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Cystic Fibrosis Liver Disease Clinical Research Workshop

June 8 - 9, 2009 Lister HiLL Auditorium

niH CAmpus, BetHesdA, md

Cystic Fibrosis Liver Disease | Clinical Research Workshop

Michael Narkewicz, M.D. University of Colorado Denver

Michael Knowles, M.D. The University of North Carolina at Chapel Hill

Patricia Robuck, Ph.D., M.P.H. National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health

Catherine McKeon, Ph.D. National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health

Jay H. Hoofnagle, M.D. National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health

Bruce Marshall, M.D. Cystic Fibrosis Foundation

Marilyn Siegel, M.D. Washington University Medical Center

Edward Doo, M.D. National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health

SponSorS: The National Institute of Diabetes and Digestive and Kidney Diseases, the Office of Rare Diseases, and the Cystic Fibrosis Foundation.

The lunch and poster session are sponsored by the Cystic Fibrosis Foundation.

organizing Committee

iii


Table of Contents

Organizing Committee iii

Agenda ix

Speaker Abstracts

Cystic Fibrosis Liver Disease: Clinical Features and Definitions Michael R. Narkewicz 3

Prevalence and Epidemiology of Cystic Fibrosis Liver Disease Jean Pappas Molleston 6

Cystic Fibrosis Liver Disease: Clinical Outcomes Peter Durie, Susanne Schibli, Rakesh Battacharjee, Mary Corey 8

Pathogenesis of Cystic Fibrosis Liver Disease Steven D. Freedman 10

The Pathology of Cystic Fibrosis Liver Disease David E. Kleiner 15

Genetic Modifiers of Cystic Fibrosis Liver Disease Michael R. Knowles for Jaclyn Bartlett, Peter Durie, and the Gene Modifier Study Group 17

Protein Folding Defects in Liver Disease: Alpha-1-antitrypsin Deficiency Jeffrey Teckman 19

Cystic Fibrosis Liver Disease: CFTR and the Liver Andrew P. Feranchak 21

The CFTR-/- Pig: Liver and Gallbladder Disease David K. Meyerholz, David A. Stoltz, Michael J. Welsh 24

Medical and Surgical Therapy of CF-associated Liver Disease Saul J. Karpen 25

Liver Transplantation in Children With Cystic Fibrosis: Status and Unanswered Questions

George V. Mazariegos 27

Biomarkers as Tools for Early Disease Detection Nicole Mayer Hamblett 29

v

Table of Contents

Emerging Techniques for Developing Novel Serum Biomarkers Keyur Patel 31

Biomarkers of Cystic Fibrosis Liver Disease Ross W. Shepherd, Grant Ramm, Peter Lewindon 33

Ultrasound and CT Imaging of Liver Fibrosis and Other Disease Marilyn J. Siegel 35

MRI Methods of Assessing Liver Fibrosis and Disease Claude B. Sirlin 37

Elastography to Assess Liver Fibrosis and Cirrhosis Detlef Schuppan 39

Poster Abstracts

Long-term Effect of Simultaneous Liver-Pancreas Transplant on Hepatic, Pulmonary, and Pancreatic Function in Three Cystic Fibrosis Patients

Molly Bozic, Jean Molleston, M. Howenstine, J. Fridell 43

Paracrine Induction of Sulfotransferase 1E1 in HepG2 Hepatocytes During Co-culture With CFTR-deficient MMNK-1 Cholangiocytes

Charles N. Falany, Dongning He, Li Li, Teresa W. Wilborn, Melissa Runge-Morris 45

Quantitative Imaging of Biliary Fibrosis Progression Using a Small Molecule α v β6 Integrin Ligand With Nanomolar Affinity

Yury Popov, Preeti Misra, Kumar Ranjan Bhushan, Deanna Y. Sverdlov, Nezam H. Afdhal, John V. Frangioni, Deflef Schuppan 46

Cystic Fibrosis Related Diabetes (CFRD) in CF Patients With Cirrhosis Kara Sullivan, A. Moran, B. Holm, S.J. Schwarzenberg 47

Design of a Pilot Study to Identify Optimum Markers of Liver Fibrosis in Children With Cystic Fibrosis Liver Disease (CFLD)

Alexandra Vasilescu, Shruti Paranjape, Wikrom Karnsakul, Kathleen B. Schwarz 48

Participants List 51

Notes 65

Agenda

Cystic Fibrosis Liver Disease | Clinical Research Workshop vi

Agenda

Monday, June 8, 2009

Lister HiLL Auditorium

niH CAmpus, BetHesdA, md

8:00 a.m. – 8:30 a.m. Registration

8:30 a.m. – 8:40 a.m. Welcoming Remarks Jay Hoofnagle

SeSSion i. CLiniCAL ASpeCTS AnD prevALenCe oF CF Liver DiSeASe

8:40 a.m. – 9:00 a.m. Cystic Fibrosis Liver Disease: Clinical Features and Definitions Michael Narkewicz

9:00 a.m. – 9:20 a.m. Prevalence and Epidemiology of Cystic Fibrosis Liver Disease Jean Molleston

9:20 a.m. – 9:40 a.m. Cystic Fibrosis Liver Disease: Clinical Outcomes Peter Durie

9:40 a.m. – 10:10 a.m. Discussion

10:10 a.m. – 10:40 a.m. Break

SeSSion ii. pAThogeneSiS oF CF Liver DiSeASe

10:40 a.m. – 11:00 a.m. Pathogenesis of CF Liver Disease Stephen Freedman

11:00 a.m. – 11:20 a.m. The Pathology of Cystic Fibrosis Liver Disease* David Kleiner

11:20 a.m. – 11:40 a.m. Genetic Modifiers of Cystic Fibrosis Liver Disease Michael Knowles

DAY ONE

* Results of a premeeting working group of clinicians and pathologists who will review pathology of CF liver disease and propose diagnostic criteria and staging and grading systems.

Cystic Fibrosis Liver Disease | Clinical Research Workshop ix

Agenda Agenda

Monday, June 8, 2009

Continued

Tuesday, June 9, 2009

Lister HiLL Auditorium

niH CAmpus, BetHesdA, md DAY TWO

11:40 a.m. – 12:00 p.m.

12:00 p.m. – 12:30 p.m.

12:30 p.m. – 2:00 p.m.

SeSSion iii. MoDeLS oF CF Liver DiSeASe AnD TherApeuTiCS

2:00 p.m. – 2:20 p.m.

2:20 p.m. – 2:40 p.m.

2:40 p.m. – 3:00 p.m.

3:00 p.m. – 3:20 p.m.

3:20 p.m. – 3:40 p.m.

3:40 p.m. – 4:10 p.m.

4:10 p.m. – 4:20 p.m.

4:20 p.m. – 5:30 p.m.

8:00 a.m. – 8:10 a.m.

SeSSion iv. noninvASive ASSeSSMenTS oF CF Liver DiSeASe

8:10 a.m. – 8:30 a.m.

8:30 a.m. – 8:50 a.m.

8:50 a.m. – 9:10 a.m.

9:10 a.m. – 9:40 a.m.

9:40 a.m. – 10:00 a.m.

10:00 a.m. – 10:20 a.m.

10:20 a.m. – 10:40 a.m.

Introduction to Day 2: Early Assessment and Identification Edward Doo

BioMArkerS

Biomarkers as Tools for Early Disease Detection Nicole Hamblett

Emerging Techniques for Developing Novel Serum Biomarkers Keyur Patel

Biomarkers of Cystic Fibrosis Liver Disease Ross Shepherd

Discussion

Break

iMAging

Ultrasound and CT Imaging of Liver Fibrosis and Other Disease Marilyn Siegel

MRI Methods of Assessing Liver Fibrosis and Disease Claude Sirlin

Protein Folding Defects in Liver Disease: Alpha-1-antitrypsin Deficiency Jeffrey Teckman

Discussion

Lunch

Cystic Fibrosis Liver Disease: CFTR and the Liver Andrew Feranchak

The CFTR-/- Pig: Liver and Gallbladder Disease David Meyerholz

Nodular Regenerative Hyperplasia: Clinical Features and Course Theo Heller

Medical and Surgical Therapy of CF-associated Liver Disease Saul Karpen

Liver Transplantation in Children With Cystic Fibrosis: Status and Unanswered Questions George Mazariegos

Discussion

Break

Poster Session

x Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop xi

Agenda

Tuesday, June 9, 2009

Continued

10:40 a.m. – 11:00 a.m.

11:00 a.m. – 11:30 a.m.

11:30 a.m. – 12:30 p.m.

12:30 p.m.

Elastography to Assess Liver Fibrosis and Cirrhosis Detlef Schuppan

Discussion

Research Needs for the Future Michael Narkewicz Michael Knowles Patricia Robuck Catherine McKeon Jay H. Hoofnagle Bruce Marshall Marilyn Siegel Edward Doo

Adjournment

iMAging

Speaker Abstracts

Cystic Fibrosis Liver Disease | Clinical Research Workshop xii

Cystic Fibrosis Liver Disease | Clinical Research Workshop 3

Michael R. Narkewicz, M.D.

Hewit-Andrews Chair in Pediatric Liver Disease, Professor of Pediatrics, University of Colorado Denver, School of Medicine, The Children’s Hospital, Aurora, CO

Cystic Fibrosis Liver Disease: Clinical Features and Definitions

Speaker Abstracts

Cystic Fibrosis (CF) is an autosomal recessive disorder that is the most common genetic disorder of Caucasian individuals, with a prevalence of 1 in 2,000 individuals of European descent. It is estimated to affect 30,000 individuals in the United States. The disease is due to a defect in the cystic fibrosis transmembrane conductance regulator (CFTR). Although the lungs and pancreas are the pathopneumonic target organs in CF, many other organs are affected. These include the small and large intestine, vas deferens, sinuses, and liver. In 2007, liver disease was the third leading cause of death in CF, accounting for 2.3 percent of deaths.

CFTR is expressed on the apical membrane of biliary epithelium and not on the hepatocyte. The putative role of CFTR is to facilitate water and solute movement via chloride secretion and therefore promote bile flow.

Clinical Features of Liver Disease in CF: The primary end result of liver disease in CF with the most significant impact is the development of fibrosis and finally cirrhosis in up to 7 percent of individuals with CF. Various theories have been proposed for the underlying pathophysiology, but no single mechanism has met with uniform acceptance. Similarly, difficulties exist in the studies of natural history and treatment, as there have not been uniform definitions applied to liver involvement in CF. As a consequence, the prevalence of liver disease in CF has been noted to be between 5 and 75 percent.

The clinical involvement of the liver is manifest in many ways. Biliary cirrhosis with or without portal hypertension is the complication with the most significant impact on outcome. Several studies have demonstrated that cirrhosis is a relatively early event, with most patients

identified by 15 years of age, affecting up to 7 percent of patients. Risk factors for cirrhosis have included male sex, Hispanic ethnicity, and pancreatic insufficiency. There has not been any identified genotype/phenotype correlation with CFTR. The pathology is that of multilobular cirrhosis, often with focal variation.

Other hepatic manifestations include:

Neonatal Cholestasis: This has been suggested to occur in 1-2 percent of infants with CF. It is more common in infants with meconium ileus. In general, the cholestasis resolves. The subsequent risk for the development of cirrhosis is probably increased, but this remains unclear.

Elevated AST, ALT, or GGT: Studies on subjects identified by newborn screening have shown that the prevalence of abnormal AST, ALT, or GGT is quite frequent. By 20 years of age, 93 percent of patients have had at least one abnormal ALT, with 50 percent having at least one ALT greater than 1.5 times the upper limit of normal (ULN) and 32 percent having an ALT greater than 2 times the ULN. Persistently abnormal ALT or AST is also quite frequent, with 42 percent of patients having a persistently abnormal ALT greater than 1.5 times the ULN by 20 years of age.

Hepatic Steatosis: This has been reported to occur in 20-60 percent of patients with CF. In many cases, this was reported in association with malnutrition related to CF. However, hepatic steatosis has been reported in well-nourished children and adults with CF. The underlying mechanism(s) is unclear and has been suggested to be related to essential fatty acid deficiency or intrinsic defects related to CFTR dysfunction. It is unclear if hepatic steatosis progresses to cirrhosis.

Focal Biliary Cirrhosis: This entity previously was described as the pathopneumonic hepatic disorder of CF. It is characterized by focal areas of fibrosis and regenerative nodules areas of normal liver. The prevalence has

Cystic Fibrosis Liver Disease | Clinical Research Workshop 4 Cystic Fibrosis Liver Disease | Clinical Research Workshop 5

Speaker Abstracts Speaker Abstracts

been reported to be between 10 and 70 percent, depending on the method of detection and the era. The highest frequency for this disorder comes from autopsy reports that predate current therapies. If and at what frequency this pathologic entity progresses to multilobular biliary cirrhosis and portal hypertension is unknown.

Given the localization of CFTR to the biliary epithelium, it is interesting to note that biliary tract disease is less frequent than might be expected. Biliary tract involvement and prevalence include: microgallbladder (30%), cholelithiasis (1-10%), common bile duct stenosis (1-2%), and a cholangiographic picture similar to sclerosing cholangitis in 1-2 percent. Microgallbladder is likely a benign entity, perhaps related to diminished bile flow. There is no agreement on the definition of common bile duct stenosis and sclerosing cholangitis in CF or their potential role in the pathophysiology of the development of cirrhosis in CF.

With the protean manifestations of liver involvement in CF, there is a need for uniform criteria for the definition of liver disease/involvement in CF. In 2007, the CF Foundation convened a group of international experts in CF-related liver disease. This group has proposed the following classification for CF-related liver disease (CFLD) to guide research in the field. The major point of the group is that blending various forms of liver involvement under one rubric of CFLD does not allow comparison of natural history or treatment studies in CF. Thus, there is a need for uniformity in the classification and definition of CF liver disease.

Proposed Classification of CF Liver Disease (CFLD):

I. CFLD with cirrhosis/portal hypertension

a. Clinical evidence of portal hypertension

b. Evidence of cirrhosis by histology, imaging, or laparoscopy

II. Liver involvement without cirrhosis/portal hypertension (patients could have more than one (i.e., II a, c)). These are primarily descriptive at this point. The abnormalities may well be multifactorial in origin, and the exact significance and relationship to each other remain to be determined.

a. Biochemical abnormalities

i. Persistent AST, ALT, GGT greater than 1.5 or 2 × ULN (These are arbitrary cutoffs that have been suggested by several groups; however, the significance of a particular value has not been studied.)

ii. Intermittently elevated

b. Steatosis (At present, this would require histological evidence: there may be imaging criteria in the future that will come from studies of other liver diseases.)

c. Fibrosis (Histology; there may be imaging criteria in the future that will come from studies of other liver diseases.)

d. Cholangiopathy (documented by abnormalities of the biliary tree on US, MRCP, CT, or ERCP)

e. Ultrasound abnormalities that are not diagnostic of cirrhosis

i. Abnormal echogenicity

III. Preclinical: No evidence of liver disease (must have all of the following: this would be used primarily in clinical studies)

a. Normal clinical exam

b. Normal imaging

c. Normal biochemistry

Future Research Challenges and Needs:

•Developmentofuniformdefinitionsforusein clinical research.

•Identificationofreproduciblemarkersof“earlycystic fibrosis liver disease.”

•Biomarkersforearlyhepaticfibrosisincysticfibrosis.

Key References:

Cohn JA, Strong TV, et al. Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells. Gastroenterology 1993;105(6):1857-64.

Colombo C, Battezzati PM, et al. Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome. Hepatology 2002;36(6):1374-82.

Narkewicz MR. Markers of cystic fibrosis-associated liver disease. J Pediatr Gastroenterol Nutr 2001;32(4):421-2.

Potter CJ, Fishbein M, et al. Can the histologic changes of cystic fibrosis-associated hepatobiliary disease be predicted by clinical criteria? J Pediatr Gastroenterol Nutr 1997;25(1):32-6.

Scott-Jupp R, Lama M, et al. Prevalence of liver disease in cystic fibrosis. Arch Dis Child 1991;66(6):698-701.

Shapira R, Hadzic N, et al. Retrospective review of cystic fibrosis presenting as infantile liver disease. Arch Dis Child 1999;81(2):125-8.

Sokol RJ, Durie PR. Recommendations for management of liver and biliary tract disease in cystic fibrosis. Cystic Fibrosis Foundation Hepatobiliary Disease Consensus Group. J Pediatr Gastroenterol Nutr 1999;28(Suppl 1):S1-13.

Vawter GF, Shwachman H. Cystic fibrosis in adults: an autopsy study. Pathol Annu 1979;14(Pt 2):357-82.

Woodruff SA, Sontag M, et al. Prevalence of elevated liver function tests in children with cystic fibrosis diagnosed by newborn screen. J Pediatr Gastroenterol Nutr 2007;45(4):E27-E28.



Continued Continued


Jean Pappas Molleston, M.D.

Department of Pediatric Gastroenterology, Hepatology, and Nutrition, Indiana University School of Medicine, James Whitcomb Riley Hospital for Children, Indianapolis, IN

Cystic fibrosis (CF) affects about 1 in 2,500 newborns. It is a disease that can manifest in multiple organ systems, including the lung, liver, intestine, and pancreas. Liver disease is the third most common cause of death in CF and accounts for about 2 percent of CF mortality. The prevalence of cystic fibrosis liver disease (CFLD) ranges widely, depending on how liver disease is assessed and over what period of time. Biochemical abnormalities, typically elevated transaminases, are common and intermittent. While at any given time the prevalence of elevated liver enzymes is reported at 20-50 percent, over longitudinal followup more than 80 percent of CF patients have elevated liver enzymes at some time. Sonographic abnormalities, including hepatomegaly, abnormal hepatic echotexture, or Doppler evidence of portal hypertension, are apparent at initial assessment in 35 percent, increasing to 67 percent at followup. Histologic abnormalities of focal biliary cirrhosis or steatosis are common in the few CF patients who undergo liver biopsies; autopsy studies indicate a 10 percent prevalence in infants but a greater than 70 percent prevalence in adults. Clinical liver disease is much less common, with a prevalence ranging from 2.5 to 10 percent. The most striking but consistent epidemiologic fact is that CFLD does not appear to increase with age; prevalence peaks before puberty and does not increase after that.

Several risk factors have been associated with CFLD but are not consistent in all large studies. Most studies demonstrate a male predominance in CFLD. Essentially, all patients with CFLD are pancreatic insufficient. While some studies indicate an association with severe genotypes, others do not. Many studies have found an association with meconium ileus, but this has not borne out in other reports. CFLD does not appear to be associated with severity of lung disease in most studies.

There has been considerable interest in the role of modifier genes in CFLD. Alpha-1-antitrypsin deficiency heterozygous phenotype MZ has been strongly associated with CFLD in a recent report in press. TGF-beta 1 polymorphisms have been related to CFLD as well. One report has suggested an association of CFLD with a glutathione S-transferase P1 polymorphism. Ongoing studies are likely to clarify these associations and identify other potential modifier genes.

Research Goals:

1. Determine clear definitions/criteria for CFLD to facilitate research and allow tracking of patients.

2. Identify risk factors or combinations of risk factors that predict CFLD and study potential mechanisms; expand list to include possible modifiable risk factors such as nutritional factors or drugs.

3. Study the pathophysiology of CFLD in specific age groups to better understand why liver disease presents only prior to adulthood.

References:

Ling SC, Wilkinson JD, Hollman AS, McColl J, Evans TJ, Paton JY. The evolution of liver disease in cystic fibrosis. Arch Dis Child 1999;81:129-132.

Feigelson J, Anagnostopoulos C, Poquet M, Pecau Y, Munck A, Navarro J. Liver cirrhosis in cystic fibrosis—therapeutic implications and long term follow up. Arch Dis Child 1993;68:653-657.

Colombo C, Russo MC, Zazzeron L, Romano G. Liver disease in cystic fibrosis. J Pediatr Gastroenterol Nutr 2006;43:S49-S55.

Corbett K, Kelleher S, Rowland M, Daly L, Drumm B, Canny G, Greally P, Hayes R, Bourke B. Cystic fibrosis-associated liver disease: a population-based study. J Pediatr 2004;145:327-332.

Scott-Jupp R, Lama M, Tanner MS. Prevalence of liver disease in cystic fibrosis. Arch Dis Child 1991;66:698-701.

Colombo C, Apostolo MG, Ferrari M, Seli M, Genoni S, Giunta, Sereni LP. Analysis of risk factors for the development of liver disease associated with cystic fibrosis. J Pediatr 1995;126:155-156.

Colombo C, Battezzati PM, Crosignani A, Morabito A, Costantini D, Padoan R, Giunta A. Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome. Hepatology 2002;36:1374-1382.

Lindblad A, Glaumann H, Strandvik B. Natural history of liver disease in cystic fibrosis. Hepatology 1999;30:1151-1158.

