirrhosis

and hepatocellular carcinoma. Current treatments for NASH

are poorly tolerated or have little long-term data to support their

use. A growing body of evidence suggests that bariatric surgery

not only leads to weight loss but may also improve NASH.

Using a Markov model, we determined the gain in life years

(LYs) and quality-adjusted life years (QALYs) following laparoscopic

Roux-en-Y gastric bypass (L-RYGB) for patients in various

weight classes with varying degrees of NASH fibrosis (F0-F3).

After surgery, a percentage of patients who benefit (69.7% in

base-case, from Mathurin et al., 2009) enter a ‘NASH remission’

state and stop progressing toward cirrhosis, although

they may later relapse (Fig. 1). The primary analysis does not

model surgery in F4 patients due to a lack of data in this group.

The model incorporated life year and quality-of-life decrements

associated with L-RYGB and complications. Extensive sensitivity

analyses examined the impact of model input uncertainty on

results. For all classes of obesity (mild, moderate and severe),

surgery led to a gain in LYs and QALYs. When we excluded

benefits of weight loss to explore the potential impact of surgery

on non-obese NASH patients, surgery reduced life expectancy

by 0.02 LYs for F0 patients, but increased LYs for F1-F3

patients. Surgery also decreased quality-adjusted survival for

F0 and F1 patients by 0.28 and 0.22 QALYs, respectively,

but increased QALYs for F2 and F3 patients. The number of

F3 patients needed to treat (NNT) to prevent one liver-related

death was seven; for F2 patients, the NNT was 17. Conclusions:

Bariatric surgery is effective as a treatment for patients

with NASH in all obesity classes. When we focused only on the

benefit due to NASH remission by eliminating any weight loss

benefit from the model, bariatric surgery improved life expectancy

for F1-F3 patients, but increased quality-adjusted survival

only for patients with more advanced fibrosis (F2 and F3), with

relatively favorable NNTs.


Kathleen E. Corey - Advisory Committees or Review Panels: Gilead; Speaking

and Teaching: Synageva

Lee M. Kaplan - Consulting: Johnson and Johnson, GI Dynamics, Novo Nordisk;

Grant/Research Support: Ethicon

Raymond T. Chung - Grant/Research Support: Gilead, Mass Biologics, Abbvie,

Merck, BMS

The following authors have nothing to disclose: Matthew Klebanoff, Chin Hur


A Trial-based Model of Liver Transplant and Liver-related

Death in Patients with Primary Biliary Cirrhosis


Marco Carbone 1 , Richard Pencek 2 , Tracy J. Mayne 2 , Tonya Marmon

2 , George F. Mells 3 , David Shapiro 2 ; 1 Division of Gastroenterology

and Hepatology, Department of Medicine, Addenbrooke’s

Hospital, Cambridge, United Kingdom; 2 Intercept Pharmaceuticals,

Inc., San Diego, CA; 3 Division of Gastroenterology and

Hepatology, Department of Medicine, University of Cambridge,

Cambridge, United Kingdom

Background: The UK-PBC research group derived and validated

a predictive model for liver transplant or liver-related

death in PBC patients based on Cox proportional hazards

regression analysis of the UK-PBC cohort (n=4022). Predictors

included ALP, bilirubin, ALT, albumin and platelet count. Obeticholic

acid (OCA) is a selective FXR agonist in development for

the treatment of PBC. In a recent Phase 3 trial (POISE), OCA

treatment was associated with significant improvements in ALP,

bilirubin and transaminases compared to placebo +UDCA.

Objective: To apply the UK-PBC risk algorithm to the POISE

trial data to predict effect of OCA on liver transplant and liver-related

death. Methods: In POISE, 216 PBC patients with

inadequate response or intolerance to UDCA were randomly

assigned to: OCA 10 mg, OCA titration (starting at 5 mg and

adjusted to 10 mg at 6 months based on clinical response) or

placebo; pre-trial UDCA continued. Treatment effect on liver

biochemistry was assessed at 12 months. The UK-PBC algorithm

assessed risk for liver-transplant or liver-related death at

5, 10 and 15 years based on 12-month change from baseline.

Results: The demographics of the POISE cohort were: mean age

56 years, 91% female, 94% white, 93% on UDCA. At baseline,

the UK-PBC algorithm indicated that placebo patients were

at slightly higher risk for events compared to both OCA arms.

At 12 months, predicted risk had significantly improved with

OCA, primarily due to improved ALP and bilirubin. Risk worsened

in the placebo arm, primarily due to increased bilirubin.

Predicted risk at 5, 10 and 15 years is shown below. Based on

absolute risk reduction at 15 years, the number needed to treat

with OCA to avoid liver transplant or liver-related death was

13 (OCA 10 mg) and 11 (OCA titration). Conclusions: The

UKPBC risk model is a validated prognostic indicator of liver

transplant-free survival. When applied to patients in POISE,


the UK-PBC algorithm predicts a significant decrease in risk for

liver transplant and liver-related death in patients treated with

OCA+UDCA relative to placebo+UDCA. OCA has the potential

to be an important new treatment option for PBC patients

unable to achieve adequate response with UDCA.


Richard Pencek - Employment: Intercept Pharmaceuticals; Stock Shareholder:

Intercept Pharmaceuticals

Tracy J. Mayne - Employment: Intercept Pharmaceuticals

Tonya Marmon - Employment: Intercept Pharmaceuticals, Inc; Stock Shareholder:

Intercept Pharmaceuticals, Inc

David Shapiro - Employment: Inttercept Pharmaceuticals; Management Position:

Intercept Pharmaceuticals; Stock Shareholder: Intercept Pharmaceuticals

The following authors have nothing to disclose: Marco Carbone, George F. Mells


Costs per Diagnosed Liver Disease Stage Among Individuals

with Chronic Hepatitis C Virus in the Veterans

Health Administration

Jennifer R. Kramer 1 , Peter Richardson 1 , Danielle Liffmann 2 , Hashem

B. El-Serag 1 , David B. Rein 2 ; 1 Department of Medicine, Michael E.

DeBakey VA Medical Center and Baylor College of Medicine,

Houston, TX; 2 NORC at the University of Chicago, Chicago, IL

Background: The prevalence of hepatitis C virus (HCV) infection

in the Veteran Health Administration (VHA) is high, but

previous research has not evaluated the costs of medical care

for diagnosed patients. Methods: We used the VHA HCV

Clinical Case Registry and Health Economics Resource Center

average cost data of inpatient, outpatient, and long-term care

to estimate the incremental annual cost of diagnosed HCV.

We compared the total healthcare costs of HCV antibody and

RNA + patients (cases) to HCV antibody + but HCV RNA -

patients (controls) identified from FY 1999 to 2009. Cases

were matched 1:1 to all available controls using a propensity

score that included demographic and clinical characteristics.

Patients were required to have an available AST/platelet ratio

(APRI) value in the index year (year of the earliest RNA test)

and one subsequent year. In each year, we classified patients

using ICD-9 codes and lab data (highest APRI in year) into 8

clinical stages: APRI 2, decompensated

cirrhosis (DCC), hepatocellular carcinoma (HCC),

transplant/post-transplant care, and death while infected with

HCV. Patients with DCC or HCC prior to HCV index date

were excluded. We estimated the incremental annual costs

for the 6 years after index date of each stage of HCV using

a hierarchical longitudinal two-part health expenditure model

(logistic, generalized linear model assuming a gamma distribution

and log link) and adjusted for clustering by incorporating

patient level random effects in both the logistic and gamma

models allowing these effects to be correlated. Results: There

were 157,303 patients (140,169 cases/17,134 controls) in

our source population; 52.8 years old (sd=8.0), 50.6% white,

25.9% Black, 96.7% male. The analysis set included 17,134

cases that were propensity matched to the 17,134 controls.

In 2009, 52.1% had APRI 2, 5.6% DCC, 0.7% HCC, 0.1% transplant,

and 3.9% died. Using 2015 dollars, on average, HCV RNA

- patients with an APRI


with the new balloon score. Most showed a stepwise increase

across the range of the score. Conclusion: The new balloon

score shows excellent correlation with clinical disease features.

Replacement of the old balloon score with the new balloon

score in the NAS would give more weight to ballooning as well

as increasing the dynamic range of the NAS.

NASH CRN New Balloon Score


Elizabeth M. Brunt - Consulting: Synageva; Independent Contractor: Rottapharm

Brent A. Neuschwander-Tetri - Advisory Committees or Review Panels: Nimbus

Therapeutics, Bristol Myers Squibb, Janssen, Mitsubishi Tanabe, Conatus,

Scholar Rock

Arun J. Sanyal - Advisory Committees or Review Panels: Bristol Myers, Gilead,

Genfit, Abbott, Ikaria, Exhalenz; Consulting: Salix, Immuron, Exhalenz, Nimbus,

Genentech, Echosens, Takeda, Merck, Enanta, Zafgen, JD Pharma, Islet

Sciences; Grant/Research Support: Salix, Genentech, Intercept, Ikaria, Takeda,

GalMed, Novartis, Gilead, Tobira; Independent Contractor: UpToDate, Elsevier

The following authors have nothing to disclose: David E. Kleiner, Patricia H. Belt,

Cynthia A. Behling, Ryan M. Gill, Cynthia D. Guy, Mark L. Van Natta


Overweight in late adolescence is associated with

decompensated liver disease later in life after 39 years

of follow-up

Hannes Hagström 1 , Per Stål 1 , Rolf W. Hultcrantz 1 , Tomas Hemmingsson

2,3 , Anna Andreasson 4,5 ; 1 Center for Digestive Diseases,

Unit of Hepatology, Karolinska University Hospital. Department

of Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden,

Stockholm, Sweden; 2 Institute of Environmental Medicine, Karolinska

Institutet, Stockholm, Sweden; 3 Centre for Social Research

on Alcohol and Drugs, Stockholm University, Stockholm, Sweden;

4 Stress Research Institute, Stockholm University, Stockholm, Sweden;

5 Division of Family Medicine, Karolinska Institutet, Stockholm,


Background: The prevalence of both obesity and liver diseases

is increasing globally. Obesity is associated with a worse prognosis

in different liver diseases. If overweight and obesity in

late adolescence is an independent predictor of liver disease

later in life is not studied. We investigated if overweight per se

predicts development of liver disease and decompensated liver

disease later in life with a up to 39 year follow-up. Materials

and methods: Data from 49,321 men (18-21 years) conscribed

to military service in Sweden between 1969 and 1970 was

used. Conscription during this time was mandatory and >97%

of the male population was available for this study. Weight and

height measured at conscription were used to calculate body

mass index (BMI). Data was collected from the national patient

register to identify any diagnosis of liver disease at a Swedish

hospital from time of conscription until the end of 2009.

