Cystic Fibrosis Liver Disease Clinical Research Workshop

June 8 - 9, 2009 Lister HiLL Auditorium niH CAmpus, BetHesdA, md organizing Committee

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.

Cystic Fibrosis Liver Disease | Clinical Research Workshop 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

Cystic Fibrosis Liver Disease | Clinical Research Workshop 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 Agenda 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 αv 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

vi Cystic Fibrosis Liver Disease | Clinical Research Workshop Agenda

Monday, June 8, 2009

Lister HiLL Auditorium niH CAmpus, BetHesdA, md DAY ONE

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

* 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 Tuesday, June 9, 2009

Lister HiLL Auditorium Continued niH CAmpus, BetHesdA, md DAY TWO

11:40 a.m. – 12:00 p.m. Protein Folding Defects in Liver Disease: 8:00 a.m. – 8:10 a.m. Introduction to Day 2: Alpha-1-antitrypsin Deficiency Early Assessment and Identification Jeffrey Teckman Edward Doo

12:00 p.m. – 12:30 p.m. Discussion SeSSion iv. noninvASive ASSeSSMenTS oF CF Liver DiSeASe 12:30 p.m. – 2:00 p.m. Lunch BioMArkerS SeSSion iii. MoDeLS oF CF Liver DiSeASe AnD TherApeuTiCS 8:10 a.m. – 8:30 a.m. Biomarkers as Tools for Early Disease Detection Nicole Hamblett 2:00 p.m. – 2:20 p.m. Cystic Fibrosis Liver Disease: CFTR and the Liver Andrew Feranchak 8:30 a.m. – 8:50 a.m. Emerging Techniques for Developing Novel Serum Biomarkers 2:20 p.m. – 2:40 p.m. The CFTR-/- Pig: Liver and Gallbladder Disease Keyur Patel David Meyerholz

8:50 a.m. – 9:10 a.m. Biomarkers of Cystic Fibrosis Liver Disease 2:40 p.m. – 3:00 p.m. Nodular Regenerative Hyperplasia: Ross Shepherd Clinical Features and Course Theo Heller 9:10 a.m. – 9:40 a.m. Discussion

3:00 p.m. – 3:20 p.m. Medical and Surgical Therapy of CF-associated 9:40 a.m. – 10:00 a.m. Break Liver Disease Saul Karpen iMAging 3:20 p.m. – 3:40 p.m. Liver Transplantation in Children With Cystic Fibrosis: Status and Unanswered Questions 10:00 a.m. – 10:20 a.m. Ultrasound and CT Imaging of Liver Fibrosis and George Mazariegos Other Disease Marilyn Siegel 3:40 p.m. – 4:10 p.m. Discussion 10:20 a.m. – 10:40 a.m. MRI Methods of Assessing Liver 4:10 p.m. – 4:20 p.m. Break Fibrosis and Disease Claude Sirlin

4:20 p.m. – 5:30 p.m. Poster Session

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

Tuesday, June 9, 2009

Continued

iMAging

10:40 a.m. – 11:00 a.m. Elastography to Assess Liver Fibrosis and Cirrhosis Detlef Schuppan

11:00 a.m. – 11:30 a.m. Discussion

11:30 a.m. – 12:30 p.m. Research Needs for the Future Michael Narkewicz Michael Knowles Patricia Robuck Catherine McKeon Speaker Abstracts Jay H. Hoofnagle Bruce Marshall Marilyn Siegel Edward Doo

12:30 p.m. Adjournment

xii Cystic Fibrosis Liver Disease | Clinical Research Workshop Speaker Abstracts

Cystic Fibrosis Liver Disease: Clinical Features and Definitions

Michael R. Narkewicz, M.D. identified by 15 years of age, affecting up to 7 percent Hewit-Andrews Chair in Pediatric Liver Disease, Professor of patients. Risk factors for cirrhosis have included male of Pediatrics, University of Colorado Denver, School of sex, Hispanic ethnicity, and pancreatic insufficiency. Medicine, The Children’s Hospital, Aurora, CO There has not been any identified genotype/phenotype correlation with CFTR. The pathology is that of multi­ lobular cirrhosis, often with focal variation. Cystic Fibrosis (CF) is an autosomal recessive disorder that is the most common genetic disorder of Caucasian Other hepatic manifestations include: individuals, with a prevalence of 1 in 2,000 individuals of European descent. It is estimated to affect 30,000 Neonatal Cholestasis: This has been suggested to individuals in the United States. The disease is due to a occur in 1-2 percent of infants with CF. It is more defect in the cystic fibrosis transmembrane conductance common in infants with meconium ileus. In general, the regulator (CFTR). Although the lungs and pancreas cholestasis resolves. The subsequent risk for the develop­ are the pathopneumonic target organs in CF, many ment of cirrhosis is probably increased, but this remains other organs are affected. These include the small and unclear. large intestine, vas deferens, sinuses, and liver. In 2007, liver disease was the third leading cause of death in CF, Elevated AST, ALT, or GGT: Studies on subjects accounting for 2.3 percent of deaths. identified by newborn screening have shown that the prevalence of abnormal AST, ALT, or GGT is quite CFTR is expressed on the apical membrane of biliary frequent. By 20 years of age, 93 percent of patients have epithelium and not on the hepatocyte. The putative role had at least one abnormal ALT, with 50 percent hav­ of CFTR is to facilitate water and solute movement via ing at least one ALT greater than 1.5 times the upper chloride secretion and therefore promote bile flow. limit of normal (ULN) and 32 percent having an ALT greater than 2 times the ULN. Persistently abnormal Clinical Features of Liver Disease in CF: The ALT or AST is also quite frequent, with 42 percent of primary end result of liver disease in CF with the patients having a persistently abnormal ALT greater than most significant impact is the development of fibrosis 1.5 times the ULN by 20 years of age. and finally cirrhosis in up to 7 percent of individuals with CF. Various theories have been proposed for the Hepatic Steatosis: This has been reported to occur underlying pathophysiology, but no single mechanism in 20-60 percent of patients with CF. In many cases, has met with uniform acceptance. Similarly, difficulties this was reported in association with malnutrition related exist in the studies of natural history and treatment, as to CF. However, hepatic steatosis has been reported in there have not been uniform definitions applied to liver well-nourished children and adults with CF. The under­ involvement in CF. As a consequence, the prevalence of lying mechanism(s) is unclear and has been suggested to liver disease in CF has been noted to be between 5 and be related to essential fatty acid deficiency or intrinsic 75 percent. defects related to CFTR dysfunction. It is unclear if hepatic steatosis progresses to cirrhosis. The clinical involvement of the liver is manifest in many ways. Biliary cirrhosis with or without portal hyperten­ Focal Biliary Cirrhosis: This entity previously was sion is the complication with the most significant impact described as the pathopneumonic hepatic disorder of CF. on outcome. Several studies have demonstrated that It is characterized by focal areas of fibrosis and regenera­ cirrhosis is a relatively early event, with most patients tive nodules areas of normal liver. The prevalence has

Cystic Fibrosis Liver Disease | Clinical Research Workshop 3 Speaker Abstracts Speaker Abstracts

Cystic Fibrosis Liver Disease: Cystic Fibrosis Liver Disease: Clinical Features and Definitions Clinical Features and Definitions

Continued Continued been reported to be between 10 and 70 percent, depend­ II. Liver involvement without cirrhosis/portal hyperten­ Future Research Challenges and Needs: ing on the method of detection and the era. The highest sion (patients could have more than one (i.e., II a, frequency for this disorder comes from autopsy reports c)). These are primarily descriptive at this point. The • Development of uniform definitions for use in that predate current therapies. If and at what frequency abnormalities may well be multifactorial in origin, clinical research. this pathologic entity progresses to multilobular biliary and the exact significance and relationship to each • Identification of reproducible markers of “early cystic cirrhosis and portal hypertension is unknown. other remain to be determined. fibrosis liver disease.”

• Biomarkers for early hepatic fibrosis in cystic fibrosis. a. Biochemical abnormalities Given the localization of CFTR to the biliary epithe­ lium, it is interesting to note that biliary tract disease Key References: i. Persistent AST, ALT, GGT greater than 1.5 is less frequent than might be expected. Biliary tract or 2 × ULN (These are arbitrary cutoffs involvement and prevalence include: microgallblad­ Cohn JA, Strong TV, et al. Localization of the cystic fibrosis transmembrane that have been suggested by several groups; conductance regulator in human bile duct epithelial cells. Gastroenterology der (30%), cholelithiasis (1-10%), common bile duct 1993;105(6):1857-64. however, the significance of a particular value stenosis (1-2%), and a cholangiographic picture similar has not been studied.) Colombo C, Battezzati PM, et al. Liver disease in cystic fibrosis: a to sclerosing cholangitis in 1-2 percent. Microgallbladder prospective study on incidence, risk factors, and outcome. Hepatology 2002;36(6):1374-82. is likely a benign entity, perhaps related to diminished ii. Intermittently elevated bile flow. There is no agreement on the definition of Narkewicz MR. Markers of cystic fibrosis-associated liver disease. J Pediatr common bile duct stenosis and sclerosing cholangitis in b. Steatosis (At present, this would require histologi­ Gastroenterol Nutr 2001;32(4):421-2. CF or their potential role in the pathophysiology of the cal evidence: there may be imaging criteria in the Potter CJ, Fishbein M, et al. Can the histologic changes of cystic fibrosis- development of cirrhosis in CF. future that will come from studies of other liver associated hepatobiliary disease be predicted by clinical criteria? J Pediatr diseases.) Gastroenterol Nutr 1997;25(1):32-6.

With the protean manifestations of liver involvement in c. Fibrosis (Histology; there may be imaging criteria Scott-Jupp R, Lama M, et al. Prevalence of liver disease in cystic fibrosis. Arch CF, there is a need for uniform criteria for the definition Dis Child 1991;66(6):698-701. in the future that will come from studies of other of liver disease/involvement in CF. In 2007, the CF liver diseases.) Shapira R, Hadzic N, et al. Retrospective review of cystic fibrosis presenting Foundation convened a group of international experts in as infantile liver disease. Arch Dis Child 1999;81(2):125-8. CF-related liver disease. This group has proposed the fol­ d. Cholangiopathy (documented by abnormalities of lowing classification for CF-related liver disease (CFLD) the biliary tree on US, MRCP, CT, or ERCP) Sokol RJ, Durie PR. Recommendations for management of liver and biliary tract disease in cystic fibrosis. Cystic Fibrosis Foundation Hepatobiliary Dis­ to guide research in the field. The major point of the ease Consensus Group. J Pediatr Gastroenterol Nutr 1999;28(Suppl 1):S1-13. group is that blending various forms of liver involvement e. Ultrasound abnormalities that are not diagnostic of cirrhosis Vawter GF, Shwachman H. Cystic fibrosis in adults: an autopsy study. Pathol under one rubric of CFLD does not allow comparison Annu 1979;14(Pt 2):357-82. of natural history or treatment studies in CF. Thus, there i. Abnormal echogenicity Woodruff SA, Sontag M, et al. Prevalence of elevated liver function tests in is a need for uniformity in the classification and defini­ children with cystic fibrosis diagnosed by newborn screen. J Pediatr Gastroen­ tion of CF liver disease. III. Preclinical: No evidence of liver disease (must have terol Nutr 2007;45(4):E27-E28. all of the following: this would be used primarily in Proposed Classification of CF clinical studies) Liver Disease (CFLD): a. Normal clinical exam I. CFLD with cirrhosis/portal hypertension b. Normal imaging a. Clinical evidence of portal hypertension c. Normal biochemistry b. Evidence of cirrhosis by histology, imaging, or laparoscopy

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

Prevalence and Epidemiology Prevalence and Epidemiology of Cystic Fibrosis Liver Disease of Cystic Fibrosis Liver Disease

Continued

Jean Pappas Molleston, M.D. There has been considerable interest in the role of modifier Lamireau T, Monnereau S, Martin S, Marcotte J-E, Winnock M, Alvarez F. genes in CFLD. Alpha-1-antitrypsin deficiency hetero­ Epidemiology of liver disease in cystic fibrosis: a longitudinal study. J Hepatol Department of Pediatric Gastroenterology, Hepatology, 2004;41:920-925. and Nutrition, Indiana University School of Medicine, zygous phenotype MZ has been strongly associated with James Whitcomb Riley Hospital for Children, CFLD in a recent report in press. TGF-beta 1 polymor­ Brazova J, Sismova K, Vavrova V, Bartosova J, Macek M Jr, Lauschman H, Sediva A. Polymorphisms of TGF-beta1 in cystic fibrosis patients. Clin Indianapolis, IN phisms have been related to CFLD as well. One report Immunol 2006;121:350-357. has suggested an association of CFLD with a glutathione S-transferase P1 polymorphism. Ongoing studies are 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 (CF) affects about 1 in 2,500 newborns. likely to clarify these associations and identify other cystic fibrosis is associated with glutathione S-transferase P1 polymorphism. It is a disease that can manifest in multiple organ sys­ potential modifier genes. Hepatology 1992;36:913-917. tems, including the lung, liver, intestine, and pancreas. Research Goals: Liver disease is the third most common cause of death in CF and accounts for about 2 percent of CF mortality. 1. Determine clear definitions/criteria for CFLD to The prevalence of cystic fibrosis liver disease (CFLD) facilitate research and allow tracking of patients. ranges widely, depending on how liver disease is assessed 2. Identify risk factors or combinations of risk factors and over what period of time. Biochemical abnormali­ that predict CFLD and study potential mechanisms; ties, typically elevated transaminases, are common and expand list to include possible modifiable risk fac­ intermittent. While at any given time the prevalence tors such as nutritional factors or drugs. of elevated liver enzymes is reported at 20-50 percent, 3. Study the pathophysiology of CFLD in specific over longitudinal followup more than 80 percent of age groups to better understand why liver disease CF patients have elevated liver enzymes at some time. presents only prior to adulthood. Sonographic abnormalities, including hepatomegaly, abnormal hepatic echotexture, or Doppler evidence of References: portal hypertension, are apparent at initial assessment in Ling SC, Wilkinson JD, Hollman AS, McColl J, Evans TJ, Paton JY. The 35 percent, increasing to 67 percent at followup. Histo­ evolution of liver disease in cystic fibrosis. Arch Dis Child 1999;81:129-132. logic abnormalities of focal biliary cirrhosis or steatosis Feigelson J, Anagnostopoulos C, Poquet M, Pecau Y, Munck A, Navarro are common in the few CF patients who undergo liver J. Liver cirrhosis in cystic fibrosis—therapeutic implications and long term biopsies; autopsy studies indicate a 10 percent prevalence follow up. Arch Dis Child 1993;68:653-657. in infants but a greater than 70 percent prevalence in Colombo C, Russo MC, Zazzeron L, Romano G. Liver disease in cystic adults. Clinical liver disease is much less common, with fibrosis. J Pediatr Gastroenterol Nutr 2006;43:S49-S55. a prevalence ranging from 2.5 to 10 percent. The most Corbett K, Kelleher S, Rowland M, Daly L, Drumm B, Canny G, Greally P, striking but consistent epidemiologic fact is that CFLD Hayes R, Bourke B. Cystic fibrosis-associated liver disease: a population-based does not appear to increase with age; prevalence peaks study. J Pediatr 2004;145:327-332. before puberty and does not increase after that. Scott-Jupp R, Lama M, Tanner MS. Prevalence of liver disease in cystic fibrosis. Arch Dis Child 1991;66:698-701. Several risk factors have been associated with CFLD Colombo C, Apostolo MG, Ferrari M, Seli M, Genoni S, Giunta, Sereni LP. but are not consistent in all large studies. Most studies Analysis of risk factors for the development of liver disease associated with demonstrate a male predominance in CFLD. Essentially, cystic fibrosis. J Pediatr 1995;126:155-156. all patients with CFLD are pancreatic insufficient. Colombo C, Battezzati PM, Crosignani A, Morabito A, Costantini D, Padoan While some studies indicate an association with severe R, Giunta A. Liver disease in cystic fibrosis: a prospective study on incidence, genotypes, others do not. Many studies have found risk factors, and outcome. Hepatology 2002;36:1374-1382. an association with meconium ileus, but this has not Lindblad A, Glaumann H, Strandvik B. Natural history of liver disease in borne out in other reports. CFLD does not appear to be cystic fibrosis. Hepatology 1999;30:1151-1158. associated with severity of lung disease in most studies.

6 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 7 Speaker Abstracts Speaker Abstracts

Cystic Fibrosis Liver Disease: Cystic Fibrosis Liver Disease: Clinical Outcomes Clinical Outcomes

Continued

Peter Durie, M.D., F.R.C.P.C. 1, Susanne Schibli, M.D.2, Accordingly, we have used prudent criteria in a well- CFLD was diagnosed in 53 patients, giving an overall while listed for combined liver/lung transplant. There Rakesh Battacharjee1, and Mary Corey1 defined patient cohort to evaluate the prevalence and prevalence of 4.6 percent. The prevalence increased were 15 deaths, with a median age of mortality of 27.3 during the study period (1986-89 1.7%; 1990-94 3.1%; 1Hospital for Sick Children and Department of Pediatrics, natural history of clinically significant CFLD (notably, (range 17.5-52) years. Only one death was attributed to University of Toronto, Toronto, Ontario, Canada; 2University evidence of biliary cirrhosis with or without portal 1995-99 4.7%; 2000-04 6.0%). Retrospective analysis to complications of liver failure. of Bern, Bern, Switzerland hypertension and/or hepatocellular failure). The purpose explore the natural history of CFLD was accomplished of this analysis was to help decipher the risk of compli­ in 52 of 53 patients. Pancreatic insufficiency was signifi­ Conclusions: cations and timing and necessity of interventions such as cantly associated with CFLD (OR 3.3, 95% CI 1.03 to The cystic fibrosis transmembrane conductance regulator liver transplantation. 10.85). There were a greater number of CFLD patients Since most patients with CFLD maintain excellent protein (CFTR) functions across the apical domain of who were male (65%) compared to female. CFLD was hepatocellular function for many years, even decades, the cholangiocytes and gallbladder epithelial cells. CFTR Methods: diagnosed in 81 percent of patients under the age of principles of care for CFLD patients should be targeted maintains an apical Cl– gradient and fluid flow into the 18. The mean duration of follow-up from diagnosis toward recognizing and managing complications of ductal lumen. It also alkalinizes intralumenal contents Patients were identified retrospectively as having CFLD of CFLD was 10.6 (range 1.1 to 32.2) years. Of the through direct extrusion of HCO –, as well as through portal hypertension. Liver and/or combined liver/lung 3 from a prospectively established database consisting of hepatic complications of CFLD, portal hypertension was a Cl–/HCO – exchanger, which solubilizes the organic transplantation should be reserved for the few patients 3 data collected for all patients diagnosed and followed most prevalent. Hypersplenism was seen in 23 patients components of bile. Absent or dysfunctional CFTR who develop synthetic liver failure. with CF in Toronto, Canada between 1986 and 2004. (44%). Variceal bleeding was seen in 14 patients (27%). results in reduced bile fluidity and alkalinity, with plug­ A diagnosis of CFLD or query CFLD was determined Duration of disease did not seem to impact the preva­ ging of intrahepatic bile ducts leading to biliary stasis, Future Research Challenges and Needs: using specific search criteria that included evidence lence of these complications. Endoscopic management of which in turn induces inflammatory damage secondary of hepatocellular failure and/or biliary cirrhosis and/ variceal bleeding was needed in 12 patients. to cholangiocyte and collateral hepatocyte injury. • Identify the genetic and environmental factors that or portal hypertension (esophageal varices and/or confer increased susceptibility to CFLD. splenomegaly). Verification of the search strategy was Hypoalbuminemia, which developed in 12 patients, was The wide range of prevalence of cystic fibrosis liver dis­ • Test the ability of genetic factors or other biomarkers accomplished through chart review of all subjects identi­ persistent in only six (12%). Coagulopathy developed in ease (CFLD) reported in the medical literature (2-7%) to identify liver disease in newborn screen positive fied as having CFLD or query CFLD as well as review eight patients (17%), and was persistent in three (7%). can be attributed to several factors. The wide spectrum CF infants. of a random sample of subjects without CFLD. A case Persistent hyperbilirubinemia developed in only seven of hepatobiliary lesions that are known to occur in patients (13%). In the majority of cases, hypoalbumin­ • Develop novel approaches to preventing or ame­ CF disease, differing definitions of what constitutes report form was used to record parameters of relevance, including demographics, diagnosis of CF, diagnosis of emia (with and without ascites), persistent coagulopathy, liorating liver disease in CF infants with increased CFLD, and the lack of sensitive and specific methods and mild hyperbilirubinemia were harbingers of hepato­ susceptibility to CFLD. of establishing or excluding a diagnosis have contrib­ CFLD, results of laboratory and imaging studies, and clinical course of CF disease and CFLD. cellular failure. uted to considerable confusion and misunderstanding. References: The majority of patients with CF exhibit some degree Only six patients (12%) developed hepatocellular failure, of hepatobiliary disease, but the nature of the lesions Results: at 16, 25, 28, 44, 45, and 52 years of age, respectively. Corbett K, Kelleher S, Rowland M, Daly L, et al. Cystic fibrosis-associated and severity of disease vary considerably from patient J Pediatr. Three underwent isolated liver transplant; they survived liver disease: a population-based study. 2004;145:327-32. to patient. Postmortem analysis of hepatic specimens In total, 1,108 CF patients attended the Toronto CF for 3, 5, and 10 years, respectively. One other was from adult CF patients has reported the prevalence to clinics from 1986 to 2004. According to the database Debray D, Lykavieris P, Gauthier F, et al. Outcome of cystic fibrosis-associated listed for liver transplantation, and one patient died liver cirrhosis: management of portal hypertension. J Hepatol. 1999;31:77-83. be as high as 70 percent. However, the vast majority of search strategies, 17 patients were identified as having subjects have clinically insignificant hepatosteatosis, focal CFLD, 157 as query CFLD, and 934 as no CFLD. portal tract bile proliferation with cholangitis, or focal Chart reviews confirmed CFLD in all 17 patients biliary cirrhosis. identified by the searching strategies and 32/157 patients from the query group. A random sample of 111 patients Studies exploring the natural evolution of CFLD have from the non-CFLD group did not reveal any patients been sparse and are difficult to interpret in view of with CFLD. Three additional patients with CFLD were different criteria to establish a diagnosis for CFLD. identified from previous studies.