Prevalence and Epidemiology of Cystic Fibrosis Liver Disease


Prevalence and Epidemiology of Cystic Fibrosis Liver Disease

Continued

Lamireau T, Monnereau S, Martin S, Marcotte J-E, Winnock M, Alvarez F. Epidemiology of liver disease in cystic fibrosis: a longitudinal study. J Hepatol 2004;41:920-925.

Brazova J, Sismova K, Vavrova V, Bartosova J, Macek M Jr, Lauschman H, Sediva A. Polymorphisms of TGF-beta1 in cystic fibrosis patients. Clin Immunol 2006;121:350-357.

Henrion-Caude A, Flamant C, Roussey M, Housset C, Flahault A, Fryer AA, Chadelat K, Strange RC, Clement A. Liver disease in pediatric patients with cystic fibrosis is associated with glutathione S-transferase P1 polymorphism. Hepatology 1992;36:913-917.


Peter Durie, M.D., F.R.C.P.C. 1, Susanne Schibli, M.D.2, Rakesh Battacharjee1, and Mary Corey1

1Hospital for Sick Children and Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada; 2University of Bern, Bern, Switzerland

The cystic fibrosis transmembrane conductance regulator protein (CFTR) functions across the apical domain of cholangiocytes and gallbladder epithelial cells. CFTR maintains an apical Cl– gradient and fluid flow into the ductal lumen. It also alkalinizes intralumenal contents through direct extrusion of HCO3

–, as well as through a Cl–/HCO3

– exchanger, which solubilizes the organic components of bile. Absent or dysfunctional CFTR results in reduced bile fluidity and alkalinity, with plugging of intrahepatic bile ducts leading to biliary stasis, which in turn induces inflammatory damage secondary to cholangiocyte and collateral hepatocyte injury.

The wide range of prevalence of cystic fibrosis liver disease (CFLD) reported in the medical literature (2-7%) can be attributed to several factors. The wide spectrum of hepatobiliary lesions that are known to occur in CF disease, differing definitions of what constitutes CFLD, and the lack of sensitive and specific methods of establishing or excluding a diagnosis have contributed to considerable confusion and misunderstanding. The majority of patients with CF exhibit some degree of hepatobiliary disease, but the nature of the lesions and severity of disease vary considerably from patient to patient. Postmortem analysis of hepatic specimens from adult CF patients has reported the prevalence to be as high as 70 percent. However, the vast majority of subjects have clinically insignificant hepatosteatosis, focal portal tract bile proliferation with cholangitis, or focal biliary cirrhosis.

Studies exploring the natural evolution of CFLD have been sparse and are difficult to interpret in view of different criteria to establish a diagnosis for CFLD.

Accordingly, we have used prudent criteria in a well-defined patient cohort to evaluate the prevalence and natural history of clinically significant CFLD (notably, evidence of biliary cirrhosis with or without portal hypertension and/or hepatocellular failure). The purpose of this analysis was to help decipher the risk of complications and timing and necessity of interventions such as liver transplantation.

Methods:

Patients were identified retrospectively as having CFLD from a prospectively established database consisting of data collected for all patients diagnosed and followed with CF in Toronto, Canada between 1986 and 2004. A diagnosis of CFLD or query CFLD was determined using specific search criteria that included evidence of hepatocellular failure and/or biliary cirrhosis and/ or portal hypertension (esophageal varices and/or splenomegaly). Verification of the search strategy was accomplished through chart review of all subjects identified as having CFLD or query CFLD as well as review of a random sample of subjects without CFLD. A case report form was used to record parameters of relevance, including demographics, diagnosis of CF, diagnosis of CFLD, results of laboratory and imaging studies, and clinical course of CF disease and CFLD.

Results:

In total, 1,108 CF patients attended the Toronto CF clinics from 1986 to 2004. According to the database search strategies, 17 patients were identified as having CFLD, 157 as query CFLD, and 934 as no CFLD. Chart reviews confirmed CFLD in all 17 patients identified by the searching strategies and 32/157 patients from the query group. A random sample of 111 patients from the non-CFLD group did not reveal any patients with CFLD. Three additional patients with CFLD were identified from previous studies.

Cystic Fibrosis Liver Disease: Clinical Outcomes

CFLD was diagnosed in 53 patients, giving an overall prevalence of 4.6 percent. The prevalence increased during the study period (1986-89 1.7%; 1990-94 3.1%; 1995-99 4.7%; 2000-04 6.0%). Retrospective analysis to explore the natural history of CFLD was accomplished in 52 of 53 patients. Pancreatic insufficiency was significantly associated with CFLD (OR 3.3, 95% CI 1.03 to 10.85). There were a greater number of CFLD patients who were male (65%) compared to female. CFLD was diagnosed in 81 percent of patients under the age of 18. The mean duration of follow-up from diagnosis of CFLD was 10.6 (range 1.1 to 32.2) years. Of the hepatic complications of CFLD, portal hypertension was most prevalent. Hypersplenism was seen in 23 patients (44%). Variceal bleeding was seen in 14 patients (27%). Duration of disease did not seem to impact the prevalence of these complications. Endoscopic management of variceal bleeding was needed in 12 patients.

Hypoalbuminemia, which developed in 12 patients, was persistent in only six (12%). Coagulopathy developed in eight patients (17%), and was persistent in three (7%). Persistent hyperbilirubinemia developed in only seven patients (13%). In the majority of cases, hypoalbuminemia (with and without ascites), persistent coagulopathy, and mild hyperbilirubinemia were harbingers of hepatocellular failure.

Only six patients (12%) developed hepatocellular failure, at 16, 25, 28, 44, 45, and 52 years of age, respectively. Three underwent isolated liver transplant; they survived for 3, 5, and 10 years, respectively. One other was listed for liver transplantation, and one patient died

while listed for combined liver/lung transplant. There were 15 deaths, with a median age of mortality of 27.3 (range 17.5-52) years. Only one death was attributed to complications of liver failure.

Conclusions:

Since most patients with CFLD maintain excellent hepatocellular function for many years, even decades, the principles of care for CFLD patients should be targeted toward recognizing and managing complications of portal hypertension. Liver and/or combined liver/lung transplantation should be reserved for the few patients who develop synthetic liver failure.


•Identifythegeneticandenvironmentalfactorsthat confer increased susceptibility to CFLD.

•Testtheabilityofgeneticfactorsorotherbiomarkers to identify liver disease in newborn screen positive CF infants.

•Developnovelapproachestopreventingorameliorating liver disease in CF infants with increased susceptibility to CFLD.

References:

Corbett K, Kelleher S, Rowland M, Daly L, et al. Cystic fibrosis-associated liver disease: a population-based study. J Pediatr. 2004;145:327-32.

Debray D, Lykavieris P, Gauthier F, et al. Outcome of cystic fibrosis-associated liver cirrhosis: management of portal hypertension. J Hepatol. 1999;31:77-83.

Cystic Fibrosis Liver Disease: Clinical Outcomes

Continued



Steven D. Freedman, M.D., Ph.D.

Associate Professor of Medicine, Harvard Medical School, Associate Dean for Clinical and Translational Research, Director, The Pancreas Center, Beth Israel Deaconess Medical Center, Boston, MA

Pathogenesis of Cystic Fibrosis Liver Disease

Cystic Fibrosis Liver Disease (CFLD) is poorly characterized both at a clinical level as well as with regard to its pathophysiology. The explanation for why 5-10 percent of CF patients develop CFLD, as well as its underlying pathophysiology, remain unknown. The clinical course, predisposing genetic factors, and the molecular mechanisms at play remain to be defined. At the Cystic Fibrosis Foundation Williamsburg Conference in 2007, a workshop on CFLD led by Drs. Steven Freedman and Michael Narkewicz brought together basic scientists and clinical experts in this area with a focus on understanding mechanisms of fibrogenesis and inflammation in the liver. It was concluded that there is no agreed upon definition of CFLD in the literature. Most would classify CFLD as cirrhosis with portal hypertension and do not include asymptomatic CF patients with isolated abnormal liver blood tests or abnormal imaging studies. Because of the lack of a consistent definition, the prevalence and outcome data in CFLD are poorly characterized. However, the prevalence of advanced CFLD, as defined by cirrhosis/portal hypertension, is 5-10 percent, and CFLD usually presents around or before puberty (1). Median age of recognition is around 10 years of age, indicating that this is an early event. Progression to liver failure is rare, although complications of portal hypertension are not uncommon. Risk factors are severe (pancreatic insufficient) CFTR mutations, male gender, the alpha-1 antitrypsin Z allele, and perhaps TGF-β polymorphisms.

I. Overall Pathophysiology: This is primarily a disorder of fibrogenesis within the liver and involving the bile ductules. CFTR is localized to the cholangiocyte apical membrane, where it is central to fluid secretion

into the biliary tract. With loss of CFTR function, there is decreased bile flow and inspissations, leading to an obstructive process. However, the fact that most CF patients have this abnormality, yet only 5-10 percent develop cirrhosis, implies that other factors must be in play to cause CFLD.

If we examine the known facts in the pathophysiology of liver fibrosis in general, the critical gaps are as follows:

1. Inflammation in the liver is tightly linked to fibrosis in humans as well as in all experimental models of fibrosis (2). Gap: The link between inflammation and hepatic fibrogenesis remains elusive.

2. Liver injury (acute or chronic) increases intestinal permeability, leading to increased bacterial translocation. This results in increased portal and plasma levels of LPS, with the liver being the prime target (2). Hence, this normal response mechanism to acute liver injury perpetuates the inflammatory/ fibrogenic process in chronic liver injury, potentially resulting in cirrhosis. Gap: The mechanisms that underlie this process are poorly understood.

3. Cholestasis is the inciting event in most experimental models of liver fibrosis. These can be categorized by: a. Mechanical: bile duct ligation (BDL). b. Chemical: carbon tetrachloride,

thioacetamide. c. Mechanical and Chemical: Xenobiotics such as

3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) lead to biliary plugs and cholangiocyte transformation to a reactive phenotype. Gap: Is the mechanism of cholestasis in these models the same as in CF, and is transformation of the cholangiocyte to a reactive phenotype critical in inflammation/fibrogenesis?

4. Intestinal bacterial products are critical in the genesis of fibrosis: a. Prior administration of antibiotics (ampicillin,

metronidazole, neomycin, vancomycin) severely attenuates liver fibrogenesis in the BDL and in the chemical carbon tetrachloride and thioacetamide models (2).

b. Attenuation of fibrosis in response to BDL is also seen in CD14 and LPS-binding protein deficient mice, indicating that LPS signaling from the intestinal flora plays a major role in activation of hepatic fibrosis.

c. LPS from the bacterial flora activates TLR4 receptors on Hepatic Stellate Cells (HSCs) but not Kupffer cells as the first critical step in the liver for activation of the fibrogenesis sequence. Gap: Is the intestinal microflora in CF critical to the pathogenesis of CFLD?

II. Overlap With Other Cholangiopathies: There are other biliary tract diseases in both the pediatric and adult populations that are similar to CFLD. Thus, understanding the molecular basis of these other disorders, which lead to biliary fibrosis and cirrhosis, may shed light on the mechanisms that underlie CFLD. These disorders include α-1 antitrypsin deficiency and the cholestatic diseases Primary Biliary Cirrhosis and more pertinent, Primary Sclerosing Cholangitis (PSC).

CFLD and PSC share many similarities, including clinical presentation, imaging abnormalities with biliary strictures in the intra- and/or extrahepatic biliary tree, histopathologic findings of bile duct injury (bile duct proliferation, peri-cholangitic inflammation, bile ductular plugs), and cirrhosis. PSC occurs in 5-7 percent of patients with colitis, typically with ulcerative colitis. Although the mechanism is unknown, PSC has been postulated to be linked to the portal vein bacteremia that results from the colitis. Although several hypotheses have been put forward, including genetic abnormalities in bile salt transporters, autoimmune dysregulation, and

aberrant toll-like signaling, none of these have been definitively corroborated.

Freedman’s and Durie’s group has shown that 34 percent of adult patients with PSC have a single CFTR allelic variant, as analyzed by multiplex heteroduplex gel shift analysis with directed sequencing, compared to patients with inflammatory bowel disease alone (3). Nasal Potential Difference Testing revealed a high prevalence of blunted isoproterenol stimulated CFTR chloride channel function in both children and adults with PSC compared to patients with inflammatory bowel disease alone (3,17). This would suggest that loss of normal CFTR function explains why a subset of patients with underlying colitis develop PSC. Supporting the link between colitis and CFTR dysfunction, induction of colitis with oral dextran sodium sulfate in CF knockout mice resulted in the genesis of bile duct injury (bile duct proliferation with a peri-cholangitic mononuclear cell infiltrate) compared to no disease being seen in wild-type littermates (4). This was due to a lack of induction in PPARα, which was corrected with docosahexaenoic acid (DHA), a known PPARα agonist, resulting in both partial prevention and reversal of established disease (5). However, the liver disease in this model did not progress to fibrosis.

III. Abnormalities in Bile Juice: Martin Cary’s group demonstrated in ∆F508 mice that a) 53 percent of these mice develop histologic features of bile ductular proliferation and mild portal fibrosis by 100 days of age but no inspissated bile; and b) the mice with histologic changes had increased secretion rates of bilirubin monoglucuronoside, resulting in an increased bile salt to phospholipid ratio (6). They concluded that increased fecal bile acid excretion increases bilirubin enterohepatic cycling, leading to increased bilirubin secretion into bile. In conjunction with secretion of more hydrophobic bile salts, this may result in toxic damage to cholangiocytes. Furthermore, unconjugation of bilirubin via β-glucuronidase hydrolysis generates insoluble metal salts

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that may be deposited within cholangiocytes. However, it is unclear from these data how toxic damage to cholangiocytes leads to downstream inflammation and fibrosis.

IV. Other Animal Models of Biliary Fibrosis: There are several approaches that have been taken to induce fibrosis in the liver. The critical element appears to be the induction of cholestasis, either chemically or mechanically.

• Chemical inducers of fibrosis: Carbon tetra-chloride and thioacetamide have been used to generate cirrhosis through their toxic effects and do not primarily involve the cholangiocytes.

• Mechanical inducer of fibrosis: Bile duct ligation leads to secondary biliary fibrosis but may not be representative of the early events leading to CFLD and PSC.

• MDR2 knockout mice demonstrate loss of phospholipid secretion into bile, leading to biliary ductal fibrosis. However, there is no significant inflammatory component. Mutations or polymorphisms in MDR3, the human genetic correlate, have not been found in patients with PSC.

• CFTR mouse models: Both ∆F508 and exon 10 congenic cftr-/- mice variably demonstrate pericholangitic inflammation and minimal fibrosis after 90 days of age (6,7).

• Alpha-1 antitrypsin deficient transgenic mice: Friedman and Knowles have presented evidence suggesting that a single α-1 antitrypsin Z allele mutation (also called PiZ) may be a gene modifier in CFLD (8). In collaboration with Mencin’s and Brenner’s group at Columbia, Knowles et al. showed that bile duct ligation in C57BL/6 transgenic for the human α-1 antitrypsin mutation (PiZ) leads to stellate cell activation and fibrosis, along with increased expression of ER stress markers where

misfolded PiZ accumulates (9). Taken together with human data, the α-1 antitrypsin Z mutation by itself is not sufficient to induce fibrosis but may do so when combined with other factors such as obstruction of biliary flow.

• Xenobiotic-induced mouse model of biliary fibrosis: Michael Trauner’s group in Austria, world experts in the pathophysiology of biliary fibrosis, recently developed a highly reproducible novel model of xenobiotic-induced cholangiopathy by supplementing the standard diet of mice with 0.1 percent (~10 mg/day) DDC (10). As a result of increased biliary porphyrin secretion, by 4 weeks there were scattered pigmented biliary plugs with bile ductular proliferation, and periductal onion skin fibrosis with a predominantly neutrophilic infiltration. Intra- and extra-hepatic segmental strictures were observed. Similar results were seen in Swiss albino, FVB/N, C57BL/6, and 129/SV mice. Thus, this model incorporates both inflammation and fibrosis.

DDC led to transformation of cholangiocytes into a reactive phenotype as assessed by increased VCAM expression in cholangiocytes (immunohistochemistry and Western blotting) and TNFα by in situ hybridization. By 8 weeks, bridging portal fibrosis was present, along with proliferation and activation of myofibroblasts (Ki-67 and α-SMA positive). Fibrosis was confirmed by increased levels of hepatic hydroxyproline content and the expression of the pro-fibrogenic genes collagen-1α2, TIMP-1, and TGF-β. The expression of osteopontin was increased in both cholangiocytes and zone 1 hepatocytes. This cytokine with potent chemoattractant and pro-fibrogenic properties has been implicated in the pathogenesis of lung fibrosis and is postulated to be central to the induction of bile ductular reaction and portal-portal bridging fibrosis. Although there was no difference in bile acid output or composition based on gas chromatography/mass spectrometry, glutathione and phospholipid secretion was significantly reduced.

Continued

V. Cholangiocyte Transport Abnormalities: The principal function of the cholangiocyte is to regulate the secretion of fluids into the bile duct. Drew Feranchak has played a sentinel role in identifying these ion channels. This includes CFTR, the Ca2+-activated K+

channel (SK2), as well as the role of purinergic signaling to regulate cholangiocyte transport and bile formation (11,12). In addition, both human and rat biliary cells exhibit flow-stimulated, PKCζ-dependent ATP release, which results in increased [Ca2+]i and Cl secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases (13). Whether impairment of purinergic signaling in the setting of loss of CFTR function explains the predilection to CFLD remains unknown. However, this could potentially explain the mechanism of action of DDC, which through biliary obstruction and increased ductal pressure, inhibits purinergic-driven alternative chloride channel secretion, resulting in biliary inflammation and fibrosis.

VI. Activation of Hepatic Stellate Cells (HSCs): These cells play a pivotal role in the activation of fibrogenesis in the liver. Brenner’s and Schwabe’s group (2) have recently demonstrated a fundamental role for intestinal microflora in the initial activation of HSCs. Induction of liver injury induced in animal models through either mechanical (BDL) or chemical sensitizers (carbon tetrachloride or thioacetamide) led to increased LPS levels in the plasma and in the portal circulation. The binding of LPS to TLR4 receptors on HSCs led to two important events: i) induction of Kupffer cell chemotaxis and ii) increased sensitivity of HSCs to TGF-β secreted from Kupffer cells through downregulation of TGF-β pseudoreceptor Bambi on HSCs.

VII. Summary: The following conclusions can be made:

•LossofCFTRaloneisnotsufficientinbothanimal models and in humans to result in CFLD.

•Geneticmodifiersand/orotherproducts(chemical, toxin, bacterial) are required for disease expression.

•Understandingwhatleadstoi)cholangiocyteactivation and ii) downstream fibrogenesis is critical in developing therapeutics and developing strategies to prevent CFLD. Thus, robust animal models need to be generated that demonstrate these features of CFLD.

Literature Cited:

1. Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver disease. Semin Liver Dis. 2001 Nov;21(4):471-88.

2. Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, Schwabe RF. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med. 2007 Nov;13(11):1324-32.

3. Sheth S, Shea JC, Bishop MD, Chopra S, Regan MM, Malmberg E, Walker C, Ricci R, Tsui LC, Durie PR, Zielenski J, Freedman SD. Increased prevalence of CFTR mutations and variants and decreased chloride secretion in primary sclerosing cholangitis. Hum Genet. 2003 Aug;3(3):286-92.

4. Blanco PG, Zaman MM, Junaidi O, Sheth S, Yantiss RK, Nasser IA, Freedman SD. Induction of colitis in cftr-/- mice results in bile duct injury. Am J Physiol Gastrointest Liver Physiol. 2004 Aug;287(2):G491-6.

5. Pall H, Zaman MM, Andersson C, Freedman SD. Decreased peroxisome proliferator activated receptor alpha is associated with bile duct injury in cystic fibrosis transmembrane conductance regulator-/- mice. J Pediatr Gastroenterol Nutr. 2006 Mar;42(3):275-81.

6. Freudenberg F, Broderick AL, Yu BB, Leonard MR, Glickman JN, Carey MC. Pathophysiological basis of liver disease in cystic fibrosis employing a DeltaF508 mouse model. Am J Physiol Gastrointest Liver Physiol. 2008 Jun;294(6):G1411-20.

7. Beharry S, Ackerley C, Corey M, Kent G, Heng YM, Christensen H, Luk C, Yantiss RK, Nasser IA, Zaman M, Freedman SD, Durie PR. Long-term docosahexaenoic acid therapy in a congenic murine model of cystic fibrosis. Am J Physiol Gastrointest Liver Physiol. 2007 Mar;292(3):G839-48

8. Friedman KJ, Ling SC, Lange EM, Macek M, Handler AJ, Pace RG, et al. Genetic modifiers of severe liver disease in cystic fibrosis. 55th Annual Meeting of The American Society of Human Genetics, 2005;395:2176/F.