A multivariate logistic regression model was used to estimate

odds ratios (OR) of decompensated liver disease (hepatorenal

syndrome [HRS], hepatocellular carcinoma [HCC], ascites,

varices or hepatic encephalopathy [HE]) for persons with BMI

over as compared to under 25. The model was adjusted for

alcohol use, smoking, use of narcotics, social status and blood

pressure at the time of conscription. Results: Mean BMI at conscription

was 20.97 kg/m 2 , where 6.6% had a BMI > 25 and

0.8% had a BMI > 30. During a follow-up period of in mean

37.9 (+/-4.8 years, range 0-39) years of follow-up, 525 persons

were diagnosed with liver disease. Of these, 206 persons

developed decompensated liver disease (18 cases of HRS, 31

HCC, 79 ascites, 124 varices and 7 HE as first manifestation

of decompensation, where 43 persons had multiple diagnoses

at presentation). Mean time to decompensation was 29.8

years (95% Confidence Interval [CI] 28.8-30.8 years). BMI >

25 was significantly associated with development of decompensated

liver disease both in the univariate (OR 2.09, 95%CI

1.38-3.16, p


than children with Zone 3 steatosis (73% vs 58%, p


olites predictive of fibrosis stage. RESULTS: Age, gender, BMI,

current/past alcohol use/smoking, total HDL/LDL cholesterol

or triglycerides did not differ with fibrosis stage. In univariate

testing, plasma concentrations of alpha-glutamyltyrosine N-acetylcitrulline

palmitoyl-palmitoyl-glycerophosphocholine, glycocholate

glutarate, fucose and fumarate associated with fibrosis

stage (p=0.0007). After controlling for age, gender and DM,

N-acetylcitrulline, alpha glutamytyrosine, palmitoyl-palmitoyl-glycerophosphocholine,

lignoceric acid, glycocholate, and

1-arachidonoyl associated with fibrosis (p=0.0009). Multivariate

analysis using logistic regression yielded an Area Under

the Receiver Operating Characteristic (AUROC) of 0.8804

for I-urobilinogen, malate, glutarate (pentanedioate), taurochenodeoxycholate,

3-hydroxyoctanoate, fucose, isoleucine,

palmitoyl-palmitoyl-glycerophosphocholine, N-acetylcitrulline.

Sparse ordinal regression was used to predict fibrosis stage,

resulting in a correlation coefficient of 0.3757 (p=5x10 -8 )

after leave-one-out cross-validation. The model identified a

69 metabolite signature predictive of fibrosis stage. CONCLU-

SIONS: Analysis of serum from NAFLD patients revealed alterations

in bile acids, steroids hormones, branched-chain amino

acid catabolism, TCA cycle metabolism, and mitochondrial

function in stage 3-4 fibrosis. The role of these metabolites as

potential biomarkers for hepatic fibrosis requires validation.

Pathways involved in bile acid and/or steroid hormone metabolism,

branched-chain amino acids, and/or mitochondrial function

may offer novel targets for anti-fibrotic therapies in NAFLD.


Lauren N. Bell - Employment: Metabolon, Inc.

Regis Perichon - Employment: Metabolon

Manal F. Abdelmalek - Consulting: Islet Sciences; Grant/Research Support:

Tobira, Gilead Sciences, NIH/NIDDK, Synageva, Genfit Pharmaceuticals,

Immuron, Galmed, TaiwanJ Pharma, Intercept, NGM Pharmaceuticals

The following authors have nothing to disclose: Ricardo Henao, Nigar Hasanaliyeva,

Jacob Wulff, Cynthia A. Moylan, James T. Lu, Cynthia D. Guy, Anna Mae



Beneficial effects of the dual PPAR α-δ agonist, GFT505,

on hepatic and cardiometabolic markers in adult NASH


Stephen A. Harrison 3 , Arun J. Sanyal 2 , Sven M. Francque 4 , Pierre

Bedossa 5 , Lawrence Serfaty 6 , Manuel Romero-Gomez 7 , Paul

Cales 8 , Manal F. Abdelmalek 9 , Stephen H. Caldwell 10 , Joost

Drenth 11 , Quentin M. Anstee 12 , Dean W. Hum 13 , Rémy Hanf 13 ,

Alice Roudot 13 , Sophie Megnien 13 , Bart Staels 14 , Vlad Ratziu 1 ;

1 Hepatology, Hopital Pitie Salpetriere, Paris, France; 2 Internal

Medicine/Division of Gastroenterology, Hepatology and Nutrition,

Virginia Commonwealth University, Richmond, VA; 3 Gastroenterology,

Brooke Army Medical Center, Fort Worth, TX; 4 Antwerp

Univesrity Hospital, Gastroenterology Hepatology, Antwerp, Belgium;

5 Pathology, Hopital Beaujon, Clichy, France; 6 Hepatology

Department, Hospital Saint-Antoine, Paris, France; 7 Valme University

Hospital, Digestive Diseases, Sevilla, Spain; 8 Hepatology

Department, Centre Hospitalier Universitaire d’ Angers, Angers,

France; 9 Medicine, Duke University, Durham, NC; 10 Hepatology

Department, University of Virginia, Charlotesville, VA; 11 Hepatology

Department, RUNMC, Nijmegen, Netherlands; 12 Institute

of Cellular Medicine, Newcastle University, Newcastle, United

Kingdom; 13 Genfit, Loos, France; 14 INSERM U1011, European

Genomic Institute for Diabetes (EGID), Université Lille 2, Lille,


Introduction: NASH patients (pts) have a high prevalence of cardiometabolic

risk factors, hence cardiovascular disease is the

leading cause of death in this population. Having demonstrated

the efficacy of GFT505 on the histological reversal of NASH,

here we evaluated the biochemical and cardiometabolic effects

of the drug in in this large, international, phase 2 randomized,

controlled trial of adult pts with biopsy-proven NASH. Methods:

Pts from the ITT population of the GOLDEN505 trial randomized

to the GFT505 120 mg (GFT120) and placebo (PBO)

arms were compared for treatment effects on plasma lipid,

glycemic, insulin resistance, inflammatory and liver markers.

Results are expressed as effect size vs. placebo (LS mean±SE).

Analyses were conducted for different stages of disease severity

based on the NAFLD Activity Score (NAS) at baseline (NAS 3:

mild, NAS 4-5: moderate, NAS>5: severe), and fibrosis stage

(F0-F1 vs. F2-F3 according to Kleiner et al). Results: Compared

to PBO, GFT120 significantly improved GGT (-29.31±6.36

U/L, p


Vlad Ratziu - Advisory Committees or Review Panels: GalMed, Abbott, Genfit,

Enterome, Gilead; Consulting: Tobira, Intercept, Exalenz, Sanofi-Synthelabo,


The following authors have nothing to disclose: Sven M. Francque, Pierre

Bedossa, Joost Drenth


Osteopontin is involved in chronic HBV infection and

enhances HBV replication and HBsAg secretion.

Sandra Phillips, Jason D. Coombes, Sameer Mistry, Roger Williams,

Wing-Kin Syn, Shilpa Chokshi; Institute of Hepatology,

Foundation for Liver Research, London, United Kingdom

Background/Aims: Hepatitis B virus (HBV) requires host cellular

machinery such as cyclophilins to support its ongoing propagation

(Phillips et al, Gastroenterology 2014). These host proteins

represent ideal candidates for therapeutic interventions

as they are generally expected to have a lower frequency of

drug-resistance and antiviral efficacy across genotypes. We

have also recently described a role for the host protein Osteopontin

(OPN), a pro-fibrogenic downstream effector of the

Hedgehog pathway, in enhancing HCV replication (Choi et al,

Clinical Sciences 2014). Whilst increased levels of blood OPN

have similarly been reported in chronic Hepatitis B (CHB), its

role in viral replication remains unknown. This study aimed

to evaluate the role of OPN in HBV replication, HBsAg secretion

and HBV-driven liver injury. Material and Methods: Stably

(HepG2215), transiently (HUH-7) transfected and infected

(HepaRG) cell lines, producing full HBV virions and HBsAg

particles were cultured over 72h in the presence/absence of

several concentrations of recombinant OPN (recOPN). In addition,

secreted OPN was neutralized using OPN-specific aptamers.

Cells and supernatants were harvested at baseline, 24, 48

and 72 hours. Intracellular OPN mRNA and HBV-DNA levels

were quantitated by Real-Time qPCR. HBsAg levels were measured

by ELISA. OPN levels were also measured by ELISA in

cell culture supernatants and in sera of controls and HBeAg(+)

CHB patients who were either treatment naive or treated with

potent antiviral agents. In addition, expression of OPN was

assessed in explanted livers from healthy and HBV-cirrhotic

patients. Results: Serum levels and intrahepatic expression of

OPN were significantly increased in CHB patients compared

to controls (up to 7 fold; p



The HBx-DLEU2 lncRNA complex regulates transcription

from cellular genes and the HBV cccDNA

Francesca Guerrieri 1,2 , Letizia Chiodo 1 , Debora Salerno 1,2 , Safaa

Jeddari 2 , Giancarlo Ruocco 1 , Massimo Levrero 2,1 ; 1 CLNS@SAPI-

ENZA, Istituto Italiano di Tecnologia (IIT), Rome, Italy; 2 Dept of

Internal Medicine, La Sapienza University, Rome, Italy

Background: HBx regulatory protein is required for HBV

cccDNA transcription/viral replication and contributes to HBV

oncogenicity. HBx affects the epigenetic control of HBV viral

chromatin, by preventing HDACs and PMRT1 recruitment onto

the cccDNA, as well as of cellular chromatin, by modulating

the recruitment of chromatin modifying enzymes. We previously

showed that HBx binds to the regulatory regions of several

ncRNAs (75 miRNAs and 34 lncRNAs). DLEU2 lncRNA

overlaps with the autophagy TRIM13 gene in the opposite

orientation. Upregulation of specific DLEU2 splicing variants

correlates with the development of solid and onco-hematolocic

malignancies. Objectives: Aim of this study is to clarify the role

of HBx in the regulation of the DLEU2/TRIM13 transcriptional

unit and their functions. Methods: The anti-HBx ChIPSeq was

performed in mock and HBV-replicating HepG2 cells using an

Illumina NGS platform. Independent ChIPs were analyzed by

TaqMan real-time PCR using lncRNA and gene specific primers.

The nCounter Nanostring technology and real-time RT-PCR

were used to evaluate lncRNAs and target gene expression,

respectively. Specific LNA longRNA GapmeRs (Exiqon) were

used for highly efficient inhibition of DLEU2 lncRNA function.

Results: ChIPSeq analysis of HBx global chromatin recruitment

revealed a specific binding to 34 lncRNAs regulatory

regions. Using a custom Nanostring panel we found that all

putative lncRNAs targets were modulated in HBV-replicating

HepG2 cells. Focusing on DLEU2 lncRNA, we demonstrated

that HBx is able to deregulate both its expression and the overlapping

gene TRIM13. HBx binding to the DLEU2 promoter

affects its epigenetic control, changes DLEU2 splicing profile

and up-regulates the antisense gene TRIM13. Selective degradation

of DLEU2 RNA resulted in a reduced H4 acetylation

on the TRIM13 promoter and a ~50% reduction of TRIM13

expression in HBV replicating HepG2 cells. In silico analysis of

a model HBx protein tertiary structure and DLEU2 tertiary structure

suggested a direct interaction. Using a RIP (RNA Immune

Precipitation) approach we confirmed HBx-DLEU2 interaction

in vivo. Finally, we found that DLEU2 inactivation inhibits the

expression of the HBx targets SREBP2 and miR138 as well as

HBV cccDNA transcription, with a sharp decrease of pgRNA

levels, thus suggesting a functional relevance of the DLEU2-

HBx interaction of HBV replication. Conclusion: HBx binds to

the DLEU2 promoter region to modify its epigenetic regulation

change the DLEU2 splicing profile. HBx forms a functional complex

with DLEU2 and regulates HBV replication.