8 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 9 Speaker Abstracts Speaker Abstracts

Pathogenesis of Cystic Pathogenesis of Cystic Fibrosis Liver Disease Fibrosis Liver Disease

Continued

Steven D. Freedman, M.D., Ph.D. into the biliary tract. With loss of CFTR function, 4. Intestinal bacterial products are critical in the aberrant toll-like signaling, none of these have been there is decreased bile flow and inspissations, leading to genesis of fibrosis: definitively corroborated. Associate Professor of Medicine, Harvard Medical School, Associate Dean for Clinical and Translational Research, an obstructive process. However, the fact that most CF a. Prior administration of antibiotics (ampicillin, Director, The Pancreas Center, Beth Israel Deaconess patients have this abnormality, yet only 5-10 percent metronidazole, neomycin, vancomycin) severely Freedman’s and Durie’s group has shown that 34 percent Medical Center, Boston, MA develop cirrhosis, implies that other factors must be in attenuates liver fibrogenesis in the BDL and in of adult patients with PSC have a single CFTR allelic play to cause CFLD. the chemical carbon tetrachloride and thioacet­ variant, as analyzed by multiplex heteroduplex gel shift amide models (2). analysis with directed sequencing, compared to patients Cystic Fibrosis Liver Disease (CFLD) is poorly character­ If we examine the known facts in the pathophysiology of b. Attenuation of fibrosis in response to BDL is also with inflammatory bowel disease alone (3). Nasal ized both at a clinical level as well as with regard to its liver fibrosis in general, the critical gaps are as follows: seen in CD14 and LPS-binding protein deficient Potential Difference Testing revealed a high prevalence pathophysiology. The explanation for why 5-10 percent mice, indicating that LPS signaling from the of blunted isoproterenol stimulated CFTR chloride of CF patients develop CFLD, as well as its underly­ 1. Inflammation in the liver is tightly linked to fibrosis intestinal flora plays a major role in activation of channel function in both children and adults with PSC ing pathophysiology, remain unknown. The clinical in humans as well as in all experimental models of hepatic fibrosis. compared to patients with inflammatory bowel disease course, predisposing genetic factors, and the molecular fibrosis (2). c. LPS from the bacterial flora activates TLR4 alone (3,17). This would suggest that loss of normal mechanisms at play remain to be defined. At the Cystic Gap: The link between inflammation and hepatic receptors on Hepatic Stellate Cells (HSCs) but CFTR function explains why a subset of patients with Fibrosis Foundation Williamsburg Conference in 2007, fibrogenesis remains elusive. not Kupffer cells as the first critical step in the underlying colitis develop PSC. Supporting the link a workshop on CFLD led by Drs. Steven Freedman liver for activation of the fibrogenesis sequence. between colitis and CFTR dysfunction, induction of and Michael Narkewicz brought together basic scientists 2. Liver injury (acute or chronic) increases intestinal Gap: Is the intestinal microflora in CF critical to colitis with oral dextran sodium sulfate in CF knockout and clinical experts in this area with a focus on under­ permeability, leading to increased bacterial transloca­ the pathogenesis of CFLD? mice resulted in the genesis of bile duct injury (bile duct standing mechanisms of fibrogenesis and inflammation tion. This results in increased portal and plasma proliferation with a peri-cholangitic mononuclear cell in the liver. It was concluded that there is no agreed levels of LPS, with the liver being the prime target II. Overlap With Other Cholangiopathies: There infiltrate) compared to no disease being seen in wild- upon definition of CFLD in the literature. Most would (2). Hence, this normal response mechanism to are other biliary tract diseases in both the pediatric type littermates (4). This was due to a lack of induction classify CFLD as cirrhosis with portal hypertension acute liver injury perpetuates the inflammatory/ and adult populations that are similar to CFLD. Thus, in PPARα, which was corrected with docosahexaenoic and do not include asymptomatic CF patients with fibrogenic process in chronic liver injury, potentially understanding the molecular basis of these other disor­ acid (DHA), a known PPARα agonist, resulting in both isolated abnormal liver blood tests or abnormal imaging resulting in cirrhosis. ders, which lead to biliary fibrosis and cirrhosis, may partial prevention and reversal of established disease (5). studies. Because of the lack of a consistent definition, Gap: The mechanisms that underlie this process are shed light on the mechanisms that underlie CFLD. However, the liver disease in this model did not progress to the prevalence and outcome data in CFLD are poorly poorly understood. These disorders include α-1 antitrypsin deficiency and fibrosis. characterized. However, the prevalence of advanced the cholestatic diseases Primary Biliary Cirrhosis and CFLD, as defined by cirrhosis/portal hypertension, is 3. Cholestasis is the inciting event in most experimen­ more pertinent, Primary Sclerosing Cholangitis (PSC). III. Abnormalities in Bile Juice: Martin Cary’s 5-10 percent, and CFLD usually presents around or tal models of liver fibrosis. These can be categorized group demonstrated in ∆F508 mice that a) 53 percent before puberty (1). Median age of recognition is around by: CFLD and PSC share many similarities, including of these mice develop histologic features of bile ductular 10 years of age, indicating that this is an early event. a. Mechanical: bile duct ligation (BDL). clinical presentation, imaging abnormalities with bili­ proliferation and mild portal fibrosis by 100 days of age Progression to liver failure is rare, although complica­ b. Chemical: carbon tetrachloride, ary strictures in the intra- and/or extrahepatic biliary but no inspissated bile; and b) the mice with histo­ tions of portal hypertension are not uncommon. Risk thioacetamide. tree, histopathologic findings of bile duct injury (bile logic changes had increased secretion rates of bilirubin factors are severe (pancreatic insufficient) CFTR muta­ c. Mechanical and Chemical: Xenobiotics such as duct proliferation, peri-cholangitic inflammation, bile monoglucuronoside, resulting in an increased bile salt to tions, male gender, the alpha-1 antitrypsin Z allele, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) ductular plugs), and cirrhosis. PSC occurs in 5-7 percent phospholipid ratio (6). They concluded that increased perhaps TGF-β polymorphisms. lead to biliary plugs and cholangiocyte transfor­ of patients with colitis, typically with ulcerative colitis. fecal bile acid excretion increases bilirubin enterohepatic mation to a reactive phenotype. Although the mechanism is unknown, PSC has been cycling, leading to increased bilirubin secretion into I. Overall Pathophysiology: This is primarily a Gap: Is the mechanism of cholestasis in these models postulated to be linked to the portal vein bacteremia bile. In conjunction with secretion of more hydrophobic disorder of fibrogenesis within the liver and involving the same as in CF, and is transformation of the that results from the colitis. Although several hypotheses bile salts, this may result in toxic damage to cholan­ the bile ductules. CFTR is localized to the cholangiocyte cholangiocyte to a reactive phenotype critical in have been put forward, including genetic abnormalities giocytes. Furthermore, unconjugation of bilirubin via apical membrane, where it is central to fluid secretion inflammation/fibrogenesis? in bile salt transporters, autoimmune dysregulation, and β-glucuronidase hydrolysis generates insoluble metal salts

10 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 11 Speaker Abstracts Speaker Abstracts

Pathogenesis of Cystic Pathogenesis of Cystic Fibrosis Liver Disease Fibrosis Liver Disease

Continued Continued that may be deposited within cholangiocytes. However, it misfolded PiZ accumulates (9). Taken together with V. Cholangiocyte Transport Abnormalities: The • Genetic modifiers and/or other products (chemical, is unclear from these data how toxic damage to cholangio­ human data, the α-1 antitrypsin Z mutation by principal function of the cholangiocyte is to regulate the toxin, bacterial) are required for disease expression. cytes leads to downstream inflammation and fibrosis. itself is not sufficient to induce fibrosis but may secretion of fluids into the bile duct. Drew Feranchak do so when combined with other factors such as has played a sentinel role in identifying these ion • Understanding what leads to i) cholangiocyte activa­ IV. Other Animal Models of Biliary Fibrosis: obstruction of biliary flow. channels. This includes CFTR, the Ca2+-activated K+ tion and ii) downstream fibrogenesis is critical in There are several approaches that have been taken to channel (SK2), as well as the role of purinergic signaling developing therapeutics and developing strategies to induce fibrosis in the liver. The critical element appears • Xenobiotic-induced mouse model of biliary to regulate cholangiocyte transport and bile formation prevent CFLD. Thus, robust animal models need to be to be the induction of cholestasis, either chemically or fibrosis: Michael Trauner’s group in Austria, world (11,12). In addition, both human and rat biliary cells generated that demonstrate these features of CFLD. mechanically. experts in the pathophysiology of biliary fibrosis, exhibit flow-stimulated, PKCζ-dependent ATP release, recently developed a highly reproducible novel which results in increased [Ca2+]i and Cl­ secretion. The Literature Cited: • Chemical inducers of fibrosis: Carbon tetra- model of xenobiotic-induced cholangiopathy by finding that fluid flow can regulate membrane transport chloride and thioacetamide have been used to supplementing the standard diet of mice with 0.1 suggests that mechanosensitive ATP release may be a key 1. Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver disease. Semin Liver Dis. 2001 Nov;21(4):471-88. generate cirrhosis through their toxic effects and do percent (~10 mg/day) DDC (10). As a result of regulator of biliary secretion and an important target to increased biliary porphyrin secretion, by 4 weeks modulate bile flow in the treatment of cholestatic liver 2. Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner not primarily involve the cholangiocytes. DA, Schwabe RF. TLR4 enhances TGF-beta signaling and hepatic there were scattered pigmented biliary plugs with diseases (13). Whether impairment of purinergic signaling fibrosis. Nat Med. 2007 Nov;13(11):1324-32. bile ductular proliferation, and periductal onion skin in the setting of loss of CFTR function explains the predi­ 3. Sheth S, Shea JC, Bishop MD, Chopra S, Regan MM, Malmberg E, • Mechanical inducer of fibrosis: Bile duct Walker C, Ricci R, Tsui LC, Durie PR, Zielenski J, Freedman SD. fibrosis with a predominantly neutrophilic infiltra­ lection to CFLD remains unknown. However, this could ligation leads to secondary biliary fibrosis but may Increased prevalence of CFTR mutations and variants and decreased tion. Intra- and extra-hepatic segmental strictures potentially explain the mechanism of action of DDC, which chloride secretion in primary sclerosing cholangitis. Hum Genet. 2003 not be representative of the early events leading to were observed. Similar results were seen in Swiss through biliary obstruction and increased ductal pressure, Aug;3(3):286-92. CFLD and PSC. 4. Blanco PG, Zaman MM, Junaidi O, Sheth S, Yantiss RK, Nasser albino, FVB/N, C57BL/6, and 129/SV mice. Thus, inhibits purinergic-driven alternative chloride channel secre­ IA, Freedman SD. Induction of colitis in cftr-/- mice results in this model incorporates both inflammation and fibrosis. tion, resulting in biliary inflammation and fibrosis. bile duct injury. Am J Physiol Gastrointest Liver Physiol. 2004 • MDR2 knockout mice demonstrate loss of phos­ Aug;287(2):G491-6. pholipid secretion into bile, leading to biliary ductal 5. Pall H, Zaman MM, Andersson C, Freedman SD. Decreased peroxi­ DDC led to transformation of cholangiocytes into a VI. Activation of Hepatic Stellate Cells (HSCs): some proliferator activated receptor alpha is associated with bile duct fibrosis. However, there is no significant inflamma­ reactive phenotype as assessed by increased VCAM These cells play a pivotal role in the activation of injury in cystic fibrosis transmembrane conductance regulator-/- mice. J tory component. Mutations or polymorphisms in Pediatr Gastroenterol Nutr. 2006 Mar;42(3):275-81. expression in cholangiocytes (immunohistochem­ fibrogenesis in the liver. Brenner’s and Schwabe’s group MDR3, the human genetic correlate, have not been 6. Freudenberg F, Broderick AL, Yu BB, Leonard MR, Glickman JN, istry and Western blotting) and TNFα by in situ (2) have recently demonstrated a fundamental role for Carey MC. Pathophysiological basis of liver disease in cystic fibrosis found in patients with PSC. hybridization. By 8 weeks, bridging portal fibrosis intestinal microflora in the initial activation of HSCs. employing a DeltaF508 mouse model. Am J Physiol Gastrointest Liver Physiol. 2008 Jun;294(6):G1411-20. was present, along with proliferation and activation Induction of liver injury induced in animal models • CFTR mouse models: Both ∆F508 and exon 7. Beharry S, Ackerley C, Corey M, Kent G, Heng YM, Christensen of myofibroblasts (Ki-67 and α-SMA positive). through either mechanical (BDL) or chemical sensitizers H, Luk C, Yantiss RK, Nasser IA, Zaman M, Freedman SD, Durie 10 congenic cftr-/- mice variably demonstrate peri­ Fibrosis was confirmed by increased levels of hepatic (carbon tetrachloride or thioacetamide) led to increased PR. Long-term docosahexaenoic acid therapy in a congenic murine cholangitic inflammation and minimal fibrosis after model of cystic fibrosis. Am J Physiol Gastrointest Liver Physiol. 2007 hydroxyproline content and the expression of the LPS levels in the plasma and in the portal circulation. Mar;292(3):G839-48 90 days of age (6,7). pro-fibrogenic genes collagen-1α2, TIMP-1, and The binding of LPS to TLR4 receptors on HSCs led to 8. Friedman KJ, Ling SC, Lange EM, Macek M, Handler AJ, Pace RG, et al. Genetic modifiers of severe liver disease in cystic fibrosis. TGF-β. The expression of osteopontin was increased two important events: i) induction of Kupffer cell che­ • Alpha-1 antitrypsin deficient transgenic mice: 55th Annual Meeting of The American Society of Human Genetics, in both cholangiocytes and zone 1 hepatocytes. motaxis and ii) increased sensitivity of HSCs to TGF-β 2005;395:2176/F. Friedman and Knowles have presented evidence This cytokine with potent chemoattractant and secreted from Kupffer cells through downregulation of 9. Mencin A, Seki E, Osawa Y, Kodama Y, De Minicis S, Knowles M, α Brenner DA. Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibro­ suggesting that a single -1 antitrypsin Z allele pro-fibrogenic properties has been implicated in the TGF-β pseudoreceptor Bambi on HSCs. mutation (also called PiZ) may be a gene modifier sis in a murine model of cholestasis. Hepatology. 2007 Nov;6(5):443-52 pathogenesis of lung fibrosis and is postulated to 10. Fickert P, Stöger U, Fuchsbichler A, Moustafa T, Marschall HU, Wei­ in CFLD (8). In collaboration with Mencin’s and be central to the induction of bile ductular reaction VII. Summary: The following conclusions can be glein AH, Tsybrovskyy O, Jaeschke H, Zatloukal K, Denk H, Trauner Brenner’s group at Columbia, Knowles et al. showed M. A new xenobiotic-induced mouse model of sclerosing cholangitis and portal-portal bridging fibrosis. Although there made: and biliary fibrosis. Am J Pathol. 2007 Aug;171(2):525-36. that bile duct ligation in C57BL/6 transgenic for was no difference in bile acid output or composition 11. Feranchak AP, Doctor RB, Troetsch M, Brookman K, Johnson SM, α Fitz JG. Calcium-dependent regulation of secretion in biliary epithelial the human -1 antitrypsin mutation (PiZ) leads based on gas chromatography/mass spectrometry, • Loss of CFTR alone is not sufficient in both animal to stellate cell activation and fibrosis, along with cells: the role of apamin-sensitive SK channels. Gastroenterology. 2004 glutathione and phospholipid secretion was signifi­ models and in humans to result in CFLD. Sep;127(3):903-13. increased expression of ER stress markers where cantly reduced.