9. Mencin A, Seki E, Osawa Y, Kodama Y, De Minicis S, Knowles M, Brenner DA. Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis. Hepatology. 2007 Nov;6(5):443-52

10. Fickert P, Stöger U, Fuchsbichler A, Moustafa T, Marschall HU, Weiglein AH, Tsybrovskyy O, Jaeschke H, Zatloukal K, Denk H, Trauner M. A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis. Am J Pathol. 2007 Aug;171(2):525-36.

11. Feranchak AP, Doctor RB, Troetsch M, Brookman K, Johnson SM, Fitz JG. Calcium-dependent regulation of secretion in biliary epithelial cells: the role of apamin-sensitive SK channels. Gastroenterology. 2004 Sep;127(3):903-13.




Continued

12. Feranchak AP, Fitz JG. Adenosine triphosphate release and purinergic regulation of cholangiocyte transport. Semin Liver Dis. 2002 Aug;22(3):251-62

13. Woo K, Dutta AK, Patel V, Kresge C, Feranchak AP. Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl– transport in biliary epithelial cells through a PKCζ-dependent pathway. J Physiol. 2008;586:2779-2798.

14. Norkina O, Burnett TG, De Lisle RC. Bacterial overgrowth in the cystic fibrosis transmembrane conductance regulator null mouse small intestine. Infect Immun. 2004 Oct;72(10):6040-9.

15. Cox KL, Cox KM. Oral vancomycin: treatment of primary sclerosing cholangitis in children with inflammatory bowel disease. J Pediatr Gastroenterol Nutr. 1998 Nov;27(5):580-3.

16. Fiorotto R, Spirlì C, Fabris L, Cadamuro M, Okolicsanyi L, Strazzabosco M. Ursodeoxycholic acid stimulates cholangiocyte fluid secretion in mice via CFTR-dependent ATP secretion. Gastroenterology. 2007 Nov;133(5):1603-13.

17. Pall H, Zielenski J, Jonas MM, DaSilva DA, Potvin KM, Yuan XW, Huang Q, Freedman SD. Primary sclerosing cholangitis in childhood is associated with abnormalities in cystic fibrosis-mediated chloride channel function. J Pediatr. 2007 Sep;151(3):255-9.


David E. Kleiner, M.D., Ph.D.

Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD

Liver disease has long been recognized as a component of the injury in cystic fibrosis. Liver disease may present with subtle clinical findings, such as liver enzyme abnormalities or irregularities noted on ultrasound. As with many chronic liver diseases, progressive fibrosis is the major threat, with the development of portal hypertension and biliary cirrhosis. Steatosis is the most commonly observed pathologic change, with approximately 60 percent of patients in the early study of Craig et al. demonstrating it. In that study, it was noted that the steatosis in mild cases tended to be at the periphery of hepatic lobules, or periportal/zone 1 in more modern terms. In severe cases, the steatosis becomes pan-acinar. Steatohepatitis has not been reported, although the variant of fatty liver disease seen more often in children has a zone 1 distribution of steatosis coupled with periportal fibrosis.

The pathognomonic hepatic lesion of cystic fibrosis is focal biliary fibrosis (cirrhosis), which is found in up to one third of patients. This lesion is characterized by the accumulation of amorphous pink, PAS-positive material in bile ducts, associated with ductular reaction, periportal fibrous expansion, and chronic inflammation. The lesions often are irregularly distributed in the liver, with variation in severity from portal area to portal area. These lesions may progress to form bridges, isolating regenerative nodules of varying sizes. The resulting cirrhosis is often macronodular, and there can be extreme variation in the sizes of nodules. Duct rupture can occur, as can other changes of large duct obstruction. A pattern of duct injury mimicking sclerosing cholangitis has been reported. Other complications that have been reported include amyloidosis, biliary obstruction of large ducts

requiring the Kasai procedure, and a neonatal hepatitis-like presentation with duct paucity. Bradford et al. performed a detailed ultrastructural examination of the hepatocytes in cystic fibrosis and demonstrated dilation of canaliculi as well as ducts and ductules. The canaliculi contained heterogeneous electron-dense material with myelin, cell debris, and fibrillar material. Distention of rough endoplasmic reticulum by a finely granular material was also seen. Membrane-bound vacuoles with an electron-dense core resembling mucus was found in hepatocytes as well as in bile duct epithelial cells.

The factors influencing progression of liver disease are incompletely understood, and a number of studies have been devoted to pathogenesis of fibrosis as well as to the development of biomarkers that would allow non-invasive monitoring of patients with cystic fibrosis. Part of the problem lies in the heterogeneity of the liver disease itself. Similar to the situation of primary sclerosing cholangitis, a liver biopsy may miss diagnostic lesions or underestimate the degree of liver injury due to sampling variability. It would be helpful to develop standard criteria for evaluating the liver lesions in cystic fibrosis similar to systems that have been developed for fatty liver disease and chronic hepatitis. Data from blinded evaluation of liver biopsies from the Cystic Fibrosis Liver Disease Network will be presented at the workshop, along with a proposal for semi-quantitative evaluation of liver lesions that can be used in prospective clinical studies.

Future Directions:

•Developmentofastandardizedfeaturerecording system for evaluation of liver biopsies in cystic fibrosis.

•Correlationofsystematicobservationsofpathology with clinical, biochemical, and molecular features of liver disease in cystic fibrosis.

The Pathology of Cystic Fibrosis Liver Disease



Selected References:

Craig JM, Haddad H, Schwachman H. The pathological changes in the liver in cystic fibrosis of the pancreas. Am J Dis Child 1957;93:357-369.

di Sant’Agnese PA, Blanc WA. A distinctive type of biliary cirrhosis of the liver associated with cystic fibrosis of the pancreas. Recognition through signs of portal hypertension. Pediatrics 1956;18:387-409.

Bradford W, Allen D, Shelburne J, Spock A. Hepatic parenchymal cells in cystic fibrosis: ultrastructural evidence for abnormal intracellular transport. Pediatr Pathol 1983;1:269-279.

Columbo C, Battezzati PM, Crosignani A, Morabito A, Costantini D, Padoan R, Giunta A. Liver disease in cystic fibrosis: a prospective study on incidence, risk factors and outcome. Hepatology 2002;36:1374-1382.

Lewindon PJ, Pereira TN, Hoskins AC, Bridle KR, Williamson RM, Shepherd RW, Ramm GA. The role of hepatic stellate cells and transforming growth factor beta 1 in cystic fibrosis liver disease. Am J Pathol 2002;160:1705-1715.

Continued

The Pathology of Cystic Fibrosis Liver Disease

Michael R. Knowles, M.D.1, for Jaclyn Bartlett, Ph.D.1, Peter Durie, M.D.2, and the Gene Modifier Study Group

1University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Hospital for Sick Children, Toronto, Ontario, Canada

Cystic fibrosis (CF) is a recessive monogenic disorder characterized by clinical heterogeneity that is not explained by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1,2). Patients with CF exhibit a wide range of lung disease severity, and genetic variability in non-CFTR genes contributes to risk for severity of pulmonary disease (3-5).

Dysfunction in the CF liver reflects loss of CFTR (Cl channel) activity on the apical membrane of cholangiocytes (6). This dysfunction results in reduced bile flow and is associated with a cholangiocyte-induced inflammatory response, with activation and proliferation of hepatic stellate cells, which results in cholangitis and fibrosis in focal portal tracts (7,8). However, only a small fraction (~5%) of CF patients develops severe liver disease characterized by cirrhosis with portal hypertension (CFLD); thus, non-CFTR genetic variability may contribute to risk for severe liver disease (9).

To test for association between non-CFTR genetic polymorphisms and severe liver disease in CF with portal hypertension (CFLD), we studied nine variants in five genes previously studied in CF liver disease, including α1-antitrypsin (also known as α1-antiprotease, α1AP, SERPINA1) (10) angiotensin-converting enzyme (ACE) (11), glutathione S-transferase (GSTP1) (12), mannose-binding lectin 2 (MBL2) (13), and transforming growth factor β1 (TGFβ1) (11). A two-stage study design was used. In the initial study, we compared polymorphic genotypes in these candidate modifier genes in CF subjectswithCFLD(n=124)and“control”CFpatients (n = 843) without CFLD who were at least 15 years of age. In the second stage, we tested our initial associations (SERPINA1 Z allele and TGFβ1 codon 10 genotype) in

a second study in different populations of CF patients with (n = 136) and without (n = 1,088) CFLD.

Clinical features of the CF patients with severe liver disease, compared to controls, showed more males (~65%) than females. The mean and median age of recognition (diagnosis) of portal hypertension in all CFLD patients was approximately 10-11 years, and more than 90 percent of the CFLD patients were diagnosed by age 20 years. More than 95 percent of patients with CFLD had two pancreatic insufficient mutations in CFTR. The prevalence of meconium ileus at birth in CFLD patients (~18%) is typical for the general CF population with pancreatic insufficient CFTR mutations. Abnormalities in biochemical tests of the liver (AST, ALT, and GGT) were not predictive of CFLD and showed no correlation with markers of hepatocellular synthetic dysfunction, such as international normalized ratio (INR) and serum albumin.

In the initial study, significant association was seen for the SERPINA1 Z allele and variants of TGFβ1. The genotypes and minor allele frequencies for genetic variants in patients without CFLD were similar to those previously reported (3-5,10-13). In the replication study, the strong association was replicated for the SERPINA1 Z allele. The association of TGFβ1 variants in the initial population was not replicated for codon 10.

When the initial and replication populations were combined for analysis, the SERPINA1 Z allele displayed very robust association with CFLD; similar evidence for association was observed in analyses restricted to Caucasian subjects in the initial and replication populations. We also performed logistic regression in the combined initial and replication groups, and estimated the odds of CFLD for the SERPINA1 Z allele, adjusting for the covariates of ethnicity, gender, and CFTR genotype. Results remained consistent when using all subjects or Caucasian subjects only, with respect to statistical significance estimates and odds ratio estimates. We also combined the initial and replication groups, and esti-

Genetic Modifiers of Cystic Fibrosis Liver Disease



Genetic Modifiers of Cystic Fibrosis Liver Disease

Continued

mated a population attributable risk due to the Z allele of approximately 7 percent.

The mechanism of the Z allele as an adverse modifier of liver disease in CF patients likely reflects stimulation of hepatic stellate cells by inflammatory mediators from both CFTR-deficient cholangiocytes and hepatocytes containing the misfolded Z allele protein. These dual inflammatory stimuli induce hepatic stellate cells to migrate and proliferate in the bile duct regions in a pro-fibrogenic manner (7-9,14). Bile duct ligation with resultant cholestasis stimulates activated stellate cells and fibrosis in the liver of homozygous transgenic PiZ versus wild-type mice, which is compatible with this proposed mechanism of the Z allele as an adverse modifier in CF (15).

This two-stage study shows that the SERPINA1 Z allele is strongly associated with CFLD and portal hypertension. This polymorphism is relatively uncommon in CF (~2.2% of CF patients are carriers), but the odds ratio for association with severe liver disease is relatively high (~5) for the contribution of a genetic modifier to a Mendelian disorder. The availability of this first genetic marker, SERPINA1 Z allele, for the development of severe liver disease in CF illustrates the concept of identifying risk factors in CF patients early in life.

Future research challenges for studying the genetics of CF liver disease relate primarily to the characterization, recruitment, and more extensive genetic studies of patients with CFLD. A recent GWAS study of nearly 300 patients with CFLD is undergoing analysis. Association of single nucleotide polymorphisms with CFLD in this large, collaborative GWAS study will require replication in a separate cohort and extensive followup studies to identify causative variants and define mechanism(s) of pathobiology of CFLD.

References:

1. Mekus F, et al. Twin Res 2000;3:277. 2. Vanscoy LL, et al. Am J Respir Crit Care Med 2007;175:1036. 3. Drumm ML, et al. N Engl J Med 2005;353:1443. 4. Bremer LA, et al. Hum Gene Ther 2008;17:2228. 5. Dorfman R, et al. J Clin Invest 2008;118:1040. 6. Cohn JA, et al. Gastroenterology 1993;105:1857. 7. Feranchak AP, Sokol RJ. Semin Liver Dis 2001;21:471. 8. Schuppan D, Alfhal NH. Lancet 2008;371:838. 9. Feranchak AP. Curr Gastroenterol Rep 2004;6:231. 10. Frangolias DD, et al. Am J Respir Cell Mol Biol 2003;29:390. 11. Arkwright PD, et al. Am J Respir Crit Care Med 2003;167:384. 12. Henrion-Caude A, et al. Hepatology 2002;36:913. 13. Gabolde M, et al. J Med Genet 2001;38:310. 14. Perlmutter DH. Pediatr Res 2006;60:233. 15. Mencin A, et al. Hepatology 2007;46:1443.

Jeffrey Teckman, M.D.

Associate Professor of Pediatrics, and Biochemistry and Molecular Biology, Saint Louis University School of Medi-cine, St. Louis, MO; Cardinal Glennon Children’s Medical Center, St. Louis, MO

Genetically determined abnormalities in protein folding are responsible for a variety of diseases, and are sometimes referred to as conformational diseases (1). In some cases, abnormal conformation inhibits normal physiologic function of the protein as part of disease pathophysiology. In other cases, accumulation of abnormally folded proteins, either directly or indirectly, triggers cell injury. Alpha-1-antitrypsin (a1AT) deficiency is commonly viewed as a prototypical conformational disease, and is caused by homozygosity for the a1AT mutant Z gene (2,3). ZZ homozygotes occur in approximately 1 in 2,000 births in North American and European populations. The Z mutant, the most common disease-associated allele, arises from a point mutation that encodes a single amino acid substitution. This mutation confers an abnormal conformation on the a1AT mutant Z protein, resulting in accumulation within the endoplasmic reticulum of hepatocytes rather than the appropriate, high magnitude secretion of the wild type protein. Some homozygous ZZ children and adults develop chronic liver disease and hepatocellular carcinoma, which is thought to result from this intracellular accumulation of the a1AT mutant Z protein. The profoundly low serum levels of a1AT found in ZZ homozygotes leaves patients susceptible to the development of chronic obstructive pulmonary disease as adults.

The accumulation of the mutant Z protein in the liver in a1AT deficiency is strikingly heterogeneous, although the mechanisms involved in accumulation and the reasons for the heterogeneity are still being investigated (4,5). Previous reports based on in vitro studies

have suggested that autophagy, mitochondrial injury, apoptosis, and other pathways may be involved in the mechanism of hepatocyte injury, although the interplay of these mechanisms in vivo is still being described (6,7). Recent studies have examined a well-characterized in vivo model of a1AT mutant Z liver injury, the PiZ mouse, and made comparisons to human liver, to better understand the in vivo mechanisms of liver injury involved in this disease. Results have shown a moderate but chronic increase in the rate of liver cell death and an associated compensatory hepatocellular proliferation (8). The results also show an increase in stimulation of the apoptotic cascade in hepatocytes, the magnitude of which strongly correlates to the absolute amount of a1AT mutant Z protein accumulated within the individual cell. It appears that the small population of hepatocytes with the largest intracellular accumulations of the mutant Z protein undergoes apoptotic cell death at an increased rate and that cells without large accumulations of the mutant Z protein proliferate in response. This chronic cycle of injury and repair leads to organ injury. Some of these insights have depended on new methods in which livers from the PiZ transgenic mouse were harvested and hepatocytes with the largest mutant Z protein accumulations were isolated from cells with less accumulation, so that these populations of cells from within the same liver could be analyzed separately. These data revealed heterogeneity in injury on the cellular level, which correlated with the magnitude of a1AT mutant Z protein intracellular accumulation (9). These studies also permitted the construction of a hypothetical hepatocellular injury cascade for this disease involving caspase activation, apoptosis, and mitochondrial injury, which takes into account the heterogeneous nature of the accumulation within the liver. Further development of this hypothetical cascade, and continued study of sub-populations of cells from within the same liver, will likely be needed to fully describe the mechanisms of injury in this disease and to design future therapies.

Protein Folding Defects in Liver Disease: Alpha-1-antitrypsin Deficiency



Protein Folding Defects in Liver Disease: Alpha-1-antitrypsin Deficiency

Continued

References:

1. Carrell RW, Lomas DA. Alpha1-antitrypsin deficiency—a model for conformational diseases. N Engl J Med 2002;346(1):45-53.

2. Perlmutter DH, Brodsky JL, Balistreri WF, Trapnell BC. Molecular pathogenesis of alpha-1-antitrypsin deficiency-associated liver disease: a meeting review. Hepatology 2007;45(5):1313-23.

3. Teckman JH. Alpha1-antitrypsin deficiency in childhood. Semin Liver Dis 2007;27(3):274-81.

4. Chappell S, Hadzic N, Stockley R, Guetta-Baranes T, Morgan K, Kalsheker N. A polymorphism of the alpha1-antitrypsin gene represents a risk factor for liver disease. Hepatology 2008;47(1):127-32.

5. Greene CM, Miller SD, Carroll T, McLean C, O’Mahony M, Lawless MW, O’Neill SJ, Taggart CC, McElvaney NG. Alpha-1 antitrypsin deficiency: a conformational disease associated with lung and liver manifestations. J Inherit Metab Dis 2008;31(1):21-34.

6. Rudnick DA, Shikapwashya O, Blomenkamp K, Teckman JH. Indomethacin increases liver damage in a murine model of liver injury from alpha-1-antitrypsin deficiency. Hepatology 2006;44(4):976-82.

7. Sifers RN. Cell biology. Protein degradation unlocked. Science 2003;299(5611):1330-1.

8. Rudnick DA, Liao Y, An JK, Muglia LJ, Perlmutter DH, Teckman JH. Analyses of hepatocellular proliferation in a mouse model of alpha-1antitrypsin deficiency. Hepatology 2004;39(4):1048-55.

9. Lindblad D, Blomenkamp K, Teckman J. Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model. Hepatology 2007;46(4):1228-35.

Andrew P. Feranchak, M.D.

Associate Professor of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX

The pathophysiology underlying the development of cystic fibrosis (CF)-associated liver disease is still only speculative, and while several hypotheses have been proposed, studies directly assessing the effects of abnormal CFTR in hepatobiliary function are limited. This reality is disappointing since the genetic underpinnings responsible for cystic fibrosis have been defined for more than 15 years. One conventional model proposes that an abnormal or absent CFTR protein in the apical membrane of biliary epithelial cells initiates a series of events, including: abnormal secretin/cAMP-stimulated Cl and HCO3

- secretion, a decrease in bile flow, subsequent bile duct plugging by thickened secretions, cholangiocyte and hepatocyte injury, activation of hepatic stellate cells, collagen deposition, and ultimately the development of focal biliary cirrhosis leading to multilobular cirrhosis (1). The histologic finding of eosinophilic material plugging the intrahepatic bile ducts of patients with cystic fibrosis-associated liver disease provides some evidence for this “bileplugging”modelbutfailstoexplainwhyonlya minority of patients with CF develop clinically significant liver disease.

Biliary Models: In the liver, CFTR is found only on intrahepatic biliary epithelial cells, known as cholangiocytes, and not on the more abundant parenchymal cells known as hepatocytes (2). Cholangiocytes contribute to the volume and composition of bile through absorption and secretion of fluid and electrolytes. However, their small size and intrahepatic location have made the direct study of cholangiocytes arduous. Several cell and epithelial models, including isolated primary rat cholangiocytes, immortalized (SV40) mouse cholangiocytes, human biliary adenocarcinoma cells, human and mouse gallbladder, isolated bile duct units, normal rat cholangiocyte (NRC) monolayers, and the isolated-perfused liver, have been developed and together have contributed

to our knowledge of biliary function. Unfortunately, the Cftr -/- mouse has not been a reliable model for the study of CF-associated liver disease due to viability issues and the inconsistent development of liver pathology. Therefore, the development of a long-living congenic Cftr -/- mouse (C57BL/6J background) or the CF pig, which appear to develop liver disease similar to humans, may provide powerful experimental models (3,4).

Role of CFTR in Hepatobiliary Function: Studies utilizing the available cell and epithelial models have revealed several important observations regarding the role of CFTR in cholangiocyte transport. First, CFTR is located on the apical membrane of cholangiocytes and functions as a cAMP-activated Cl channel (2,5). Second, binding of the hormone secretin to basolateral receptors results in an increase in intracellular cAMP and activation of CFTR through a PKA-dependent mechanism (6,7). Third, secretin-stimulated increases in cAMP result in a parallel increase in HCO3

- transport (8,9). Fourth, increases in cAMP are a stimulus for regulated exocytosis, including movement and insertion of Aquaporin-1 channels to the apical membrane (10,11). Together, these findings support a working model whereby secretin binding basolateral cholangiocyte receptors increases intracellular cAMP levels and stimulates apical membrane Cl-, HCO3

-, and H2O transport, thus modulating alkalinization and dilution of bile. Although these findings support an important role of CFTR in cAMPmediated biliary secretion, the identification of other membrane Cl channels, which contribute to ductular secretion via cAMP-independent mechanisms, adds complexity to the mechanisms responsible for bile formation.