Massimo Levrero - Advisory Committees or Review Panels: BMS, Jansen, Gilead,

Tekmira, Galapagos, Medimmune; Speaking and Teaching: MSD, Roche

The following authors have nothing to disclose: Francesca Guerrieri, Letizia Chiodo,

Debora Salerno, Safaa Jeddari, Giancarlo Ruocco


Viral Expression and Molecular Profiling of Liver Tissue

and Microdissected Hepatocytes in Hepatitis D Virus

(HDV) - Associated Hepatocellular Carcinoma (HCC)

Patrizia Farci 1 , Ashley B. Tice 1 , Ronald E. Engle 1 , Fausto Zamboni

2 , Marta Melis 1 , Zhifeng Long 3 , Jaime Rodriguez-Canales 4 ,

Jeffrey Hanson 4 , Michael R. Emmert-Buck 4 , David E. Kleiner 4 ,

Giacomo Diaz 5 ; 1 Laboratory of Infectious Diseases, National Inst

of Health, Bethesda, MD; 2 Liver Transplantation Center, Brotzu

Hospital, Cagliari, Italy; 3 Personal Diagnostix, Gaithersburg, MD;

4 Laboratory of Pathology, National Cancer Institute, Bethesda,

MD; 5 Department of Biomedical Sciences, University of Cagliari,

Cagliari, Italy

Although HCC develops in a high proportion of patients

with chronic hepatitis D, there are no data on the molecular

mechanisms of HDV-induced hepatocarcinogenesis. Because

of the vital dependence of HDV on HBV, the role of HDV in

promoting HCC is unknown. We used genomic techniques

to dissect the role of HDV and HBV. Microarray (Affymetrix

Human U133 Plus2) was performed on 33 specimens of

whole liver tissue (WLT, tumor vs. non-tumor) and selected

laser capture-microdissected hepatocytes (LCM, malignant vs.

non-malignant hepatocytes) obtained at liver transplant (LT)

from 5 patients with HDV-HCC; 29 WLT specimens from 7

patients with non-HCC HDV cirrhosis served as controls. A

parallel analysis was performed on WLT and LCM from 11

patients with HBV-HCC without HDV. Microarray data were

analyzed using BRB-Array Tools, with multivariate permutation

F-tests (FDR



Characterisation of the immune profile in Chronic Hepatitis

B, with CyTOF, to identify biomarkers of immune

control following NUC therapy discontinuation

Upkar S. Gill 1 , Laura Rivino 2 , Nina Le Bert 3 , Kamini Kunasegaran

2 , Damien Tan 3 , Sarene Koh 3 , Machteld Van Den Berg 2 , Yang

Cheng 4 , Navjyot K. Hansi 1 , Graham R. Foster 1 , Evan W. Newell

4 , Antonio Bertoletti 2,3 , Patrick T. Kennedy 1 ; 1 Hepatology Unit,

Centre for Immunobiology, Blizard Institute, Barts and The London,

School of Medicine & Dentistry, QMUL, London, United Kingdom;

2 Program Emerging Viral Diseases, Duke-NUS Graduate Medical

School, Singapore, Singapore; 3 Infection & Immunity Program,

Singapore Institute for Clinical Sciences, Agency for Science, Technology

& Research (A*STAR), Singapore, Singapore; 4 Singapore

Immunology Network, Singapore Agency for Science, Technology

& Research (A*STAR), Singapore, Singapore

INTRODUCTION: The absence/functional exhaustion of

HBV-specific T cells is the hallmark of chronic HBV infection

(CHB); conversely a robust functional response of these cells

is associated with viral control. Current therapies are limited

in their ability to restore the functional HBV-specific repertoire.

Consequently, Nucleot(s)ide analogue (NUC) therapy remains

indefinite in the majority. Clinical parameters alone cannot

distinguish in which patients NUC therapy can be safely discontinued,

with durable immune control from those who will

relapse and develop hepatic flares (HF). We studied a cohort

of virally suppressed patients on potent NUC therapy prior to

and after treatment discontinuation to characterise the immune

profile associated with viral control. PATIENTS & METHODS:

PBMC were analysed at 4-weekly intervals prior to and after

NUC discontinuation. The frequency of HBV-specific T cells

was assessed by IFNγ ELISPOT after 10 day expansion in the

presence of HBV peptides spanning the entire viral proteome.

Phenotypic and functional characteristics of T & NK cells were

studied with an in-depth longitudinal analysis of the expression

of >35 markers involved in cell activation, differentiation and

exhaustion, by cytometry time of flight (CyTOF), a novel mass

cytometry technology. Serum cytokine and chemokine levels

(IL-1β, IL-6, TNF-α, IL-10, CXCL-8, CXCL-10) were also measured

using Luminex. RESULTS: Prior to NUC discontinuation,

patients could be divided into 2 groups based on the presence/absence

of HBV-specific T cells. Upon NUC discontinuation,

those patients with detectable frequencies of circulating

HBV-specific T cells were able to control HBV replication and

did not demonstrate HF’s. In these patients HBV-specific T cells

preferentially target HBV polymerase followed by core proteins.

In patients without viral control who developed HF, we

noted a significant increase in serum CXCL10 and IL10 levels,

correlating with ALT. Patients without HF’s were characterised

by increased frequencies of a CD8+ T cell subset expressing

CD56, CD107, CCR5, CD127, CD28, 2B4, KLRG1, IFNγ,

TNFα, MIP1β & IL2. NK cell profiles did not differ depending

on viral rebound/HF upon NUC discontinuation, but all CHB

patients expressed significantly higher levels of the exhaustion

marker, KLRG1 on NK cells, compared with healthy controls.

CONCLUSIONS: Our data suggest that HBV-polymerase

specific T cells represent a potential biomarker that can be

used to predict those patients who will control HBV after NUC

discontinuation. These results further support the concept that

HBV-specific T cells are associated with viral control rather than

contributing to immunopathology.


Graham R. Foster - Advisory Committees or Review Panels: GlaxoSmithKline,

Novartis, Boehringer Ingelheim, Tibotec, Chughai, Gilead, Janssen, Idenix,

GlaxoSmithKline, Novartis, Roche, Tibotec, Chughai, Gilead, Merck, Janssen,

Idenix, BMS; Board Membership: Boehringer Ingelheim; Grant/Research Support:

Chughai, Roche, Chughai; Speaking and Teaching: Roche, Gilead, Tibotec,

Merck, BMS, Boehringer Ingelheim, Gilead, Janssen

Patrick T. Kennedy - Grant/Research Support: Roche, Gilead; Speaking and

Teaching: BMS, Roche, Gilead

The following authors have nothing to disclose: Upkar S. Gill, Laura Rivino, Nina

Le Bert, Kamini Kunasegaran, Damien Tan, Sarene Koh, Machteld Van Den Berg,

Yang Cheng, Navjyot K. Hansi, Evan W. Newell, Antonio Bertoletti


Hepatitis B virus X protein stimulates HBV replication by

regulating transcription factors associated with DNA or

histone methylation

Naoki Oishi 1,2 , Xuyang Wang 2 , Kazunori Kawaguchi 1,2 , Masao

Honda 1,2 , Seishi Murakami 2 , Shuichi Kaneko 1,2 ; 1 Department of

Gastroenterology, Kanazawa University Hospital, Kanazawa,

Japan; 2 Department of Disease Control and Homeostasis,

Kanazawa University Hospital, Kanazawa, Japan

Background: Hepatitis B virus (HBV), a small enveloped DNA

virus, chronically infects more than 350 million people worldwide

and causes liver diseases, from hepatitis to cirrhosis and

liver cancer. HBx is a multifunctional protein encoded by the

HBV genome that stimulates HBV replication. Previously, we

found that HBx has an important role in stimulating HBV transcription

and replication and that the transcriptional transactivation

function of HBx may be critical for its augmentation

effect on HBV replication. However, the molecular mechanism

of HBx in transcriptional coactivation remains unclear. In this

study, we provide a new insight into the coactivator mechanism

and the possibility of a new treatment target for HBV replication

and HBV-related hepatocarcinogenesis. Methods: We

used a retroviral vector to introduce wild-type HBx (HBxwt) or

empty vector (EV) into HepG2 cells. Gene expression profiling

was performed using Affymetrix GeneChip Human U133A2.0

ver.2.0 arrays according to the manufacturer’s protocol. Unsupervised

hierarchical clustering analysis and class comparison

analysis were performed with BRB-Array Tools software version

4.2.2. Transcription factor analysis was performed using

Ingenuity Pathway Analysis (IPA; Ingenuity Systems) to identify

potential upstream transcription factors (TFs). We used array

data from 244 chronic hepatitis B patients for analyzing clinical

features. Results: Unsupervised hierarchical clustering analysis

of HepG2-HBxwt or HepG2-EV cells revealed that the gene

expression profiles of these cell lines were significantly different.

Class comparison analysis between both cell lines identified

803 HBx-related genes. Six HBx-related activated TFs that

affected HBV replication were identified by IPA, small interfering

RNA, and inhibitor analyses. Three of these six TFs control

HBV replication by modifying DNA or histone methylation. By

analysis of 244 hepatitis B patients, these three TFs were found

to be expressed at a significantly highly level in HBeAg(+)

HBeAb(-) cases than in HBeAg(-)HBeAb(+) cases. Moreover,

these TFs up-regulated the expression of stemness markers

(EpCAM, AFP, and SOX9) and epithelial-mesenchymal transition

markers (ZEB1, ZEB2, and VIM) and were associated with

a poor prognosis of HCC. Conclusions: HBx upregulated three

TFs associated with DNA or histone methylation. Moreover,

HBx stimulates HBV replication and hepatocarcinogenesis by

modifying DNA or histone methylation. Our study suggests that

TFs activated by HBx will be an important therapeutic target

against both virus replication and hepatocarcinogenesis aimed

at the prevention of HCC.



Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,

Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,

Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,

Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer

Japan, Eli lilly Japan

The following authors have nothing to disclose: Naoki Oishi, Xuyang Wang,

Kazunori Kawaguchi, Masao Honda, Seishi Murakami


DNA Methylation Markers for Detection of Extrahepatic

Cholangiocarcinoma: Discovery, Tissue validation, and

Pilot Testing in Biliary Brush Samples

Hassan M. Ghoz 1 , Mohammed M. Aboelsoud 5 , Tracy C. Yab 1 ,

Calise K. Berger 1 , William R. Taylor 1 , Xiaoming Cao 1 , Patrick

H. Foote 1 , Nasra H. Giama 1 , Catherine D. Moser 1 , Douglas W.

Mahoney 4 , Emily G. Barr Fritcher 2 , Benjamin R. Kipp 3 , Thomas C.