12 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 13 Speaker Abstracts Speaker Abstracts

Pathogenesis of Cystic The Pathology of Cystic Fibrosis Liver Disease Fibrosis Liver Disease

Continued

12. Feranchak AP, Fitz JG. Adenosine triphosphate release and puriner­ David E. Kleiner, M.D., Ph.D. requiring the Kasai procedure, and a neonatal hepatitis- gic regulation of cholangiocyte transport. Semin Liver Dis. 2002 like presentation with duct paucity. Bradford et al. Aug;22(3):251-62 Laboratory of Pathology, National Cancer Institute, 13. Woo K, Dutta AK, Patel V, Kresge C, Feranchak AP. Fluid flow induces National Institutes of Health, Bethesda, MD performed a detailed ultrastructural examination of the mechanosensitive ATP release, calcium signalling and Cl– transport in hepatocytes in cystic fibrosis and demonstrated dilation biliary epithelial cells through a PKCζ-dependent pathway. J Physiol. 2008;586:2779-2798. of canaliculi as well as ducts and ductules. The canaliculi 14. Norkina O, Burnett TG, De Lisle RC. Bacterial overgrowth in the contained heterogeneous electron-dense material with cystic fibrosis transmembrane conductance regulator null mouse small Liver disease has long been recognized as a component intestine. Infect Immun. 2004 Oct;72(10):6040-9. of the injury in cystic fibrosis. Liver disease may pres­ myelin, cell debris, and fibrillar material. Distention 15. Cox KL, Cox KM. Oral vancomycin: treatment of primary scleros­ ent with subtle clinical findings, such as liver enzyme of rough endoplasmic reticulum by a finely granular ing cholangitis in children with inflammatory bowel disease. J Pediatr material was also seen. Membrane-bound vacuoles with Gastroenterol Nutr. 1998 Nov;27(5):580-3. abnormalities or irregularities noted on ultrasound. As 16. Fiorotto R, Spirlì C, Fabris L, Cadamuro M, Okolicsanyi L, Strazza­ with many chronic liver diseases, progressive fibrosis an electron-dense core resembling mucus was found in bosco M. Ursodeoxycholic acid stimulates cholangiocyte fluid secretion hepatocytes as well as in bile duct epithelial cells. in mice via CFTR-dependent ATP secretion. Gastroenterology. 2007 is the major threat, with the development of portal Nov;133(5):1603-13. hypertension and biliary cirrhosis. Steatosis is the most 17. Pall H, Zielenski J, Jonas MM, DaSilva DA, Potvin KM, Yuan XW, commonly observed pathologic change, with approxi­ The factors influencing progression of liver disease are Huang Q, Freedman SD. Primary sclerosing cholangitis in childhood is incompletely understood, and a number of studies associated with abnormalities in cystic fibrosis-mediated chloride channel mately 60 percent of patients in the early study of Craig function. J Pediatr. 2007 Sep;151(3):255-9. et al. demonstrating it. In that study, it was noted that have been devoted to pathogenesis of fibrosis as well the steatosis in mild cases tended to be at the periphery as to the development of biomarkers that would allow of hepatic lobules, or periportal/zone 1 in more modern non-invasive monitoring of patients with cystic fibrosis. terms. In severe cases, the steatosis becomes pan-acinar. Part of the problem lies in the heterogeneity of the Steatohepatitis has not been reported, although the vari­ liver disease itself. Similar to the situation of primary ant of fatty liver disease seen more often in children has sclerosing cholangitis, a liver biopsy may miss diagnostic a zone 1 distribution of steatosis coupled with periportal lesions or underestimate the degree of liver injury due fibrosis. to sampling variability. It would be helpful to develop standard criteria for evaluating the liver lesions in cystic The pathognomonic hepatic lesion of cystic fibrosis is fibrosis similar to systems that have been developed focal biliary fibrosis (cirrhosis), which is found in up for fatty liver disease and chronic hepatitis. Data from to one third of patients. This lesion is characterized blinded evaluation of liver biopsies from the Cystic by the accumulation of amorphous pink, PAS-positive Fibrosis Liver Disease Network will be presented at the material in bile ducts, associated with ductular reaction, workshop, along with a proposal for semi-quantitative periportal fibrous expansion, and chronic inflammation. evaluation of liver lesions that can be used in prospective The lesions often are irregularly distributed in the liver, clinical studies. with variation in severity from portal area to portal area. These lesions may progress to form bridges, isolating Future Directions: regenerative nodules of varying sizes. The resulting cir­ rhosis is often macronodular, and there can be extreme • Development of a standardized feature recording variation in the sizes of nodules. Duct rupture can occur, system for evaluation of liver biopsies in cystic fibrosis. as can other changes of large duct obstruction. A pattern of duct injury mimicking sclerosing cholangitis has been • Correlation of systematic observations of pathology reported. Other complications that have been reported with clinical, biochemical, and molecular features of include amyloidosis, biliary obstruction of large ducts liver disease in cystic fibrosis.

14 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 15 Speaker Abstracts Speaker Abstracts

The Pathology of Cystic Genetic Modifiers of Cystic Fibrosis Liver Disease Fibrosis Liver Disease

Continued

Selected References: Michael R. Knowles, M.D.1, for Jaclyn Bartlett, Ph.D.1, a second study in different populations of CF patients Peter Durie, M.D.2, and the Gene Modifier Study with (n = 136) and without (n = 1,088) CFLD. Craig JM, Haddad H, Schwachman H. The pathological changes in the liver Group in cystic fibrosis of the pancreas. Am J Dis Child 1957;93:357-369. 1University of North Carolina at Chapel Hill, Chapel Hill, NC; Clinical features of the CF patients with severe liver dis­ di Sant’Agnese PA, Blanc WA. A distinctive type of biliary cirrhosis of the 2Hospital for Sick Children, Toronto, Ontario, Canada ease, compared to controls, showed more males (~65%) liver associated with cystic fibrosis of the pancreas. Recognition through signs of portal hypertension. Pediatrics 1956;18:387-409. than females. The mean and median age of recognition (diagnosis) of portal hypertension in all CFLD patients Bradford W, Allen D, Shelburne J, Spock A. Hepatic parenchymal cells in Cystic fibrosis (CF) is a recessive monogenic disorder was approximately 10-11 years, and more than 90 cystic fibrosis: ultrastructural evidence for abnormal intracellular transport. characterized by clinical heterogeneity that is not Pediatr Pathol 1983;1:269-279. percent of the CFLD patients were diagnosed by age explained by mutations in the cystic fibrosis transmem­ 20 years. More than 95 percent of patients with CFLD Columbo C, Battezzati PM, Crosignani A, Morabito A, Costantini D, Padoan brane conductance regulator (CFTR) gene (1,2). Patients R, Giunta A. Liver disease in cystic fibrosis: a prospective study on incidence, had two pancreatic insufficient mutations in CFTR. The risk factors and outcome. Hepatology 2002;36:1374-1382. with CF exhibit a wide range of lung disease severity, prevalence of meconium ileus at birth in CFLD patients and genetic variability in non-CFTR genes contributes to (~18%) is typical for the general CF population with Lewindon PJ, Pereira TN, Hoskins AC, Bridle KR, Williamson RM, risk for severity of pulmonary disease (3-5). Shepherd RW, Ramm GA. The role of hepatic stellate cells and trans­ pancreatic insufficient CFTR mutations. Abnormalities forming growth factor beta 1 in cystic fibrosis liver disease. Am J Pathol in biochemical tests of the liver (AST, ALT, and GGT) 2002;160:1705-1715. Dysfunction in the CF liver reflects loss of CFTR were not predictive of CFLD and showed no correlation ­ (Cl channel) activity on the apical membrane of chol­ with markers of hepatocellular synthetic dysfunction, angiocytes (6). This dysfunction results in reduced bile such as international normalized ratio (INR) and serum flow and is associated with a cholangiocyte-induced albumin. inflammatory response, with activation and prolifera­ tion of hepatic stellate cells, which results in cholangitis In the initial study, significant association was seen and fibrosis in focal portal tracts (7,8). However, only a for the SERPINA1 Z allele and variants of TGFβ1. small fraction (~5%) of CF patients develops severe liver The genotypes and minor allele frequencies for genetic disease characterized by cirrhosis with portal hyperten­ variants in patients without CFLD were similar to those sion (CFLD); thus, non-CFTR genetic variability may previously reported (3-5,10-13). In the replication study, contribute to risk for severe liver disease (9). the strong association was replicated for the SERPINA1 Z allele. The association of TGFβ1 variants in the initial To test for association between non-CFTR genetic poly­ population was not replicated for codon 10. morphisms and severe liver disease in CF with portal hypertension (CFLD), we studied nine variants in five When the initial and replication populations were genes previously studied in CF liver disease, including combined for analysis, the SERPINA1 Z allele displayed α1-antitrypsin (also known as α1-antiprotease, α1AP, very robust association with CFLD; similar evidence for SERPINA1) (10) angiotensin-converting enzyme (ACE) association was observed in analyses restricted to Cauca­ (11), glutathione S-transferase (GSTP1) (12), mannose- sian subjects in the initial and replication populations. binding lectin 2 (MBL2) (13), and transforming growth We also performed logistic regression in the combined factor β1 (TGFβ1) (11). A two-stage study design was initial and replication groups, and estimated the odds used. In the initial study, we compared polymorphic of CFLD for the SERPINA1 Z allele, adjusting for the genotypes in these candidate modifier genes in CF sub­ covariates of ethnicity, gender, and CFTR genotype. jects with CFLD (n = 124) and “control” CF patients Results remained consistent when using all subjects (n = 843) without CFLD who were at least 15 years of or Caucasian subjects only, with respect to statistical age. In the second stage, we tested our initial associations significance estimates and odds ratio estimates. We also (SERPINA1 Z allele and TGFβ1 codon 10 genotype) in combined the initial and replication groups, and esti-

16 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 17 Speaker Abstracts Speaker Abstracts

Genetic Modifiers of Cystic Protein Folding Defects in Liver Disease: Fibrosis Liver Disease Alpha-1-antitrypsin Deficiency

Continued mated a population attributable risk due to the Z allele References: Jeffrey Teckman, M.D. have suggested that autophagy, mitochondrial injury, of approximately 7 percent. apoptosis, and other pathways may be involved in the Associate Professor of Pediatrics, and Biochemistry and 1. Mekus F, et al. Twin Res 2000;3:277. mechanism of hepatocyte injury, although the interplay 2. Vanscoy LL, et al. Am J Respir Crit Care Med 2007;175:1036. Molecular Biology, Saint Louis University School of Medi- The mechanism of the Z allele as an adverse modifier 3. Drumm ML, et al. N Engl J Med 2005;353:1443. cine, St. Louis, MO; Cardinal Glennon Children’s Medical of these mechanisms in vivo is still being described (6,7). of liver disease in CF patients likely reflects stimulation 4. Bremer LA, et al. Hum Gene Ther 2008;17:2228. Center, St. Louis, MO Recent studies have examined a well-characterized in vivo of hepatic stellate cells by inflammatory mediators from 5. Dorfman R, et al. J Clin Invest 2008;118:1040. model of a1AT mutant Z liver injury, the PiZ mouse, 6. Cohn JA, et al. Gastroenterology 1993;105:1857. both CFTR-deficient cholangiocytes and hepatocytes 7. Feranchak AP, Sokol RJ. Semin Liver Dis 2001;21:471. and made comparisons to human liver, to better under­ containing the misfolded Z allele protein. These dual 8. Schuppan D, Alfhal NH. Lancet 2008;371:838. Genetically determined abnormalities in protein fold­ stand the in vivo mechanisms of liver injury involved in 9. Feranchak AP. Curr Gastroenterol Rep 2004;6:231. ing are responsible for a variety of diseases, and are inflammatory stimuli induce hepatic stellate cells to 10. Frangolias DD, et al. Am J Respir Cell Mol Biol 2003;29:390. this disease. Results have shown a moderate but chronic migrate and proliferate in the bile duct regions in a 11. Arkwright PD, et al. Am J Respir Crit Care Med 2003;167:384. sometimes referred to as conformational diseases (1). increase in the rate of liver cell death and an associated pro-fibrogenic manner (7-9,14). Bile duct ligation with 12. Henrion-Caude A, et al. Hepatology 2002;36:913. In some cases, abnormal conformation inhibits normal compensatory hepatocellular proliferation (8). The results 13. Gabolde M, et al. J Med Genet 2001;38:310. resultant cholestasis stimulates activated stellate cells and 14. Perlmutter DH. Pediatr Res 2006;60:233. physiologic function of the protein as part of disease also show an increase in stimulation of the apoptotic fibrosis in the liver of homozygous transgenic PiZ versus 15. Mencin A, et al. Hepatology 2007;46:1443. pathophysiology. In other cases, accumulation of cascade in hepatocytes, the magnitude of which strongly wild-type mice, which is compatible with this proposed abnormally folded proteins, either directly or indirectly, correlates to the absolute amount of a1AT mutant mechanism of the Z allele as an adverse modifier in CF triggers cell injury. Alpha-1-antitrypsin (a1AT) deficiency Z protein accumulated within the individual cell. It (15). is commonly viewed as a prototypical conformational appears that the small population of hepatocytes with disease, and is caused by homozygosity for the a1AT the largest intracellular accumulations of the mutant Z This two-stage study shows that the SERPINA1 Z allele mutant Z gene (2,3). ZZ homozygotes occur in protein undergoes apoptotic cell death at an increased is strongly associated with CFLD and portal hyperten­ approximately 1 in 2,000 births in North American rate and that cells without large accumulations of the sion. This polymorphism is relatively uncommon in and European populations. The Z mutant, the most mutant Z protein proliferate in response. This chronic CF (~2.2% of CF patients are carriers), but the odds common disease-associated allele, arises from a point cycle of injury and repair leads to organ injury. Some of ratio for association with severe liver disease is relatively mutation that encodes a single amino acid substitution. these insights have depended on new methods in which high (~5) for the contribution of a genetic modifier This mutation confers an abnormal conformation on livers from the PiZ transgenic mouse were harvested and to a Mendelian disorder. The availability of this first the a1AT mutant Z protein, resulting in accumulation hepatocytes with the largest mutant Z protein accumula­ genetic marker, SERPINA1 Z allele, for the development within the endoplasmic reticulum of hepatocytes rather tions were isolated from cells with less accumulation, so of severe liver disease in CF illustrates the concept of than the appropriate, high magnitude secretion of the that these populations of cells from within the same liver identifying risk factors in CF patients early in life. wild type protein. Some homozygous ZZ children and could be analyzed separately. These data revealed hetero­ adults develop chronic liver disease and hepatocellular geneity in injury on the cellular level, which correlated Future research challenges for studying the genetics of carcinoma, which is thought to result from this intra­ with the magnitude of a1AT mutant Z protein intracel­ CF liver disease relate primarily to the characteriza­ cellular accumulation of the a1AT mutant Z protein. lular accumulation (9). These studies also permitted tion, recruitment, and more extensive genetic studies of The profoundly low serum levels of a1AT found in ZZ the construction of a hypothetical hepatocellular injury patients with CFLD. A recent GWAS study of nearly homozygotes leaves patients susceptible to the develop­ cascade for this disease involving caspase activation, 300 patients with CFLD is undergoing analysis. Associa­ ment of chronic obstructive pulmonary disease as adults. apoptosis, and mitochondrial injury, which takes into tion of single nucleotide polymorphisms with CFLD in account the heterogeneous nature of the accumulation this large, collaborative GWAS study will require replica­ The accumulation of the mutant Z protein in the within the liver. Further development of this hypotheti­ tion in a separate cohort and extensive followup studies liver in a1AT deficiency is strikingly heterogeneous, cal cascade, and continued study of sub-populations of to identify causative variants and define mechanism(s) of although the mechanisms involved in accumulation and cells from within the same liver, will likely be needed to pathobiology of CFLD. the reasons for the heterogeneity are still being investi­ fully describe the mechanisms of injury in this disease gated (4,5). Previous reports based on in vitro studies and to design future therapies.

18 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 19 Speaker Abstracts Speaker Abstracts

Protein Folding Defects in Liver Disease: Cystic Fibrosis Liver Disease: Alpha-1-antitrypsin Deficiency CFTR and the Liver

Continued

References: Andrew P. Feranchak, M.D. to our knowledge of biliary function. Unfortunately, the Cftr -/- mouse has not been a reliable model for the Associate Professor of Pediatrics, University of Texas 1. Carrell RW, Lomas DA. Alpha1-antitrypsin deficiency—a model for study of CF-associated liver disease due to viability issues conformational diseases. N Engl J Med 2002;346(1):45-53. Southwestern Medical Center, Dallas, TX 2. Perlmutter DH, Brodsky JL, Balistreri WF, Trapnell BC. Molecular and the inconsistent development of liver pathology. pathogenesis of alpha-1-antitrypsin deficiency-associated liver disease: a Therefore, the development of a long-living congenic meeting review. Hepatology 2007;45(5):1313-23. Cftr -/- 3. Teckman JH. Alpha1-antitrypsin deficiency in childhood. Semin Liver The pathophysiology underlying the development of mouse (C57BL/6J background) or the CF pig, Dis 2007;27(3):274-81. cystic fibrosis (CF)-associated liver disease is still only which appear to develop liver disease similar to humans, 4. Chappell S, Hadzic N, Stockley R, Guetta-Baranes T, Morgan K, may provide powerful experimental models (3,4). Kalsheker N. A polymorphism of the alpha1-antitrypsin gene represents speculative, and while several hypotheses have been a risk factor for liver disease. Hepatology 2008;47(1):127-32. proposed, studies directly assessing the effects of abnor­ 5. Greene CM, Miller SD, Carroll T, McLean C, O’Mahony M, Lawless Role of CFTR in Hepatobiliary Function: Studies MW, O’Neill SJ, Taggart CC, McElvaney NG. Alpha-1 antitrypsin mal CFTR in hepatobiliary function are limited. This deficiency: a conformational disease associated with lung and liver reality is disappointing since the genetic underpinnings utilizing the available cell and epithelial models have manifestations. J Inherit Metab Dis 2008;31(1):21-34. responsible for cystic fibrosis have been defined for more revealed several important observations regarding the 6. Rudnick DA, Shikapwashya O, Blomenkamp K, Teckman JH. Indo­ role of CFTR in cholangiocyte transport. First, CFTR is methacin increases liver damage in a murine model of liver injury from than 15 years. One conventional model proposes that an alpha-1-antitrypsin deficiency. Hepatology 2006;44(4):976-82. abnormal or absent CFTR protein in the apical mem­ located on the apical membrane of cholangiocytes and 7. Sifers RN. Cell biology. Protein degradation unlocked. Science functions as a cAMP-activated Cl­ channel (2,5). Second, 2003;299(5611):1330-1. brane of biliary epithelial cells initiates a series of events, ­ 8. Rudnick DA, Liao Y, An JK, Muglia LJ, Perlmutter DH, Teckman JH. including: abnormal secretin/cAMP-stimulated Cl and binding of the hormone secretin to basolateral receptors Analyses of hepatocellular proliferation in a mouse model of alpha-1­ - results in an increase in intracellular cAMP and activa­ HCO3 secretion, a decrease in bile flow, subsequent bile antitrypsin deficiency. Hepatology 2004;39(4):1048-55. tion of CFTR through a PKA-dependent mechanism 9. Lindblad D, Blomenkamp K, Teckman J. Alpha-1-antitrypsin mutant Z duct plugging by thickened secretions, cholangiocyte protein content in individual hepatocytes correlates with cell death in a and hepatocyte injury, activation of hepatic stellate cells, (6,7). Third, secretin-stimulated increases in cAMP result mouse model. Hepatology 2007;46(4):1228-35. - collagen deposition, and ultimately the development of in a parallel increase in HCO3 transport (8,9). Fourth, focal biliary cirrhosis leading to multilobular cirrhosis (1). increases in cAMP are a stimulus for regulated exocyto­ The histologic finding of eosinophilic material plugging sis, including movement and insertion of Aquaporin-1 the intrahepatic bile ducts of patients with cystic fibrosis- channels to the apical membrane (10,11). Together, associated liver disease provides some evidence for this these findings support a working model whereby secretin “bile plugging” model but fails to explain why only a binding basolateral cholangiocyte receptors increases minority of patients with CF develop clinically signifi­ intracellular cAMP levels and stimulates apical mem­ - - cant liver disease. brane Cl , HCO3 , and H2O transport, thus modulating alkalinization and dilution of bile. Although these Biliary Models: In the liver, CFTR is found only on findings support an important role of CFTR in cAMP­ intrahepatic biliary epithelial cells, known as cholangio­ mediated biliary secretion, the identification of other cytes, and not on the more abundant parenchymal cells membrane Cl­ channels, which contribute to ductular known as hepatocytes (2). Cholangiocytes contribute secretion via cAMP-independent mechanisms, adds com­ to the volume and composition of bile through absorp­ plexity to the mechanisms responsible for bile formation. tion and secretion of fluid and electrolytes. However, their small size and intrahepatic location have made the Alternate Secretory Pathways: Other Cl­ channels direct study of cholangiocytes arduous. Several cell and have been identified in cholangiocytes and appear to epithelial models, including isolated primary rat chol­ contribute to ductular secretion independent of CFTR, angiocytes, immortalized (SV40) mouse cholangiocytes, including those activated by Ca2+/calmodulin kinase human biliary adenocarcinoma cells, human and mouse (5,12), increases in cell volume (13), and extracellular gallbladder, isolated bile duct units, normal rat cholan­ nucleotides (14-16). The contribution of these alternate giocyte (NRC) monolayers, and the isolated-perfused secretory pathways to biliary secretion has been hard to liver, have been developed and together have contributed assess. It is attractive to speculate that these alternate