Alternate Secretory Pathways: Other Cl channels have been identified in cholangiocytes and appear to contribute to ductular secretion independent of CFTR, including those activated by Ca2+/calmodulin kinase (5,12), increases in cell volume (13), and extracellular nucleotides (14-16). The contribution of these alternate secretory pathways to biliary secretion has been hard to assess. It is attractive to speculate that these alternate

Cystic Fibrosis Liver Disease: CFTR and the Liver




Continued

Cl channels may partly compensate for the CF secretory defect in the liver and may serve to bypass the secretory defect associated with CF. In fact, in the Cftr -/- mouse model, increased expression of alternate Cl channels modulates organ-level disease (17). Thus, Cl channels other than those encoded by CFTR can sustain biliary secretion.

Luminal Nucleotides Regulate Bile Formation: Recently, secretion mediated by extracellular nucleotides (e.g., ATP) acting on purinergic (P2) receptors on the luminal membrane of biliary epithelial cells has emerged as functionally important. ATP is present in bile, and binding of ATP to P2 receptors increases Cl efflux from isolated cholangiocytes (14) and dramatically increases transepithelial secretion in biliary epithelial monolayers (15). Indeed, the magnitude of the secretory response to ATP is two- to threefold greater than that to cAMP (16). Interestingly, recent evidence suggests that even cAMP-stimulated bile flow is mediated by ATP release into the duct lumen and stimulation of apical P2 receptors (18). Together, these studies challenge and extend the conventional model that centers on the concept that cAMP-dependent opening of CFTR-related Cl channels is the driving force for cholangiocyte secretion. Rather, the operative regulatory pathways appear to take place within the lumen of intrahepatic ducts, where release of ATP into bile is a final common pathway controlling ductular bile formation. In light of recent studies demonstrating that the mechanical effects of fluid-flow or shear stress at the apical membrane of biliary epithelial cells is a robust stimulus for ATP release, a model emerges in which mechanosensitive ATP release and Cl secretion is a dominant pathway regulating biliary secretion (19). Thus, a decrease in bile flow associated with CF may be accompanied by alterations in mechanosensitive pathways, which may exacerbate abnormalities in Cl secretion and bile formation.

Role of Abnormal CFTR in the Development of Liver Disease: How does an absent or abnormal CFTR protein on the cholangiocyte membrane lead to

the clinical manifestations of liver disease? Functional studies of primary CF cholangiocytes are limited. A study of regulated transport in isolated human cholangiocytes with a compound heterozygous mutation (∆F508/G542X) demonstrated abnormal cAMPstimulated Cl-, K+, and HCO3

- secretion, but normal Ca2+-activated Cl secretion (20). Studies in human CF gallbladder revealed similar abnormalities in cAMPstimulated Cl transport, but an intact ATP-stimulated Cl conductance, presumably through a Ca2+-dependent pathway (21). Although these studies are in agreement with the working model, demonstrating an important role of CFTR in ductular Cl and HCO3

- secretion, they fail to provide a mechanistic explanation for why only a subset of patients develops clinically significant liver disease (even with the same CFTR mutation); however, the existence of alternate Cl secretory pathways in these studies is intriguing. Indeed, although CF biliary epithelium has abnormal secretin/cAMP-stimulated Cl

secretion, it maintains a robust Cl secretory response to exogenous nucleotides (21,22), suggesting that this may represent a therapeutic strategy to improve the abnormal biliary secretion associated with CF liver disease.

Summary: Several observations can be made concerning the role of CFTR in hepatobiliary function and the development of CF-associated liver disease. First, an abnormal or absent CFTR protein is necessary but not sufficient for the development of clinically significant CF liver disease. Second, while CFTR functions as a cAMPstimulated Cl channel in the apical membrane of biliary epithelium,itsroleasa“regulator”ofothermembrane transporters and transport pathways may surpass its role as a Cl channel. Third, mechanosensitive pathways, including ATP release and P2 receptor binding, may be a predominant pathway, independent from CFTR, contributing to bile formation. Further studies are clearly needed to: define the role of CFTR in normal liver function and during cholestasis, understand the potential regulatory roles of alternate secretory pathways, and elucidate the potential genetic and environmental modifiers of disease progression.

Continued

Future Research Challenges/Needs:

Development of hepatobiliary models of CF (cholangiocytes, polarized biliary epithelium, whole animals)

•ClearelucidationoftheroleofCFTRinhepatobiliary function. •Identificationoftheinitiatingfactorsinthedevelop

ment of CF liver disease. •Characterizationofalternatesecretorypathwaysalong

the bile duct that may serve as potential therapeutic targets.

Acknowledgments: Studies in our laboratory are supported by the Cystic Fibrosis Foundation and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health.

References:

1. Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver disease. Sem Liv Dis 2001;21:471-88.

2. Cohn JA, Strong TA, Picciotto MA, Nairn AC, Collins FS, Francis S, Fitz JG. Localization of CFTR in human bile duct epithelial cells. Gastroenterology 1993;105:1857-64.

3. Durie PR, Kent G, Phillips MJ, Ackerley CA. Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model. Am J Pathol 2004 Apr;164(4):1481-93.

4. Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, et al. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 2008 Sep 26;321(5897):1837-41.

5. Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regulation of membrane chloride currents in rat bile duct epithelial cells. J Clin Invest 1993;91:319-28.

6. Levine RA, Hall RC. Cyclic AMP in secretin choleresis. Evidence for a regulatory role in man and baboons but not in dogs. Gastroenterology 1976;70:537-44.

7. McGill J, Gettys TW, Basavappa S, Fitz JG. Secretin activates Cl- channels in bile duct epithelial cells through a cAMP-dependent mechanism. Hepatology 1992;16:125A.

8. Alvaro D, Cho WK, Mennone A, Boyer JL. Effect of secretin on intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 1993;92:1314-25.

9. Strazzabosco M, Mennone A, Boyer JL. Intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 1991;87:1503-12.

10. Roberts SK, Yano M, Ueno Y, Pham L, Alpini G, Agre P, LaRusso N. Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism. Proc Natl Acad Sci U S A 1994;91:13009-13.

11. Tietz PS, Marinelli RA, Chen XM, Huang B, Cohn J, Kole J, McNiven MA, Alper S, LaRusso NF. Agonist-induced coordinated trafficking of functionally related transport proteins for water and ions in cholangiocytes. J Biol Chem 2003 May 30;278(22):20413-9.

12. Schlenker T, Fitz JG. Calcium-activated chloride channels in a human biliary cell line: regulation by calcium/calmodulin-dependent protein kinase. Am J Physiol 1996;271:G304-G310.

13. Roman RM, Wang Y, Fitz JG. Regulation of cell volume in a human biliary cell line: calcium-dependent activation of K+ and Cl- currents. Am J Physiol 1996;271:G239-G248.

14. McGill J, Basavappa S, Shimokura GH, Middleton JP, Fitz JG. Adenosine triphosphate activates ion permeabilities in biliary epithelial cells. Gastroenterology 1994;107:236-43.

15. Schlenker T, Romac JMJ, Sharara A, Roman RM, Kim S, LaRusso N, Liddle R, Fitz JG. Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am J Physiol 1997;273:G1108-G1117.

16. Dutta AK, Woo K, Doctor RB, Fitz JG, Feranchak AP. Extracellular nucleotides stimulate Cl currents in biliary epithelia through receptor-mediated IP3 and Ca2+ release. Am J Physiol Gastrointest Liver Physiol 2008 (in press).

17. Clarke LL, Grubb BR, Yankaskas J, Cotton CU, McKenzie A, Boucher RC. Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in cftr(-/) mice. Proc Natl Acad Sci U S A 1994;91:479-83.

18. Minagawa N, Nagata J, Shibao K, Masyuk AI, Gomes DA, Rodrigues MA, Lesage G, Akiba Y, Kaunitz JD, Ehrlich BE, et al. Cyclic AMP regulates bicarbonate secretion in cholangiocytes through release of ATP into bile. Gastroenterology 2007 Nov;133(5):1592-602.

19. Woo K, Dutta AK, Patel V, Kresge C, Feranchak AP. Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl transport in biliary epithelial cells through a PKCzeta-dependent pathway. J Physiol 2008 Jun 1;586(Pt 11):2779-98.

20. Zsembery A, Jessner W, Sitter G, Spirli C, Strazzabosco M, Graf J. Correction of CFTR malfunction and stimulation of Ca-activated Cl channels restore HCO3

- secretion in cystic fibrosis bile ductular cells. Hepatology 2002 Jan;35(1):95-104.

21. Chinet T, Fouassier L, Dray-Charier N, Imam-Ghali M, Morel H, Mergey M, Dousset B, Parc R, Housset C. Regulation of electrogenic anion secretion in normal and cystic fibrosis gallbladder mucosa. Hepatology (forthcoming).

22. Clarke LL, Harline MC, Gawenis LR, Walker NM, Turner JT, Weisman GA. Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium. Am J Physiol Gastrointest Liver Physiol 2000 Jul;279(1):G132-G138.




David K. Meyerholz, D.V.M., Ph.D.1, David A. Stoltz, M.D., Ph.D.2,and Michael J. Welsh, M.D.2-4

1Departments of Pathology, 2Internal Medicine, and 3Mo-lecular Physiology and Biophysics, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA; 4Howard Hughes Medical Institute, Chevy Chase, MD

The CFTR-/- Pig: Liver and Gallbladder Disease

Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the cystic fibrosis transmembrane conductance (CFTR) regulator. Liver disease is the second leading cause of CF mortality, and liver disease can occur in neonatal infants, suggesting lesion initiation and development in utero. Recently, we described a novel CFTR-/- pig model that has multiorgan disease at birth, including meconium ileus and pancreatic destruction, with gallbladder and liver lesions. In neonatal CFTR-/- pigs, microgallbladder is a ubiquitous gross lesion and its lumen is variably filled with mucoid to inspissated bile and uncommon cellular aggregates of neutrophils. Alcian Blue/PyronineY as well as Periodic Acid Schiff stains detect the luminal inspissated mucus, and the walls of the gallbladder are thickened by mucosal proliferation with circumferential accentuation of the gallbladder wall by fibrosis and smooth muscle. Inspissated bile and mucus are variably seen obstructing lumens of the cystic ducts. Large extrahepatic and intrahepatic ducts are uncommonly filled by mucus with scattered neutrophils, cellular plugs, or inspissated bile. In the liver, triads are focally accentuated by variable ductular hyperplasia, inflammation, and fibrosis. Rarely, triads have widespread bridging, predominantly by biliary hyperplasia, with lesser inflammation and fibrosis. These cases also have microscopic retraction of the surface serosa creating a lobular surface appearance. Steatosis is not present in neonatal CFTR-/- pigs.

Future challenges include optimization of the surgical and medical therapies required for resolution of the meconium ileus that is universally seen in the CFTR-/- pig model. To characterize the development and pathogenesis of hepatobiliary disease, in utero studies

are needed to define the gestational timing and anatomic changes associated with initial lesion development. Furthermore, long-term studies following the progression of liver disease are needed to define the clinical pathology, the incidence of multilobular cirrhosis, and what modifier genes contribute to its development.

References:

Bodian M. Fibrocystic Disease of the Pancreas: A Congenital Disorder of Mucus Production-Mucosis. New York: Grune & Stratton, 1953.

Diwakar V, Pearson L, Beath S. Liver disease in children with cystic fibrosis. Paediatric Respiratory Reviews 2001;2:340-349.

Feranchak AP. Cystic fibrosis liver disease. In: Liver Disease in Children, 3rd ed. Suchy FJ, Sokol RJ, Balistreri WF, eds. Cambridge, United Kingdom: Cambridge University Press, 2007, pp. 572-594.

Lindblad A, Hultcrantz R, Strandvik B. Bile-duct destruction and collagen deposition: a prominent ultrastructural feature of the liver in cystic fibrosis. Hepatology 1992;16:372-381.

Oppenheimer EH, Esterley JR. Hepatic changes in young infants with cystic fibrosis: possible relation to focal biliary cirrhosis. Journal of Pediatrics 1975;86:683-689.

Oppenheimer EH, Esterley JR. Pathology of cystic fibrosis: review of the literature and comparison with 146 autopsied cases. Perspectives in Pediatric Pathology 1975;2:241-278.

Porta EA, Stein AA, Patterson P. Ultrastructural changes of the pancreas and liver in cystic fibrosis. American Journal of Clinical Pathology 1964;42:451-465.

Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyansky J, McCray PB Jr, Zabner J, Prather RS, Welsh MJ. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn piglets. Science 2008 Sep 26;321(5897):1837-1841.

Saul J. Karpen, M.D., Ph.D.

Director, Texas Children’s Liver Center, Texas Children’s Hospital/Baylor College of Medicine, Houston, TX

With recent improvements in the care of patients with Cystic Fibrosis (CF) leading to enhanced longevity, the manifestations of non-pulmonary features of CF are becoming increasingly recognized as components linked to outcomes. Among the more serious non-pulmonary manifestations of CF are those that involve the liver, affecting upwards of 20 percent of CF patients (1-3). CF-associated liver disease (CFLD) is considered the second leading cause of death in CF patients, and progression of CFLD has an impact on CF patients’ quality of life and function, indicating a much-needed role for safe and effective therapeutics. However, there are disappointingly few effective therapeutic choices for most cholestatic or fibrotic liver disease, including CFLD. Despite these current caveats, there are medical, nutritional, radiological, surgical, and transplant options for CFLD.

1. Cholestasis and Malnutrition in the Infant With CF: The clinical features of CFLD in infancy typically involve prolonged neonatal jaundice and poor growth, with hepatomegaly and laboratory values notable for elevated serum conjugated bilirubin and GGT levels. The overlapping nutritional and metabolic needs of infants with CF are multiplied during this period of life, especially when there is short gut or cholestasis. Treatment options in the first year of life are primarily directed toward appropriate nutritional supplementation, even in those whose jaundice has resolved. Periodic verification of adequate fat-soluble vitamin nutriture is essential. Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is employed by many caregivers, although evidence for its efficacy in CFLD at this age is lacking (4). There are some infants with CFLD whose disease progresses rapidly, mainly those born prematurely who require parenteral nutrition or have bowel resections

due to intestinal obstruction, leading to persistence of cholestasis, biliary tract damage/obstruction, and in some, progression to liver insufficiency and death. In these high-risk patients, medical treatment options are limited, with emerging roles for single- or multi-organ transplants in select infants.

2. The Older Child With CFLD: The clinical features of the older child with CFLD may involve hepatomegaly, splenomegaly, or elevated liver enzymes, or the child may be asymptomatic (5). A proportion of these patients have gall bladder dysmotility and propensity to form gallstones. Treatment options include UDCA, which may improve gall bladder kinetics, but many times, cholecystectomy is the preferred, long-term treatment option (6). A main clinical feature of progression of biliary fibrosis and cirrhosis in CFLD is the development of portal hypertension (PHTN) and sequelae of varices, ascites, and splenomegaly. The splenomegaly can be massive, with associated sequestration of leukocytes and platelets, as well as potential impairments in pulmonary dynamics. At times, the spleen seems to take on a major focus for some clinicians, but the contributions of thrombocytopenia and abdominal distension to survival and lung function have yet to be fully evaluated (7). Non-medical treatments for varices include therapeutic endoscopic banding of varices and surgical or radiological porto-systemic shunts (8,9). UDCA therapy has not been shown to affect the development of PHTN in CFLD.

3. Isolated Liver and Multi-organ Transplantation: Consideration for liver transplantation in CF patients is complex because clear guidelines for proper patient selection are lacking. Currently, those with minimal lung dysfunction who progress to liver insufficiency or severe sequelae of PHTN warrant consideration for liver transplantation (10-12). Given the multiple organs involved in patients with CF, reports of lung-liver, liver-pancreas, heart-lung-liver and liver-intestine transplants suggest generally positive outcomes, although the number of cases is small (13,14).

Medical and Surgical Therapy of CF-associated Liver Disease



Summary and Future Directions: The complexity of making treatment decisions rests not only with the inherently individual patient-to-patient manifestations of pulmonary and liver disease in CF, but also with the lack of suitable markers for progression of CFLD. Identification of markers associated with disease progression and severity is needed. Current therapeutic options are limited for CFLD, perhaps mainly due to a lack of basic knowledge of the clinical and genetic underpinnings for its cause. The current key questions include: identifying those patients who may benefit from bile-focused therapies (e.g., UDCA or other bile acid-related therapies, 15); appropriate management and treatment of PHTN; and the identification of those who would benefit from surgical interventions and transplantation.

References:

1. di Sant’Agnese PA, Blanc WA. A distinctive type of biliary cirrhosis of the liver associated with cystic fibrosis of the pancreas: recognition through signs of portal hypertension. Pediatrics 1956;18:387-409.

2. Colombo C, Russo MC, Zazzeron L, Romano G. Liver disease in cystic fibrosis. J Pediatr Gastroenterol Nutr 2006;43:S49-55.

3. Brigman C, Feranchak A. Liver involvement in cystic fibrosis. Curr Treat Options Gastroenterol 2006;9:484-96.

4. Beuers U. Drug insight: Mechanisms and sites of action of ursodeoxycholic acid in cholestasis. Nat Clin Pract Gastroenterol Hepatol 2006;3:318-28.

5. Colombo C, Battezzati PM, Crosignani A, et al. Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome. Hepatology 2002;36:1374-82.

6. Colombo C, Crosignani A, Battezzati PM, et al. Delayed intestinal visualization at hepatobiliary scintigraphy is associated with response to long-term treatment with ursodeoxycholic acid in patients with cystic fibrosis-associated liver disease. J Hepatol 1999;31:672-7.

7. Louis D, Duc ML, Reix P, et al. Partial splenectomy for portal hypertension in cystic fibrosis related liver disease. Pediatr Pulmonol 2007;42:1173-80.

8. Robberecht E, Van Biervliet S, Vanrentergem K, Kerremans I. Outcome of total splenectomy with portosystemic shunt for massive splenomegaly and variceal bleeding in cystic fibrosis. J Pediatr Surg 2006;41:1561-5.

9. Pozler O, Krajina A, Vanicek H, et al. Transjugular intrahepatic portosystemic shunt in five children with cystic fibrosis: long-term results. Hepatogastroenterology 2003;50:1111-4.

10. Jonas MM. The role of liver transplantation in cystic fibrosis reexamined. Liver Transpl 2005;11:1463-5.

11. Melzi ML, Kelly DA, Colombo C, et al. Liver transplant in cystic fibrosis: a poll among European centers. A study from the European Liver Transplant Registry. Transpl Int 2006;19:726-31.

12. Nash KL, Collier JD, French J, et al. Cystic fibrosis liver disease: to transplant or not to transplant? Am J Transplant 2008;8:162-9.

13. Barshes NR, DiBardino DJ, McKenzie ED, et al. Combined lung and liver transplantation: the United States experience. Transplantation 2005;80:1161-7.

14. Faro A, Shepherd R, Huddleston CB, et al. Lower incidence of bronchiolitis obliterans in pediatric liver-lung transplant recipients with cystic fibrosis. Transplantation 2007;83:1435-9.

15. Trauner M, Fickert P, Halilbasic E, Moustafa T. Lessons from the toxic bile concept for the pathogenesis and treatment of cholestatic liver diseases. Wien Med Wochenschr 2008;158:542-8.

Continued

Medical and Surgical Therapy of CF-associated Liver Disease

George V. Mazariegos, M.D., F.A.C.S.

Hillman Center for Pediatric Transplantation, Children’s Hospital of Pittsburgh at University of Pittsburgh Medical Center, Pittsburgh, PA

Twenty to 50 percent of CF patients develop liver disease, which may range from minor biochemical liver abnormalities to focal biliary fibrosis or multilobular cirrhosis with portal hypertension and chronic liver failure. Advanced liver disease is most common in adolescents and young adults with CF, with an overall incidence of 4 to 10 percent of all CF patients (1,2). The rate of clinical decompensation, however, is difficult to predict and leads to challenges in timing of therapies, including liver transplantation (LT). True decompensated disease with manifestations of hepatic insufficiency is a very late manifestation; often, complications of portal hypertension (hypersplenism, cytopenias, ascites, and variceal hemorrhage) are the predominant and clinically relevant morbidity. Optimal timing of LT in CF is complicated by the systemic nature of the disease process—in particular, the progressive pulmonary disease, which is exacerbated by nutritional problems. Often, patients who are stable enough from a pulmonary perspective do not have clear indications for a liver transplant, and by the time a liver transplant is indicated, the pulmonary disease may have progressed and precludes LT.

Initial concerns about LT in CF centered around the potential infectious complications in the lung in the setting of immunosuppression, with the potential for rapid decline in pulmonary function. Fortunately, since initial case reports of LT for CF (3,4), single center experience has been favorable. Fridell et al. (5) reported on 12 children transplanted for CF with 1- and 5-year survival of 92 and 75 percent, respectively. There were five deaths, at a mean interval of 6.8 ± 6.3 years. All of these deaths were related to pulmonary disease. Pulmonary function did not worsen but improved or remained stable in eight of nine patients tested, likely as a result of improved nutritional status and better pulmonary mechanics.