Smyrk 3 , Lewis R. Roberts 1 , David Ahlquist 1 , John B. Kisiel 1 ; 1 Division

of Gastroenterology and Hepatology, Mayo Clinic, Rochester,

MN; 2 Molecular Anatomic Pathology, Mayo Clinic, Rochester,

MN; 3 Anatomic Pathology, Mayo Clinic, Rochester, MN; 4 Biomedical

Statistics and Infomatics, Mayo Clinic, Rochester, MN;

5 Memorial Hospital, Pawtucket, RI

Background & Aims: Cholangiocarcinoma (CCA) has poor

prognosis due to late stage presentation. Molecular markers

may offer improved accuracy for early detection. We have

identified discriminant DNA methylation markers for intrahepatic

CCA by next-generation sequencing. We now explore

this discovery approach further with extrahepatic CCA (eCCA)

and pilot best markers on brush cytology specimens. Methods:

Reduced-representation bisulfite sequencing (RRBS) was performed

on DNA extracted from 18 frozen eCCA tissue samples

and matched, adjacent benign biliary epithelia or liver parenchyma.

Differentially methylated regions (DMRs) with at least

3 CpGs were ranked by area under the receiver operating

characteristics curve (AUC) & by tumor:normal ratio and then

technically validated by methylation specific PCR (MSP) on

DNA from same samples. Best DMRs were selected for biological

validation on DNA from independent tissues comprising

15 eCCA cases and 60 controls (6 adjacent bile duct, 18

adjacent liver, 18 white blood cell samples, 18 normal colon

epithelia) using MSP. Biologically valid DMRs were then blindly

assayed on DNA extracted from independent archival biliary

brushing specimens including 14 perihilar (pCCA) & 4 distal

(dCCA) cases and 18 matched cytology-negative controls

(CTRL), 4 of which had primary sclerosing cholangitis (CTRL-

PSC). Results: From 5.5 million CpGs, 3674 significant DMRs

were mapped; 46 were selected for technical validation from

which 18 DMRs had an AUC of 0.75–1.0. In biological validation,

8 of these showed an AUC >0.75 in eCCA tissues. In

brushings, methylated EMX1, HOXA1, VSTM2B.764, KC01,

BMP3, SALL1, PTGDR, and RYR2, showed sensitivities of 100%

89%, 83%, 78%, 72%, 72%, 72%, and 72%, respectively,

at 90% specificity. Accuracy of EMX1 on brushings is shown

(figure). Conclusion: Whole-methylome discovery by next-generation

DNA sequencing yielded novel, highly-discriminant

methylation markers for eCCA. Results were validated in independent

tissues as well as cytology brushings. Further clinical

evaluation is clearly warranted.


Tracy C. Yab - Patent Held/Filed: Exact Sciences; Stock Shareholder: Exact


William R. Taylor - Patent Held/Filed: Exact Sciences; Stock Shareholder: Exact


Douglas W. Mahoney - Patent Held/Filed: Exact Sciences

Benjamin R. Kipp - Grant/Research Support: Abbott Molecular Inc.

Lewis R. Roberts - Grant/Research Support: Bristol Myers Squibb, ARIAD Pharmaceuticals,

BTG, Wako Diagnostics, Inova Diagnostics, Gilead Sciences, Five

Prime Therapeutics

David Ahlquist - Advisory Committees or Review Panels: exact sciences; Consulting:

exact sciences; Grant/Research Support: exact sciences; Stock Shareholder:

exact sciences

John B. Kisiel - Grant/Research Support: Exact Sciences Corporation

The following authors have nothing to disclose: Hassan M. Ghoz, Mohammed M.

Aboelsoud, Calise K. Berger, Xiaoming Cao, Patrick H. Foote, Nasra H. Giama,

Catherine D. Moser, Emily G. Barr Fritcher, Thomas C. Smyrk


Circulating Osteopontin and Prediction of Hepatocellular

Carcinoma Development in a Large European Population

Talita Duarte-Salles 2 , Sandeep Misra 1 , Magdalena Stepien 2 ,

Mazda Jenab 2 , Pierre Hainaut 2 , Laura Beretta 1 ; 1 Molecular and

Cellular Oncology, MD Anderson Cancer Center, Houston, TX;

2 International Agency for Research on Cancer (IARC-WHO), Lyon,


Background: Prevention and early detection strategies are

urgently needed to reduce the burden and mortality of hepatocellular

carcinoma (HCC), the second leading cause of cancer

death worldwide. We previously identified osteopontin

(OPN) as a promising marker for the early detection of HCC.

In this study, we investigated the association between pre-diagnostic

circulating OPN levels and HCC incidence and OPN

performance for early detection of HCC in a large population-based

cohort. Methods: A nested-case control study was

conducted within the European Prospective Investigation into

Cancer and Nutrition (EPIC) cohort study, a large longitudinal

cohort of >520,000 participants from 10 Western European

countries. During a mean follow-up of 4.8 years, 100 HCC

cases were identified. Each case was matched to two controls

by incidence density sampling. OPN levels were measured in

baseline plasma samples. Hepatitis infection status and other

relevant biomarkers such as alpha-fetoprotein (AFP) were also

measured. Conditional logistic regression models were used to

calculate multivariable odds ratio (OR) and 95% confidence

intervals (95%CI) for tertiles and continuous OPN levels in relation

to HCC. Receiver operating characteristics curves (ROC)

were constructed to determine the discriminatory accuracy of

OPN alone or in combination with other liver function biomarkers

in the prediction of HCC. Behavior of OPN in relation to

time of HCC diagnosis was also evaluated. Results: Circulating

OPN level was significantly associated with increased HCC

risk (per 10% increment in OPN level, OR multivariable



95%CI: 1.14-1.48), also among hepatitis-free participants

(OR multivariable

=1.19; 95%CI: 1.05-1.34). Adding liver function

tests to OPN improved the discrimination of those subjects who

developed HCC (AUC=0.86) while AFP didn’t improve the

model. In contrast, within two years of HCC diagnosis, the

combination of OPN and AFP was best able to predict HCC

risk (AUC=0.88) while further adjustment for liver function tests

or chronic hepatitis B/C infection did not improve the model.

Conclusions: In this study, pre-diagnostic serum OPN concentration

was associated with higher risk of first incident HCC

and the association was stronger among cases diagnosed

during the first two years of follow-up. OPN in combination

with liver function tests improved the detection of HCC in this

low-risk population. However, OPN in combination with AFP

was the best predictive model for detection of HCC within

two years of follow-up and did not change substantially after

excluding hepatitis positive participants, or after adjustment for

biomarkers of liver function.


The following authors have nothing to disclose: Talita Duarte-Salles, Sandeep

Misra, Magdalena Stepien, Mazda Jenab, Pierre Hainaut, Laura Beretta


Risk factors of complications in liver adenomatosis.

Barbara Willandt 1 , Ragna Vanslembrouck 2 , Raymond Aerts 3 ,

Baki Topal 3 , Wim Laleman 1 , David Cassiman 1 , Schalk Van Der

Merwe 1 , Werner Van Steenbergen 1 , Chris Verslype 1 , Frederik

Nevens 1 ; 1 Hepatology, UZ Leuven, Leuven, Belgium; 2 Radiology,

UZ Leuven, Leuven, Belgium; 3 Adominal Surgery, UZ Leuven, Leuven,


Introduction & aims: Hepatic adenomatosis is a disorder characterized

by multiple adenomas in an otherwise normal liver,

originally defined as a distinct entity from a solitary hepatocellular

adenoma. Due to the rarity of the disorder there is

lack of consensus about treatment strategies. We explored the

risk factors of complications and whether the genotype-phenotype

classification used for solitary hepatic adenomas has the

same prognostic significance in this disorder. Methods: We

re-analyzed 35 patients with liver adenomatosis : > 10-20

nodules (n=23pts) and > 20 (n=12pts); 6 patients with 5-10

nodules were evaluated separately; 2 pts excluded because

of glycogen storage disease. Biopsies, taken in the largest

nodules, were available in 30 patients. Results: The median

follow-up period was 6 years (range 1-24 years). All patients

(median age 38 year, range 18-54) were female except one

and 91% of them had a history of long term use of oral contraceptives

(median 17 years, range 5-34). BMI was > 25 kg/m 2

in 61% of patients and steatosis of the surrounding liver tissue

was present in 60%. Histological subtype analysis showed: 17

patients (63%) with inflammatory adenomas - in one patient

associated with β-catenin activated lesions, 9 patients (33%)

with HNF1-α mutation positive lesions of which 2 patients also

had a β-catenin activated lesion and in 1 patient only β-catenin

activated adenomata were identified. Inflammatory adenomas

were clearly associated with high BMI (> 25 kg/m 2 ) and steatosis

(13/16 (p


The following authors have nothing to disclose: Barry Schlansky, Willscott E.



Vitamin K dosing during sorafenib treatment for hepatocellular

carcinoma markedly prolonged overall survival

as well as progression free survival probably by

augmented ischemic tumor cell damage

Yoshimichi Haruna, Atsuo Inoue; Department of Gastroenterology

and Hepatology, Osaka General Medical Center, Osaka, Japan

Backgrounds and Aims. Sorafenib is the only oral anticancer

agent for advanced hepatocellular carcinoma (HCC). However,

its anticancer effects are not satisfying. It was reported

that combination of vitamin K and sorafenib drastically inhibited

HCC growth in vitro and in animal experiments. In this

study, we examined advantage of vitamin K dosing during

sorafenib treatment for HCC, focusing on different manners

of changes in serum des-γ-carboxy prothrombin (DCP) levels

during vitamin K combination and sorafenib alone groups.

Patients and Methods. Fifty-five patients (male/female 42/13,

age (mean ± SD) 73.9 ± 8.3, the Barcelona Clinic Liver Cancer

(BCLC) stage B/C 21/34) were retrospectively studied.

Twenty out of them were orally given vitamin K2 (45mg daily)

during sorafenib treatment. There were no relevant differences

between the vitamin K combination and sorafenib alone groups

in characteristics at baseline. We compared the time to radiologic

progression and overall survival (OS) in the two groups.

The radiologic assessment was performed according to modified

RECIST. Serum DCP levels was tested before and 8 weeks

after the start of treatment. Results. The disease control rate was

78.9% vs. 22.9% in sorafenib + vitamin K group and sorafenib

alone one, respectively (P < 0.001). Median progression free

survival (PFS) was 8.0 months vs. 2.5 months in sorafenib +

vitamin K group and sorafenib alone one, respectively (P <

0.001). Furthermore, vitamin K dosing markedly prolonged OS

(median survival: 23.0 months vs. 10.0 months, P = 0.002).