20 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 21 Speaker Abstracts Speaker Abstracts

Cystic Fibrosis Liver Disease: Cystic Fibrosis Liver Disease: CFTR and the Liver CFTR and the Liver

Continued Continued

Cl­ channels may partly compensate for the CF secretory the clinical manifestations of liver disease? Functional Future Research Challenges/Needs: 10. Roberts SK, Yano M, Ueno Y, Pham L, Alpini G, Agre P, LaRusso N. defect in the liver and may serve to bypass the secretory studies of primary CF cholangiocytes are limited. A Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism. Proc Natl Acad Sci U S A 1994;91:13009-13. defect associated with CF. In fact, in the Cftr -/- mouse study of regulated transport in isolated human chol­ Development of hepatobiliary models of CF (cholangio­ 11. Tietz PS, Marinelli RA, Chen XM, Huang B, Cohn J, Kole J, McNiven ­ model, increased expression of alternate Cl channels angiocytes with a compound heterozygous mutation cytes, polarized biliary epithelium, whole animals) MA, Alper S, LaRusso NF. Agonist-induced coordinated trafficking of modulates organ-level disease (17). Thus, Cl­ channels (∆F508/G542X) demonstrated abnormal cAMP­ functionally related transport proteins for water and ions in cholangio­ - + - cytes. J Biol Chem 2003 May 30;278(22):20413-9. other than those encoded by CFTR can sustain biliary stimulated Cl , K , and HCO3 secretion, but normal • Clear elucidation of the role of CFTR in hepatobiliary 2+ ­ 12. Schlenker T, Fitz JG. Calcium-activated chloride channels in a human secretion. Ca -activated Cl secretion (20). Studies in human CF function. biliary cell line: regulation by calcium/calmodulin-dependent protein gallbladder revealed similar abnormalities in cAMP­ • Identification of the initiating factors in the develop­ kinase. Am J Physiol 1996;271:G304-G310. Luminal Nucleotides Regulate Bile Formation: stimulated Cl­ transport, but an intact ATP-stimulated ment of CF liver disease. 13. Roman RM, Wang Y, Fitz JG. Regulation of cell volume in a human Recently, secretion mediated by extracellular nucleotides Cl­ conductance, presumably through a Ca2+-dependent • Characterization of alternate secretory pathways along biliary cell line: calcium-dependent activation of K+ and Cl- currents. Am J Physiol 1996;271:G239-G248. (e.g., ATP) acting on purinergic (P2) receptors on the pathway (21). Although these studies are in agreement the bile duct that may serve as potential therapeutic 14. McGill J, Basavappa S, Shimokura GH, Middleton JP, Fitz JG. luminal membrane of biliary epithelial cells has emerged with the working model, demonstrating an important targets. Adenosine triphosphate activates ion permeabilities in biliary epithelial ­ - cells. Gastroenterology 1994;107:236-43. as functionally important. ATP is present in bile, and role of CFTR in ductular Cl and HCO3 secretion, they binding of ATP to P2 receptors increases Cl­ efflux from fail to provide a mechanistic explanation for why only Acknowledgments: Studies in our laboratory are 15. Schlenker T, Romac JMJ, Sharara A, Roman RM, Kim S, LaRusso supported by the Cystic Fibrosis Foundation and the N, Liddle R, Fitz JG. Regulation of biliary secretion through api­ isolated cholangiocytes (14) and dramatically increases a subset of patients develops clinically significant liver cal purinergic receptors in cultured rat cholangiocytes. Am J Physiol transepithelial secretion in biliary epithelial monolayers disease (even with the same CFTR mutation); however, National Institute of Diabetes and Digestive and Kidney 1997;273:G1108-G1117. (15). Indeed, the magnitude of the secretory response the existence of alternate Cl­ secretory pathways in Diseases (NIDDK) of the National Institutes of Health. 16. Dutta AK, Woo K, Doctor RB, Fitz JG, Feranchak AP. Extracellular to ATP is two- to threefold greater than that to cAMP these studies is intriguing. Indeed, although CF biliary nucleotides stimulate Cl­ currents in biliary epithelia through receptor- ­ References: mediated IP3 and Ca2+ release. Am J Physiol Gastrointest Liver Physiol (16). Interestingly, recent evidence suggests that even epithelium has abnormal secretin/cAMP-stimulated Cl 2008 (in press). ­ cAMP-stimulated bile flow is mediated by ATP release secretion, it maintains a robust Cl secretory response to 17. Clarke LL, Grubb BR, Yankaskas J, Cotton CU, McKenzie A, Boucher 1. Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver RC. Relationship of a non-cystic fibrosis transmembrane conductance into the duct lumen and stimulation of apical P2 recep­ exogenous nucleotides (21,22), suggesting that this may disease. Sem Liv Dis 2001;21:471-88. tors (18). Together, these studies challenge and extend represent a therapeutic strategy to improve the abnormal 2. Cohn JA, Strong TA, Picciotto MA, Nairn AC, Collins FS, Francis regulator-mediated chloride conductance to organ-level disease in cftr(-/­ the conventional model that centers on the concept that biliary secretion associated with CF liver disease. S, Fitz JG. Localization of CFTR in human bile duct epithelial cells. ) mice. Proc Natl Acad Sci U S A 1994;91:479-83. 18. Minagawa N, Nagata J, Shibao K, Masyuk AI, Gomes DA, Rodrigues ­ Gastroenterology 1993;105:1857-64. cAMP-dependent opening of CFTR-related Cl channels 3. Durie PR, Kent G, Phillips MJ, Ackerley CA. Characteristic multiorgan MA, Lesage G, Akiba Y, Kaunitz JD, Ehrlich BE, et al. Cyclic AMP is the driving force for cholangiocyte secretion. Rather, Summary: Several observations can be made concern­ pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulates bicarbonate secretion in cholangiocytes through release of ATP the operative regulatory pathways appear to take place ing the role of CFTR in hepatobiliary function and regulator knockout murine model. Am J Pathol 2004 Apr;164(4):1481-93. into bile. Gastroenterology 2007 Nov;133(5):1592-602. 4. Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft 19. Woo K, Dutta AK, Patel V, Kresge C, Feranchak AP. Fluid flow induces within the lumen of intrahepatic ducts, where release the development of CF-associated liver disease. First, an PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, et al. Disruption of the mechanosensitive ATP release, calcium signalling and Cl­ transport in CFTR gene produces a model of cystic fibrosis in newborn pigs. Science of ATP into bile is a final common pathway control­ abnormal or absent CFTR protein is necessary but not biliary epithelial cells through a PKCzeta-dependent pathway. J Physiol 2008 Sep 26;321(5897):1837-41. ling ductular bile formation. In light of recent studies sufficient for the development of clinically significant CF 5. Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regulation of 2008 Jun 1;586(Pt 11):2779-98. demonstrating that the mechanical effects of fluid- liver disease. Second, while CFTR functions as a cAMP­ membrane chloride currents in rat bile duct epithelial cells. J Clin Invest 20. Zsembery A, Jessner W, Sitter G, Spirli C, Strazzabosco M, Graf J. flow or shear stress at the apical membrane of biliary stimulated Cl­ channel in the apical membrane of biliary 1993;91:319-28. Correction of CFTR malfunction and stimulation of Ca-activated Cl 6. Levine RA, Hall RC. Cyclic AMP in secretin choleresis. Evidence for a channels restore HCO - secretion in cystic fibrosis bile ductular cells. 3 epithelial cells is a robust stimulus for ATP release, a epithelium, its role as a “regulator” of other membrane regulatory role in man and baboons but not in dogs. Gastroenterology Hepatology 2002 Jan;35(1):95-104. model emerges in which mechanosensitive ATP release transporters and transport pathways may surpass its role 1976;70:537-44. 21. Chinet T, Fouassier L, Dray-Charier N, Imam-Ghali M, Morel H, Mer­ ­ secretion is a dominant pathway regulating as a Cl­ channel. Third, mechanosensitive pathways, 7. McGill J, Gettys TW, Basavappa S, Fitz JG. Secretin activates Cl- chan­ gey M, Dousset B, Parc R, Housset C. Regulation of electrogenic anion and Cl nels in bile duct epithelial cells through a cAMP-dependent mechanism. secretion in normal and cystic fibrosis gallbladder mucosa. Hepatology biliary secretion (19). Thus, a decrease in bile flow including ATP release and P2 receptor binding, may Hepatology 1992;16:125A. (forthcoming). associated with CF may be accompanied by alterations be a predominant pathway, independent from CFTR, 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 22. Clarke LL, Harline MC, Gawenis LR, Walker NM, Turner JT, Weisman in mechanosensitive pathways, which may exacerbate contributing to bile formation. Further studies are clearly Invest 1993;92:1314-25. GA. Extracellular UTP stimulates electrogenic bicarbonate secretion abnormalities in Cl­ secretion and bile formation. needed to: define the role of CFTR in normal liver 9. Strazzabosco M, Mennone A, Boyer JL. Intracellular pH regulation in across CFTR knockout gallbladder epithelium. Am J Physiol Gastrointest function and during cholestasis, understand the potential isolated rat bile duct epithelial cells. J Clin Invest 1991;87:1503-12. Liver Physiol 2000 Jul;279(1):G132-G138. Role of Abnormal CFTR in the Development regulatory roles of alternate secretory pathways, and elu­ of Liver Disease: How does an absent or abnormal cidate the potential genetic and environmental modifiers CFTR protein on the cholangiocyte membrane lead to of disease progression.

22 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 23 Speaker Abstracts Speaker Abstracts

The CFTR-/- Pig: Medical and Surgical Therapy Liver and Gallbladder Disease of CF-associated Liver Disease

David K. Meyerholz, D.V.M., Ph.D.1, are needed to define the gestational timing and anatomic Saul J. Karpen, M.D., Ph.D. due to intestinal obstruction, leading to persistence of David A. Stoltz, M.D., Ph.D.2,and changes associated with initial lesion development. cholestasis, biliary tract damage/obstruction, and in Michael J. Welsh, M.D.2-4 Director, Texas Children’s Liver Center, Texas Children’s Furthermore, long-term studies following the progression Hospital/Baylor College of Medicine, Houston, TX some, progression to liver insufficiency and death. In 1Departments of Pathology, 2Internal Medicine, and 3Mo- of liver disease are needed to define the clinical pathol­ these high-risk patients, medical treatment options are lecular Physiology and Biophysics, University of Iowa Roy ogy, the incidence of multilobular cirrhosis, and what limited, with emerging roles for single- or multi-organ J. and Lucille A. Carver College of Medicine, Iowa City, modifier genes contribute to its development. transplants in select infants. IA; 4Howard Hughes Medical Institute, Chevy Chase, MD With recent improvements in the care of patients with Cystic Fibrosis (CF) leading to enhanced longevity, the References: 2. The Older Child With CFLD: The clinical manifestations of non-pulmonary features of CF are features of the older child with CFLD may involve Cystic fibrosis (CF) is a multiorgan disease caused by becoming increasingly recognized as components linked Bodian M. Fibrocystic Disease of the Pancreas: A Congenital Disorder of Mucus hepatomegaly, splenomegaly, or elevated liver enzymes, mutations in the cystic fibrosis transmembrane con­ Production-Mucosis. New York: Grune & Stratton, 1953. to outcomes. Among the more serious non-pulmonary or the child may be asymptomatic (5). A proportion ductance (CFTR) regulator. Liver disease is the second manifestations of CF are those that involve the liver, Diwakar V, Pearson L, Beath S. Liver disease in children with cystic fibrosis. of these patients have gall bladder dysmotility and leading cause of CF mortality, and liver disease can Paediatric Respiratory Reviews 2001;2:340-349. affecting upwards of 20 percent of CF patients (1-3). propensity to form gallstones. Treatment options occur in neonatal infants, suggesting lesion initiation and CF-associated liver disease (CFLD) is considered the Feranchak AP. Cystic fibrosis liver disease. In: Liver Disease in Children, 3rd include UDCA, which may improve gall bladder kinet­ development in utero. Recently, we described a novel second leading cause of death in CF patients, and ed. Suchy FJ, Sokol RJ, Balistreri WF, eds. Cambridge, United Kingdom: ics, but many times, cholecystectomy is the preferred, CFTR-/- pig model that has multiorgan disease at birth, Cambridge University Press, 2007, pp. 572-594. progression of CFLD has an impact on CF patients’ long-term treatment option (6). A main clinical feature including meconium ileus and pancreatic destruction, quality of life and function, indicating a much-needed Lindblad A, Hultcrantz R, Strandvik B. Bile-duct destruction and collagen of progression of biliary fibrosis and cirrhosis in CFLD with gallbladder and liver lesions. In neonatal CFTR-/- deposition: a prominent ultrastructural feature of the liver in cystic fibrosis. role for safe and effective therapeutics. However, there is the development of portal hypertension (PHTN) pigs, microgallbladder is a ubiquitous gross lesion and its Hepatology 1992;16:372-381. are disappointingly few effective therapeutic choices and sequelae of varices, ascites, and splenomegaly. The lumen is variably filled with mucoid to inspissated bile Oppenheimer EH, Esterley JR. Hepatic changes in young infants with for most cholestatic or fibrotic liver disease, including splenomegaly can be massive, with associated sequestra­ and uncommon cellular aggregates of neutrophils. Alcian cystic fibrosis: possible relation to focal biliary cirrhosis. Journal of Pediatrics CFLD. Despite these current caveats, there are medical, tion of leukocytes and platelets, as well as potential Blue/PyronineY as well as Periodic Acid Schiff stains 1975;86:683-689. nutritional, radiological, surgical, and transplant options impairments in pulmonary dynamics. At times, the detect the luminal inspissated mucus, and the walls of Oppenheimer EH, Esterley JR. Pathology of cystic fibrosis: review of the for CFLD. spleen seems to take on a major focus for some clini­ the gallbladder are thickened by mucosal proliferation literature and comparison with 146 autopsied cases. Perspectives in Pediatric cians, but the contributions of thrombocytopenia and with circumferential accentuation of the gallbladder Pathology 1975;2:241-278. 1. Cholestasis and Malnutrition in the Infant abdominal distension to survival and lung function have wall by fibrosis and smooth muscle. Inspissated bile and Porta EA, Stein AA, Patterson P. Ultrastructural changes of the pancreas and With CF: The clinical features of CFLD in infancy yet to be fully evaluated (7). Non-medical treatments mucus are variably seen obstructing lumens of the cystic liver in cystic fibrosis. American Journal of Clinical Pathology 1964;42:451-465. typically involve prolonged neonatal jaundice and poor for varices include therapeutic endoscopic banding of ducts. Large extrahepatic and intrahepatic ducts are Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft growth, with hepatomegaly and laboratory values notable varices and surgical or radiological porto-systemic shunts uncommonly filled by mucus with scattered neutrophils, PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford- for elevated serum conjugated bilirubin and GGT (8,9). UDCA therapy has not been shown to affect the cellular plugs, or inspissated bile. In the liver, triads are Lenane CL, Davis GJ, Smith TL, Samuel M, Wax D, Murphy CN, Rieke levels. The overlapping nutritional and metabolic needs development of PHTN in CFLD. focally accentuated by variable ductular hyperplasia, 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 of infants with CF are multiplied during this period inflammation, and fibrosis. Rarely, triads have wide­ produces a model of cystic fibrosis in newborn piglets. Science 2008 Sep of life, especially when there is short gut or cholestasis. 3. Isolated Liver and Multi-organ Transplantation: spread bridging, predominantly by biliary hyperplasia, 26;321(5897):1837-1841. Treatment options in the first year of life are primarily Consideration for liver transplantation in CF patients with lesser inflammation and fibrosis. These cases also directed toward appropriate nutritional supplementa­ is complex because clear guidelines for proper patient have microscopic retraction of the surface serosa creating tion, even in those whose jaundice has resolved. Periodic selection are lacking. Currently, those with minimal lung a lobular surface appearance. Steatosis is not present in verification of adequate fat-soluble vitamin nutriture is dysfunction who progress to liver insufficiency or severe neonatal CFTR-/- pigs. essential. Ursodeoxycholic acid (UDCA), a hydrophilic sequelae of PHTN warrant consideration for liver trans­ bile acid, is employed by many caregivers, although plantation (10-12). Given the multiple organs involved Future challenges include optimization of the surgi­ evidence for its efficacy in CFLD at this age is lacking in patients with CF, reports of lung-liver, liver-pancreas, cal and medical therapies required for resolution of heart-lung-liver and liver-intestine transplants suggest the meconium ileus that is universally seen in the (4). There are some infants with CFLD whose disease generally positive outcomes, although the number of CFTR-/- pig model. To characterize the development progresses rapidly, mainly those born prematurely who and pathogenesis of hepatobiliary disease, in utero studies require parenteral nutrition or have bowel resections cases is small (13,14).