Molmenti et al. (6) reported 60 percent patient survival in their cohort.

Clear indications and timing for LT, however, have not been well described. The European Transplant Registry reported good outcomes for LT in patients with CF but noted that most centers performed LT for evidence of portal hypertension and hypersplenism but prior to the development of end-stage liver disease (7). Portal hypertension and variceal bleeding have most commonly been used as indications for LT. However, the long-term impact of variceal bleeding on mortality was reported to be low in a small cohort of patients (8), raising questions on whether portal hypertension, even complicated by variceal bleeding, is the right indicator for LT (9).

Recently, children with cystic fibrosis who were enrolled in the Studies of Pediatric Liver Transplantation (SPLIT) registry were compared to non-CF patients in terms of pre- and post-liver transplant (LT) outcomes (10). Sixty-two CF patients in the SPLIT registry were listed for LT, and neither waitlist deaths nor the probability of death from time of listing was different from non-CF children. Overall, 2,702 children underwent LT in the study period. Forty-two CF patients (1.6%) underwent LT, with an average follow-up of 3 yrs (0-10 yrs). Transplanted patients had a mean PELD of 5.4 (86.5% had a PELD < 10), and a median age of 11.9 yrs (0.7 to 17.5).

Post-LT, the 1-, 3-, and 5-yr patient survival in CF patients was 92.9, 88.6, and 88.6 percent, compared to 90.8, 88.0, and 86.0 percent in non-CF patients. Graft survival in CF patients was 85.0, 81.2, and 81.2 percent at 1, 3, and 5 yrs compared to 85.6, 80.7, and 78.1 percent. Rejection rates were not different (50.6% CF vs. 56.4% non-CF at 5 yrs). Total re-operations (< 30 day) were less (21.4 versus 38%; p = 0.03). However, renal complications (< 30 day) were increased (14.3 vs. 6.1%; p = 0.04), with 50 percent of these patients requiring dialysis. Standardized height and weight scores showed no improvement over 2 years of




follow-up in the CF patients (height Z 1.4 at Tx to 1.4 at 2 yrs, weight Z 1.2 to 1.5), but tended to improve in the non-CF group (height Z 1.3 at Tx to 0.8 at 2 yrs, weight Z 1.0 to 0.1). It is of note that the nutritional improvement seen in smaller single center reports (11) were not seen in this initial SPLIT analysis; however, there was not the decline in nutritional status that is often seen in patients with CF and severe liver disease.

These data suggest several unresolved areas of clinical understanding that will benefit from additional investigation. These critical focus areas include optimizing liver allocation with potential alternatives to PELD in children with CF, better methods for determining those patients most likely to decompensate so that early LT would be warranted, and the role of surgical options such as splenectomy (12,13), splenic embolization, and portal hypertensive surgery in the management of these patients prior to or in lieu of LT.

Additional issues to be addressed in this challenging patient population include the indications for multi-organ transplant, including LT-pancreas, heart-lung-liver or lung-liver, and multivisceral transplantation, as well as changes in the best clinical management once the CF patient reaches adulthood. Finally, optimizing outcomes in the post-LT period is critical, with focus needed on improving nutritional and growth outcomes, minimizing significant morbidity such as renal complications, proper pulmonary management post LT, and analysis of the effects of LT on pulmonary disease progression.

References:

1. Colombo C, Battezzati PM, Crosignani A, Morabito A, Costantini D, Padoan R, et al. Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome. Hepatology 2002;36(6):1374-1382.

2. Hansen K, Horslen S. Metabolic liver disease in children. Liver Transpl 2008;14(4):391-411.

3. Cox KL, Ward RE, Furgiuele TL, Cannon RA, Sanders KD, Kurland G. Orthotopic liver transplantation in patients with cystic fibrosis. Pediatrics 1987;80(4):571-574.

4. Mieles LA, Orenstein D, Teperman L, Podesta L, Koneru B, Starzl TE. Liver transplantation in cystic fibrosis. Lancet 1989;1(8646):1073.

5. Fridell JA, Bond GJ, Mazariegos GV, Orenstein DM, Jain A, Sindhi R, et al. Liver transplantation in children with cystic fibrosis: a long-term longitudinal review of a single center’s experience. J Pediatr Surg 2003;38(8):1152-1156.

6. Molmenti EP, Squires RH, Nagata D, Roden JS, Molmenti H, Fasola CG, et al. Liver transplantation for cholestasis associated with cystic fibrosis in the pediatric population. Pediatr Transplant 2003;7(2):93-97.

7. Melzi ML, Kelly DA, Colombo C, Jara P, Manzanares J, Colledan M, et al. Liver transplant in cystic fibrosis: a poll among European centers. A study from the European Liver Transplant Registry. Transpl Int 2006;19(9):726-731.

8. Gooding I, Dondos V, Gyi KM, Hodson M, Westaby D. Variceal hemorrhage and cystic fibrosis: outcomes and implications for liver transplantation. Liver Transpl 2005;11(12):1522-1526.

9. Jonas MM. The role of liver transplantation in cystic fibrosis reexamined. Liver Transpl 2005;11(12):1463-1465.

10. Christopher D. Anderson JAL, Turmelle YP, Anand R, Song C, Shepherd RW, McDiarmid SV. The SPLIT Research Group. Excellent outcome after liver transplantation for cystic fibrosis in children. Accessed at SPLIT Web site https://webemmescom/study/lvr/ April 24, 2009 [Abstract] 2008.

11. Colombo C, Costantini D, Rocchi A, Romano G, Rossi G, Bianchi ML, et al. Effects of liver transplantation on the nutritional status of patients with cystic fibrosis. Transpl Int 2005;18(2):246-255.

12. Louis D, Duc ML, Reix P, Chazalette JP, Durieu I, Feigelson J et al. Partial splenectomy for portal hypertension in cystic fibrosis related liver disease. Pediatr Pulmonol 2007;42(12):1173-1180.

13. Linnane B, Oliver MR, Robinson PJ. Does splenectomy in cystic fibrosis related liver disease improve lung function and nutritional status? A case series. Arch Dis Child 2006;91(9):771-773.

Continued


Nicole Mayer Hamblett, Ph.D.

Research Assistant Professor, Department of Pediatrics, University of Washington, Seattle, WA; Director, Biostatis-tics and Clinical Data Management, Cystic Fibrosis Thera-peutics Development Network, Seattle Children’s Hospital, Seattle, WA

Biomarkers as Tools for Early Disease Detection

Developing biomarkers as predictors for disease is a multifaceted problem requiring collaboration among clinicians, scientists, and biostatisticians. In the setting of Cystic Fibrosis (CF) liver disease, it is an even more challenging problem because of limited patient populations and an evolving, non-standardized definition of the disease (1,2). Despite this challenge, there are several strategies that can be employed to make progress in biomarker development, and the key to this progress will be to implement these strategies in a unified and consistent manner across the research field.

It is often tempting to start biomarker development focused on the end goal, which in this case is to evaluate the biomarker as a diagnostic predictor for early liver disease. While it is important to stay focused on the end goal, and even necessary in some cases to elicit funding opportunities, there are several principal characteristics of a biomarker that should not be overlooked in the evaluation process. These include, but are not limited to, establishing the marker as both repeatable within a patient and reproducible across centers and laboratories. The terms repeatability and reproducibility often are used interchangeably but in fact point to different sources of variability in the biomarker. Repeatability captures the inherent biological variation within a patient, whereas reproducibility captures variability due to the measurement process. Biomarkers that satisfy these basic criteria have manageable variation within patients, which directly impacts their accuracy and precision for predicting disease within an individual patient (3).

Candidate biomarkers that have been established as robust, reliable measures of biologic activity within a patient are those for which future studies are

warranted to establish their association with known markers of disease. There are many study designs with which to demonstrate this association, with the main designs focusing on: 1) the objective to demonstrate cross-sectional correlation between the biomarker and either existing or future disease, or 2) the objective to demonstrate longitudinal correlation between changes in the biomarker and progression of disease. These studies provide important proof-of-concept during the biomarker development process by confirming hypothesized mechanisms of action of the biomarker in relation to the disease process. Importantly, these correlation studies are focused on population-level results, and not all correlates of disease in a population make reliable predictors of disease within an individual.

Biomarkers that have been established as correlates of disease are those for which the most rigorous of evaluations should occur, and for which examples already exist in the setting of CF liver disease (4). These evaluations are necessary for the translation of a biomarker from a correlate of disease in the population to a predictor of disease in an individual patient. The most common framework for evaluating a biomarker as a predictor of disease is based on methodology for diagnostic testing procedures, which includes the computation of familiar measures such as sensitivity, specificity, and positive and negative predictive values. This framework provides a blueprint for evaluating biomarkers in the CF liver disease setting, with the exception that there will be unique challenges to address, including the application of this framework to a small patient population and to a disease for which there is no gold standard diagnosis (5). A successful strategy will build awareness of these challenges and set both the standards and the expectations that will ultimately define how these biomarkers will be identified and utilized as tools for early disease detection.

References:

1. Sokol RJ, Durie PR. Recommendations for management of liver and biliary tract disease in cystic fibrosis. Cystic Fibrosis Foundation Hepatobiliary Disease Consensus Group. J Pediatr Gastroenterol Nutr. 1999;28 Suppl 1:S1-13.



Continued

2. Colombo C, Russo MC, Zazzeron L, Romano G. Liver disease in cystic fibrosis. J Pediatr Gastroenterol Nutr. 2006 Jul;43 Suppl 1:S49-55.

3. Mayer-Hamblett N, Ramsey BW, Kronmal RA. Advancing outcome measures for the new era of drug development in cystic fibrosis. Proc Am Thorac Soc. 2007 Aug 1;4(4):370-7.

4. Pereira TN, Lewindon PJ, Smith JL, Murphy TL, Lincoln DJ, Shepherd RW, Ramm GA. Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease. J Hepatol. 2004 Oct;41(4):576-83.

5. Hui SL, Zhou XH. Evaluation of diagnostic tests without gold standards. Stat Methods Med Res. 1998 Dec;7(4):354-70.

Biomarkers as Tools for Early Disease Detection

Keyur Patel

Assistant Professor in Medicine, Division of Gastroenterol-ogy, Duke Clinical Research Institute and Duke University Medical Center, Durham, NC

Emerging Techniques for Developing Novel Serum Biomarkers

Over the last decade, there has been considerable progress in our understanding of the various biological pathways leading to liver fibrosis in response to injury. We now recognize that fibrosis is a dynamic process mediated by factors that regulate matrix synthesis and degradation. Clinical studies have indicated that successful treatment of the underlying source of injury has led to regression of even advanced stages of fibrosis (1). However, a cross-sectional biopsy sample is unable to reflect matrix turnover, and the semi-quantitative histologic grading used in practice does not reflect linearity of fibrosis deposition. These categorical grading scores were developed to standardize and improve observer variability, and provide some assessment of the severity of chronic liver injury that could be used to determine thresholds for therapy (2). Currently available noninvasive serum marker panel algorithms include simple inflammatory markers and extracellular matrix proteins that were developed to separate mild from advanced histological grades. These marker panels were not designed to assess the dynamic nature of fibrogenesis or follow longitudinal changes in disease. As such, these noninvasive tests appear to have only limited clinical utility in the cross-sectional differentiation between mild and severe fibrosis in a proportion of patients (3). Accurate staging of fibrosis remains important for guiding therapy, providing prognosis, and following disease progression in chronic liver disease patients. This requires the development of reliable noninvasive alternatives to liver biopsy. The sequencing of the human genome and the subsequent advent of high-throughput technologies and the integrated systems biology approach have provided an opportunity to further improve our understanding of the fibrogenesis pathway. This will potentially help to develop a molecular signature that may be more disease specific and reflect early alterations in biological path

ways of fibrogenesis that may occur prior to appreciable changes in liver histology. For example, gene expression studies using microarray platforms in fibrosis have noted upregulation of various genes related to hepatic stellate cell activation, signal transduction, and immune response. A genome-wide association study that evaluated over 24,000 single nucleotide polymorphisms (SNPs) in chronic hepatitis C-infected patients subsequently developed an algorithm based on a seven-SNP signature that identified the risk of developing cirrhosis (4). Integration of gene expression profiles with functional protein expression is important for determining biological function and relevance. Mass spectrometry (MS) proteomics to develop blood protein signatures for diagnostic purposes continues to evolve rapidly (5). However, there are significant challenges with blood proteome analysis. There are likely millions of polypeptides spanning at least 10 orders of magnitude, with only a small proportion that have been identified and quantified to date (6,7). At this stage, 2D-gel or matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) methods for serum proteomic analysis in CHC patients with fibrosis have not provided a significant improvement over existing serum biomarkers for differentiating fibrosis stages (8,9). DNA sequencer-based assessment of the serum protein-linked N-glycans also has been evaluated to assess fibrosis and appears promising (10). However, issues relating to cost, reproducibility, and failure to identify proteins of interest for targeted high-throughput approaches have limited their utility. Quantitative protein profiling techniques continue to evolve, with improved separation methods, accurate mass and time peptide tagging, and high resolution electrospray ionization (ESI) liquid-chromatography (LC) MS techniques for complex samples such as serum. Future application of targeted MS-based metabolite profiling also may provide an integrated view of the disease process. The integration of clinical, functional gene and protein expression data remains a significant bioinformatics challenge but will be important for improving our understanding of complex biological processes and translation of novel biomarker signatures of fibrosis into clinical practice.



References:

1. Poynard T, McHutchison J, Manns M, Trepo C, Lindsay K, Goodman Z, Ling MH, et al. Impact of pegylated interferon alfa-2b and ribavirin on liver fibrosis in patients with chronic hepatitis C. Gastroenterology 2002;122:1303-13.

2. Standish RA, Cholongitas E, Dhillon A, Burroughs AK, Dhillon AP. An appraisal of the histopathological assessment of liver fibrosis. Gut 2006;55:569-78.

3. Rockey DC, Bissell DM. Noninvasive measures of liver fibrosis. Hepatology 2006;43:S113-20.

4. Huang H, Shiffman ML, Friedman S, Venkatesh R, Bzowej N, Abar OT, Rowland CM, et al. A 7 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis C. Hepatology 2007;46:297-306.

5. Diamond DL, Proll SC, Jacobs JM, Chan EY, Camp DG 2nd, Smith RD, Katze MG. HepatoProteomics: applying proteomic technologies to the study of liver function and disease. Hepatology 2006;44:299-308.

6. States DJ, Omenn GS, Blackwell TW, Fermin D, Eng J, Speicher DW, Hanash SM. Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study. Nat Biotechnol 2006;24:333-8.

7. Hanash SM, Pitteri SJ, Faca VM. Mining the plasma proteome for cancer biomarkers. Nature 2008;452:571-9.

8. Morra R, Munteanu M, Bedossa P, Dargere D, Janneau JL, Paradis V, Ratziu V, et al. Diagnostic value of serum protein profiling by SELDITOF ProteinChip compared with a biochemical marker, FibroTest, for the diagnosis of advanced fibrosis in patients with chronic hepatitis C. Aliment Pharmacol Ther 2007;26:847-58.

9. White IR, Patel K, Symonds WT, Dev A, Griffin P, Tsokanas N, Skehel M, et al. Serum proteomic analysis focused on fibrosis in patients with hepatitis C virus infection. J Transl Med 2007;5:33.

10. Vanderschaeghe D, Laroy W, Sablon E, Halfon P, Van Hecke A, Delanghe J, Callewaert N. GlycoFibroTest: DNA sequencer-based serum protein glycomics yields a highly performant liver fibrosis biomarker. Mol Cell Proteomics 2009 Jan 29 [Epub ahead of print].

Continued

Emerging Techniques for Developing Novel Serum Biomarkers

Ross W. Shepherd, M.D.1,2, Grant Ramm, Ph.D.2, and Peter Lewindon, M.D., F.R.A.C.P.2

1Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO; 2Hepatic Fibrosis Group, Queensland Institute of Medical Research, Brisbane, Queensland, Australia

Biomarkers of Cystic Fibrosis Liver Disease

Optimal evaluation of patients with clinically significant CF liver disease (CFLD) is unclear: clinical hepatosplenomegaly and elevated transaminases are non-specific or late. Many patients escape detection until signs of cirrhosis develop. Although commonly used, screening ultrasound (US) and isotope scintigraphy (HIDA) have not been subject to rigorous analysis in predicting CFLD. Invasive liver biopsy might be considered the gold standard, although early lesions are focal, raising issues of sampling error and interobserver variability. The challenge thus exists to develop sensitive, reliable, and preferably noninvasive biomarkers to identify CF patients who are at risk of developing clinically significant CFLD and to assess progress of liver injury in patients with CFLD.

Studies of basic mechanisms of liver fibrogenesis in CFLD perhaps provide a basis for biomarker study (1-4): The cholangiocyte is the only cell type in the liver that expresses the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In our liver biopsy studies (1,2), focal peribiliary fibrosis mediated by induction of Hepatic Stellate Cell (HSC) collagen gene expression appears to be a key early event (1,2). We have proposed that altered cholangiocyte transport and altered bile flow in CF result in upregulation of key HSC-responsive chemokines, which spill to the sinusoidal blood supply, thus having a paracrine effect on HSC chemotaxis (2). The role of steatosis, which occurs in one-third of biopsies, is unknown. Over time, active fibrogenesis, along the expanding scar interface, leads to multilobular biliary cirrhosis, events associated with elevated serum levels of bile acids, chemokines, collagen proteins, and components of hepatic matrix remodeling (2-4).

In 40 children (age 8.4 ± x yr, followed 3-8 yrs) with suspected CFLD, subject to dual pass liver biopsy with blinded staging of liver fibrosis, more patients with fibrosis were documented using two passes (P = 0.045), there being non-concordance for Scheuer Fibrosis Scores in 35 percent of biopsy pairs. Clinical exam (sensitivity = 0.66, specificity = 0.62, PPV = 0.87, NPV = 0.31), ALT (sensitivity = 0.69, specificity = 0.37, PPV = 0.81, NPV = 0.23), HIDA scan (sensitivity = 0.71, specificity = 0.0, PPV = 0.77, NPV = 0.0), and US (sensitivity = 0.80, specificity = 0.5, PPV = 0.86, NPV = 0.4) were poor predictors of liver fibrosis. The development of portal hypertension over time was associated with higher stage of fibrosis on biopsy and a younger age of CFLD onset (Cox proportional hazards model, P = 0.002).

Of serum total bile acids, hyaluronic acid (HA), collagen type IV (CL-IV), prolyl hydroxylase (PH), matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs), only levels of PH, CL-IV, and TIMP-1 were higher in CFLD (n = 36) versus both CfnoLD (n = 30) and controls (n = 39), P < 0.001. The fibrosis score was negatively correlated with TIMP-1 (r = −0.34, P = 0.06) and PH (r = −0.48, P = 0.008). Receiver-operating characteristics analysis showed CL-IV (AUC 0.785, P < 0.0001) and TIMP-1 (AUC 0.765, P < 0.0001) differentiated CFLD from CfnoLD and controls, while PH (AUC 0.814, P < 0.0001) predicted early fibrogenesis. Diagnostic accuracy improved using logistic regression combining i) CL-IV, TIMP-1, PH to identify CFLD (AUC 0.831, P < 0.0001) and ii) TIMP-1, PH to identify CFLD patients with no fibrosis (AUC 0.852, P < 0.02).

From these detailed but limited prospective cohort studies, we conclude that clinical, biochemical, and US evaluations for CFLD without dual pass biopsy are insensitive and non-specific for early CFLD fibrosis; they detect significant liver fibrosis/cirrhosis with reasonable sensitivity, but lack specificity. Liver fibrosis correlates



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Biomarkers of Cystic Fibrosis Liver Disease

with occurrence of portal hypertension. Three of seven selected serum fibrosis markers (CLIV, PH, and TIMP) were found to discriminate CFLD from no CFLD, and two of seven serum fibrosis markers (PH and TIMP) were found to discriminate fibrosis severity. Early diagnosis of CFLD still requires liver biopsy, enhanced by dual pass and specialized stains. US clearly lacks sensitivity. Selective noninvasive serum markers are deserving of wider application and prospective serial study in a larger cohort of CFLD patients.


• Should liver biopsy be performed routinely as part of a CFLD study (with development of disease-specific definitions of fibrosis for use in clinical research)?

• Because we do not seem to be able to rely on US, other more specific imaging techniques such as DFMRI or elastography require development.

• Reproduciblemarkersof“earlycysticfibrosis liver disease” identified in these studies require prospective serial study in a larger cohort of CFLD patients.

Potential Conflict of Interest: Nothing to report.

Acknowledgments: This project was funded by the National Health and Medical Research Council (NHMRC) of Australia; Grant 290220, Australian Cystic Fibrosis Research Trust, Royal Children’s Hospital Foundation, Brisbane, Australia; Grant 913-003, Senior Research Fellowship from the NHMRC of Australia; and Grant 241913.