Interestingly, serum DCP levels were increased in partial

response (PR) + stable disease (SD) cases of sorafenib alone

group (2.42±0.92 Ç 2.78±0.95 Log mAU/mL, P = 0.051)

despite suppressed tumor growth. In contrast, those of vitamin

K-dosing group showed remarkable decline (2.35±0.56

Ç 1.38±0.20 Log mAU/mL, P = 0.001). Discussion. DCP,

a tumor marker, is also known as an indicator of ischemic

status of HCC. The DCP elevation observed in sorafenib alone

group seems to show ischemic status of tumor cells in deteriorated

angiogenesis caused by sorafenib. On the other hand,

there are many reports suggesting that the DCP is autologous

growth factor augmenting HCC growth and a paracrine factor

enhancing tumor angiogenesis. Thus, DCP elevation resulting

from sorafenib treatment could incomplete the antitumor effect.

In contrast, the suppressed DCP production under pharmacological

vitamin K dosing could complete antitumor action of

sorafenib. Conclusion. The nontoxic agent, vitamin K, dosing

during sorafenib treatment for HCC markedly prolonged OS

as well as PFS probably by augmented ischemic damage of

tumor cells.


The following authors have nothing to disclose: Yoshimichi Haruna, Atsuo Inoue


Outcomes following Radiofrequency Ablation of small

HCC: Impact of etiology

Suraj Sharma 2 , Katherine Pratte 1 , Matthew Kowgier 2 , Morris Sherman

2 ; 1 General Internal Medicine, University of Toronto, Toronto,

ON, Canada; 2 Toronto Liver Clinic, Toronto Western Hospital,

Toronto, ON, Canada

Background: Among patients who have undergone surgical

resection, etiology of HCC may be an important predictor of

transplant-free survival (TFS), with some studies showing worse

survival in HCV, compared to HBV. However, there is a paucity

of data on the impact of etiology on outcomes in patients

undergoing RFA. This study aims to characterize a cohort of

patients undergoing RFA for small HCCs, and determine the

impact of etiology of HCC on TFS. Methods: Chart review was

conducted on all patients undergoing RFA at the University

Health Network between January 1, 2008 and December 31,

2011. Patients were followed for outcomes until January 1,

2014. Data collected included etiology of chronic liver disease,

severity of liver disease and size and location of HCC.

Complete ablation was defined as the absence of residual disease

at 3 months post RFA. Recurrence of HCC was defined as

tumor foci developing at a location of prior RFA treatment – this

may be early (1 year). Other treatment

modalities offered to patients were noted. Outcome measures

included transplant-free survival and tumor recurrence. Results:

256 patients underwent RFA during the study period. 87% of

the patients had documented evidence of cirrhosis. 81 patients

with HBV (79% on treatment), 112 patients with HCV (54%

treated, 20% SVR), and 63 patients with nonviral etiologies

were included. 24% of the cohort was female. The mean age

at diagnosis of HCC was 62.3 (±10.1) years. The mean size

of the largest lesion at diagnosis was 2.8cm (±1.6cm). All

patients underwent at least one session of RFA (mean 1.9 treatments).

The average size of RFA-treated lesions was 2.4cm

(±0.96cm). 79% of the patients had complete ablation. Recurrence

occurred in 74 patients (29%), with mean recurrence-free

interval of 498 days (±383d). 109 (42%) patients died or

underwent liver transplant during the follow-up period. The

primary outcome (transplant or death) was reached in 27%

of HBV patients (median TFS 1.5 years), 53% of HCV patients

(median TFS 1.7 years) (p=0.0003 HBV vs. HCV), and 40%

of nonviral patients (median TFS 1.5 years). In a multivariable

analysis including age, etiology and MELD score, patients

with HCV had significantly worse survival than patients with

HBV (HR 2.6, p



Transcriptomic Analysis Of Human Hepatocytes In

3-Dimensional Cultures With Maintained Perfusion And

Transport Offers Insights Into The Pharmacotoxicology

Of Clinically Relevant Concentrations Of Obeticholic


Arun J. Sanyal 2 , Robert Figler 1 , Brett R. Blackman 1 , Svetlana

Marukian 1 , Sol Collado 1 , Mark Lawson 1 , Aaron J. Mackey 1 ,

David Manka 1 , Brian R. Wamhoff 1 , Ajit Dash 1 ; 1 HemoShear Therapeutics,

Charlottesville, VA; 2 Virginia Commonwealth University,

Richmond, VA

NASH has emerged as the most common cause of chronic liver

disease in the Western world. Recently, obeticholic acid (OCA)

a semi-synthetic bile acid derivative and farnesoid-X-receptor

(FXR) agonist has been shown to improve insulin sensitivity in

humans with type 2 diabetes and liver histology in subjects

with NASH. The precise mechanisms by which these effects

are mediated are not fully known. AIMS: To use an unbiased

transcriptomic analysis of hepatocytes exposed to clinically relevant

doses of OCA to evaluate its effects on pathophysiologically

relevant pathways. METHODS: We previously described

a dynamic 3-dimensional system using liver-derived blood flow

and transport parameters to restore primary human hepatocyte

biology, allowing for culture at close to physiological insulin/

glucose conditions and eliciting drug responses at clinically-relevant

concentrations. This system represents an advance over

existing in vitro systems, which require supra-physiological insulin

concentrations and are associated with loss of polarity and

transport functions. We applied this system to gain insights into

the pharmacotoxicological effects of OCA. Primary hepatocytes

from 5 human donors were exposed to OCA for 48 hours

at concentrations approximating clinical therapeutic (0.5 μM)

and supratherapeutic (10 μM) levels. Whole genome transcriptomics

was performed by RNAseq, and the data analyzed

using both unbiased algorithms and biased interrogation of

specific pathways. RESULTS: A dose-dependent suppression

of Cyp7a and Cyp27a along with upregulation of bile salt

canalicular export transporters ABCB4 and ABCB11 and basolateral

transporters OSTA and OSTB were noted as expected.

Interestingly, FGF19, not known to be highly expressed in

hepatocytes, was strongly upregulated in our system by OCA

suggesting an autocrine suppression of FXR expression. A concomitant

suppression of LXR and HNF4α signaling associated

with NROB2 activation was also seen. Pleiotropic effects of

OCA included suppression of TGFβ as well as TNFα signaling

pathways, and could explain its beneficial effect of reducing

inflammatory and fibrotic changes in NASH. Consistent with

the hypercholesterolemia reported with OCA treatment was the

upregulation of ApoB, the major lipoprotein of LDL. CONCLU-

SIONS: OCA, in clinically relevant conditions, induces metabolic,

anti-inflammatory and anti-fibrotic/oncogenic signaling

in primary hepatocytes. These findings provide a mechanistic

basis for OCA effects in NASH.


Arun J. Sanyal - Advisory Committees or Review Panels: Bristol Myers, Gilead,

Genfit, Abbott, Ikaria, Exhalenz; Consulting: Salix, Immuron, Exhalenz, Nimbus,

Genentech, Echosens, Takeda, Merck, Enanta, Zafgen, JD Pharma, Islet

Sciences; Grant/Research Support: Salix, Genentech, Intercept, Ikaria, Takeda,

GalMed, Novartis, Gilead, Tobira; Independent Contractor: UpToDate, Elsevier

Robert Figler - Employment: HemoShear Therapeutics, LLC

Brett R. Blackman - Board Membership: HemoShear LLC; Management Position:

HemoShear LLC; Patent Held/Filed: HemoShear LLC; Stock Shareholder:

HemoShear LLC

Svetlana Marukian - Employment: HemoShear LLC

Mark Lawson - Employment: Hemoshear

Aaron J. Mackey - Employment: HemoShear, LLC

David Manka - Employment: HemoShear Therapeutics; Stock Shareholder:

HemoShear Therapeutics

Brian R. Wamhoff - Stock Shareholder: HemoShear, LLC

Ajit Dash - Employment: HemoShear LLC

The following authors have nothing to disclose: Sol Collado


Inhibition of NF-kB by deoxycholic acid induces miR-

21/PDCD4-dependent hepatocelular apoptosis

Pedro M. Rodrigues 2 , Marta B. Afonso 2 , André L. Simão 2 , Pedro

M. Borralho 1 , Cecília M. Rodrigues 1 , Rui E. Castro 1 ; 1 iMed.

ULisboa & Dep. of Biochemistry and Human Biology, Faculty of

Pharmacy, University of Lisbon, Lisboa, Portugal; 2 iMed.ULisboa,

Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal

MicroRNAs (miRNAs/miRs) play a key regulatory role in

metabolic liver function. In particular, miR-21 deregulated

expression has been associated with a wide spectrum of liver

disorders, contributing to disease development and progression.

We have demonstrated that deoxycholic acid (DCA), a

cytotoxic bile acid implicated in the pathogenesis of non-alcoholic

fatty liver disease, inhibits miR-21 expression in hepatocytes.

Still, the regulatory mechanisms behind miR-21 inhibition

and its contribution for cell demise are lacking. We aimed to

unveil the mechanisms underlying modulation of miR-21 by

DCA and to evaluate their exact contribution to DCA-induced

cell death. Primary rat hepatocytes were treated with 25-200

mM DCA for 24 h. Cell death, viability and caspase-3 activity

were measured by the ApoTox-Glo Triplex Assay. miR-21

expression was measured by qRT-PCR. Programmed cell death

4 (PDCD4), a miR-21 pro-apoptotic target, was evaluated by

immunoblotting and after transfecting cells with a reporter luciferase

plasmid. NF-kB, IkB and active caspase-2 levels were

also measured by immunoblotting, while NF-kB activation was

evaluated using a specific luciferase assay and by analyzing

NF-kB subcellular localization. Reactive oxygen species (ROS)

levels were analysed using the fluorescent probe 2’,7’-dichlorodihydrofluorescein

diacetate. Finally, for functional studies,

miR-21, PDCD4, caspase-2 and NF-kB were modulated using

both genetic and pharmacologic approaches, and a well-established

antioxidant, N-acetyl-L-cysteine (NAC). Our results

show that DCA inhibits miR-21 expression in a dose-dependent

manner, while increasing PDCD4 protein levels, with a

concomitant decrease in cell viability and an increase in cell

death, caspase-2/-3 activation and ROS production. Either

miR-21 overexpression or PDCD4 silencing hampered DCA-induced

cell death. Furthermore, NF-kB activity was decreased in

a similar pattern to miR-21 expression in DCA-treated hepatocytes.

In fact, NF-kB inhibition, using a selective chemical inhibitor

(BAY 11-7085), further decreased miR-21 and increased

PDCD4 expression levels and apoptosis by DCA. In agreement,

opposite results were observed in cells overexpressing

NF-kB or incubated with NAC. In conclusion, NF-kB represents

a major target of DCA in regulating the miR-21/PDCD4 pathway

whereas, in turn, oxidative stress and caspase-2 activation

are two key upstream mechanisms leading to inhibition of

NF-kB transcriptional activity by DCA. As such, these signaling

circuits constitute appealing targets for bile acid- and/or

miR-21-associated liver pathologies.


The following authors have nothing to disclose: Pedro M. Rodrigues, Marta B.