24 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 25 Speaker Abstracts Speaker Abstracts

Medical and Surgical Therapy Liver Transplantation in Children of CF-associated Liver Disease With Cystic Fibrosis: Status and Unanswered Questions Continued

Summary and Future Directions: The complexity 5. Colombo C, Battezzati PM, Crosignani A, et al. Liver disease in cystic George V. Mazariegos, M.D., F.A.C.S. Molmenti et al. (6) reported 60 percent patient survival of making treatment decisions rests not only with the fibrosis: a prospective study on incidence, risk factors, and outcome. in their cohort. Hepatology 2002;36:1374-82. Hillman Center for Pediatric Transplantation, Children’s inherently individual patient-to-patient manifestations 6. Colombo C, Crosignani A, Battezzati PM, et al. Delayed intestinal Hospital of Pittsburgh at University of Pittsburgh Medical of pulmonary and liver disease in CF, but also with the visualization at hepatobiliary scintigraphy is associated with response to Center, Pittsburgh, PA Clear indications and timing for LT, however, have not lack of suitable markers for progression of CFLD. Iden­ long-term treatment with ursodeoxycholic acid in patients with cystic been well described. The European Transplant Registry fibrosis-associated liver disease. J Hepatol 1999;31:672-7. reported good outcomes for LT in patients with CF tification of markers associated with disease progression 7. Louis D, Duc ML, Reix P, et al. Partial splenectomy for portal and severity is needed. Current therapeutic options are hypertension in cystic fibrosis related liver disease. Pediatr Pulmonol Twenty to 50 percent of CF patients develop liver but noted that most centers performed LT for evidence limited for CFLD, perhaps mainly due to a lack of basic 2007;42:1173-80. disease, which may range from minor biochemical liver of portal hypertension and hypersplenism but prior to knowledge of the clinical and genetic underpinnings for 8. Robberecht E, Van Biervliet S, Vanrentergem K, Kerremans I. Outcome abnormalities to focal biliary fibrosis or multilobular cir­ the development of end-stage liver disease (7). Portal of total splenectomy with portosystemic shunt for massive splenomegaly its cause. The current key questions include: identifying and variceal bleeding in cystic fibrosis. J Pediatr Surg 2006;41:1561-5. rhosis with portal hypertension and chronic liver failure. hypertension and variceal bleeding have most commonly those patients who may benefit from bile-focused thera­ 9. Pozler O, Krajina A, Vanicek H, et al. Transjugular intrahepatic Advanced liver disease is most common in adolescents been used as indications for LT. However, the long-term pies (e.g., UDCA or other bile acid-related therapies, portosystemic shunt in five children with cystic fibrosis: long-term and young adults with CF, with an overall incidence impact of variceal bleeding on mortality was reported to 15); appropriate management and treatment of PHTN; results. Hepatogastroenterology 2003;50:1111-4. of 4 to 10 percent of all CF patients (1,2). The rate of be low in a small cohort of patients (8), raising ques­ 10. Jonas MM. The role of liver transplantation in cystic fibrosis re­ and the identification of those who would benefit from examined. Liver Transpl 2005;11:1463-5. clinical decompensation, however, is difficult to predict tions on whether portal hypertension, even complicated surgical interventions and transplantation. 11. Melzi ML, Kelly DA, Colombo C, et al. Liver transplant in cystic and leads to challenges in timing of therapies, including by variceal bleeding, is the right indicator for LT (9). fibrosis: a poll among European centers. A study from the European liver transplantation (LT). True decompensated disease References: Liver Transplant Registry. Transpl Int 2006;19:726-31. with manifestations of hepatic insufficiency is a very late Recently, children with cystic fibrosis who were enrolled 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. manifestation; often, complications of portal hyperten­ in the Studies of Pediatric Liver Transplantation (SPLIT) 1. di Sant’Agnese PA, Blanc WA. A distinctive type of biliary cirrhosis 13. Barshes NR, DiBardino DJ, McKenzie ED, et al. Combined lung and sion (hypersplenism, cytopenias, ascites, and variceal registry were compared to non-CF patients in terms of the liver associated with cystic fibrosis of the pancreas: recognition liver transplantation: the United States experience. Transplantation through signs of portal hypertension. Pediatrics 1956;18:387-409. hemorrhage) are the predominant and clinically relevant of pre- and post-liver transplant (LT) outcomes (10). 2005;80:1161-7. 2. Colombo C, Russo MC, Zazzeron L, Romano G. Liver disease in morbidity. Optimal timing of LT in CF is complicated Sixty-two CF patients in the SPLIT registry were listed 14. Faro A, Shepherd R, Huddleston CB, et al. Lower incidence of cystic fibrosis. J Pediatr Gastroenterol Nutr 2006;43:S49-55. bronchiolitis obliterans in pediatric liver-lung transplant recipients with by the systemic nature of the disease process—in for LT, and neither waitlist deaths nor the probability of 3. Brigman C, Feranchak A. Liver involvement in cystic fibrosis. Curr Treat Options Gastroenterol 2006;9:484-96. cystic fibrosis. Transplantation 2007;83:1435-9. particular, the progressive pulmonary disease, which is death from time of listing was different from non-CF 4. Beuers U. Drug insight: Mechanisms and sites of action of urso­ 15. Trauner M, Fickert P, Halilbasic E, Moustafa T. Lessons from the toxic exacerbated by nutritional problems. Often, patients children. Overall, 2,702 children underwent LT in the deoxycholic acid in cholestasis. Nat Clin Pract Gastroenterol Hepatol bile concept for the pathogenesis and treatment of cholestatic liver who are stable enough from a pulmonary perspective do study period. Forty-two CF patients (1.6%) underwent 2006;3:318-28. diseases. Wien Med Wochenschr 2008;158:542-8. not have clear indications for a liver transplant, and by LT, with an average follow-up of 3 yrs (0-10 yrs). the time a liver transplant is indicated, the pulmonary Transplanted patients had a mean PELD of 5.4 disease may have progressed and precludes LT. (86.5% had a PELD < 10), and a median age of 11.9 yrs (0.7 to 17.5). Initial concerns about LT in CF centered around the potential infectious complications in the lung in the set­ Post-LT, the 1-, 3-, and 5-yr patient survival in CF ting of immunosuppression, with the potential for rapid patients was 92.9, 88.6, and 88.6 percent, compared to decline in pulmonary function. Fortunately, since initial 90.8, 88.0, and 86.0 percent in non-CF patients. Graft case reports of LT for CF (3,4), single center experience survival in CF patients was 85.0, 81.2, and 81.2 percent has been favorable. Fridell et al. (5) reported on 12 chil­ at 1, 3, and 5 yrs compared to 85.6, 80.7, and 78.1 dren transplanted for CF with 1- and 5-year survival of percent. Rejection rates were not different (50.6% CF 92 and 75 percent, respectively. There were five deaths, vs. 56.4% non-CF at 5 yrs). Total re-operations at a mean interval of 6.8 ± 6.3 years. All of these deaths (< 30 day) were less (21.4 versus 38%; p = 0.03). were related to pulmonary disease. Pulmonary function However, renal complications (< 30 day) were increased did not worsen but improved or remained stable in eight (14.3 vs. 6.1%; p = 0.04), with 50 percent of these of nine patients tested, likely as a result of improved patients requiring dialysis. Standardized height and nutritional status and better pulmonary mechanics. weight scores showed no improvement over 2 years of

26 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 27 Speaker Abstracts Speaker Abstracts

Liver Transplantation in Children Biomarkers as Tools for With Cystic Fibrosis: Early Disease Detection Status and Unanswered Questions Continued follow-up in the CF patients (height Z 1.4 at Tx to 1.4 References: Nicole Mayer Hamblett, Ph.D. warranted to establish their association with known at 2 yrs, weight Z 1.2 to 1.5), but tended to improve in markers of disease. There are many study designs with Research Assistant Professor, Department of Pediatrics, the non-CF group (height Z 1.3 at Tx to 0.8 at 2 yrs, 1. Colombo C, Battezzati PM, Crosignani A, Morabito A, Costantini D, which to demonstrate this association, with the main Padoan R, et al. Liver disease in cystic fibrosis: a prospective study on University of Washington, Seattle, WA; Director, Biostatis- weight Z 1.0 to 0.1). It is of note that the nutritional incidence, risk factors, and outcome. Hepatology 2002;36(6):1374-1382. tics and Clinical Data Management, Cystic Fibrosis Thera- designs focusing on: 1) the objective to demonstrate improvement seen in smaller single center reports (11) 2. Hansen K, Horslen S. Metabolic liver disease in children. Liver Transpl peutics Development Network, Seattle Children’s Hospital, cross-sectional correlation between the biomarker and were not seen in this initial SPLIT analysis; however, 2008;14(4):391-411. Seattle, WA 3. Cox KL, Ward RE, Furgiuele TL, Cannon RA, Sanders KD, Kurland either existing or future disease, or 2) the objective to there was not the decline in nutritional status that is G. Orthotopic liver transplantation in patients with cystic fibrosis. demonstrate longitudinal correlation between changes in often seen in patients with CF and severe liver disease. Pediatrics 1987;80(4):571-574. the biomarker and progression of disease. These studies 4. Mieles LA, Orenstein D, Teperman L, Podesta L, Koneru B, Starzl TE. Developing biomarkers as predictors for disease is a provide important proof-of-concept during the bio­ Liver transplantation in cystic fibrosis. Lancet 1989;1(8646):1073. multifaceted problem requiring collaboration among These data suggest several unresolved areas of clinical 5. Fridell JA, Bond GJ, Mazariegos GV, Orenstein DM, Jain A, Sindhi R, marker development process by confirming hypothesized clinicians, scientists, and biostatisticians. In the setting understanding that will benefit from additional inves­ et al. Liver transplantation in children with cystic fibrosis: a long- mechanisms of action of the biomarker in relation to the term longitudinal review of a single center’s experience. J Pediatr Surg of Cystic Fibrosis (CF) liver disease, it is an even more tigation. These critical focus areas include optimizing disease process. Importantly, these correlation studies are 2003;38(8):1152-1156. challenging problem because of limited patient popula­ liver allocation with potential alternatives to PELD in 6. Molmenti EP, Squires RH, Nagata D, Roden JS, Molmenti H, Fasola focused on population-level results, and not all correlates CG, et al. Liver transplantation for cholestasis associated with cystic tions and an evolving, non-standardized definition of children with CF, better methods for determining those of disease in a population make reliable predictors of fibrosis in the pediatric population. Pediatr Transplant 2003;7(2):93-97. the disease (1,2). Despite this challenge, there are several patients most likely to decompensate so that early LT 7. Melzi ML, Kelly DA, Colombo C, Jara P, Manzanares J, Colledan M, disease within an individual. would be warranted, and the role of surgical options et al. Liver transplant in cystic fibrosis: a poll among European centers. strategies that can be employed to make progress in bio­ A study from the European Liver Transplant Registry. Transpl Int marker development, and the key to this progress will be such as splenectomy (12,13), splenic embolization, and 2006;19(9):726-731. Biomarkers that have been established as correlates of portal hypertensive surgery in the management of these 8. Gooding I, Dondos V, Gyi KM, Hodson M, Westaby D. Variceal to implement these strategies in a unified and consistent disease are those for which the most rigorous of evalua­ patients prior to or in lieu of LT. hemorrhage and cystic fibrosis: outcomes and implications for liver manner across the research field. tions should occur, and for which examples already exist transplantation. Liver Transpl 2005;11(12):1522-1526. 9. Jonas MM. The role of liver transplantation in cystic fibrosis re­ in the setting of CF liver disease (4). These evaluations Additional issues to be addressed in this challenging examined. Liver Transpl 2005;11(12):1463-1465. It is often tempting to start biomarker development are necessary for the translation of a biomarker from patient population include the indications for multi- 10. Christopher D. Anderson JAL, Turmelle YP, Anand R, Song C, focused on the end goal, which in this case is to evaluate Shepherd RW, McDiarmid SV. The SPLIT Research Group. Excel­ a correlate of disease in the population to a predictor organ transplant, including LT-pancreas, heart-lung-liver lent outcome after liver transplantation for cystic fibrosis in children. the biomarker as a diagnostic predictor for early liver of disease in an individual patient. The most common or lung-liver, and multivisceral transplantation, as well Accessed at SPLIT Web site https://webemmescom/study/lvr/ April 24, disease. While it is important to stay focused on the end 2009 [Abstract] 2008. framework for evaluating a biomarker as a predictor of as changes in the best clinical management once the CF 11. Colombo C, Costantini D, Rocchi A, Romano G, Rossi G, Bianchi goal, and even necessary in some cases to elicit funding disease is based on methodology for diagnostic testing patient reaches adulthood. Finally, optimizing outcomes ML, et al. Effects of liver transplantation on the nutritional status of opportunities, there are several principal characteristics procedures, which includes the computation of familiar in the post-LT period is critical, with focus needed on patients with cystic fibrosis. Transpl Int 2005;18(2):246-255. of a biomarker that should not be overlooked in the 12. Louis D, Duc ML, Reix P, Chazalette JP, Durieu I, Feigelson J et al. measures such as sensitivity, specificity, and positive and improving nutritional and growth outcomes, minimizing Partial splenectomy for portal hypertension in cystic fibrosis related liver evaluation process. These include, but are not limited negative predictive values. This framework provides a significant morbidity such as renal complications, proper disease. Pediatr Pulmonol 2007;42(12):1173-1180. to, establishing the marker as both repeatable within a blueprint for evaluating biomarkers in the CF liver dis­ pulmonary management post LT, and analysis of the 13. Linnane B, Oliver MR, Robinson PJ. Does splenectomy in cystic fibrosis related liver disease improve lung function and nutritional status? patient and reproducible across centers and laboratories. ease setting, with the exception that there will be unique effects of LT on pulmonary disease progression. A case series. Arch Dis Child 2006;91(9):771-773. The terms repeatability and reproducibility often are challenges to address, including the application of this used interchangeably but in fact point to different framework to a small patient population and to a disease sources of variability in the biomarker. Repeatability cap­ for which there is no gold standard diagnosis (5). A suc­ tures the inherent biological variation within a patient, cessful strategy will build awareness of these challenges whereas reproducibility captures variability due to the and set both the standards and the expectations that will measurement process. Biomarkers that satisfy these basic ultimately define how these biomarkers will be identified criteria have manageable variation within patients, which and utilized as tools for early disease detection. directly impacts their accuracy and precision for predict­ ing disease within an individual patient (3). References:

Candidate biomarkers that have been established 1. Sokol RJ, Durie PR. Recommendations for management of liver and biliary tract disease in cystic fibrosis. Cystic Fibrosis Foundation as robust, reliable measures of biologic activity within Hepatobiliary Disease Consensus Group. J Pediatr Gastroenterol Nutr. a patient are those for which future studies are 1999;28 Suppl 1:S1-13.

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Biomarkers as Tools for Emerging Techniques for Developing Early Disease Detection Novel Serum Biomarkers

Continued

2. Colombo C, Russo MC, Zazzeron L, Romano G. Liver disease in cystic Keyur Patel ways of fibrogenesis that may occur prior to appreciable fibrosis. J Pediatr Gastroenterol Nutr. 2006 Jul;43 Suppl 1:S49-55. changes in liver histology. For example, gene expression 3. Mayer-Hamblett N, Ramsey BW, Kronmal RA. Advancing outcome Assistant Professor in Medicine, Division of Gastroenterol- measures for the new era of drug development in cystic fibrosis. Proc ogy, Duke Clinical Research Institute and Duke University studies using microarray platforms in fibrosis have Am Thorac Soc. 2007 Aug 1;4(4):370-7. Medical Center, Durham, NC noted upregulation of various genes related to hepatic 4. Pereira TN, Lewindon PJ, Smith JL, Murphy TL, Lincoln DJ, Shepherd RW, Ramm GA. Serum markers of hepatic fibrogenesis in cystic fibrosis stellate cell activation, signal transduction, and immune liver disease. J Hepatol. 2004 Oct;41(4):576-83. response. A genome-wide association study that evaluated 5. Hui SL, Zhou XH. Evaluation of diagnostic tests without gold standards. Stat Methods Med Res. 1998 Dec;7(4):354-70. Over the last decade, there has been considerable over 24,000 single nucleotide polymorphisms (SNPs) in progress in our understanding of the various biological chronic hepatitis C-infected patients subsequently devel­ pathways leading to liver fibrosis in response to injury. oped an algorithm based on a seven-SNP signature that We now recognize that fibrosis is a dynamic process identified the risk of developing cirrhosis (4). Integration mediated by factors that regulate matrix synthesis and of gene expression profiles with functional protein expres­ degradation. Clinical studies have indicated that suc­ sion is important for determining biological function and cessful treatment of the underlying source of injury has relevance. Mass spectrometry (MS) proteomics to develop led to regression of even advanced stages of fibrosis (1). blood protein signatures for diagnostic purposes continues However, a cross-sectional biopsy sample is unable to to evolve rapidly (5). However, there are significant reflect matrix turnover, and the semi-quantitative histo­ challenges with blood proteome analysis. There are likely logic grading used in practice does not reflect linearity millions of polypeptides spanning at least 10 orders of of fibrosis deposition. These categorical grading scores magnitude, with only a small proportion that have been were developed to standardize and improve observer identified and quantified to date (6,7). At this stage, variability, and provide some assessment of the severity 2D-gel or matrix-assisted laser desorption/ionization of chronic liver injury that could be used to determine time-of-flight (MALDI-ToF) methods for serum pro­ thresholds for therapy (2). Currently available nonin­ teomic analysis in CHC patients with fibrosis have not vasive serum marker panel algorithms include simple provided a significant improvement over existing serum inflammatory markers and extracellular matrix proteins biomarkers for differentiating fibrosis stages (8,9). DNA that were developed to separate mild from advanced his­ sequencer-based assessment of the serum protein-linked tological grades. These marker panels were not designed N-glycans also has been evaluated to assess fibrosis and to assess the dynamic nature of fibrogenesis or follow appears promising (10). However, issues relating to cost, longitudinal changes in disease. As such, these nonin­ reproducibility, and failure to identify proteins of interest vasive tests appear to have only limited clinical utility for targeted high-throughput approaches have limited in the cross-sectional differentiation between mild and their utility. Quantitative protein profiling techniques severe fibrosis in a proportion of patients (3). Accurate continue to evolve, with improved separation methods, staging of fibrosis remains important for guiding therapy, accurate mass and time peptide tagging, and high resolu­ providing prognosis, and following disease progres­ tion electrospray ionization (ESI) liquid-chromatography sion in chronic liver disease patients. This requires the (LC) MS techniques for complex samples such as serum. development of reliable noninvasive alternatives to liver Future application of targeted MS-based metabolite pro­ biopsy. The sequencing of the human genome and the filing also may provide an integrated view of the disease subsequent advent of high-throughput technologies and process. The integration of clinical, functional gene and the integrated systems biology approach have provided protein expression data remains a significant bioinfor­ an opportunity to further improve our understanding matics challenge but will be important for improving of the fibrogenesis pathway. This will potentially help to our understanding of complex biological processes and develop a molecular signature that may be more disease translation of novel biomarker signatures of fibrosis into specific and reflect early alterations in biological path­ clinical practice.