References:

1. Lewindon PJ, Pereira TN, Hoskins AC, Bridle KR, Williamson RM, Shepherd RW, Ramm GA. The role of hepatic stellate cells and transforming growthfactor-beta(1) in cystic fibrosis liver disease. Am J Pathol. 2002 May;160(5):1705-15.

2. Ramm GA, Shepherd RW, Hoskins AC, Greco SA, Ney AD, Pereira TN, Bridle KR, Doecke JD, Meikle PJ, Turlin B, Lewindon PJ. Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment. Hepatology. 2009 Feb;49(2):533-44.

3. Pereira TN, Lewindon PJ, Smith JL, Murphy TL, Lincoln DJ, Shepherd RW, Ramm GA. Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease. J Hepatol. 2004 Oct;41(4):576-83.

4. Smith JL, Lewindon PJ, Hoskins AC, Pereira TN, Setchell KD, O’Connell NC, Shepherd RW, Ramm GA. Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis. Hepatology. 2004 Jun;39(6):1673-82.

5. Mueller-Abt PR, Frawley KJ, Greer RM, Lewindon PJ. Comparison of ultrasound and biopsy findings in children with cystic fibrosis related liver disease. J Cyst Fibros. 2008 May;7(3):215-21.

Marilyn J. Siegel, M.D.

Professor of Radiology and Pediatrics, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO

Ultrasound and CT Imaging of Liver Fibrosis and Other Disease

Ultrasonography (US)

Fibrosis: US findings of fibrosis are a heterogeneous echo pattern, either diffuse or in a periportal distribution (1,2). In a single center study, 67 percent of patients with cystic fibrosis with a heterogeneous echo pattern progressed to have US features of cirrhosis, and 46 percent progressed to portal hypertension (2). In another series comparing US and liver biopsy in children with cystic fibrosis, an abnormal US predicted moderate-tosevere liver disease with a specificity of 84 percent, but a normal US did not preclude significant liver fibrosis (3). The sensitivity of US to detect normal histology was 57 percent, with a specificity of 65 percent (3). Approximately 60 percent of patients with normal US had fibrosis. US detection of early liver disease remains a challenging problem.

Cirrhosis: The US findings of cirrhosis are a nodular hepatic surface and coarse echotexture. Based on these findings, the sensitivity of US to detect marked fibrosis or cirrhosis (METAVIR 3 or 4 score) is approximately 55 percent, with a specificity of 85 percent to 95 percent (3,4). US diagnosis of portal hypertension is based on findings of cirrhosis along with findings of splenomegaly, thickened omentum, collateral circulation, and ascites. US sensitivity for diagnosis of portal hypertension is 80 percent to 90 percent (5,6).

Significant correlations have been found between Doppler measurements of hepatic arterial resistance and pulsatility indices, hepatic vein waveforms, and blood flow velocity in the portal vein in studies of adults (p < 0.5) (7,8). Portal vein velocity is lower in the cirrhosis group than in the fibrosis group (7,8), whereas hepatic artery resistive index and pulsatility index are

higher in cirrhosis than in fibrosis and control subjects. Nontriphasic flow is also more likely in cirrhosis than fibrosis. Other studies have shown that the splenic artery pulsatility increases in fibrosis (9).

Steatosis: US findings of diffuse steatosis include: hepatomegaly, increased parenchymal echogenicity (i.e., “bright”liversign),andpoorpenetrationofthesound beam, resulting in poor visualization of intrahepatic vessels, the posterior liver, and the diaphragm. The adjacent renal cortex becomes much less echogenic than liver. The US sensitivity is 90 percent and the specificity is 93 percent for diagnosing moderate-to-severe diffuse fatty infiltration (> 30% hepatocytes with fat vacuoles). Focal steatosis commonly involves the gallbladder fossa in the periportal area, falciform ligament, medial segment of the left lobe, and subcapsular regions. Focal steatosis is usually geographic or fingerlike and well-defined and can mimic fibrosis.

Computed Tomography

Computed tomography (CT) has been used to study parenchymal liver disease, but its use of ionizing radiation is a major disadvantage, particularly in children who need repeated studies.

Fibrosis: In a study by Akata et al. using US and CT in 50 CF patients, increased hepatic echogenicity was detected in 24 of 48 (50%) (10). US showed increased periportal echogenicity in 18 of 24 patients, whereas CT could not demonstrate any sign of increased periportal thickness. Findings of cirrhosis were present in five patients with both modalities.

Cirrhosis: CT features of cirrhosis include decreased hepatic size, heterogeneous parenchyma, nodular hepatic margins, regenerating nodules, and atrophy of the right hepatic lobe and medial segment of the left lobe, with relative enlargement of the caudate lobe and left lateral segment.



Continued

Steatosis: Diffuse steatosis manifests as decreased attenuation value on unenhanced CT scans. Focal hepatic steatosis can be patchy, segmental, or lobar, with margins that are well-circumscribed or ill-defined. The sensitivity of CT for diagnosis of steatosis is nearly 100 percent.

References:

1. Patriquin H, Lenaerts C, Smith L, et al. Liver disease in children with cystic fibrosis: US-biochemical comparison in 195 patients. Radiology 1999;211:229-32.

2. Lenaerts C, Lapierre C, Patriquin H, et al. Surveillance for cystic fibrosis-associated hepatobiliary disease: early ultrasound changes and predisposing factors. J Pediatr 2003;143:343-50.

3. Mueller-Abt PR, Frawley KJ, Greer RM, Lewindon PJ. Comparison of ultrasound and biopsy findings in children with cystic fibrosis related liver disease. J Cyst Fibros 2008;7:215-21.

4. Colli A, Fraquelli M, Andreoletti M, Marino B, Zuccoli E, Conte D. Severe liver fibrosis or cirrhosis: accuracy of US for detection—analysis of 300 cases. Radiology 2003;227:89-94.

5. Aube C, Winkfield B, Oberti F, et al. New Doppler ultrasound signs improve the non-invasive diagnosis of cirrhosis or severe liver fibrosis. Eur J Gastroenterol Hepatol 2004;16:743-51.

6. Gaiani S, Bramantieri L, Venturoli N, et al. What is the criterion for differentiating chronic hepatitis from compensated cirrhosis? A prospective study comparing ultrasonography and percutaneous liver biopsy. J Hepatol 1997;27:979-85.

7. Annet L, Materne R, Danse E, Jamart J, Horsmans Y, Van Beers BE. Hepatic flow parameters measured with MR imaging and Doppler US: correlations with degree of cirrhosis and portal hypertension. Radiology 2003;229:409-14.

8. Haktanir A, Cihan BS, Celenk C, Cihan S. Value of Doppler sonography in assessing the progression of chronic viral hepatitis and in the diagnosis and grading of cirrhosis. J Ultrasound Med 2005;24:311-21.

9. Liu CH, Lin JW, Tsai FC, et al. Noninvasive tests for the prediction of significant hepatic fibrosis in hepatitis C virus carriers with persistently normal alanine aminotransferases. Liver Int 2006;26:1087-94.

10. Akata D, Akhan O, Ozcelik U, et al. Hepatobiliary manifestations of cystic fibrosis in children: correlation of CT and US findings. J Radiol 2002;41:26-33.

Ultrasound and CT Imaging of Liver Fibrosis and Other Disease

Claude B. Sirlin, M.D.

Liver Imaging Group, Department of Radiology, University of California, San Diego, La Jolla, CA

MRI Methods of Assessing Liver Fibrosis and Disease

Overview: Several promising MRI methods are in development for non-invasive assessment of liver fibrosis. These are outlined below and will be discussed in the presentation. All MRI methods for liver fibrosis assessment are experimental.

Experimental MRI Techniques for Liver Fibrosis: These can be classified broadly into those that assess structure (morphology and texture) and those that assess tissue properties (apparent diffusion coefficient, perfusion, elastic modulus).

Structure: Morphology. The cirrhotic liver develops characteristic morphological alterations visible on cross-sectional imaging. These alterations are features of advanced disease. They have high specificity but low sensitivity for cirrhosis and so are not appropriate for early fibrosis detection or fibrosis staging.

Structure: Texture. Recent studies show that liver texture at MR imaging may be altered in advanced fibrosis and cirrhosis and that MR imaging can directly depict advanced liver fibrosis as a meshwork of linear reticulations and non-tumoral bands. Importantly, contrast agents can improve visualization of textural alterations caused by liver fibrosis (1,2). Clinical trials of contrast-enhanced MRI for non-invasive staging of liver fibrosis in HCV and NAFLD are in progress.

Tissue Property: Apparent Diffusion Coefficient. Diffusion-weighted (DW) imaging assesses the diffusion of protons within tissue and is quantified by the apparent diffusion coefficient (ADC). DW imaging uses motion-sensitizing gradients that cause diffusing protons to lose signal. The amount of signal loss is influenced by the ability of protons to diffuse through tissue (the ADC of the tissue), as well as the strength of the diffusion weighting

(the diffusion sensitivity parameter or the b-value of the sequence). Studies using fat-suppressed (or water-excited) DW echo planar imaging have shown reduced ADC in cirrhosis of various etiologies. These observations have led investigators to postulate that liver fibrosis restricts water diffusion, and investigators now are studying whether DW imaging can be used to stage liver fibrosis non-invasively (3).

Tissue Property: Perfusion. Perfusion imaging assesses flow alterations associated with chronic liver disease. Progressive deposition of collagen and other macromolecules within the extracellular matrix of the liver gradually obliterates the space of Disse, narrows the sinusoidal lumen, and causes sinusoisal basement membrane formation with loss of endothelial fenestrations. These abnormalities slow the passage of blood through the parenchyma, elevate intrahepatic resistance, and cause portal venous blood to be shunted to systemic veins with lower pressures. The microvascular architecture of the liver remodels in response, with development of abnormal connections between the portal vein and terminal hepatic vein, which contribute to the shunting of blood from the hepatic parenchyma. As portal flow to the liver diminishes, there is compensatory hepatic arterial vasodilation, with subsequent elevation of the hepatic arterial fraction. These flow alterations can be evaluated non-invasively with contrast-enhanced perfusion imaging (4).

Tissue Property: Elastic Modulus. MR elastography (MRE) is an MR imaging technique that non-invasively quantifies the elastic modulus (or stiffness) of liver as well as other organs by analyzing the propagation of mechanical waves through tissue. The premise of this technique for assessing liver fibrosis is that progressive deposition of a lattice-like framework of interconnecting collagen fibers throughout the liver increases parenchymal elastic modulus. Hence, measuring liver elastic modulus may provide non-invasive information on the severity of fibrosis (5). Because liver elastic modulus cannot be reliably assessed by external physical palpation,



Continued

an imaging approach is required. There are two main imaging methods to measure hepatic elastic modulus. One is ultrasound-based transient elastography. The other is MRE, discussed in this presentation.

Conclusion: Several promising MRI techniques for assessing liver fibrosis are under development. No MRI method yet has been validated in large-scale, multicenter trials as accurate, robust, or reproducible for assessment of liver fibrosis across the entire spectrum of disease severity from early fibrosis to cirrhosis. Thus, no MRI method is yet suitable as a non-invasive surrogate endpoint for fibrosis severity in clinical trials or observational studies; a definitive assessment of fibrosis stage still requires a biopsy reference standard. Moreover, most MRI methods in development assess liver fibrosis indirectly and therefore may be confounded by numerous variables, most of which may not be directly measurable. Confounding variables may include age, gender, and etiology of liver disease. Thus, there is a need to test MRI techniques in age-, gender-, and etiology-specific studies. To the author’s knowledge, no experimental MRI technique for liver fibrosis has yet been studied in children with cystic fibrosis. Finally, liver biopsy with conventional histology staging of liver fibrosis may not be an ideal reference standard for MRI-based methods, as these assess the liver volumetrically, sample all or most of the liver, and are quantitative.


There are overlapping research challenges and needs. These will need to be pursued in parallel.

1. Refine and standardize MRI techniques. 2. Test MRI techniques prospectively in children with

known or suspected CFLD. 3. Include clinical outcome measures in the study

design. 4. Disseminate negative as well as positive results of

studies. 5. Identify disease-specific targets for imaging for

future technical development. 6. Recognize that biopsy may be an imperfect refer

ence standard for MRI approaches.

References:

1. Aguirre DA, Behling CA, Alpert E, Hassanein TI, Sirlin CB. Liver fibrosis: noninvasive diagnosis with double contrast material-enhanced MR imaging. Radiology 2006;239(2):425-437.

2. Hughes-Cassidy F, Chavez AD, Schlang A, et al. Superparamagnetic iron oxides and low molecular weight gadolinium chelates are synergistic for direct visualization of advanced liver fibrosis. J Magn Reson Imaging 2007;26(3):728-737.

3. Taouli B, Tolia AJ, Losada M, et al. Diffusion-weighted MRI for quantification of liver fibrosis: preliminary experience. AJR Am J Roentgenol 2007;189(4):799-806.

4. Hagiwara M, Rusinek H, Lee VS, et al. Advanced liver fibrosis: diagnosis with 3D whole-liver perfusion MR imaging—initial experience. Radiology 2008;246(3):926-934.

5. Rouviere O, Yin M, Dresner MA, et al. MR elastography of the liver: preliminary results. Radiology 2006;240(2):440-448.

MRI Methods of Assessing Liver Fibrosis and Disease

Detlef Schuppan, M.D., Ph.D.

Division of Gastroenterology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA

Elastography to Assess Liver Fibrosis and Cirrhosis

The development of cirrhosis is a central hard endpoint in the natural history of patients with chronic liver diseases, including children with cystic fibrosis liver disease (CFLD). In this vein, advanced fibrosis and fibrosis progression are accepted surrogates for the development of cirrhosis and liver failure and other complications of cirrhosis. There has been striking progress in our understanding of the biochemistry and cell biology of liver fibrosis and cirrhosis. This includes numerous agents and emerging strategies that prevent fibrosis progression or even induce reversal of established fibrosis and cirrhosis. However, effective therapeutic intervention has essentially been limited to rodent models of liver fibrosis (1).

Translation into the clinic requires improved noninvasive markers or techniques that permit the measurement of hepatic fibrosis, and in particular an assessment of the dynamic parameters of fibrogenesis and fibrolysis (de novo deposition or removal of excess extracellular matrix, respectively). This is necessary since liver biopsy has an inherent sampling error, and progression from stage 0 to stage 4 (cirrhosis) usually takes 20-30 years, even in patients classified as fast progressors (1). In addition, biopsy is risky and cannot be repeated on a regular basis.

For practical purposes, especially for children with CFLD, who are at risk of developing cirrhosis at an early age, even the limited option to reliably diagnose advanced fibrosis or incipient cirrhosis would be highly useful. This could permit stratification of patients: 1) according to their risk of progression to (advanced) cirrhosis, 2) to assess the need for confirmation by liver biopsy, and 3) for inclusion in clinical trials aimed at inhibiting disease progression.

The current serum markers and serum marker algorithms have not been validated in children with CFLD.

Furthermore, in adult patients they permit only a crude differentiation between no to mild and moderate to severe fibrosis (Metavir stages 0-1 vs. 2-4, respectively) (1,2).

Conventional or contrast ultrasound, and conventional MRI or CT, do not allow assessment of liver fibrosis, except for the diagnosis of advanced cirrhosis.

Transient elastography (Fibroscan, Echosense, France) is a novel, noninvasive, bedside method to assess liver fibrosis by measuring hepatic stiffness. An intercostally placed probe transmits low-amplitude shear waves through the liver, the velocity of which is picked up by an integrated ultrasound device (pulse-echo ultrasound). Shear wave velocity is inversely correlated with liver stiffness. Stiffness is automatically calculated as the mean of 10 measurements and expressed as kPa. This methodology is highly attractive, since it is painless and without risk, only takes 5 min, and can be performed in a fairly standardized way after only a short training period. Furthermore, ultrasound elastography measures a mechanical quality of the liver related to fibrosis, other than current imaging techniques or serum markers, and when compared to biopsy samples a 100-fold larger volume (4 × 1 cm, ∼1/500 of the liver).

More than 100 clinical studies correlating ultrasound elastography with liver biopsy have appeared since its first description in 2003 (3), including meta-analyses (4-7). Almost all have been performed in adults and have shown a good correlation of stiffness values with the histological stage of fibrosis. Area under receiver operating curves (that reflect the diagnostic precision in select populations) yielded excellent accuracy for differentiating cirrhosis from non-cirrhosis (AUROC between 0.90 and 0.99), especially for patients with chronic hepatitis C. However, with the practical but crude histological Metavir staging system (with F1 = mild, periportal fibrosis, and F4 = cirrhosis), there is high overlap between the lower stages, only part of which can be attributed to biopsy sampling variability. In addition, severe acute inflammation and mechanical cholestasis


Speaker Abstracts

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Elastography to Assess Liver Fibrosis and Cirrhosis

can significantly increase hepatic stiffness (7-9), thus confounding the fibrosis readout.

Of note, different etiologies of liver disease require different cutoffs for the diagnosis of cirrhosis (e.g., a cutoff of 12 kPa in chronic hepatitis C and of 17.3 kPa in biliary fibrosis).

Only a few studies have been performed in children. In a small study of children with nonalcoholic steatohepatitis (n = 52), ultrasound elastography demonstrated an outstanding prediction of all stages of fibrosis (AUROC 0.992 to 1.0), but these results need confirmation in a larger cohort (10). In a larger cohort of 116 children with various chronic liver diseases, the technique diagnosed histological cirrhosis with an AUROC of 0.88, superior to serum marker algorithms (Fibrotest and APRI score, both with an AUROC of 0.73) (11). de Lédinghen et al. also showed the usefulness of a novel, smaller probe adapted to the small intercostal space of children (11).

MR elastography is based on similar principles as ultrasound elastography, but studies are few and include low numbers of patients. A larger generator transmits the shear waves, which are recorded with a 1.5 Tesla MR scanner (12,13). Advantages are examination of the whole liver, an even lower observer dependency, and applicability in patients with severe obesity or narrow intercostal spaces (which can preclude ultrasound elastography). A disadvantage is the more restricted availability. Importantly, acquisition times can be shortened tenfold (20 min to 2 min) by use of echo-planar versus spin-echo sequences, without compromise of the stiffness readout (12). Hepatic inflammation and mechanical cholestasis are expected to be confounders, as with ultrasound elastography, but studies are lacking.

Although studies in children with CFLD are lacking, ultrasound elastography is expected to be a screening tool of choice, since it will permit non-invasive detection of advanced fibrosis. This will allow selection of

patients who are in need of (antifibrotic) therapies to prevent further progression to (decompensated) cirrhosis. Furthermore, it is quick and easy to perform, with low interobserver variability. The technology should be used to screen for children or adults with significant (fibrotic) CFLD, and to stratify patients for inclusion in therapeutic studies that are aimed at inhibition of fibrosis progression or at fibrosis reversal.

References:

1. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet 2008;371:838-51. 2. Parkes J, Guha IN, Roderick P, Rosenberg W. Performance of serum

marker panels for liver fibrosis in chronic hepatitis C. J Hepatol 2006;44:462-74.

3. Sandrin L, Fourquet B, Hasquenoph JM, Yon S, Fournier C, Mal F, Christidis C, Ziol M, Poulet B, Kazemi F, Beaugrand M, Palau R. Transient elastography: a new noninvasive method for assessment of hepatic fibrosis. Ultrasound Med Biol 2003; 29:1705-13.

4. Foucher J, Chanteloup E, Vergniol J, Castéra L, Le Bail B, Adhoute X, Bertet J, Couzigou P, de Lédinghen V. Diagnosis of cirrhosis by transient elastography (FibroScan): a prospective study. Gut 2006;55:403-8.

5. Friedrich-Rust M, Ong MF, Martens S, Sarrazin C, Bojunga J, Zeuzem S, Herrmann E. Performance of transient elastography for the staging of liver fibrosis: a meta-analysis. Gastroenterology 2008;134:960-74.

6. Castera L, Forns X, Alberti A. Non-invasive evaluation of liver fibrosis using transient elastography. J Hepatol 2008;48:835-47.

7. Coco B, Oliveri F, Maina AM, Ciccorossi P, Sacco R, Colombatto P, Bonino F, Brunetto MR. Transient elastography: a new surrogate marker of liver fibrosis influenced by major changes of transaminases. J Viral Hepat 2007;14:360-9.

8. Arena U, Vizzutti F, Corti G, Ambu S, Stasi C, Bresci S, Moscarella S, Boddi V, Petrarca A, Laffi G, Marra F, Pinzani M. Acute viral hepatitis increases liver stiffness values measured by transient elastography. Hepatology 2008;47:380-4.

9. Millonig G, Reimann FM, Friedrich S, Fonouni H, Mehrabi A, Büchler MW, Seitz HK, Mueller S. Extrahepatic cholestasis increases liver stiffness (FibroScan) irrespective of fibrosis. Hepatology 2008;48:1718-23.