Afonso, André L. Simão, Pedro M. Borralho, Cecília M. Rodrigues, Rui E. Castro



Critical role of sirtuin 1-mitofusin 2 axis in ischemia/

reperfusion injury in aged livers

Sooyeon Lee, Kristina L. Go, Rebecca Y. U, Joseph A. Flores-Toro,

Ivan Zendejas, Kevin E. Behrns, Jae-Sung Kim; Surgery, University

of Florida, Gainesville, FL

INTRODUCTION: Ischemia/reperfusion (I/R) injury is a fundamental

obstacle in liver resection and transplantation surgery.

Aged livers have markedly less reparative capacity after I/R

than young livers. Impaired autophagy and consequent onset

of mitochondrial dysfunction contributes to this age-dependent

increase in I/R injury. Autophagy, a primary catabolic pathway

in the liver, is strongly modulated by sirtuin 1 (SIRT1),

a deacetylase boosting cell survival and longevity. The AIM

of this study is to investigate the role of SIRT1 in I/R injury in

aged livers. METHODS: Hepatocytes from male C57/BL6 mice

at 3 (young) and 26 months (aged) of age were subjected to

simulated I/R. Biochemical, genetic and imaging analysis were

performed to assess cell death, autophagy flux, and mitochondrial

function. RESULTS: While 2 h of ischemia has no effects

on young hepatocytes, this short-term I/R impaired autophagy,

and rapidly induced the mitochondrial permeability transition

and necrotic cell death in aged hepatocytes. Immunoblotting

analysis indicated a near-complete loss of SIRT1 in aged cells

after I/R, which did not occur in young cells. Interestingly,

adenoviral overexpression SIRT1 in aged cells failed to mitigate

I/R injury, suggesting that additional factor(s) is required

to support cell survival. Immunoprecipitation approaches

revealed an interaction of SIRT1 with mitofusin 2 (MFN2), a

mitochondrial outer membrane protein. MFN2 levels in aged

cells became undetectable after a short-term I/R, while the

depletion of MFN2 was not observed in young cells. Similar

to SIRT1, MFN2 overexpression alone did not confer cytoprotection.

However, co-overexpression of both MFN2 and SIRT1

protected aged cells against I/R, implying a critical cross-talk

between SIRT1 and MFN2. To further investigate the importance

of SIRT1-MFN2 interaction, deletion mutants of MFN2

were constructed and expressed into HEK293T cells. Consistent

with the findings in hepatocytes, SIRT1 overexpression in

wild type cells not only deacetylated MFN2 but also increased

autophagic flux. However, autophagy stimulated by SIRT1 was

subdued in the N-terminal deletion mutants (Δ2-92 and Δ262-

392), suggesting a pivotal role of the N-terminal domain of

MFN2 in SIRT1-mediated autophagy induction. CONCLUSION:

Our findings show that the depletion of both SIRT1 and MFN2

attributes to defective autophagy, mitochondrial dysfunction

and cell death in aged hepatocytes after I/R. The SIRT1-MFN2

axis may be an integral factor that governs survivability of

aged livers after reperfusion.


The following authors have nothing to disclose: Sooyeon Lee, Kristina L. Go,

Rebecca Y. U, Joseph A. Flores-Toro, Ivan Zendejas, Kevin E. Behrns, Jae-Sung



HMGB1-driven Feedforward Hepatocyte Necroptosis

Circuit in Lethal Acetaminophen-induced liver injury.

Charlotte Minsart 1 , Claire Liefferinckx 1 , Sandrine Rorive 2,3 , Arnaud

Lemmers 4,1 , Eric Quertinmont 1 , Eric Trépo 1,4 , Isabelle Salmon 2,3 ,

Jacques Devière 4,1 , Christophe Moreno 4,1 , Isabelle A. Leclercq 5 ,

Richard Moreau 6,7 , Thierry Gustot 4,1 ; 1 Laboratory of Experimental

Gastroenterology, Université Libre de Bruxelles, Brussels, Belgium;

2 Pathology, Erasme Hospital, Brussels, Belgium; 3 DIAPATH- center

for microscopy and molecular imaging (CMMI), Gosselies,

Belgium; 4 Gastroenterology and Hepato-Pancreatology, Erasme

Hospital, Brussels, Belgium; 5 Laboratory of Hepato-Gastroenterology,

Institut de Recherche Expérimentale et Clinique, Université

Catholique de Louvain, Brussels, Belgium; 6 INSERM Unité 1149,

Centre de Recherche sur l’inflammation (CRI), Paris, France; 7 UMR

S_1149, Université Paris Diderot, Paris, France

Background & Aims: Release of damage-associated molecular

patterns, in particular High-mobility group box (HMGB)

1, contributes to acetaminophen (APAP)-induced liver injury

but the mechanisms involved are currently incompletely understood.

The aim of the study is to investigate the contribution of

HMGB1 in vivo and in vitro at early time points of the APAP-induced

liver injury and its role in the propagation of necrosis

process. Methods: APAP hepatotoxicity was induced in vivo

by intraperitoneal injection in C57Bl/6 mice and in vitro on

cultured HepaRG cells. HMGB1 was quantified by ELISA or

immuno-staining. Cell death was determined by MTT, ALT, LDH

and caspase-3 assays. Glycyrrhizin (GL) and ethyly pyruvate

(EP) was used to inhibit HMGB1. Liposomal clodronate was

administrated to mice to deplete Kupffer cells (KC). Expression

of HMGB1 receptors was assessed by RT-PCR and flow

cytometry. Dabrafenib and necrostatin-1was used to inhibit

receptor-interacting protein (RIP)3 and RIP1 respectively.

Results: In APAP-challenged mice, GL inhibited the HMGB1

release (decrease of serum levels and increase in hepatocellular

retention in centrolobular area) with improved survival.

Depletion of KC in mice exacerbated APAP-induced hepatocyte

necrosis and HMGB1 release suggesting that HMGB1 did

not act through KC activation. Addition of APAP on cultured

HepaRG induced cell necrosis characterized by LDH release

without caspase-3 activation, and HMGB1 release. Moreover,

HepaRG were exposed to APAP for 6 hours and the so-conditioned

medium induced cell death of unexposed HepaRG.

Inhibition of HMGB1 by GL or EP reduced APAP- and conditioned

medium-induced HepaRG necrosis and further HMGB1

release. Exposure of HepaRG and primary human hepatocytes

to rhHMGB1 resulted in their death, underlining that HMGB1

acts directly on hepatocytes. HepaRG expressed previously

described HMGB1 receptors (TLR2, TLR4 and TLR9) at mRNA

and protein level. Pre-treatment of HepaRG by dabrafenib, a

specific RIP3 inhibitor, and not by necrostatin-1prevented this

HMGB1-induced cell death. Conclusion: HMGB1 contributes

to APAP-induced liver injury through a RIP3-dependent hepatocyte

necroptosis using a feedforward mechanism. Inhibition

of HMGB1 at the early phase of APAP-induced liver injury

improved animal survival by reducing the propagation of this

regulated hepatocyte necrosis.


Christophe Moreno - Consulting: Abbvie, Janssen, Gilead, BMS; Grant/Research

Support: Janssen, Gilead, Roche, Astellas

Isabelle A. Leclercq - Independent Contractor: Genfit

The following authors have nothing to disclose: Charlotte Minsart, Claire Liefferinckx,

Sandrine Rorive, Arnaud Lemmers, Eric Quertinmont, Eric Trépo, Isabelle

Salmon, Jacques Devière, Richard Moreau, Thierry Gustot



Oxaloacetic Acid Protects the Liver From Warm Ischemia/Reperfusion


Grégory Merlen, Benoit Lacoste, Benoît Dupont, Valérie-Ann Raymond,

Marc Bilodeau; CRCHUM, Montreal, QC, Canada

Background: Liver ischemia/reperfusion (I/R) is an important

cause of liver damage early after liver transplantation, post

liver resection or during hemorrhagic shock. One of the mechanisms

of cell death occurring in that setting is the disruption of

mitochondrial activity that lead to alterations in cellular energy

metabolism. We hypothesized that oxaloacetic acid (OAA),

which is at the crossroads of gluconeogenesis, amino acid

metabolism and the citric acid cycle, could refuel the energy

metabolism during I/R and therefore protect the liver against

injury. In vitro, we have already demonstrated that OAA was

the most potent citric acid intermediate in its capacity to protect

rat hepatocytes from hypoxia. We have also shown that the

administration of OAA considerably reduces the extent of liver

injury in the left portal vein ligation model of warm liver ischemia

in the rat. We here analyze the potential protective effect

of this compound in a model of hepatic I/R. Methods: Animals

were subjected to 1 hour of left portal pedicle ligation (common

bile duct, left hepatic artery and left portal vein) followed

by reperfusion by unclamping. Animals treated with OAA

[100mg/kg] received a bolus injection through the ileocolic

vein 30 minutes before surgery. The extent of liver I/R injury

was assessed by serum transaminase levels measurements, histological

signs of tissue damage and liver weight (edema).

Results: Treatment with OAA before reperfusion significantly

decreased the AST levels released in blood. After 2 hours of

reperfusion the AST and ALT levels were respectively decreased

by 43%±14% (p


Here, we investigated the underlying mechanisms of immune

tolerance by the gut-liver axis, focusing on interactions between

gut microbiota and hepatic immune competent cells. METHODS:

Male C57BL/6 mice were administered an initial and subsequent

sub-lethal dose of ConA to induce immunological tolerance.

Liver mononuclear cells were separated 12 h after the

final ConA injection and analyzed by flow cytometry. Immune

cell subset cytokine production stimulated with TLR ligands was

measured and the composition of intestinal bacterial flora was

evaluated by T-RFLP and comprehensive metagenomics. Intestinal

permeability was measured by FITC-dextran following

ConA injection. Mice were treated with antibiotics (ampicillin,

neomycin, metronidazole, and vancomycin) or fecal microbiota

transplantation to clarify the role of the gut-liver axis in liver

tolerance. RESULTS: A single ConA injection induced severe

liver inflammation but ConA administration 7 days after initial

injection increased immunosuppresive CD11c + DCs (D7-cDC)

and regulatory T cells in the liver and promoted immunological

tolerance. D7-cDC had regulatory characteristics and produced

IL-10 and TGF-β upon TLR9 ligand stimulation especially

from damaged hepatocytes. As an initiation of liver tolerance,

intestinal permeability was significantly increased at 4 h following

a single ConA injection. Importantly, the composition of

intestinal bacterial flora changed and the ratio of Clostridium

subcluster XIV to Bacteroides increased thereafter. Transplantation

of fecal microbiota derived from mice post-ConA administration,

but not from untreated mice, to gut sterilized mice

induced immunosuppressive CD11c + cDCs and regulatory T

cells in the liver and reduced liver injury by ConA. A similar

result was observed in germ-free and gnotobiotic mice inoculated

with fecal microbiota derived from ConA-injected SPF

mice. CONCLUSIONS: Dysbiosis with increased intestinal permeability

following ConA administration promotes the migration

of immunosuppressive cells in the liver in preparation for

further liver injury. This study identifies a novel homeostasis

pathway that regulates immune activation and tolerance in the

liver through the gut-liver axis.