30 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 31 Speaker Abstracts Speaker Abstracts

Emerging Techniques for Developing Biomarkers of Cystic Novel Serum Biomarkers Fibrosis Liver Disease

Continued

References: Ross W. Shepherd, M.D.1,2, Grant Ramm, Ph.D.2, In 40 children (age 8.4 ± x yr, followed 3-8 yrs) with and Peter Lewindon, M.D., F.R.A.C.P.2 suspected CFLD, subject to dual pass liver biopsy with 1. Poynard T, McHutchison J, Manns M, Trepo C, Lindsay K, Goodman 1 blinded staging of liver fibrosis, more patients with Z, Ling MH, et al. Impact of pegylated interferon alfa-2b and ribavirin Division of Gastroenterology, Hepatology and Nutrition, on liver fibrosis in patients with chronic hepatitis C. Gastroenterology Department of Pediatrics, Washington University School fibrosis were documented using two passes (P = 0.045), 2002;122:1303-13. of Medicine, St. Louis, MO; 2Hepatic Fibrosis Group, there being non-concordance for Scheuer Fibrosis 2. Standish RA, Cholongitas E, Dhillon A, Burroughs AK, Dhillon AP. Queensland Institute of Medical Research, Brisbane, Scores in 35 percent of biopsy pairs. Clinical exam An appraisal of the histopathological assessment of liver fibrosis. Gut Queensland, Australia 2006;55:569-78. (sensitivity = 0.66, specificity = 0.62, PPV = 0.87, 3. Rockey DC, Bissell DM. Noninvasive measures of liver fibrosis. Hepatol­ NPV = 0.31), ALT (sensitivity = 0.69, specificity = 0.37, ogy 2006;43:S113-20. 4. Huang H, Shiffman ML, Friedman S, Venkatesh R, Bzowej N, Abar Optimal evaluation of patients with clinically significant PPV = 0.81, NPV = 0.23), HIDA scan OT, Rowland CM, et al. A 7 gene signature identifies the risk of CF liver disease (CFLD) is unclear: clinical hepato­ (sensitivity = 0.71, specificity = 0.0, PPV = 0.77, developing cirrhosis in patients with chronic hepatitis C. Hepatology NPV = 0.0), and US (sensitivity = 0.80, specificity = 0.5, 2007;46:297-306. splenomegaly and elevated transaminases are non-specific 5. Diamond DL, Proll SC, Jacobs JM, Chan EY, Camp DG 2nd, Smith or late. Many patients escape detection until signs of PPV = 0.86, NPV = 0.4) were poor predictors of liver RD, Katze MG. HepatoProteomics: applying proteomic technologies to cirrhosis develop. Although commonly used, screen­ fibrosis. The development of portal hypertension over the study of liver function and disease. Hepatology 2006;44:299-308. time was associated with higher stage of fibrosis on 6. States DJ, Omenn GS, Blackwell TW, Fermin D, Eng J, Speicher DW, ing ultrasound (US) and isotope scintigraphy (HIDA) Hanash SM. Challenges in deriving high-confidence protein identifica­ have not been subject to rigorous analysis in predicting biopsy and a younger age of CFLD onset (Cox propor­ tions from data gathered by a HUPO plasma proteome collaborative CFLD. Invasive liver biopsy might be considered the tional hazards model, P = 0.002). study. Nat Biotechnol 2006;24:333-8. 7. Hanash SM, Pitteri SJ, Faca VM. Mining the plasma proteome for gold standard, although early lesions are focal, raising cancer biomarkers. Nature 2008;452:571-9. issues of sampling error and interobserver variability. Of serum total bile acids, hyaluronic acid (HA), collagen 8. Morra R, Munteanu M, Bedossa P, Dargere D, Janneau JL, Paradis V, type IV (CL-IV), prolyl hydroxylase (PH), matrix Ratziu V, et al. Diagnostic value of serum protein profiling by SELDI­ The challenge thus exists to develop sensitive, reliable, TOF ProteinChip compared with a biochemical marker, FibroTest, for and preferably noninvasive biomarkers to identify CF metalloproteinases (MMPs), and tissue inhibitors of the diagnosis of advanced fibrosis in patients with chronic hepatitis C. patients who are at risk of developing clinically sig­ MMPs (TIMPs), only levels of PH, CL-IV, and TIMP-1 Aliment Pharmacol Ther 2007;26:847-58. were higher in CFLD (n = 36) versus both CfnoLD 9. White IR, Patel K, Symonds WT, Dev A, Griffin P, Tsokanas N, Skehel nificant CFLD and to assess progress of liver injury in M, et al. Serum proteomic analysis focused on fibrosis in patients with patients with CFLD. (n = 30) and controls (n = 39), P < 0.001. The fibrosis hepatitis C virus infection. J Transl Med 2007;5:33. score was negatively correlated with TIMP-1 10. Vanderschaeghe D, Laroy W, Sablon E, Halfon P, Van Hecke A, Delanghe J, Callewaert N. GlycoFibroTest: DNA sequencer-based Studies of basic mechanisms of liver fibrogenesis in (r = −0.34, P = 0.06) and PH (r = −0.48, P = 0.008). serum protein glycomics yields a highly performant liver fibrosis CFLD perhaps provide a basis for biomarker study Receiver-operating characteristics analysis showed biomarker. Mol Cell Proteomics 2009 Jan 29 [Epub ahead of print]. (1-4): The cholangiocyte is the only cell type in the CL-IV (AUC 0.785, P < 0.0001) and TIMP-1 liver that expresses the cystic fibrosis transmembrane (AUC 0.765, P < 0.0001) differentiated CFLD conductance regulator (CFTR) protein. In our liver from CfnoLD and controls, while PH biopsy studies (1,2), focal peribiliary fibrosis mediated (AUC 0.814, P < 0.0001) predicted early fibrogenesis. by induction of Hepatic Stellate Cell (HSC) collagen Diagnostic accuracy improved using logistic regression gene expression appears to be a key early event (1,2). combining i) CL-IV, TIMP-1, PH to identify CFLD We have proposed that altered cholangiocyte transport (AUC 0.831, P < 0.0001) and ii) TIMP-1, PH to and altered bile flow in CF result in upregulation of key identify CFLD patients with no fibrosis HSC-responsive chemokines, which spill to the sinu­ (AUC 0.852, P < 0.02). soidal blood supply, thus having a paracrine effect on HSC chemotaxis (2). The role of steatosis, which occurs From these detailed but limited prospective cohort in one-third of biopsies, is unknown. Over time, active studies, we conclude that clinical, biochemical, and fibrogenesis, along the expanding scar interface, leads to US evaluations for CFLD without dual pass biopsy are multilobular biliary cirrhosis, events associated with elevated insensitive and non-specific for early CFLD fibrosis; they serum levels of bile acids, chemokines, collagen proteins, detect significant liver fibrosis/cirrhosis with reasonable and components of hepatic matrix remodeling (2-4). sensitivity, but lack specificity. Liver fibrosis correlates

32 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 33 Speaker Abstracts Speaker Abstracts

Biomarkers of Cystic Ultrasound and CT Imaging of Fibrosis Liver Disease Liver Fibrosis and Other Disease

Continued with occurrence of portal hypertension. Three of seven Potential Conflict of Interest: Nothing to report. Marilyn J. Siegel, M.D. higher in cirrhosis than in fibrosis and control subjects. selected serum fibrosis markers (CLIV, PH, and TIMP) Nontriphasic flow is also more likely in cirrhosis than Professor of Radiology and Pediatrics, Mallinckrodt were found to discriminate CFLD from no CFLD, and Acknowledgments: This project was funded by Institute of Radiology, Washington University School of fibrosis. Other studies have shown that the splenic artery two of seven serum fibrosis markers (PH and TIMP) the National Health and Medical Research Council Medicine, St. Louis, MO pulsatility increases in fibrosis (9). were found to discriminate fibrosis severity. Early diag­ (NHMRC) of Australia; Grant 290220, Australian Steatosis: nosis of CFLD still requires liver biopsy, enhanced by Cystic Fibrosis Research Trust, Royal Children’s Hospital Ultrasonography (US) US findings of diffuse steatosis include: dual pass and specialized stains. US clearly lacks sensitiv­ Foundation, Brisbane, Australia; Grant 913-003, Senior hepatomegaly, increased parenchymal echogenicity (i.e., Research Fellowship from the NHMRC of Australia; and ity. Selective noninvasive serum markers are deserving of Fibrosis: US findings of fibrosis are a heterogeneous “bright” liver sign), and poor penetration of the sound Grant 241913. wider application and prospective serial study in a larger echo pattern, either diffuse or in a periportal distribution beam, resulting in poor visualization of intrahepatic ves­ cohort of CFLD patients. (1,2). In a single center study, 67 percent of patients sels, the posterior liver, and the diaphragm. The adjacent References: with cystic fibrosis with a heterogeneous echo pattern renal cortex becomes much less echogenic than liver. Future Research Challenges and Needs: progressed to have US features of cirrhosis, and 46 The US sensitivity is 90 percent and the specificity is 93 1. Lewindon PJ, Pereira TN, Hoskins AC, Bridle KR, Williamson RM, percent for diagnosing moderate-to-severe diffuse fatty Shepherd RW, Ramm GA. The role of hepatic stellate cells and trans­ percent progressed to portal hypertension (2). In another • Should liver biopsy be performed routinely as part of forming growthfactor-beta(1) in cystic fibrosis liver disease. Am J Pathol. series comparing US and liver biopsy in children with infiltration (> 30% hepatocytes with fat vacuoles). Focal 2002 May;160(5):1705-15. steatosis commonly involves the gallbladder fossa in the a CFLD study (with development of disease-specific 2. Ramm GA, Shepherd RW, Hoskins AC, Greco SA, Ney AD, Pereira cystic fibrosis, an abnormal US predicted moderate-to­ definitions of fibrosis for use in clinical research)? TN, Bridle KR, Doecke JD, Meikle PJ, Turlin B, Lewindon PJ. Fibro­ severe liver disease with a specificity of 84 percent, but periportal area, falciform ligament, medial segment of genesis in pediatric cholestatic liver disease: role of taurocholate and a normal US did not preclude significant liver fibrosis the left lobe, and subcapsular regions. Focal steatosis is hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell usually geographic or fingerlike and well-defined and can • Because we do not seem to be able to rely on US, recruitment. Hepatology. 2009 Feb;49(2):533-44. (3). The sensitivity of US to detect normal histology other more specific imaging techniques such as 3. Pereira TN, Lewindon PJ, Smith JL, Murphy TL, Lincoln DJ, Shepherd was 57 percent, with a specificity of 65 percent (3). mimic fibrosis. DFMRI or elastography require development. RW, Ramm GA. Serum markers of hepatic fibrogenesis in cystic fibrosis Approximately 60 percent of patients with normal US liver disease. J Hepatol. 2004 Oct;41(4):576-83. Computed Tomography 4. Smith JL, Lewindon PJ, Hoskins AC, Pereira TN, Setchell KD, had fibrosis. US detection of early liver disease remains a • Reproducible markers of “early cystic fibrosis liver O’Connell NC, Shepherd RW, Ramm GA. Endogenous ursodeoxycholic challenging problem. acid and cholic acid in liver disease due to cystic fibrosis. Hepatology. Computed tomography (CT) has been used to study disease” identified in these studies require prospective 2004 Jun;39(6):1673-82. serial study in a larger cohort of CFLD patients. 5. Mueller-Abt PR, Frawley KJ, Greer RM, Lewindon PJ. Comparison of Cirrhosis: The US findings of cirrhosis are a nodular parenchymal liver disease, but its use of ionizing radia­ ultrasound and biopsy findings in children with cystic fibrosis related hepatic surface and coarse echotexture. Based on these tion is a major disadvantage, particularly in children who liver disease. J Cyst Fibros. 2008 May;7(3):215-21. findings, the sensitivity of US to detect marked fibrosis need repeated studies. or cirrhosis (METAVIR 3 or 4 score) is approximately 55 percent, with a specificity of 85 percent to 95 percent (3,4). Fibrosis: In a study by Akata et al. using US and CT US diagnosis of portal hypertension is based on findings of in 50 CF patients, increased hepatic echogenicity was cirrhosis along with findings of splenomegaly, thickened detected in 24 of 48 (50%) (10). US showed increased omentum, collateral circulation, and ascites. US sensitiv­ periportal echogenicity in 18 of 24 patients, whereas CT ity for diagnosis of portal hypertension is 80 percent to could not demonstrate any sign of increased periportal 90 percent (5,6). thickness. Findings of cirrhosis were present in five patients with both modalities. Significant correlations have been found between Doppler measurements of hepatic arterial resistance and Cirrhosis: CT features of cirrhosis include decreased pulsatility indices, hepatic vein waveforms, and blood hepatic size, heterogeneous parenchyma, nodular hepatic flow velocity in the portal vein in studies of adults margins, regenerating nodules, and atrophy of the right (p < 0.5) (7,8). Portal vein velocity is lower in the hepatic lobe and medial segment of the left lobe, with cirrhosis group than in the fibrosis group (7,8), whereas relative enlargement of the caudate lobe and left lateral hepatic artery resistive index and pulsatility index are segment.

34 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 35 Speaker Abstracts Speaker Abstracts

Ultrasound and CT Imaging of MRI Methods of Assessing Liver Fibrosis and Other Disease Liver Fibrosis and Disease

Continued

Steatosis: Diffuse steatosis manifests as decreased Claude B. Sirlin, M.D. (the diffusion sensitivity parameter or the b-value of the attenuation value on unenhanced CT scans. Focal sequence). Studies using fat-suppressed (or water-excited) Liver Imaging Group, Department of Radiology, University hepatic steatosis can be patchy, segmental, or lobar, with of California, San Diego, La Jolla, CA DW echo planar imaging have shown reduced ADC in margins that are well-circumscribed or ill-defined. The cirrhosis of various etiologies. These observations have sensitivity of CT for diagnosis of steatosis is nearly 100 led investigators to postulate that liver fibrosis restricts percent. Overview: Several promising MRI methods are in water diffusion, and investigators now are studying development for non-invasive assessment of liver fibrosis. whether DW imaging can be used to stage liver fibrosis References: These are outlined below and will be discussed in the non-invasively (3). presentation. All MRI methods for liver fibrosis assess­ 1. Patriquin H, Lenaerts C, Smith L, et al. Liver disease in children with ment are experimental. cystic fibrosis: US-biochemical comparison in 195 patients. Radiology Tissue Property: Perfusion. Perfusion imaging assesses 1999;211:229-32. flow alterations associated with chronic liver disease. 2. Lenaerts C, Lapierre C, Patriquin H, et al. Surveillance for cystic Experimental MRI Techniques for Liver Fibrosis: Progressive deposition of collagen and other macro­ fibrosis-associated hepatobiliary disease: early ultrasound changes and These can be classified broadly into those that assess predisposing factors. J Pediatr 2003;143:343-50. molecules within the extracellular matrix of the liver 3. Mueller-Abt PR, Frawley KJ, Greer RM, Lewindon PJ. Comparison of structure (morphology and texture) and those that assess gradually obliterates the space of Disse, narrows the ultrasound and biopsy findings in children with cystic fibrosis related tissue properties (apparent diffusion coefficient, perfu­ liver disease. J Cyst Fibros 2008;7:215-21. sinusoidal lumen, and causes sinusoisal basement 4. Colli A, Fraquelli M, Andreoletti M, Marino B, Zuccoli E, Conte D. sion, elastic modulus). membrane formation with loss of endothelial fenestra­ Severe liver fibrosis or cirrhosis: accuracy of US for detection—analysis tions. These abnormalities slow the passage of blood of 300 cases. Radiology 2003;227:89-94. Structure: Morphology. The cirrhotic liver develops 5. Aube C, Winkfield B, Oberti F, et al. New Doppler ultrasound signs through the parenchyma, elevate intrahepatic resistance, improve the non-invasive diagnosis of cirrhosis or severe liver fibrosis. characteristic morphological alterations visible on and cause portal venous blood to be shunted to systemic Eur J Gastroenterol Hepatol 2004;16:743-51. cross-sectional imaging. These alterations are features 6. Gaiani S, Bramantieri L, Venturoli N, et al. What is the criterion for veins with lower pressures. The microvascular architec­ differentiating chronic hepatitis from compensated cirrhosis? A prospec­ of advanced disease. They have high specificity but low ture of the liver remodels in response, with development tive study comparing ultrasonography and percutaneous liver biopsy. J sensitivity for cirrhosis and so are not appropriate for of abnormal connections between the portal vein and Hepatol 1997;27:979-85. 7. Annet L, Materne R, Danse E, Jamart J, Horsmans Y, Van Beers BE. early fibrosis detection or fibrosis staging. terminal hepatic vein, which contribute to the shunting Hepatic flow parameters measured with MR imaging and Doppler US: of blood from the hepatic parenchyma. As portal flow correlations with degree of cirrhosis and portal hypertension. Radiology Structure: Texture. Recent studies show that liver texture 2003;229:409-14. to the liver diminishes, there is compensatory hepatic 8. Haktanir A, Cihan BS, Celenk C, Cihan S. Value of Doppler sonog­ at MR imaging may be altered in advanced fibrosis arterial vasodilation, with subsequent elevation of the raphy in assessing the progression of chronic viral hepatitis and in the and cirrhosis and that MR imaging can directly depict hepatic arterial fraction. These flow alterations can be 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 advanced liver fibrosis as a meshwork of linear reticula­ evaluated non-invasively with contrast-enhanced perfu­ significant hepatic fibrosis in hepatitis C virus carriers with persistently tions and non-tumoral bands. Importantly, contrast sion imaging (4). normal alanine aminotransferases. Liver Int 2006;26:1087-94. agents can improve visualization of textural alterations 10. Akata D, Akhan O, Ozcelik U, et al. Hepatobiliary manifestations of cystic fibrosis in children: correlation of CT and US findings. J Radiol caused by liver fibrosis (1,2). Clinical trials of contrast- Tissue Property: Elastic Modulus. MR elastography 2002;41:26-33. enhanced MRI for non-invasive staging of liver fibrosis (MRE) is an MR imaging technique that non-invasively in HCV and NAFLD are in progress. quantifies the elastic modulus (or stiffness) of liver as well as other organs by analyzing the propagation of Tissue Property: Apparent Diffusion Coefficient. Diffusion- mechanical waves through tissue. The premise of this weighted (DW) imaging assesses the diffusion of protons technique for assessing liver fibrosis is that progressive within tissue and is quantified by the apparent diffusion deposition of a lattice-like framework of interconnecting coefficient (ADC). DW imaging uses motion-sensitizing collagen fibers throughout the liver increases paren­ gradients that cause diffusing protons to lose signal. chymal elastic modulus. Hence, measuring liver elastic The amount of signal loss is influenced by the ability modulus may provide non-invasive information on the of protons to diffuse through tissue (the ADC of the severity of fibrosis (5). Because liver elastic modulus tissue), as well as the strength of the diffusion weighting cannot be reliably assessed by external physical palpation,

36 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 37 Speaker Abstracts Speaker Abstracts

MRI Methods of Assessing Elastography to Assess Liver Fibrosis and Disease Liver Fibrosis and Cirrhosis