10. Nobili V, Vizzutti F, Arena U, Abraldes JG, Marra F, Pietrobattista A, Fruhwirth R, Marcellini M, Pinzani M. Accuracy and reproducibility of transient elastography for the diagnosis of fibrosis in pediatric nonalcoholic steatohepatitis. Hepatology 2008;48:442-8.

11. de Lédinghen V, Le Bail B, Rebouissoux L, Fournier C, Foucher J, Miette V, Castéra L, Sandrin L, Merrouche W, Lavrand F, Lamireau T. Liver stiffness measurement in children using FibroScan: feasibility study and comparison with Fibrotest, aspartate transaminase to platelets ratio index, and liver biopsy. J Pediatr Gastroenterol Nutr 2007;45:443-50.

12. Huwart L, Salameh N, ter Beek L, Vicaut E, Peeters F, Sinkus R, Van Beers BE. MR elastography of liver fibrosis: preliminary results comparing spin-echo and echo-planar imaging. Eur Radiol 2008;18:2535-41.

13. Talwalkar JA, Yin M, Fidler JL, Sanderson SO, Kamath PS, Ehman RL. Magnetic resonance imaging of hepatic fibrosis: emerging clinical applications. Hepatology 2008;47:332-42.

poster Abstracts



Molly Bozic1, Jean Molleston2, M. Howenstine3, and J. Fridell4

Departments of 1Pediatrics, 2Pediatric Gastroenterology, 3Pediatric Pulmonology, and 4Surgery, Indiana University School of Medicine, Indianapolis, IN

Background: Cystic fibrosis (CF), an autosomal recessive disorder affecting multiple organ systems, is the most common genetic disease among the Caucasian population. The CFTR mutation can adversely affect hepatic function in cystic fibrosis patients. Up to 10 percent of CF patients will develop cirrhosis, often with severe portal hypertension. Cystic fibrosis liver disease (CFLD) is the third most common cause of death in CF. Liver transplant has been successful in improving the outcomes of a number of CF patients with severe liver disease. Pancreatic insufficiency is a hallmark of cystic fibrosis and results in poor growth and malnutrition. Pancreatic insufficiency and CF-related diabetes (CFRD) can negatively impact pulmonary function. Insulin-dependent diabetes in CF patients is known to result in increased mortality and morbidity compared to nondiabetic CF patients. Since CFLD occurs solely in the context of pancreatic disease, cirrhosis can present in individuals with severe malabsorption and diabetes. Simultaneous liver-pancreas transplantation has been proposed to address the severe hepatic dysfunction, nutritional compromise, and hyperglycemia in diabetic pancreatic-insufficient CF patients under one immunosuppressive regimen. We report here the impact of simultaneous liver-pancreas transplants on hepatic, pulmonary, and pancreatic function in three patients with CF.

Methods: A retrospective chart review of three patients with CF who underwent liver-pancreas transplant was performed using the electronic medical record, Port CF, and OTTR transplant database. Data were gathered to describe hepatic function, pulmonary function, and

pancreatic endocrine and exocrine function, as well as nutritional status, before and after simultaneous liver-pancreas transplant.

Results: Three female patients, ages 13, 14, and 22 years, with cystic fibrosis underwent simultaneous liver-pancreas transplants. Prior to transplant, all three had cirrhosis resulting in portal hypertension with variceal bleeding, and two had some degree of encephalopathy and ascites. Each patient had pancreatic insufficiency with poor growth and malnutrition in spite of pancreatic enzyme supplementation. Insulin-dependent diabetes was present in all. Patients are currently 1½, 5, and 6 years post transplant.

Combined transplantation was tolerated well in these patients, with length of stay during initial hospitalization of 14-21 days. One year after liver/pancreas transplant, each patient showed improvement in hepatic, pancreatic, and pulmonary function as well as nutritional status. All three patients were free of diarrhea and gained weight without pancreatic enzyme supplementation. These patients demonstrated weight gains of 5-11 kg in the years after transplant. Endocrine function of the pancreas was maintained, as each patient demonstrated normal blood glucose levels without the use of insulin. Hgb A1c prior to transplant in two patients ranged from 7.3 to 9.7, with post-transplant levels improving to 4.5-5.5 in these two patients. Platelet counts normalized, and clinical symptoms of portal hypertension resolved with continued normal bilirubin and liver enzymes and low-normal albumin. There were no episodes of rejection. Pulmonary function tests 1 year post transplant improved in patient A (FVC up 5%; FEV1 up 12%), remained relatively stable in patient B (FVC up 2%; FEV1 down 1%), and improved in patient C (FVC up 11%; FEV1 up 6%). One of the three continued to require frequent hospitalizations for pulmonary infections. All three attended school or worked full-time after transplant.


Poster Abstracts


Continued


Conclusions: Simultaneous liver/pancreas transplants in three cystic fibrosis patients with CFLD and CFRD resulted in improved pancreatic endocrine function as evidenced by normalization of blood glucose levels without exogenous insulin treatment. Improved nutritional status and improved weight gain without pancreatic enzyme supplementation demonstrated improved pancreatic exocrine function as well. Liver disease was improved, as evidenced by resolution of signs/symptoms of portal hypertension. Pulmonary function was not adversely affected. Simultaneous liver-pancreas transplant appears to be a satisfactory option for CF patients with CFLD and CFRD.

Charles N. Falany1, Dongning He1, Li Li1, Teresa W. Wilborn2, and Melissa Runge-Morris3

1Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL; 2Department of Physiological Sciences, Samford University, Birming-ham, AL; 3Institute of Toxicology, Wayne State University, Detroit, MI

Although pulmonary disease is the major pathology in Cystic Fibrosis (CF), the loss of CF transmembrane receptor (CFTR) activity results in significant pathologies in other tissues. In the liver, CFTR is expressed in intrahepatic epithelial cells or cholangiocytes, forming the bile ducts as well as the gallbladder. Most patients with a severe form of CF have biliary transport problems, steatosis, and focal biliary cirrhosis, and a subgroup of patients develops cholestasis and cirrhosis, requiring liver transplantation.

In CFTR-deficient mice, the activity of hepatic sulfotransferase (SULT) 1E1 is induced up to 100-fold. SULT1E1 has a K m of 4 nM for the sulfation of β-estradiol (E2) and is the major mechanism for the inactivation of E2 at physiological concentrations. The induced SULT1E1 activity alters the expression of E2-regulated proteins in CFTR-mouse liver. Similar results are observed in human HepG2 hepatocytes with increased SULT1E1 activity. Increased SULT1E1 activity in HepG2 cells also inhibits the ability of growth hormone to stimulate IGF-1 synthesis by decreasing the activation of STAT5b.

To investigate whether the loss of CFTR expression in human cholangiocytes induces the expression of SULT1E1 in hepatocytes, a membrane separated co-culture system was developed. Human MMNK-1 cholangiocytes express CFTR but not SULT1E1. CFTR expression in the MNMNK-1 cells was repressed by more than 85 percent using siRNA and the cells plated in Transwell inserts. HepG2 hepatocytes were plated

in well plates. After 24 hr, the Transwell inserts with the MMNK-1 cells were placed in the wells containing the HepG2 cells and left for 8-24 hr. The inserts were removed, and the HepG2 cells assayed for SULT1E1 expression and activity. SULT1E1, but not SULT1A1, SULT2A1, or CYP3A4 expression, was induced 15-fold when cultured with CFTR-deficient MMNK-1 cells as compared to control cells. These results indicate a factor is secreted by the CFTR-deficient MMNK-1 cells that acts in a paracrine manner on the expression of SULT1E1 in the HepG2 cells.

To identify the paracrine factor involved in the induction of SULT1E1, expression profiling of the CFTR-deficient and control MMNK-1 cells was carried out. RNA was isolated from the cells and used for expression profiling. One pathway significantly altered in the CFTR-MMNK-1 cells involved cholesterol biosynthesis. Desmosterol (DES) reductase and 7-dehydrocholesterol (7DHC) reductase expression were significantly decreased in the CFTR-deficient cells. DES and 7DHC are oxysterols and the immediate precursors to cholesterol. Oxysterols are the physiological regulators of the liver-x-receptors (LXR). Treatment of the HepG2 cells with DES, 7DHC, and T0901317 induced SULT1E1 expression. These data suggest that the loss of CFTR activity in MMNK-1 cells results in increased levels of oxysterols that act as paracrine factors to activate LXRα in HepG2 cells and thereby induce SULT1E1 expression. In liver, LXRα acts as an oxysterol sensor to regulate cholesterol levels by reverse transport. Inappropriate LXR activation may also have a role in the development of steatosis observed in many CF patients as well as the disruption of E2 regulatory properties in the liver.

Acknowledgment: This work was supported by the University of Alabama at Birmingham Cystic Fibrosis Research Center and by a grant to Charles N. Falany from CFRI, Inc.

Paracrine Induction of Sulfotransferase 1E1 in HepG2 Hepatocytes During Co-culture With CFTR-deficient MMNK-1 Cholangiocytes

Poster Abstracts Poster Abstracts

Poster Abstracts Poster Abstracts

Yury Popov1, Preeti Misra2, Kumar Ranjan Bhushan2 , Deanna Y. Sverdlov1, Nezam H. Afdhal1, John V. Frangioni2,3, and Detlef Schuppan1

Divisions of 1Gastroenterology and 2Hematology/Oncology, Department of Medicine, and 3Department of Radiology, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA

Quantitative Imaging of Biliary Fibrosis Progression Using a Small Molecule α v β6 Integrin Ligand With Nano-molar Affinity

Kara Sullivan, A. Moran, B. Holm, and S.J. Schwarzenberg

Minnesota Cystic Fibrosis Center (MCFC), University of Minnesota, Minneapolis, MN

Cystic Fibrosis Related Diabetes (CFRD) in CF Patients With Cirrhosis

Background: Quantitative imaging of liver fibrosis or fibrogenesis has been impossible. α v β6 is a fibronectin/ tenascin receptor whose expression is highly restricted and found only on activated cholangiocytes/progenitor cells that correlate with and are instrumental in hepatic fibrogenesis. Its tissue levels are highest in active biliary fibrosis, but correlate with fibrosis progression regardless of the underlying liver disease. Therefore, high affinity α v β6-specific peptidomimetics are attractive candidates for hepatic fibrosis/fibrogenesis imaging.

Methods: A peptidomimetic small molecule ligand of the α v β6-integrin (EMD527040) with low nanomolar affinity and no relevant crossreactivity with other integrins was modified by introducing a six-carbon linker, followed by trimerization on an adamantane scaffold. This targeting molecule was converted into a radiotracer by conjugation with the pre-labeled metal chelator [Tc-99m-MAS3]-NHS. Homologous competition with nonradioactive Re derivatives of the compound was performed on HT-29 (α v β6 integrin positive) and Huh-7 (α v β6 integrin negative) epithelial cell lines. Biodistribution, clearance and specific label accumulation were quantified in 8-week-old Mdr2(abcb4)-/- mice that

have spontaneous progressive biliary fibrogenesis due to cholangiocyte activation and were compared to nonfibrotic wildtype mice.

Results: The trimerized tracer displayed fourfold increased affinity over the monomer (Bmax 2.6 × 104

receptors for αv6-positive HT-29 cells). In vivo biodistribution in normal and fibrotic mice showed that the compound cleared from the blood and most non-target tissues with an alpha half-life of 23-45 min and was excreted efficiently via the kidneys. Liver uptake at 2 h was 1.5 ± 0.3 percent of the injected dose per gram, and the α v β6-based imaging agent showed a two- to threefold enhanced uptake in the fibrogenic as compared to wildtype nonfibrotic between 2 and 20 h after injection. Retention of the imaging agent in the liver could be correlated with α v β6 expression. SPECT-CT verified prominent in vivo localization over the fibrotic liver. Specificity of α v β6-targeted imaging was confirmed by blocking of the enhanced signal when MDR2KO mice received a prior injection of a 103-fold surplus of the nonlabeled α v β6-trimer.

Conclusions: 1) A high affinity and high specificity fibrosis/fibrogenesis imaging agent was created based on trimerization of an α v β6-selective peptidomimetic small molecule ligand. 2) Our in vivo data suggest that with minor increase of affinity and more effective background suppression, noninvasive quantification of liver fibrosis/ fibrogenesis by molecular imaging will be possible.

Background: CFRD occurs in many patients with cystic fibrosis (CF), and its incidence increases with age. At our center, a previous study of 527 patients with CF revealed a prevalence of CFRD in 33 percent of patients, with 45 percent of patients over 40 years old affected. Approximately 10 percent of CF patients have cirrhosis. The purpose of this study is to determine if CF patients with cirrhosis are more likely to have CFRD.

Methods: A retrospective case control study of the MCFC database was performed. All data were collected as of September 15, 2008. Patients with cystic fibrosis and outpatient platelet counts less than 150,000 on at least two occasions that were 3 months apart were used as a surrogate for cirrhosis. The patient’s diabetic status was identified through the results of routine annual oral glucose tolerance screening of the CF population and a carefully maintained CFRD database. Patient age was also collected.

Results: Forty-five of 527 cystic fibrosis patients (8.5%) were considered to have cirrhosis and were selected for review. Thirty of the 45 had diabetes (66.7%) and were alive as of September 15, 2008. Age range was 1-65 years old. Most cases of CFRD in patients with cirrhosis were found in patients over 20 years old.

Data Analysis: A chi-square test using SAS software was constructed. It demonstrated a significant difference in the rate of CFRD in patients with cirrhosis, compared to CF patients without cirrhosis (p < .0001). The odds ratio was 4.788 (95% CI 2.49, 9.17).

Conclusions: CFRD is more common in CF patients with cirrhosis compared to the overall CF population (p < .0001). Patients with CF and cirrhosis have 4.8 times the odds of having CFRD. This may reflect a common pathogenic pathway. The presence of both complications presents a challenge in managing these patients. Our data, derived using a surrogate marker of cirrhosis, suggest the need for a more direct examination of the interaction of CF liver disease and CFRD.

Table 1. CF patients followed at the MCFC as of September 15, 2008.

Age (yr) All CF CFRD Percent Sample Cirrhosis Percent

Cirrhotic Cirrhosis and

CFRD Percent Cirrhosis

and CFRD

< 10 63 1 2 2 3.2 1 50

10–19 126 21 17 8 6.3 4 50

20–29 150 60 40 12 8 9 75

30–39 115 56 49 11 9.6 7 64

> 40 73 33 45 12 16.4 9 75

Total 527 172 33 (172/527) 45 8.5

(45/527) 30 66.7 (30/45)

Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 46 47

Poster Abstracts

Alexandra Vasilescu, Shruti Paranjape, Wikrom Karnsakul, and Kathleen B. Schwarz

Division of Pediatric Gastroenterology and Nutrition, Johns Hopkins University, Baltimore, MD

Design of a Pilot Study to Identify Optimum Markers of Liver Fibrosis in Children With Cystic Fibrosis Liver Disease (CFLD)

participants List

Background: Cystic fibrosis liver disease (CFLD) is the third leading cause of death in cystic fibrosis (CF) after pulmonary and lung transplant-related complications. The prevalence has been reported between 4 and 24 percent, with cirrhosis in approximately 15 percent. Although liver biopsy is the current gold standard of identifying fibrosis, it has a high sampling error, with a 24 percent false negative rate. There is a great need to identify noninvasive markers of liver fibrosis (NIMLF). One of the problems in trying to identify such a marker in this special population is that, as we have previously shown, healthy infants and children exhibit increased circulating cytokines, such as transforming growth factor beta 1 (TGFβ1), used as NIMF in adults. In addition, the CF patients often have pulmonary fibrosis. As we have demonstrated previously, these patients also exhibit increased plasma TGFβ1. Noninvasive methods of diagnosing degree of fibrosis have not been systematically studied in large groups of children with cystic fibrosis; however, these have been studied in various disease entities, such as hepatitis B, hepatitis C, primary sclerosing cholangitis (PSC), non-alcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD). These methods include ultrasound (U/S) identification of a heterogeneous parenchyma with a grading system of 0-5. As well, various serological assays have included direct and indirect markers of fibrosis. The indirect markers of fibrosis, including AST, ALT, and platelets, are nonspecific, whereas the direct markers of fibrosis, such as hyaluronic acid (HA), tissue inhibitors of metalloproteinase (TIMP-1), amino terminal fragment of procollagen III (P3NP), and procollagens type IV and VI, have higher sensitivity and specificity.

Objective: The selected marker/s identified in the pilot study will be sensitive and specific to distinguish CFLD cirrhosis versus CF patients without liver disease and versus those with severe lung disease.

Methods: We propose to conduct a pilot study at Johns Hopkins Hospital, one of the CF Centers that is part of the CFLD-Research Consortium, to validate the Enhanced Liver Fibrosis (ELF) Test® (HA, TIMP1, NP3P) and determine if CF patients with advanced fibrosis (U/S grade 4-5) can be clearly distinguished from two groups: CF pulmonary disease (CFPD) (FEV1 < 40% predicted) and age/sex, FEV1-matched controls, without CFPD, and with normal physical exam, normal liver enzymes, and normal liver U/S.

Results: Sample size calculation:

CF with no fibrosis versus CF with cirrhosis ELF scores: 7.92 ± 0.44 (no fibrosis)

11.08 ± 0.07 (cirrhosis) Type I error 0.05 and Type II error 0.8 SD = 1.2 Δ = 0.6 (change in outcome) n = 10

Conclusion: A sensitive and specific NIMLFCF would obviate the need for liver biopsy, would help identify patients at risk for disease progression, and ideally could be applied in future studies of antifibrotic therapies in CF patients. If the ELF test shows acceptable positive predictive value (PPV) and negative predictive value (NPV) in our pilot, then we would propose to test it in the larger cohort of the Cystic Fibrosis Network.

Funding: NIH, F32 submitted; Johns Hopkins, T32 submitted; CFLD, grant supplement.