Takanori Kanai - Grant/Research Support: Mitsubishi Tanabe Pharma Corporation

The following authors have nothing to disclose: Nobuhiro Nakamoto, Hirotoshi

Ebinuma, Po-sung Chu, Nobuhito Taniki, Takeru Amiya, Akihiro Yamaguchi,

Shunsuke Shiba, Hidetsugu Saito


Chronic ethanol feeding suppresses Con A induced T cell

response and hepatitis in ALDH2-deficient mice

Yanhang Gao 1,2 , Yong He 1 , Dechun Feng 1 , Zhou Zhou 1 , Bin

Gao 1 ; 1 NIAAA, National Institutes of Health, Rockville, MD;

2 Hepatology, The first hospital of Jilin University, Changchun,


Background and Aims: Aldehyde dehydrogenase 2 (ALDH2)

is well known about its role on detoxifying aldehydes in ethanol

metabolism. An ALDH2 inactivating mutation is the most

common single point mutation in humans, mostly found in East

Asians, which can cause acetaldehyde accumulation after

alcohol consumption. However, how acetaldehyde accumulation

affects T cell response and hepatitis in ALDH2-deficent

individuals remains unknown. Methods: Wide-type and ALDH2

knockout mice were subjected to ethanol feeding for 6 weeks,

followed by injection of a single dose of Concanavalin A (Con

A). Liver injury and serum cytokine levels were evaluated.

Results: Con A injection rapidly induced T cell hepatitis, which

recapitulates the histological and pathological sequelae of T

cell-mediated hepatitis in viral hepatitis patients. Compared

with ethanol-fed wild-type mice, ethanol-fed ALDH2 -/- mice had

lower degree of liver damage post Con A injection, as demonstrated

by the lower level of serum alanine aminotransferase

(ALT), the less infiltration of neutrophils, the fewer number of

activated macrophages, and the smaller area of necrosis in

the liver. Furthermore, serum levels of several pro-inflammatory

cytokines including IFN-γ, TNF-α, MCP-1, IL-4, IL-6, IL-10,

IL12p70, were lower in ethanol-fed ALDH2 knockout mice than

in wide-type mice post Con A injection. In agree with serum

cytokine levels, hepatic activation of the IFN-, IL-6, IL-4 downstream

signaling molecule signal transducer and activator of

transcription (STAT1, 3, 6) were lower in ALDH2 knockout mice

than in wide-type mice. Furthermore, ethanol-fed ALDH2 knockout

mice had much higher levels of plasma corticosterone than

ethanol-fed wild-type mice. Inhibition of endogenous glucocorticoid

activity by pretreatment with the glucocorticoid receptor

antagonist RU-486 restored Con A-mediated liver injury in

ALDH2-deficeint mice. Conclusions: Chronic ethanol feeding

results in greater elevation of plasma corticosterone levels in

ALDH2 knockout mice than in wild-type mice. These elevated

corticosterone levels inhibit Con A-induced T cell response and

hepatitis in ALDH2 knockout mice. ALDH2-deficient individuals

who are excessive drinkers may have reduced T cell response

and are more susceptible to hepatitis viral infection.


The following authors have nothing to disclose: Yanhang Gao, Yong He, Dechun

Feng, Zhou Zhou, Bin Gao


The antifibrotic effect of IL-4Rα signaling depends on

macrophage subsets prevalent during liver fibrosis progression

and reversal

Shih-Yen Weng 1 , Santosh Vijayan 1 , Xiaoyu Wang 1 , Yilang Tang 4 ,

Kornelius Padberg 1 , Yong Ook Kim 1 , Brombacher Frank 5 , Jeff R.

Crosby 3 , Michael L. McCaleb 3 , Ari Waisman 4 , Ernesto Bockamp 1 ,

Detlef Schuppan 1,2 ; 1 Institute of Translational Immunology, University

Medicine, Johannes Gutenberg University, Mainz, Germany,

Mainz, Germany; 2 Beth Israel Deaconess Medical Center, Boston,

MA; 3 Isis Pharmaceuticals, Carlsbad, CA; 4 Institute for Molecular

Medicine, Mainz, Germany; 5 Institute of Infectious Disease and

Molecular Medicine, Cape town, South Africa

Background and aims: In response to various stimuli, macrophages

can be functionally divided into M1 (inflammatory) and

M2 (anti-inflammatory) subsets. Alteration of the M1-M2 ratio

likely impacts liver fibrosis progression and reversal. IL-4Rα,

which is activated by IL-4 and IL-13 has been linked to M2

polarization. However, the functional role of IL-4Rα in liver

fibrosis progression and reversal remained unclear. Methods:

IL-4Rα -/- and wild type mice were treated with CCL 4

via oral

gavage for 6 weeks. Liver fibrosis reversal was investigated

after 2 weeks of CCL 4

withdrawal. Antisense oligonucleotides

(ASO) were applied intraperitoneally (40mg/kg, 3 times per

week) during the 6 week s of CCL 4

treatment or the during 2

weeks off CCL4. Results: At 6 weeks of CCL 4

treatment, IL-4Rα -/-

mice had 25% less fibrosis than their wild type littermates. Flow

cytometry analysis of the liver of IL-4Rα -/- mice showed less

inflammation indicated by a reduction of B cells, and CD4

and CD8 T cells. The proportion of resident M1 macrophages

was significantly increased and that of proinflammatory monocytic

Ly6c hi macrophages largely reduced in IL-4Rα -/- livers.

To further validate IL-4Rα function, mice with cell specific deletion

of IL-4Rα were generated and subjected to CCL 4


No overt change of liver fibrosis induction was found

in T cell-specific knockout mice (IL-4Rα ∆CD4 ). However, after 6

weeks of CCL4-treatment, fibrosis was significantly attenuated

in mice with myeloid cell-specific IL-4Rα deletion (IL-4Rα ∆LysM ).


In contrast, IL-4Rα ∆LysM mice displayed retarded fibrosis resolution

as evidenced by slower clearance of hydroxyproline

and Sirius-Red stained collagen at 2 weeks off CCL 4

. This was

accompanied by a reduction of Ly-6c lo restorative and M2

macrophages and an increase of M1 macrophages. In a therapeutic

approach, we applied an ASO targeting IL-4Rα (IL-4Rα

ASO) to CCL 4

-treated mice. IL-4Rα ASO strongly attenuated

fibrosis progression but mitigated fibrosis resolution after CCL 4

withdrawal. Conclusion: Our studies demonstrate that IL-4Rα

modulates fibrosis progression and reversal in discordant ways

through macrophages. During progression, IL-4Rα signaling

increases inflammation and fibrosis by activating Ly6c hi macrophages;

during reversal, IL-4Rα potentiates restorative (M2,

Ly6c lo ) macrophage signaling. Accordingly, IL-4Rα ASO treatment

attenuates fibrosis progression, but retards reversal.


Jeff R. Crosby - Employment: ISIS Pharmaceuticals

Michael L. McCaleb - Employment: Isis Pharmaceuticals; Stock Shareholder: Isis


The following authors have nothing to disclose: Shih-Yen Weng, Santosh Vijayan,

Xiaoyu Wang, Yilang Tang, Kornelius Padberg, Yong Ook Kim, Brombacher

Frank, Ari Waisman, Ernesto Bockamp, Detlef Schuppan


Liver sinusoidal endothelial cells induce neutrophil

extracellular traps in liver sterile inflammation via

IL-33/ST2 axis

Hai Huang, Hamza Yazdani, Patricia Loughran, Heth R. Turnquist,

Allan Tsung; Department of Suegery, University of Pittsburgh Medical

Center, Pittsburgh, PA

Both liver sinusoidal endothelial cells (LSECs) and neutrophils

are involved and interact in liver ischemia/reperfusion (I/R)

injury. Damaged LSECs, as the major sources of IL-33, play

important role in neutrophil infiltration during liver I/R. We

recently found that in response to damage associated molecular

patterns (DAMPs), infiltrated neutrophils forming neutrophil

extracellular traps (NETs), exacerbate sterile inflammatory

injury during liver I/R. We here sought to determine the role

of IL-33 released from LSECs in NET formation during liver I/R.

WT or IL-33 KO mice were subjected to a non-lethal warm liver

I/R model. Recombinant IL-33 (rIL-33) or soluble ST2 (sST2,

decoy receptor of IL-33) was administered in select groups.

IL-33 KO mice or sST2-treated WT mice were significantly protected

from I/R injury, having significantly less serum sALT and

hepatic necrosis, lower levels of systemic cytokines, abrogation

of proinflammatory signaling pathways compared to WT mice.

rIL-33 administered during I/R exacerbated hepatotoxicity and

systemic inflammation. Although significant neutrophils infiltration

was found in both IL-33 KO and WT mice, NETs formation

decreased in IL-33 KO mice during liver I/R compared

with WT mice, associated with significant less serum level of

myeloperoxidase (MPO)-DNA complexes and tissue level of

citrullinated-histone H3 (NET markers). In addition, treatment

of sST2 reduced NET formation whereas significant increased

NETs were found in rIL-33 treated mice after liver I/R. In vitro,

IL-33 released from LSECs that were subjected to hypoxia (1%

O 2

) promotes NET formation. Directly using rIL-33 stimulates

neutrophils confirmed IL-33/ST2-MyD88 signaling pathway in

NET formation. Using either IL-33 KO LSECs, or co-stimulated

neutrophil with IL-33 neutralizing antibody or sST2 blocked

NET formation. Our study demonstrates IL-33 from LSECs initiates

a feed-forward mechanism to neutrophils inducing NETs

formation in excessive inflammation and hepatotoxicity during

liver I/R.


The following authors have nothing to disclose: Hai Huang, Hamza Yazdani,

Patricia Loughran, Heth R. Turnquist, Allan Tsung


15-PGDH prevents LPS/GalN-induced acute liver injury

through inhibiting Kupffer cell activation

Lu Yao, Chang Han, Tong Wu; pathology and laboratory medicine,

tulane university, New Orleans, USA Minor Outlying Islands

The NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

(15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl

group of Prostaglandin E 2

(PGE 2

), converting the pro-inflammatory


to the anti-inflammatory 15-keto-PGE 2


endogenous ligand for peroxisome proliferator-activated receptor-gamma

[PPAR-γ]). PPAR-γ is a ligand-dependent transcriptional

factor, which plays an important role in regulation of

inflammatory cell activation. To evaluate the significance of

15-PGDH/PPAR-γ cascade in liver inflammation, we generated

transgenic mice with targeted expression of 15-PGDH in the

liver (15-PGDH Tg) and the animals were subjected to lipopolysaccharide

(LPS)/Galactosamine (GalN) induced acute liver

inflammation and tissue damage. Compared to the wild type

mice, the 15-PGDH Tg mice showed lower levels of alanine

aminotransferase (ALT) and aspartate aminotransferase (AST),

less liver tissue damage, less hepatic apoptosis/necrosis, less

macrophage activation, and lower inflammatory cytokine production.