Continued an imaging approach is required. There are two main Future Research Challenges and Needs: Detlef Schuppan, M.D., Ph.D. Furthermore, in adult patients they permit only a crude imaging methods to measure hepatic elastic modulus. differentiation between no to mild and moderate to severe Division of Gastroenterology, Beth Israel Deaconess One is ultrasound-based transient elastography. The There are overlapping research challenges and needs. Medical Center and Harvard Medical School, Boston, MA fibrosis (Metavir stages 0-1 vs. 2-4, respectively) (1,2). other is MRE, discussed in this presentation. These will need to be pursued in parallel. Conventional or contrast ultrasound, and conventional Conclusion: Several promising MRI techniques for 1. Refine and standardize MRI techniques. The development of cirrhosis is a central hard endpoint MRI or CT, do not allow assessment of liver fibrosis, assessing liver fibrosis are under development. No MRI 2. Test MRI techniques prospectively in children with in the natural history of patients with chronic liver dis­ except for the diagnosis of advanced cirrhosis. method yet has been validated in large-scale, multi­ known or suspected CFLD. eases, including children with cystic fibrosis liver disease center trials as accurate, robust, or reproducible for 3. Include clinical outcome measures in the study  (CFLD). In this vein, advanced fibrosis and fibrosis Transient elastography (Fibroscan, Echosense , France) assessment of liver fibrosis across the entire spectrum of design. progression are accepted surrogates for the development is a novel, noninvasive, bedside method to assess liver disease severity from early fibrosis to cirrhosis. Thus, no 4. Disseminate negative as well as positive results of of cirrhosis and liver failure and other complications of fibrosis by measuring hepatic stiffness. An intercostally MRI method is yet suitable as a non-invasive surrogate studies. cirrhosis. There has been striking progress in our under­ placed probe transmits low-amplitude shear waves endpoint for fibrosis severity in clinical trials or obser­ 5. Identify disease-specific targets for imaging for standing of the biochemistry and cell biology of liver through the liver, the velocity of which is picked up by vational studies; a definitive assessment of fibrosis stage future technical development. fibrosis and cirrhosis. This includes numerous agents and an integrated ultrasound device (pulse-echo ultrasound). still requires a biopsy reference standard. Moreover, most 6. Recognize that biopsy may be an imperfect refer­ emerging strategies that prevent fibrosis progression or Shear wave velocity is inversely correlated with liver MRI methods in development assess liver fibrosis indi­ ence standard for MRI approaches. even induce reversal of established fibrosis and cirrhosis. stiffness. Stiffness is automatically calculated as the rectly and therefore may be confounded by numerous However, effective therapeutic intervention has essentially mean of 10 measurements and expressed as kPa. This variables, most of which may not be directly measurable. References: been limited to rodent models of liver fibrosis (1). methodology is highly attractive, since it is painless and Confounding variables may include age, gender, and without risk, only takes 5 min, and can be performed etiology of liver disease. Thus, there is a need to test 1. Aguirre DA, Behling CA, Alpert E, Hassanein TI, Sirlin CB. Liver fibrosis: noninvasive diagnosis with double contrast material-enhanced Translation into the clinic requires improved noninvasive in a fairly standardized way after only a short training MRI techniques in age-, gender-, and etiology-specific MR imaging. Radiology 2006;239(2):425-437. markers or techniques that permit the measurement of period. Furthermore, ultrasound elastography measures a studies. To the author’s knowledge, no experimental 2. Hughes-Cassidy F, Chavez AD, Schlang A, et al. Superparamagnetic hepatic fibrosis, and in particular an assessment of the mechanical quality of the liver related to fibrosis, other MRI technique for liver fibrosis has yet been studied in iron oxides and low molecular weight gadolinium chelates are synergistic for direct visualization of advanced liver fibrosis. J Magn Reson Imaging dynamic parameters of fibrogenesis and fibrolysis (de than current imaging techniques or serum markers, and children with cystic fibrosis. Finally, liver biopsy with 2007;26(3):728-737. novo deposition or removal of excess extracellular matrix, when compared to biopsy samples a 100-fold larger conventional histology staging of liver fibrosis may not 3. Taouli B, Tolia AJ, Losada M, et al. Diffusion-weighted MRI for quanti­ fication of liver fibrosis: preliminary experience. AJR Am J Roentgenol respectively). This is necessary since liver biopsy has an volume (4 × 1 cm, ∼1/500 of the liver). be an ideal reference standard for MRI-based methods, 2007;189(4):799-806. inherent sampling error, and progression from stage 0 as these assess the liver volumetrically, sample all or most 4. Hagiwara M, Rusinek H, Lee VS, et al. Advanced liver fibrosis: diag­ to stage 4 (cirrhosis) usually takes 20-30 years, even in More than 100 clinical studies correlating ultrasound of the liver, and are quantitative. nosis with 3D whole-liver perfusion MR imaging—initial experience. Radiology 2008;246(3):926-934. patients classified as fast progressors (1). In addition, elastography with liver biopsy have appeared since its 5. Rouviere O, Yin M, Dresner MA, et al. MR elastography of the liver: first description in 2003 (3), including meta-analyses preliminary results. Radiology 2006;240(2):440-448. biopsy is risky and cannot be repeated on a regular basis. (4-7). Almost all have been performed in adults and For practical purposes, especially for children with have shown a good correlation of stiffness values with CFLD, who are at risk of developing cirrhosis at an the histological stage of fibrosis. Area under receiver early age, even the limited option to reliably diagnose operating curves (that reflect the diagnostic precision in advanced fibrosis or incipient cirrhosis would be highly select populations) yielded excellent accuracy for differ­ useful. This could permit stratification of patients: entiating cirrhosis from non-cirrhosis (AUROC between 1) according to their risk of progression to (advanced) 0.90 and 0.99), especially for patients with chronic cirrhosis, 2) to assess the need for confirmation by liver hepatitis C. However, with the practical but crude biopsy, and 3) for inclusion in clinical trials aimed at histological Metavir staging system (with F1 = mild, inhibiting disease progression. periportal fibrosis, and F4 = cirrhosis), there is high overlap between the lower stages, only part of which can The current serum markers and serum marker algo­ be attributed to biopsy sampling variability. In addition, rithms have not been validated in children with CFLD. severe acute inflammation and mechanical cholestasis

38 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 39 Speaker Abstracts

Elastography to Assess Liver Fibrosis and Cirrhosis

Continued can significantly increase hepatic stiffness (7-9), thus patients who are in need of (antifibrotic) therapies to confounding the fibrosis readout. prevent further progression to (decompensated) cirrhosis. Furthermore, it is quick and easy to perform, with low Of note, different etiologies of liver disease require differ­ interobserver variability. The technology should be used ent cutoffs for the diagnosis of cirrhosis (e.g., a cutoff of to screen for children or adults with significant (fibrotic) 12 kPa in chronic hepatitis C and of 17.3 kPa in biliary CFLD, and to stratify patients for inclusion in thera­ fibrosis). peutic studies that are aimed at inhibition of fibrosis progression or at fibrosis reversal. Only a few studies have been performed in children. In a small study of children with nonalcoholic steatohepa­ References: titis (n = 52), ultrasound elastography demonstrated an outstanding prediction of all stages of fibrosis (AUROC 1. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet 2008;371:838-51. 2. Parkes J, Guha IN, Roderick P, Rosenberg W. Performance of serum 0.992 to 1.0), but these results need confirmation in a marker panels for liver fibrosis in chronic hepatitis C. J Hepatol larger cohort (10). In a larger cohort of 116 children 2006;44:462-74. poster Abstracts with various chronic liver diseases, the technique diag­ 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. nosed histological cirrhosis with an AUROC of 0.88, Transient elastography: a new noninvasive method for assessment of superior to serum marker algorithms (Fibrotest and hepatic fibrosis. Ultrasound Med Biol 2003; 29:1705-13. APRI score, both with an AUROC of 0.73) (11). 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 transi­ de Lédinghen et al. also showed the usefulness of a ent elastography (FibroScan): a prospective study. Gut 2006;55:403-8. novel, smaller probe adapted to the small intercostal 5. Friedrich-Rust M, Ong MF, Martens S, Sarrazin C, Bojunga J, Zeuzem S, Herrmann E. Performance of transient elastography for the staging of space of children (11). liver fibrosis: a meta-analysis. Gastroenterology 2008;134:960-74. 6. Castera L, Forns X, Alberti A. Non-invasive evaluation of liver fibrosis MR elastography is based on similar principles as using transient elastography. J Hepatol 2008;48:835-47. 7. Coco B, Oliveri F, Maina AM, Ciccorossi P, Sacco R, Colombatto ultrasound elastography, but studies are few and include P, Bonino F, Brunetto MR. Transient elastography: a new surrogate low numbers of patients. A larger generator transmits marker of liver fibrosis influenced by major changes of transaminases. J the shear waves, which are recorded with a 1.5 Tesla Viral Hepat 2007;14:360-9. 8. Arena U, Vizzutti F, Corti G, Ambu S, Stasi C, Bresci S, Moscarella S, MR scanner (12,13). Advantages are examination of the Boddi V, Petrarca A, Laffi G, Marra F, Pinzani M. Acute viral hepatitis whole liver, an even lower observer dependency, and increases liver stiffness values measured by transient elastography. applicability in patients with severe obesity or nar­ Hepatology 2008;47:380-4. 9. Millonig G, Reimann FM, Friedrich S, Fonouni H, Mehrabi A, Büchler row intercostal spaces (which can preclude ultrasound MW, Seitz HK, Mueller S. Extrahepatic cholestasis increases liver elastography). A disadvantage is the more restricted avail­ stiffness (FibroScan) irrespective of fibrosis. Hepatology 2008;48:1718-23. ability. Importantly, acquisition times can be shortened 10. Nobili V, Vizzutti F, Arena U, Abraldes JG, Marra F, Pietrobattista A, Fruhwirth R, Marcellini M, Pinzani M. Accuracy and reproducibility tenfold (20 min to 2 min) by use of echo-planar versus of transient elastography for the diagnosis of fibrosis in pediatric spin-echo sequences, without compromise of the stiffness nonalcoholic steatohepatitis. Hepatology 2008;48:442-8. 11. de Lédinghen V, Le Bail B, Rebouissoux L, Fournier C, Foucher J, Miette readout (12). Hepatic inflammation and mechanical V, Castéra L, Sandrin L, Merrouche W, Lavrand F, Lamireau T. Liver cholestasis are expected to be confounders, as with stiffness measurement in children using FibroScan: feasibility study and ultrasound elastography, but studies are lacking. 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 Although studies in children with CFLD are lacking, Beers BE. MR elastography of liver fibrosis: preliminary results compar­ ultrasound elastography is expected to be a screening ing spin-echo and echo-planar imaging. Eur Radiol 2008;18:2535-41. 13. Talwalkar JA, Yin M, Fidler JL, Sanderson SO, Kamath PS, Ehman tool of choice, since it will permit non-invasive detec­ RL. Magnetic resonance imaging of hepatic fibrosis: emerging clinical tion of advanced fibrosis. This will allow selection of applications. Hepatology 2008;47:332-42.

40 Cystic Fibrosis Liver Disease | Clinical Research Workshop Poster Abstracts

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

Molly Bozic1, Jean Molleston2, M. Howenstine3, and pancreatic endocrine and exocrine function, as well as J. Fridell4 nutritional status, before and after simultaneous liver- Departments of 1Pediatrics, 2Pediatric Gastroenterology, pancreas transplant. 3Pediatric Pulmonology, and 4Surgery, Indiana University School of Medicine, Indianapolis, IN Results: Three female patients, ages 13, 14, and 22 years, with cystic fibrosis underwent simultaneous liver- pancreas transplants. Prior to transplant, all three had Background: Cystic fibrosis (CF), an autosomal cirrhosis resulting in portal hypertension with variceal recessive disorder affecting multiple organ systems, is bleeding, and two had some degree of encephalopathy the most common genetic disease among the Caucasian and ascites. Each patient had pancreatic insufficiency population. The CFTR mutation can adversely affect with poor growth and malnutrition in spite of pancreatic hepatic function in cystic fibrosis patients. Up to 10 enzyme supplementation. Insulin-dependent diabetes was percent of CF patients will develop cirrhosis, often with present in all. Patients are currently 1½, 5, and 6 years severe portal hypertension. Cystic fibrosis liver disease post transplant. (CFLD) is the third most common cause of death in CF. Liver transplant has been successful in improving Combined transplantation was tolerated well in these the outcomes of a number of CF patients with severe patients, with length of stay during initial hospitalization liver disease. Pancreatic insufficiency is a hallmark of of 14-21 days. One year after liver/pancreas transplant, cystic fibrosis and results in poor growth and malnutri­ each patient showed improvement in hepatic, pancreatic, tion. Pancreatic insufficiency and CF-related diabetes and pulmonary function as well as nutritional status. (CFRD) can negatively impact pulmonary function. All three patients were free of diarrhea and gained Insulin-dependent diabetes in CF patients is known to weight without pancreatic enzyme supplementation. result in increased mortality and morbidity compared to These patients demonstrated weight gains of 5-11 kg nondiabetic CF patients. Since CFLD occurs solely in in the years after transplant. Endocrine function of the the context of pancreatic disease, cirrhosis can present pancreas was maintained, as each patient demonstrated in individuals with severe malabsorption and diabetes. normal blood glucose levels without the use of insulin. Simultaneous liver-pancreas transplantation has been Hgb A1c prior to transplant in two patients ranged proposed to address the severe hepatic dysfunction, from 7.3 to 9.7, with post-transplant levels improving to nutritional compromise, and hyperglycemia in diabetic 4.5-5.5 in these two patients. Platelet counts normalized, pancreatic-insufficient CF patients under one immu­ and clinical symptoms of portal hypertension resolved nosuppressive regimen. We report here the impact of with continued normal bilirubin and liver enzymes and simultaneous liver-pancreas transplants on hepatic, low-normal albumin. There were no episodes of rejec­ pulmonary, and pancreatic function in three patients tion. Pulmonary function tests 1 year post transplant with CF. improved in patient A (FVC up 5%; FEV1 up 12%), remained relatively stable in patient B (FVC up 2%; Methods: A retrospective chart review of three patients FEV1 down 1%), and improved in patient C (FVC up with CF who underwent liver-pancreas transplant was 11%; FEV1 up 6%). One of the three continued to performed using the electronic medical record, Port CF, require frequent hospitalizations for pulmonary infec­ and OTTR transplant database. Data were gathered tions. All three attended school or worked full-time after to describe hepatic function, pulmonary function, and transplant.

Cystic Fibrosis Liver Disease | Clinical Research Workshop 43 Poster Abstracts Poster Abstracts

Long-term Effect of Simultaneous Liver-Pancreas Paracrine Induction of Sulfotransferase 1E1 in HepG2 Transplant on Hepatic, Pulmonary, and Pancreatic Hepatocytes During Co-culture With CFTR-deficient Function in Three Cystic Fibrosis Patients MMNK-1 Cholangiocytes Continued

1 1 1 Conclusions: Simultaneous liver/pancreas transplants Charles N. Falany , Dongning He , Li Li , Teresa W. in well plates. After 24 hr, the Transwell inserts with 2 3 in three cystic fibrosis patients with CFLD and CFRD Wilborn , and Melissa Runge-Morris the MMNK-1 cells were placed in the wells containing resulted in improved pancreatic endocrine function as 1Department of Pharmacology and Toxicology, University the HepG2 cells and left for 8-24 hr. The inserts were evidenced by normalization of blood glucose levels with­ of Alabama at Birmingham, Birmingham, AL; 2Department removed, and the HepG2 cells assayed for SULT1E1 out exogenous insulin treatment. Improved nutritional of Physiological Sciences, Samford University, Birming- expression and activity. SULT1E1, but not SULT1A1, 3 status and improved weight gain without pancre­ ham, AL; Institute of Toxicology, Wayne State University, Detroit, MI SULT2A1, or CYP3A4 expression, was induced 15-fold atic enzyme supplementation demonstrated improved when cultured with CFTR-deficient MMNK-1 cells pancreatic exocrine function as well. Liver disease was as compared to control cells. These results indicate a improved, as evidenced by resolution of signs/symptoms Although pulmonary disease is the major pathology in factor is secreted by the CFTR-deficient MMNK-1 cells of portal hypertension. Pulmonary function was not Cystic Fibrosis (CF), the loss of CF transmembrane that acts in a paracrine manner on the expression of adversely affected. Simultaneous liver-pancreas transplant receptor (CFTR) activity results in significant pathologies SULT1E1 in the HepG2 cells. appears to be a satisfactory option for CF patients with in other tissues. In the liver, CFTR is expressed in intra­ CFLD and CFRD. hepatic epithelial cells or cholangiocytes, forming the To identify the paracrine factor involved in the bile ducts as well as the gallbladder. Most patients with induction of SULT1E1, expression profiling of the a severe form of CF have biliary transport problems, CFTR-deficient and control MMNK-1 cells was car­ steatosis, and focal biliary cirrhosis, and a subgroup of ried out. RNA was isolated from the cells and used patients develops cholestasis and cirrhosis, requiring liver for expression profiling. One pathway significantly transplantation. altered in the CFTR-MMNK-1 cells involved choles­ terol biosynthesis. Desmosterol (DES) reductase and In CFTR-deficient mice, the activity of hepatic sul­ 7-dehydrocholesterol (7DHC) reductase expression were fotransferase (SULT) 1E1 is induced up to 100-fold. significantly decreased in the CFTR-deficient cells. DES

SULT1E1 has a Km of 4 nM for the sulfation of and 7DHC are oxysterols and the immediate precur­ β-estradiol (E2) and is the major mechanism for the sors to cholesterol. Oxysterols are the physiological inactivation of E2 at physiological concentrations. The regulators of the liver-x-receptors (LXR). Treatment of induced SULT1E1 activity alters the expression of the HepG2 cells with DES, 7DHC, and T0901317 E2-regulated proteins in CFTR-mouse liver. Similar induced SULT1E1 expression. These data suggest that results are observed in human HepG2 hepatocytes with the loss of CFTR activity in MMNK-1 cells results in increased SULT1E1 activity. Increased SULT1E1 activ­ increased levels of oxysterols that act as paracrine factors ity in HepG2 cells also inhibits the ability of growth to activate LXRα in HepG2 cells and thereby induce hormone to stimulate IGF-1 synthesis by decreasing the SULT1E1 expression. In liver, LXRα acts as an oxysterol activation of STAT5b. sensor to regulate cholesterol levels by reverse transport. Inappropriate LXR activation may also have a role in the To investigate whether the loss of CFTR expression development of steatosis observed in many CF patients in human cholangiocytes induces the expression of as well as the disruption of E2 regulatory properties in SULT1E1 in hepatocytes, a membrane separated the liver. co-culture system was developed. Human MMNK-1 cholangiocytes express CFTR but not SULT1E1. CFTR Acknowledgment: This work was supported by the expression in the MNMNK-1 cells was repressed by University of Alabama at Birmingham Cystic Fibrosis more than 85 percent using siRNA and the cells plated Research Center and by a grant to Charles N. Falany in Transwell inserts. HepG2 hepatocytes were plated from CFRI, Inc.

44 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 45 Poster Abstracts Poster Abstracts

Quantitative Imaging of Biliary Fibrosis Progression Cystic Fibrosis Related Diabetes (CFRD) in CF

Using a Small Molecule αv β 6 Integrin Ligand With Nano- Patients With Cirrhosis molar Affinity

1 2 2 Yury Popov , Preeti Misra , Kumar Ranjan Bhushan , have spontaneous progressive biliary fibrogenesis due to Kara Sullivan, A. Moran, B. Holm, Results: Forty-five of 527 cystic fibrosis patients 1 1 Deanna Y. Sverdlov , Nezam H. Afdhal , John V. cholangiocyte activation and were compared to nonfi­ and S.J. Schwarzenberg (8.5%) were considered to have cirrhosis and were select­ Frangioni2,3, and Detlef Schuppan1 brotic wildtype mice. Minnesota Cystic Fibrosis Center (MCFC), University of ed for review. Thirty of the 45 had diabetes (66.7%) Divisions of 1Gastroenterology and 2Hematology/Oncology, Minnesota, Minneapolis, MN and were alive as of September 15, 2008. Age range was 3 Department of Medicine, and Department of Radiology, Results: The trimerized tracer displayed fourfold 1-65 years old. Most cases of CFRD in patients with Beth Israel Deaconess Medical Center/Harvard Medical 4 cirrhosis were found in patients over 20 years old. School, Boston, MA increased affinity over the monomer (Bmax 2.6 × 10 receptors for α -positive HT-29 cells). In vivo biodis­ Background: CFRD occurs in many patients with v6 Data Analysis: A chi-square test using SAS software tribution in normal and fibrotic mice showed that the cystic fibrosis (CF), and its incidence increases with age. Background: Quantitative imaging of liver fibrosis or At our center, a previous study of 527 patients with CF was constructed. It demonstrated a significant difference compound cleared from the blood and most non-target fibrogenesis has been impossible. α β is a fibronectin/ revealed a prevalence of CFRD in 33 percent of patients, in the rate of CFRD in patients with cirrhosis, com­ v 6 tissues with an alpha half-life of 23-45 min and was tenascin receptor whose expression is highly restricted with 45 percent of patients over 40 years old affected. pared to CF patients without cirrhosis (p < .0001). The and found only on activated cholangiocytes/progenitor excreted efficiently via the kidneys. Liver uptake at Approximately 10 percent of CF patients have cirrhosis. odds ratio was 4.788 (95% CI 2.49, 9.17). cells that correlate with and are instrumental in hepatic 2 h was 1.5 ± 0.3 percent of the injected dose per gram, The purpose of this study is to determine if CF patients α β fibrogenesis. Its tissue levels are highest in active biliary and the v 6-based imaging agent showed a two- to with cirrhosis are more likely to have CFRD. Conclusions: CFRD is more common in CF patients fibrosis, but correlate with fibrosis progression regardless threefold enhanced uptake in the fibrogenic as compared with cirrhosis compared to the overall CF population of the underlying liver disease. Therefore, high affinity to wildtype nonfibrotic between 2 and 20 h after injec­ Methods: A retrospective case control study of the (p < .0001). Patients with CF and cirrhosis have 4.8 MCFC database was performed. All data were collected times the odds of having CFRD. This may reflect a αvβ 6-specific peptidomimetics are attractive candidates tion. Retention of the imaging agent in the liver could as of September 15, 2008. Patients with cystic fibrosis common pathogenic pathway. The presence of both for hepatic fibrosis/fibrogenesis imaging. be correlated with αvβ 6 expression. SPECT-CT verified prominent in vivo localization over the fibrotic liver. and outpatient platelet counts less than 150,000 on at complications presents a challenge in managing these Methods: A peptidomimetic small molecule ligand least two occasions that were 3 months apart were used patients. Our data, derived using a surrogate marker of Specificity of αvβ 6-targeted imaging was confirmed by α β as a surrogate for cirrhosis. The patient’s diabetic status cirrhosis, suggest the need for a more direct examination of the v 6-integrin (EMD527040) with low nanomo­ blocking of the enhanced signal when MDR2KO mice was identified through the results of routine annual oral of the interaction of CF liver disease and CFRD. lar affinity and no relevant crossreactivity with other received a prior injection of a 103-fold surplus of the glucose tolerance screening of the CF population and integrins was modified by introducing a six-carbon nonlabeled α β -trimer. linker, followed by trimerization on an adamantane v 6 a carefully maintained CFRD database. Patient age was scaffold. This targeting molecule was converted into a also collected. Conclusions: 1) A high affinity and high specificity radiotracer by conjugation with the pre-labeled metal fibrosis/fibrogenesis imaging agent was created based on chelator [Tc-99m-MAS3]-NHS. Homologous competi­ Table 1. CF patients followed at the MCFC as of September 15, 2008. trimerization of an α β -selective peptidomimetic small tion with nonradioactive Re derivatives of the compound v 6 was performed on HT-29 (α β integrin positive) and molecule ligand. 2) Our in vivo data suggest that with Percent Percent Cirrhosis and Percent Cirrhosis v 6 Age (yr) All CF CFRD Cirrhosis minor increase of affinity and more effective background Sample Cirrhotic CFRD and CFRD Huh-7 (αvβ 6 integrin negative) epithelial cell lines. Biodistribution, clearance and specific label accumulation suppression, noninvasive quantification of liver fibrosis/ were quantified in 8-week-old Mdr2(abcb4)-/- mice that fibrogenesis by molecular imaging will be possible. < 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

33 8.5 66.7 Total 527 172 45 30 (172/527) (45/527) (30/45)

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

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 Objective: The selected marker/s identified in the pilot Karnsakul, and Kathleen B. Schwarz study will be sensitive and specific to distinguish CFLD Division of Pediatric Gastroenterology and Nutrition, Johns cirrhosis versus CF patients without liver disease and Hopkins University, Baltimore, MD versus those with severe lung disease.