Participants List

Adil Abdalla, M.D. Clinical Fellow Liver Diseases Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Building 10, Room 4-5722 10 Center Drive, MSC 1614 Bethesda, MD 20892-1614 Telephone: (301) 451-1273 Email: [email protected]

Lisa Allwein Registered Nurse Pediatric Pulmonary Department Penn State Milton S. Hershey Medical Center 500 University Drive Hershey, PA 17033 Telephone: (814) 952-1669 Email: [email protected]

Natalia Argel Pulmonary Research Nurse Coordinator Phoenix Children’s Hospital Building B 1919 E. Thomas Road Phoenix, AZ 85016 Telephone: (602) 546-0343 Email: [email protected]

Dawn Baker, M.S.N. Advanced Registered Nurse Practitioner Department of Pediatrics University of Massachusetts Medical School Biotech 5, Suite 250 381 Plantation Street Worcester, MA 01605 Telephone: (508) 334-2339 Email: [email protected]

Ashok Batta, Ph.D. Bioanalytical Chemist Digestive Care, Inc. 1120 Win Drive Bethlehem, PA 18017 Telephone: (610) 882-5950 Email: [email protected]

Gerri Bickerton Registered Nurse Warren Grant Magnuson Clinical Center National Institutes of Health Building 10, Room 9C317 10 Center Drive, MSC 1664 Bethesda, MD 20892-1664 Telephone: (301) 496-9090 Email: [email protected]

Lynelle Boamah, M.D. Pediatric Gastroenterologist Pediatric Subspecialty Naval Medical Center San Diego 34800 Bob Wilson Drive San Diego, CA 92131 Telephone: (619) 532-9720 Email: [email protected]

Drucy Borowitz, M.D. Professor of Clinical Pediatrics Department of Pediatrics State University of New York at Buffalo Women and Children’s Hospital of Buffalo 219 Bryant Street Buffalo, NY 14222 Telephone: (716) 878-7524 Email: [email protected]

Molly Bozic, M.D. Pediatric Resident Department of Pediatrics Indiana University School of Medicine James

Whitcomb Riley Hospital for Children 702 Barnhill Drive, Room 5867 Indianapolis, IN 46202 Telephone: (317) 501-9900 Email: [email protected]

51

Cystic Fibrosis Liver Disease | Clinical Research Workshop 52 Cystic Fibrosis Liver Disease | Clinical Research Workshop

Participants List Participants List

Rebekah Brown, M.D. Clinical Fellow Department of Pediatrics Division of Pediatric Allergy, Immunology,

and Pulmonary Medicine Vanderbilt Children’s Hospital 11215 DOT 2200 Children’s Way Nashville, TN 37232-9500 Telephone: (615) 343-7617 Email: [email protected]

Karen Callahan, M.S. Research Nurse Manager Pediatric Pulmonary Johns Hopkins University 200 N. Wolfe Street, Suite 3074 Baltimore, MD 21287 Telephone: (443) 287-8983 Email: [email protected]

Maggi Campbell Marketing Manager Digestive Care, Inc. 1120 Win Drive Bethlehem, PA 18017 Telephone: (610) 882-5950 Email: [email protected]

Daniel Caplan, M.D. Professor of Pediatrics Department of Pediatrics Emory University School of Medicine 2040 Ridgewood Drive, N.E. Atlanta, GA 30322 Telephone: (404) 727-5728 Email: [email protected]

Rubin Cohen, M.D. Associate Professor of Medicine Division of Pulmonary Medicine The Albert Einstein College of Medicine 410 Lakeville Road, Suite 107 New Hyde Park, NY 11040 Telephone: (516) 465-5400 Email: [email protected]

Michael D’Amico, M.D. Pediatric Gastroenterologist Department of Pediatrics Vermont Children’s Hospital Smith 553 111 Colchester Avenue Burlington, VT 05401 Telephone: (802) 847-5670 Email: [email protected]

Pamela DeMorrow, M.S., C.P.N.P. Pediatric Pulmonology Kalamazoo Center for Medical Studies Michigan State University 1000 Oakland Drive Kalamazoo, MI 49008 Telephone: (269) 337-6476 Email: [email protected]

Tania Diaz, M.D. 4221 29th Street Mount Rainier, MD 20712 Telephone: (301) 927-4240 Email: [email protected]

Edward Doo, M.D. Program Director Liver Diseases Research Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Two Democracy Plaza, Room 651 6707 Democracy Boulevard, MSC 5450 Bethesda, MD 20892-5450 Telephone: (301) 451-4524 Email: [email protected]

Peter Durie, M.D. Professor and Senior Scientist Research Institute Hospital for Sick Children 555 University Avenue Toronto, ON M5G 1X8 Canada Telephone: (416) 813-6185 Email: [email protected]

Charles Falany, Ph.D. Professor Department of Pharmacology

and Toxicology University of Alabama at Birmingham Volker Hall G133M 1670 University Boulevard Birmingham, AL 35216 Telephone: (205) 934-9848 Email: [email protected]

Andrew Feranchak, M.D. Associate Professor Department of Pediatrics University of Texas Southwestern

Medical Center 5323 Harry Hines Boulevard Dallas, TX 75390-9063 Telephone: (214) 648-3118 Email: [email protected]

Steven Freedman, M.D. Associate Professor of Medicine Department of Medicine Beth Israel Deaconess Medical Center Dana 501 330 Brookline Avenue Boston, MA 02215 Telephone: (617) 667-5576 Email: [email protected]

Lynn Fukushima, M.S.N. Cystic Fibrosis Research Coordinator Pediatric Pulmonology Children’s Hospital Los Angeles, MS 83 4650 Sunset Boulevard Los Angeles, CA 90027 Telephone: (323) 361-5950 Email: [email protected]

Marc Ghany, M.D. Staff Physician Liver Diseases Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Building 10, Room 9B-16 10 Center Drive, MSC 1800 Bethesda, MD 20892-1800 Telephone: (301) 402-5115 Email: [email protected]

Vita Goei, M.D. Assistant Professor Pediatric Gastroenterology Children’s Subspecialty Group Children’s Hospital of the King’s Daughters 601 Children’s Lane Norfolk, VA 23507 Telephone: (757) 668-7240 Email: [email protected]

Gavin Graff, M.D. Professor of Pediatrics Department of Pediatrics Penn State College of Medicine H085 500 University Drive Hershey, PA 17033 Telephone: (717) 531-5338 Email: [email protected]

Laura Grande, M.S. Pediatric/Cystic Fibrosis Dietitian Department of Nutrition Children’s National Medical Center 111 Michigan Avenue, N.W. Washington, DC 20010 Telephone: (202) 476-2218 Email: [email protected]

53



Donna Griebel, M.D. Director Division of Gastroenterology Products Center for Drug Evaluation and Research U.S. Food and Drug Administration White Oak Building, Room 5112 22 New Hampshire Avenue Silver Spring, MD 20861 Telephone: (301) 796-4153 Email: [email protected]

Nicole Mayer Hamblett, Ph.D. Director Biostatistics and Clinical Data Management Cystic Fibrosis Therapeutics Development

Network 1100 Olive Way, Suite 500 Seattle, WA 98101 Telephone: (206) 884-7547 Email: [email protected]

Koji Hara, M.D. Fellow Liver Diseases Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Building 10, Room 9B06 10 Center Drive, MSC 1800 Bethesda, MD 20892-1800 Telephone: (301) 443-7309 Email: [email protected]

Beth Harkness, R.N. Registered Nurse Cystic Fibrosis Center Children’s National Medical Center 111 Michigan Avenue, N.W., Room #1030 Washington, DC 22201 Telephone: (202) 476-2215 Email: [email protected]

Dustin Hays Health Science Writer Information Clearinghouses National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health 8280 Greensboro Drive, Suite 300 McLean, VA 22102 Telephone: (301) 204-3249 Email: [email protected]

Theo Heller, M.D. Investigator Liver Diseases Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Building 10, Room 9B16 10 Center Drive, MSC 1800 Bethesda, MD 20892-1800 Telephone: (301) 496-1721 Email: [email protected]

James Heubi, M.D. Professor and Associate Chair Department of Pediatrics Cincinnati Children’s Hospital General Clinical Research Center 3333 Burnet Avenue Cincinnati, OH 45229 Telephone: (513) 636-8046 Email: [email protected]

Pamela Hofley, M.D. Section Chief Section of Gastroenterology

and Hepatology Department of General Pediatrics Dartmouth-Hitchcock Medical Center One Medical Center Drive Lebanon, NH 03756 Telephone: (603) 695-2745 Email: [email protected]

Douglas Holsclaw, M.D. Director Adult Cystic Fibrosis Center University of Pennsylvania PennLung Center-PHI First Floor 39th and Market Streets Philadelphia, PA 19104 Telephone: (215) 662-8766 Email: [email protected]

Jay Hoofnagle, M.D. Director Liver Disease Research Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Building 31, Room 9A27 31 Center Drive, MSC 2560 Bethesda, MD 20892-2560 Telephone: (301) 496-1333 Email: [email protected]

Stephen James, M.D. Director Division of Digestive Diseases and Nutrition National Institute of Diabetes and Digestive


Arthur Jarrett, M.D. Director of Paediatrics Department of Paediatrics Liverpool Hospital Liverpool Health Service Locked Bag 7103 Liverpool BC Sydney, NSW 1871 Australia Telephone: 61-298285680 Email: [email protected]

Karen Jones Project Manager Biometrics and Outcomes Research Core University of Michigan 1420 Washington Heights, M4523 Ann Arbor, MI 18109 Telephone: (737) 763-7738 Email: [email protected]

Binita Kamath, M.D. Assistant Professor Fellowship Training Program in Pediatric

Gastroenterology Division of Gastroenterology, Hepatology,

and Nutrition Children’s Hospital of Philadelphia 34th Street and Civic Center Boulevard Philadelphia, PA 19103 Telephone: (215) 590-3247 Email: [email protected]

Wikrom Karnsakul, M.D. Assistant Professor Department of Pediatrics Johns Hopkins University Hospital 600 N. Wolfe Street, Brady 320 Baltimore, MD 21287 Telephone: (410) 502-9522 Email: [email protected]

Saul Karpen, M.D., Ph.D. Director, Liver Center Department of Pediatrics Baylor College of Medicine One Baylor Plaza Houston, TX 77030 Telephone: (832) 824-3754 Email: [email protected]

55



David Kleiner, M.D., Ph.D. Chief, Postmortem Section Laboratory of Pathology National Cancer Institute National Institutes of Health Building 10, Room 2B50 10 Center Drive, MSC 1500 Bethesda, MD 20892-1500 Telephone: (301) 594-2942 Email: [email protected]

Michael Knowles, M.D. Professor of Medicine Division of Pulmonary/Critical Care University of North Carolina at Chapel Hill 7019 Thurston Bowles Building, CB 7248 Chapel Hill, NC 27599-7248 Telephone: (919) 966-6780 Email: [email protected]

Christopher Koh, M.D. Clinical Hepatology Fellow Liver Diseases Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Building 10, Room 4-5722 10 Center Drive, MSC 1800 Bethesda, MD 20892-1800 Telephone: (301) 451-7009 Email: [email protected]

Debra Kroeker, R.N., C.P.N. Department of Pulmonology Children’s Hospital Central California 9300 Valley Children’s Place Madera, CA 93636 Telephone: (559) 353-5550 Email: [email protected]

Jalayne Lapke, M.D. Associate Professor of Clinical Pediatrics Department of Pediatrics University of Illinois College of Medicine 320 E. Armstrong Avenue Peoria, IL 61603 Telephone: (309) 303-1236 Email: [email protected]

Susan Lehto, R.N. Cystic Fibrosis Clinic Children’s Hospital Central California 9300 Valley Children’s Place Madera, CA 93636 Telephone: (559) 353-5550 Email: [email protected]

Simon Ling, M.D. Division of Gastroenterology, Hepatology,

and Nutrition The Hospital for Sick Children 555 University Avenue Toronto, ON M4E 1C3 Canada Telephone: (416) 813-7734 Email: [email protected]

John Magee, M.D. Associate Professor Department of Surgery University of Michigan 2922 Taubman Center 1500 E. Medical Center Drive Ann Arbor, MI 48109 Telephone: (734) 936-9623 Email: [email protected]

Bruce Marshall, M.D. Vice President of Clinical Affairs Cystic Fibrosis Foundation 6931 Arlington Road Bethesda, MD 20814 Telephone: (800) 344-4823 Email: [email protected]

George Mazariegos, M.D. Chief, Pediatric Transplantation Division of Transplant Surgery Hillman Center for Pediatric Transplantation Children’s Hospital of Pittsburgh of UPMC Faculty Pavilion, Sixth Floor 45th Street and Penn Avenue Pittsburgh, PA 15201 Telephone: (412) 692-6110 Email: [email protected]

Melissa McClure Pediatric Cystic Fibrosis Coordinator Pediatric Pulmonology Hershey Medical Center 500 University Drive Hershey, PA 17033 Telephone: (717) 531-5338 Email: [email protected]

Debbie McCormick, R.N. Customer Service Manager Digestive Care, Inc. 1120 Win Drive Bethlehem, PA 18017 Telephone: (610) 882-5950 Email: [email protected]

Catherine McKeon, Ph.D. Senior Advisor for Genetic Research National Institute of Diabetes and Digestive


David Meyerholz, D.V.M., Ph.D. Assistant Professor Department of Pathology University of Iowa Carver College

of Medicine 200 Hawkins Drive, 1165ML Iowa City, IA 52242 Telephone: (319) 353-4589 Email: [email protected]

Suzanne Michel, M.P.H., R.D. Registered Dietitian Department of Clinical Nutrition The Children’s Hospital of Philadelphia 358 Trevor Lane Bala Cynwyd, PA 19004 Telephone: (267) 426-2858 Email: [email protected]

Mohamad Miqdady, M.D. Pediatric Gastroenterologist Department of Pediatrics Sheikh Khalifa Medical City P. O. Box 51900 Abu Dhabi United Arab Emirates Telephone: 971-50-1234-283 Email: [email protected]

Paul Mohabir, M.D. Outpatient Director, Cystic Fibrosis Division of Pulmonary and Critical

Care Medicine Stanford University Medical Center 300 Pasteur Drive, H3143 Stanford, CA 94305-5236 Telephone: (650) 804-4811 Email: [email protected]

57



Jean Molleston, M.D. Professor of Clinical Pediatrics Division of Pediatric Gastroenterology,

Hepatology, and Nutrition Indiana University School of Medicine Riley Hospital for Children 702 Barnhill Drive, Room ROC 4210 Indianapolis, IN 46202 Telephone: (317) 274-3774 Email: [email protected]

Veronique Morinville, M.D. Assistant Professor of Pediatrics Division of Pediatric Gastroenterology

and Nutrition Montreal Children’s Hospital D-562 2300 Tupper Street Montreal, QC H3H 1P3 Canada Telephone: (514) 412-4474 Email: [email protected]

Rosemary Nagy, M.B.A. Clinical Research Coordinator Department of Pediatrics Washington University School of Medicine CB 8208 660 S. Euclid Avenue Saint Louis, MO 63110 Telephone: (314) 454-2295 Email: [email protected]

Michael Narkewicz, M.D. Professor of Pediatrics Division of Pediatric Gastroenterology,

Hepatology, and Nutrition University of Colorado Denver School of

Medicine and The Children’s Hospital 13123 E. 16th Avenue Aurora, CO 80045 Telephone: (720) 777-6669 Email: [email protected]

Joel Nations, M.D. Staff Physician Pulmonary Medicine Service National Naval Medical Center 8901 Wisconsin Avenue Bethesda, MD 20889 Telephone: (301) 295-4217 Email: [email protected]

Andrea Nepa, M.S. Clinical Dietitian Department of Clinical Nutrition The Children’s Hospital of Philadelphia 34th Street and Civic Center Boulevard Philadelphia, PA 19104 Telephone: (215) 590-3217 Email: [email protected]

Joe Palermo, M.D., Ph.D. Instructor Department of Pediatrics Washington University in St. Louis One Children’s Place St. Louis, MO 63110 Telephone: (314) 454-6173 Email: [email protected]

Shruti Paranjape, M.D. Assistant Professor of Pediatrics Eudowood Division of Pediatric Respiratory

Sciences Johns Hopkins University DM Rubenstein Child Health Building 200 N. Wolfe Street Baltimore, MD 21287 Telephone: (410) 502-5792 Email: [email protected]

Keyur Patel, M.D. Assistant Professor Duke Gastroenterology/Hepatology

Site Coordination Duke Clinical Research Institute 2400 Pratt Street Durham, NC 27705 Telephone: (919) 668-7186 Email: [email protected]

Thankam Paul, M.D. Assistant Professor Department of Pediatrics Rhode Island Hospital 593 Eddy Street Providence, RI 02903 Telephone: (401) 258-1949 Email: [email protected]

Elizabeth Perkett, M.D. Professor Department of Pediatrics Division of Pediatric Allergy, Immunology,

and Pulmonary Medicine Vanderbilt Medical School 11215 DOT 2200 Children’s Way Nashville, TN 37232-9500 Telephone: (615) 343-7617 Email: [email protected]

Peggy Radford, M.D. Director of Pulmonary Research Pediatric Pulmonary Phoenix Children’s Hospital 1919 E. Thomas Road Phoenix, AZ 85016 Telephone: (602) 546-0985 Email: [email protected]

Elizabeth Reid, M.S. Clinical Nutrition Specialist Pediatric Pulmonology Penn State Milton S. Hershey Medical Center P.O. Box 850, HO 85 500 University Drive Hershey, PA 17033 Telephone: (717) 531-4679 Email: [email protected]

Patricia Robuck, Ph.D., M.P.H. Director, Clinical Trials Program Division of Digestive Diseases

and Nutrition National Institute of Diabetes and Digestive


Rene Romero, M.D. Associate Professor Department of Pediatrics Emory University School of Medicine 2015 Uppergate Drive Atlanta, GA 30322 Telephone: (404) 785-1832 Email: [email protected]

Carl Rountree, M.D. Assistant Professor Department of Pediatrics and

Pharmacology Penn State College of Medicine 500 University Drive, H085 Hershey, PA 17033 Telephone: (717) 531-5901 Email: [email protected]

59



Wael Sayej, M.D. Fellow Pediatric Gastroenterology Digestive Diseases and Nutrition Center Women and Children’s Hospital of Buffalo 219 Bryant Street, Second Floor Buffalo, NY 14222 Telephone: (201) 921-1312 Email: [email protected]

Detlef Schuppan, M.D., Ph.D. Professor Department of Medicine Division of Gastroenterology Beth Israel Deaconess Medical Center 330 Brookline Avenue Boston, MA 02215 Telephone: (617) 667-2371 Email: [email protected]

Kathleen Schwarz, M.D. Professor Department of Pediatrics Johns Hopkins University School

of Medicine Brady 320 600 N. Wolfe Street Baltimore, MD 21287 Telephone: (410) 955-8769 Email: [email protected]

Sarah Jane Schwarzenberg, M.D. Associate Professor Department of Pediatrics University of Minnesota MMC 185 420 Delaware Street, S.E. Minneapolis, MN 55455 Telephone: (612) 624-4669 Email: [email protected]

Leonard Seeff, M.D. Senior Scientist Liver Disease Research Branch National Institute of Diabetes and Digestive

and Kidney Diseases National Institutes of Health Building 31, Room 9A27 31 Center Drive, MSC 2560 Bethesda, MD 20892-2560 Telephone: (301) 435-3338 Email: [email protected]

Girish Sharma, M.D. Cystic Fibrosis Center Director Department of Pediatrics Rush University Medical Center 1653 W. Congress Parkway, Suite 452 Chicago, IL 60612 Telephone: (312) 563-2291 Email: [email protected]

Ross Shepherd, M.D. Professor Department of Pediatrics Washington University School of Medicine Queensland Institute for Medical Research One Children’s Place St Louis, MO 63110 Telephone: (314) 420-2553 Email: [email protected]

Marilyn Siegel, M.D. Professor of Radiology and Pediatrics Mallinckrodt Institute of Radiology Washington University Medical Center 510 S. Kingshighway Boulevard St. Louis, MO 63110 Telephone: (314) 454-6229 Email: [email protected]

Claude Sirlin, M.D. Department of Radiology University of California, San Diego 408 Dickinson Street, MC 8226 San Diego, CA 92103 Telephone: (619) 471-0513 Email: [email protected]

Ronald Sokol, M.D. Vice Chair of Pediatrics and Chief

Gastroenterologist Department of Gastroenterology The Children’s Hospital/University

of Colorado Denver 13123 E. 16th Avenue, B290 Aurora, CO 80045 Telephone: (720) 777-6669 Email: [email protected]

Sean Sullivan, M.D. Pediatric Gastroenterologist Department of Pediatrics Naval Medical Center Portsmouth 620 John Paul Jones Circle Portsmouth, VA 23708 Telephone: (757) 953-2645 Email: [email protected]

Horst-Dietmar Tauschel, Ph.D. Medical Sciences Dr. Falk Pharma GmbH Leinenweberstrasse 5, PO Box 6529 Freiburg D-79041 Germany Telephone: 49-761-1514-127 Email: [email protected]

Jeffrey Teckman, M.D. Associate Professor Department of Pediatrics Division of Gastroenterology

and Hepatology Cardinal Glennon Children’s Medical Center Saint Louis University 1465 S. Grand Boulevard St. Louis, MO 63104 Telephone: (314) 577-5647 Email: [email protected]

Yumi Turmelle, M.D. Transplant Hepatologist Division of Pediatric Gastroenterology

and Nutrition St. Louis Children’s Hospital Washington University School of Medicine Campus Box 8116 One Children’s Place St. Louis, MO 63110 Telephone: (314) 454-6173 Email: [email protected]

Hubert van der Doef, M.D. Pediatric Gastroenterology University Medical Center Utrecht Postbox 85090, KE 04.133.1 Utrecht 3508 AB The Netherlands Telephone: (31) 887555294 Email: [email protected]

Alexandra Vasilescu, D.O. Pediatric Gastroenterology Fellow Pediatric Gastroenterology Johns Hopkins University 900 N. Wolfe Street Baltimore, MD 21287 Telephone: (410) 614-4621 Email: [email protected]

61

Participants List

Xavier Villa, M.D., M.S.P.H. Director Division of Gastroenterology, Hepatology,

and Nutrition Department of Pediatrics University of Texas Medical Branch

at Galveston 301 University Boulevard Houston, TX 77555-0352 Telephone: (713) 838-9687 Email: [email protected]

Diana Wetmore, Ph.D. Vice President Alliance Management Cystic Fibrosis Foundation Therapeutics 6931 Arlington Road, Suite 200 Bethesda, MD 20814 Telephone: (301) 907-2535 Email: [email protected]

Ronni Wetmore, M.S. Coordinator, Adult Cystic Fibrosis Center Pulmonary and Critical Care Associates LaVilla Medical Building, Suite 202 425 N. Lee Street Jacksonville, FL 32204 Telephone: (904) 318-8807 Email: [email protected]

Marlyn Woo, M.D. Director, Cystic Fibrosis Clinical Research Pediatric Pulmonology Children’s Hospital Los Angeles 4650 Sunset Boulevard, MS #83 Los Angeles, CA 90027 Telephone: (323) 361-2101 Email: [email protected]

Patti Yanchuk Adult Cystic Fibrosis Program Coordinator Pulmonary Medicine Penn State Milton S. Hershey Medical Center Box 850, Mailcode H041 500 University Drive Hershey, PA 17033 Telephone: (717) 531-6525 Email: [email protected]

Patrick Young, M.D. Director of Clinical Research Department of Gastroenterology National Naval Medical Center Building 9, First Floor 8901 Wisconsin Avenue Bethesda, MD 20889 Telephone: (301) 295-6044 Email: [email protected]

ConTrACTor SupporT:

Denise Hoffman, CMP The Scientific Consulting Group, Inc. 656 Quince Orchard Road, Suite 210 Gaithersburg, MD 20878 Telephone: (301) 670-4990 Email: [email protected]

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