In cultured Kupffer cells, treatment with 15-keto-PGE 2

or the conditioned medium (CM) from 15-PGDH Tg hepatocyes

inhibited LPS-induced cytokine production. Both 15-keto-PGE 2

and the CM from15-PGDH Tg hepatocyes also significantly

up-regulated the expression of PPAR-γ down-stream genes in

Kupffer cells. On the other hand, 15-PGDH overexpression

or 15-keto-PGE 2

treatment of hepatocytes did not influence

TNF-α-induced hepatocyte apoptosis. These results suggest

that the resistance of 15-PGDH Tg mice to LPS/GalN-induced

liver injury is mediated through 15-keto-PGE 2

, which activates

PPAR-γ in Kupffer cells and inhibited inflammatory cytokine

production. Consistent with this assertion, we observed that the

PPAR-γ antagonist, GW9662, reversed the effect of 15-keto-


in Kupffer cell (in vitro) and restored the susceptibility

of 15-PGDH Tg mice to LPS/GalN-induced acute liver injury

(in vivo). Taken together, our findings provide novel evidence

that hepatic overexpression of 15-PGDH protects against

LPS/GalN-induced acute liver injury by inhibiting Kupffer cell

inflammatory response. Thus, 15-keto-PGE 2

is an endogenous

PPAR-γ ligand that may be utilized as a pharmacological agent

to ameliorate liver inflammation and tissue damage.



The following authors have nothing to disclose: Lu Yao, Chang Han, Tong Wu


IL-17 signaling in hepatocellular carcinoma promoted

by ethanol.

Hsiao-Yen Ma 2,1 , Jun Xu 1,2 , Mengxi Sun 1,2 , David A. Brenner 2 ,

Tatiana Kisseleva 1 ; 1 Department of Surgery, UC San Diego, La

Jolla, CA; 2 Department of Medicine, UC San Diego, La Jolla, CA

Hepatocellular carcinoma (HCC) is a malignant tumor made of

cells dedifferentiated from mature hepatocytes, which usually

arises in patients with end stage cirrhosis. Progression of HCC

is associated with upregulation of inflammation and constitutive

activation of STAT3. Meanwhile, prolonged alcohol consumption

has strong immunosuppressive and hepatotoxic effects,

implying chronic alcohol consumption would promote HCC

development. Furthermore, ALD patients showed significant

increase in plasma and hepatic IL-17A expression. Blockage

of IL-17 signaling significantly reduced alcohol induced steatohepatitis

and fibrosis in preclinical mouse models. However,

the role of IL-17 signaling in alcohol promoted HCC remains

unclear. AIM: To determine the role of IL-17A signaling pathway

during alcohol promoted HCC development and evaluate

IL-17A as a therapeutic target for HCC. METHODS: Two different

models were used to assess the IL-17 signaling in alcohol

promoted HCC. First, hepatic carcinogen (DEN) induced

HCC in which WT and IL-17RA null mice (KO) were injected

with single dose of DEN at 14 days old. Second, steatohepatitis

associated HCC in which MUP-uPA transgenic mice were

crossed with WT and IL-17RA KO to generate MUP-uPA|WT

(MWT) and MUP-uPA|IL-17RA KO (MKO) mice. By 12 weeks

old, each genotype was assigned to pair-fed group and EtOH

group to receive Liber-Decarli HFD or alcohol diet for 18 weeks

respctively. The HCC development in both WT and KO mice

were compared and Stat3 signal pathway was evaluated. To

investigate how IL-17A signal regulating HCC associated fibroblasts

and macrophage, MC-38-GFP cancer cells were injected

into the spleen of WT and KO recipients for 10 days and the

tumor size and numbers were compared. RESULTS: Chronic

administration of the Liber-DeCarli alcohol diet accelerated

tumorogenesis in both WT/MWT and KO/MKO mice. Blockade

of IL-17 signaling significantly suppressed tumor size and

tumor number in both pair-fed and EtOH group. In MUP-uPA

mice, liver fibrosis was increased after EtOH feeding, which

was decreased by blockade of IL-17 signaling. Blockade of

IL-17 signaling showed less inflammation (reduced TNFa, IL1b,

TGFb1), mediators of metastasis (MMP7, MMP9) and ROS

production (Nox1, Nox2). As expected, CYP2E1 was elevated

in EtOH group, but suppressed in KO mice. CONCLUSION:

Chronic alcohol consumption accelerated tumor development

which was mediated by IL-17 signaling pathway. The role of

IL-17 signaling during alcohol promoted HCC may be via regulating

STAT3 signaling pathway and upregulation of CYP2E1.


The following authors have nothing to disclose: Hsiao-Yen Ma, Jun Xu, Mengxi

Sun, David A. Brenner, Tatiana Kisseleva


Deletion of fibrocytes in mice attenuates experimental

liver fibrosis.

Jun Xu 1,2 , Min Cong 1,2 , Tae Jun Park 1,2 , David A. Brenner 1 , Tatiana

Kisseleva 2 ; 1 Department of Medicine, UCSD, San Diego, CA;

2 Department of Surgery, UC San Diego, La Jolla, CA

BACKGROUND:Bone marrow (BM) fibrocytes, designated as

CD45 + and Collagen type I + (Col1a1) cells, are recruited to

the injured liver, however, their role in liver fibrosis remains

unclear. AIM: To determine the role of fibrocytes in pathogenesis

of liver fibrosis. METHODS: 1The contribution of fibrocytes to

liver fibrosis was studied in BM chimeric mice devoid of fibrocytes

(DFibrocyte mice), in which fibrocyte death was induced

by expression of Diphtheria toxin a (DTA) in CD45 + Col1a1 +

fibrocytes. Specifically, tamoxifen-inducible Col1a1 ER-Cre

mice were crossed with Rosa26 flox-Stop-flox-YFP reporter mice ±

Rosa26 flox-Stop-flox-DTA mice, and used as donors for BM transplantation

into lethally irradiated wt mice to generate wt and

DFibrocyte mice. 2The role of Col1a1 in regulation of fibrocyte

function was studied in BM chimeric Col1a1 5’SL-/- -into-wt

mice, in which mutation of 5’stemloop (5’SL -/- mice) prevented

proper Col1a1 translation in fibrocytes. 3)All mice were subjected

to CCl 4

for 6 w. The therapeutic potential of Serum

Amyloid P (SAP), a natural inhibitor of fibrocytes, was tested.

4To translate our findings to humans, the role of fibrocytes

as prognostic biomarker was evaluated in patients with liver

cirrhosis. RESULTS: Cell fate mapping revealed that 20% of

hepatic fibrocytes become a-SMA myofibroblasts, while 80%

become myeloid cells, which serve as a significant source of

TGFβ1, IL-6, and IL-1b1 in CCl 4

treated wt mice. Deletion of

fibrocytes/progeny in DFibrocyte mice resulted in inhibition

of CCl 4

induced liver fibrosis by 50%, as shown by reduced

Col1a1, a-SMA, and TGFb1 mRNA expression. Interestingly,

fibrocyte ablation also resulted in suppression of hepatic pro-inflammatory

macrophages (M1) and promote proliferation of

anti-inflammatory macrophages (M2), suggesting that fibrocytes

regulate M1/M2 macrophage polarization. Next, we

tested if Col1a1 expression affects fibrocyte function. Indeed,

liver fibrosis was reduced in Col1a1 5’SL-/- into-wt mice by >30%,

and was associated with impaired proliferation of BM fibrocytes.

Surprisingly, activation of non-fibrocyte-derived myeloid

lineages was also reduced, indicating that fibrocytes have an

important immunoregulatory function. In support, the number

of circulating fibrocytes in patients correlated with the stage

of liver fibrosis, and with low levels of serum SAP. Administration

of SAP significantly ameliorated liver fibrosis in CCl 4


mice. CONCLUSION: Fibrocytes contribute to liver fibrosis indirectly

by increasing liver inflammation and secreting fibrogenic

agonists. Targeting fibrocytes with SAP may become a novel

therapy of liver fibrosis in patients with reduced serum levels

of SAP.


The following authors have nothing to disclose: Jun Xu, Min Cong, Tae Jun Park,

David A. Brenner, Tatiana Kisseleva



Splenectomy attenuates advanced liver fibrosis stimulating

hepatic accumulation of anti-fibrogenic monocytes/


Yuji Iimuro 1,2 , Akito Yada 1 , Toshihiro Okada 1 , Naoki Uyama 1 ,

Jiro Fujimoto 1 ; 1 Department of Surgery, Hyogo College of Medicine,

Hyogo, Japan; 2 Surgery, Nirasaki Municipal Hospital, Nirasaki,


Splenectomy (SP), which is performed in cirrhotic patients with

hypersplenism, has been reported to improve liver function;

however the underlying mechanism remains obscure. The AIM

of the present study was to investigate the mechanism using

a murine model and human liver tissues. Methods: C57BL/6

male mice were allowed to drink water including thioacetamide

(TAA: 300 mg/l) ad libitum for 32 weeks. After SP at

32 weeks, mice were sacrificed on days 1, 7, and 28, respectively,

while TAA-administration was continued. Perioperative

changes in peripheral blood and liver tissues were analyzed.

In cirrhotic patients with hepatocellular carcinoma (HCC), who

underwent laparoscopic SP in advance, non-tumorous liver

tissues obtained from resected specimen were histologically

analyzed. Results: TAA treatment of mice for 32 weeks reproducibly

achieved advanced liver fibrosis with splenomegaly,

thrombocytopenia, and leukocytopenia, well representing

the compensated liver cirrhosis in humans. After SP, thrombocytopenia

and leukocytopenia were quickly improved, and

liver fibrosis was obviously attenuated. Among the leukocytes,

monocytes/macrophages were selectively increased in peripheral

blood, as well as in the liver. Meanwhile, progenitor-like

cells expressing CK-19, EpCAM, or CD-133 appeared along

the fibrous scar in the liver after TAA treatment, and gradually

disappeared after SP. Monocytes/macrophages accumulated

in the liver, most of which were negative for Ly-6C, existed

adjacent to the hepatic progenitor-like cells. qRT-PCR indicated

increased canonical Wnt and decreased Notch signals in

the liver, and Wnt2 protein expression in monocytes/macrophages

was confirmed. A significant amount of β-catenin accumulated

in the adjacent progenitor-like cells, and Ki67-positive

relatively small hepatic cells were significantly increased. Protein

expression of MMP-9, to which Ly-6G-positive neutrophils

contributed, was also increased in the liver after SP. In cirrhotic

patients with HCC, thrombocytopenia and lekocytopenia were

quickly attenuated after SP, and the percentage of monocytes

in peripheral blood was selectively increased. In the their liver

tissues, remarkable accumulation of round- or oval-shaped macrophages/monocytes

positive for CD163 was detected after

SP, and they existed closely to the CK19-positive cells, which

spread out from the ductular reactions, suggesting interaction

between these cells. Conclusions: The hepatic accumulation of

monocytes/macrophages, the reduction of fibrosis, and the