Methods: We propose to conduct a pilot study at Background: Cystic fibrosis liver disease (CFLD) is Johns Hopkins Hospital, one of the CF Centers that the third leading cause of death in cystic fibrosis (CF) is part of the CFLD-Research Consortium, to validate after pulmonary and lung transplant-related complica­ the Enhanced Liver Fibrosis (ELF) Test® (HA, TIMP1, tions. The prevalence has been reported between 4 and NP3P) and determine if CF patients with advanced 24 percent, with cirrhosis in approximately 15 percent. fibrosis (U/S grade 4-5) can be clearly distinguished Although liver biopsy is the current gold standard of from two groups: CF pulmonary disease (CFPD) identifying fibrosis, it has a high sampling error, with a (FEV1 < 40% predicted) and age/sex, FEV1-matched 24 percent false negative rate. There is a great need to controls, without CFPD, and with normal physical participants List identify noninvasive markers of liver fibrosis (NIMLF). exam, normal liver enzymes, and normal liver U/S. One of the problems in trying to identify such a marker in this special population is that, as we have previously Results: Sample size calculation: shown, healthy infants and children exhibit increased circulating cytokines, such as transforming growth factor CF with no fibrosis versus CF with cirrhosis beta 1 (TGFβ1), used as NIMF in adults. In addition, ELF scores: 7.92 ± 0.44 (no fibrosis) the CF patients often have pulmonary fibrosis. As we 11.08 ± 0.07 (cirrhosis) have demonstrated previously, these patients also exhibit Type I error 0.05 and Type II error 0.8 increased plasma TGFβ1. Noninvasive methods of SD = 1.2 diagnosing degree of fibrosis have not been systematically Δ = 0.6 (change in outcome) studied in large groups of children with cystic fibrosis; n = 10 however, these have been studied in various disease entities, such as hepatitis B, hepatitis C, primary scleros­ Conclusion: A sensitive and specific NIMLFCF would ing cholangitis (PSC), non-alcoholic fatty liver disease obviate the need for liver biopsy, would help identify (NAFLD), and alcoholic liver disease (ALD). These patients at risk for disease progression, and ideally could methods include ultrasound (U/S) identification of a be applied in future studies of antifibrotic therapies in heterogeneous parenchyma with a grading system of 0-5. CF patients. If the ELF test shows acceptable positive As well, various serological assays have included direct predictive value (PPV) and negative predictive value and indirect markers of fibrosis. The indirect markers of (NPV) in our pilot, then we would propose to test it in fibrosis, including AST, ALT, and platelets, are non­ the larger cohort of the Cystic Fibrosis Network. specific, whereas the direct markers of fibrosis, such as hyaluronic acid (HA), tissue inhibitors of metalloprotein­ Funding: NIH, F32 submitted; Johns Hopkins, T32 ase (TIMP-1), amino terminal fragment of procollagen submitted; CFLD, grant supplement. III (P3NP), and procollagens type IV and VI, have higher sensitivity and specificity.

48 Cystic Fibrosis Liver Disease | Clinical Research Workshop Participants List

Adil Abdalla, M.D. Ashok Batta, Ph.D. Clinical Fellow Bioanalytical Chemist Liver Diseases Branch Digestive Care, Inc. National Institute of Diabetes and Digestive 1120 Win Drive and Kidney Diseases Bethlehem, PA 18017 National Institutes of Health Telephone: (610) 882-5950 Building 10, Room 4-5722 Email: [email protected] 10 Center Drive, MSC 1614 Bethesda, MD 20892-1614 Gerri Bickerton Telephone: (301) 451-1273 Registered Nurse Email: [email protected] Warren Grant Magnuson Clinical Center National Institutes of Health Lisa Allwein Building 10, Room 9C317 Registered Nurse 10 Center Drive, MSC 1664 Pediatric Pulmonary Department Bethesda, MD 20892-1664 Penn State Milton S. Hershey Medical Center Telephone: (301) 496-9090 500 University Drive Email: [email protected] Hershey, PA 17033 Telephone: (814) 952-1669 Lynelle Boamah, M.D. Email: [email protected] Pediatric Gastroenterologist Pediatric Subspecialty Natalia Argel Naval Medical Center San Diego Pulmonary Research Nurse Coordinator 34800 Bob Wilson Drive Phoenix Children’s Hospital San Diego, CA 92131 Building B Telephone: (619) 532-9720 1919 E. Thomas Road Email: [email protected] Phoenix, AZ 85016 Telephone: (602) 546-0343 Drucy Borowitz, M.D. Email: [email protected] Professor of Clinical Pediatrics Department of Pediatrics Dawn Baker, M.S.N. State University of New York at Buffalo Advanced Registered Nurse Practitioner Women and Children’s Hospital of Buffalo Department of Pediatrics 219 Bryant Street University of Massachusetts Medical School Buffalo, NY 14222 Biotech 5, Suite 250 Telephone: (716) 878-7524 381 Plantation Street Email: [email protected] Worcester, MA 01605 Telephone: (508) 334-2339 Molly Bozic, M.D. Email: [email protected] 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]

Cystic Fibrosis Liver Disease | Clinical Research Workshop 51 Participants List Participants List

Rebekah Brown, M.D. Michael D’Amico, M.D. Charles Falany, Ph.D. Marc Ghany, M.D. Clinical Fellow Pediatric Gastroenterologist Professor Staff Physician Department of Pediatrics Department of Pediatrics Department of Pharmacology Liver Diseases Branch Division of Pediatric Allergy, Immunology, Vermont Children’s Hospital and Toxicology National Institute of Diabetes and Digestive and Pulmonary Medicine Smith 553 University of Alabama at Birmingham and Kidney Diseases Vanderbilt Children’s Hospital 111 Colchester Avenue Volker Hall G133M National Institutes of Health 11215 DOT Burlington, VT 05401 1670 University Boulevard Building 10, Room 9B-16 2200 Children’s Way Telephone: (802) 847-5670 Birmingham, AL 35216 10 Center Drive, MSC 1800 Nashville, TN 37232-9500 Email: [email protected] Telephone: (205) 934-9848 Bethesda, MD 20892-1800 Telephone: (615) 343-7617 Email: [email protected] Telephone: (301) 402-5115 Email: [email protected] Pamela DeMorrow, M.S., C.P.N.P. Email: [email protected] Pediatric Pulmonology Andrew Feranchak, M.D. Karen Callahan, M.S. Kalamazoo Center for Medical Studies Associate Professor Vita Goei, M.D. Research Nurse Manager Michigan State University Department of Pediatrics Assistant Professor Pediatric Pulmonary 1000 Oakland Drive University of Texas Southwestern Pediatric Gastroenterology Johns Hopkins University Kalamazoo, MI 49008 Medical Center Children’s Subspecialty Group 200 N. Wolfe Street, Suite 3074 Telephone: (269) 337-6476 5323 Harry Hines Boulevard Children’s Hospital of the King’s Daughters Baltimore, MD 21287 Email: [email protected] Dallas, TX 75390-9063 601 Children’s Lane Telephone: (443) 287-8983 Telephone: (214) 648-3118 Norfolk, VA 23507 Email: [email protected] Tania Diaz, M.D. Email: [email protected] Telephone: (757) 668-7240 4221 29th Street Email: [email protected] Maggi Campbell Mount Rainier, MD 20712 Steven Freedman, M.D. Marketing Manager Telephone: (301) 927-4240 Associate Professor of Medicine Gavin Graff, M.D. Digestive Care, Inc. Email: [email protected] Department of Medicine Professor of Pediatrics 1120 Win Drive Beth Israel Deaconess Medical Center Department of Pediatrics Bethlehem, PA 18017 Edward Doo, M.D. Dana 501 Penn State College of Medicine Telephone: (610) 882-5950 Program Director 330 Brookline Avenue H085 Email: [email protected] Liver Diseases Research Branch Boston, MA 02215 500 University Drive National Institute of Diabetes and Digestive Telephone: (617) 667-5576 Hershey, PA 17033 Daniel Caplan, M.D. and Kidney Diseases Email: [email protected] Telephone: (717) 531-5338 Professor of Pediatrics National Institutes of Health Email: [email protected] Department of Pediatrics Two Democracy Plaza, Room 651 Lynn Fukushima, M.S.N. Emory University School of Medicine 6707 Democracy Boulevard, MSC 5450 Cystic Fibrosis Research Coordinator Laura Grande, M.S. 2040 Ridgewood Drive, N.E. Bethesda, MD 20892-5450 Pediatric Pulmonology Pediatric/Cystic Fibrosis Dietitian Atlanta, GA 30322 Telephone: (301) 451-4524 Children’s Hospital Los Angeles, MS 83 Department of Nutrition Telephone: (404) 727-5728 Email: [email protected] 4650 Sunset Boulevard Children’s National Medical Center Email: [email protected] Los Angeles, CA 90027 111 Michigan Avenue, N.W. Peter Durie, M.D. Telephone: (323) 361-5950 Washington, DC 20010 Rubin Cohen, M.D. Professor and Senior Scientist Email: [email protected] Telephone: (202) 476-2218 Associate Professor of Medicine Research Institute Email: [email protected] Division of Pulmonary Medicine Hospital for Sick Children The Albert Einstein College of Medicine 555 University Avenue 410 Lakeville Road, Suite 107 Toronto, ON M5G 1X8 New Hyde Park, NY 11040 Canada Telephone: (516) 465-5400 Telephone: (416) 813-6185 Email: [email protected] Email: [email protected]

52 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 53 Participants List Participants List

Donna Griebel, M.D. Dustin Hays Douglas Holsclaw, M.D. Karen Jones Director Health Science Writer Director Project Manager Division of Gastroenterology Products Information Clearinghouses Adult Cystic Fibrosis Center Biometrics and Outcomes Research Core Center for Drug Evaluation and Research National Institute of Diabetes and Digestive University of Pennsylvania University of Michigan U.S. Food and Drug Administration and Kidney Diseases PennLung Center-PHI First Floor 1420 Washington Heights, M4523 White Oak Building, Room 5112 National Institutes of Health 39th and Market Streets Ann Arbor, MI 18109 22 New Hampshire Avenue 8280 Greensboro Drive, Suite 300 Philadelphia, PA 19104 Telephone: (737) 763-7738 Silver Spring, MD 20861 McLean, VA 22102 Telephone: (215) 662-8766 Email: [email protected] Telephone: (301) 796-4153 Telephone: (301) 204-3249 Email: [email protected] Email: [email protected] Email: [email protected] Binita Kamath, M.D. Jay Hoofnagle, M.D. Assistant Professor Nicole Mayer Hamblett, Ph.D. Theo Heller, M.D. Director Fellowship Training Program in Pediatric Director Investigator Liver Disease Research Branch Gastroenterology Biostatistics and Clinical Data Management Liver Diseases Branch National Institute of Diabetes and Digestive Division of Gastroenterology, Hepatology, Cystic Fibrosis Therapeutics Development National Institute of Diabetes and Digestive and Kidney Diseases and Nutrition Network and Kidney Diseases National Institutes of Health Children’s Hospital of Philadelphia 1100 Olive Way, Suite 500 National Institutes of Health Building 31, Room 9A27 34th Street and Civic Center Boulevard Seattle, WA 98101 Building 10, Room 9B16 31 Center Drive, MSC 2560 Philadelphia, PA 19103 Telephone: (206) 884-7547 10 Center Drive, MSC 1800 Bethesda, MD 20892-2560 Telephone: (215) 590-3247 Email: [email protected] Bethesda, MD 20892-1800 Telephone: (301) 496-1333 Email: [email protected] Telephone: (301) 496-1721 Email: [email protected] Koji Hara, M.D. Email: [email protected] Wikrom Karnsakul, M.D. Fellow Stephen James, M.D. Assistant Professor Liver Diseases Branch James Heubi, M.D. Director Department of Pediatrics National Institute of Diabetes and Digestive Professor and Associate Chair Division of Digestive Diseases and Nutrition Johns Hopkins University Hospital and Kidney Diseases Department of Pediatrics National Institute of Diabetes and Digestive 600 N. Wolfe Street, Brady 320 National Institutes of Health Cincinnati Children’s Hospital and Kidney Diseases Baltimore, MD 21287 Building 10, Room 9B06 General Clinical Research Center National Institutes of Health Telephone: (410) 502-9522 10 Center Drive, MSC 1800 3333 Burnet Avenue Two Democracy Plaza, Room 677 Email: [email protected] Bethesda, MD 20892-1800 Cincinnati, OH 45229 6707 Democracy Boulevard, MSC 5450 Telephone: (301) 443-7309 Telephone: (513) 636-8046 Bethesda, MD 20892-5450 Saul Karpen, M.D., Ph.D. Email: [email protected] Email: [email protected] Telephone: (301) 594-7680 Director, Liver Center Email: [email protected] Department of Pediatrics Beth Harkness, R.N. Pamela Hofley, M.D. Baylor College of Medicine Registered Nurse Section Chief Arthur Jarrett, M.D. One Baylor Plaza Cystic Fibrosis Center Section of Gastroenterology Director of Paediatrics Houston, TX 77030 Children’s National Medical Center and Hepatology Department of Paediatrics Telephone: (832) 824-3754 111 Michigan Avenue, N.W., Room #1030 Department of General Pediatrics Liverpool Hospital Email: [email protected] Washington, DC 22201 Dartmouth-Hitchcock Medical Center Liverpool Health Service Locked Bag 7103 Telephone: (202) 476-2215 One Medical Center Drive Liverpool BC Email: [email protected] Lebanon, NH 03756 Sydney, NSW 1871 Telephone: (603) 695-2745 Australia Email: [email protected] Telephone: 61-298285680 Email: [email protected]

54 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 55 Participants List Participants List

David Kleiner, M.D., Ph.D. Jalayne Lapke, M.D. George Mazariegos, M.D. David Meyerholz, D.V.M., Ph.D. Chief, Postmortem Section Associate Professor of Clinical Pediatrics Chief, Pediatric Transplantation Assistant Professor Laboratory of Pathology Department of Pediatrics Division of Transplant Surgery Department of Pathology National Cancer Institute University of Illinois College of Medicine Hillman Center for Pediatric Transplantation University of Iowa Carver College National Institutes of Health 320 E. Armstrong Avenue Children’s Hospital of Pittsburgh of UPMC of Medicine Building 10, Room 2B50 Peoria, IL 61603 Faculty Pavilion, Sixth Floor 200 Hawkins Drive, 1165ML 10 Center Drive, MSC 1500 Telephone: (309) 303-1236 45th Street and Penn Avenue Iowa City, IA 52242 Bethesda, MD 20892-1500 Email: [email protected] Pittsburgh, PA 15201 Telephone: (319) 353-4589 Telephone: (301) 594-2942 Telephone: (412) 692-6110 Email: [email protected] Email: [email protected] Susan Lehto, R.N. Email: [email protected] Cystic Fibrosis Clinic Suzanne Michel, M.P.H., R.D. Michael Knowles, M.D. Children’s Hospital Central California Melissa McClure Registered Dietitian Professor of Medicine 9300 Valley Children’s Place Pediatric Cystic Fibrosis Coordinator Department of Clinical Nutrition Division of Pulmonary/Critical Care Madera, CA 93636 Pediatric Pulmonology The Children’s Hospital of Philadelphia University of North Carolina at Chapel Hill Telephone: (559) 353-5550 Hershey Medical Center 358 Trevor Lane 7019 Thurston Bowles Building, CB 7248 Email: [email protected] 500 University Drive Bala Cynwyd, PA 19004 Chapel Hill, NC 27599-7248 Hershey, PA 17033 Telephone: (267) 426-2858 Telephone: (919) 966-6780 Simon Ling, M.D. Telephone: (717) 531-5338 Email: [email protected] Email: [email protected] Division of Gastroenterology, Hepatology, Email: [email protected] and Nutrition Mohamad Miqdady, M.D. Christopher Koh, M.D. The Hospital for Sick Children Debbie McCormick, R.N. Pediatric Gastroenterologist Clinical Hepatology Fellow 555 University Avenue Customer Service Manager Department of Pediatrics Liver Diseases Branch Toronto, ON M4E 1C3 Digestive Care, Inc. Sheikh Khalifa Medical City National Institute of Diabetes and Digestive Canada 1120 Win Drive P. O. Box 51900 and Kidney Diseases Telephone: (416) 813-7734 Bethlehem, PA 18017 Abu Dhabi National Institutes of Health Email: [email protected] Telephone: (610) 882-5950 United Arab Emirates Building 10, Room 4-5722 Email: [email protected] Telephone: 971-50-1234-283 10 Center Drive, MSC 1800 John Magee, M.D. Email: [email protected] Bethesda, MD 20892-1800 Associate Professor Catherine McKeon, Ph.D. Telephone: (301) 451-7009 Department of Surgery Senior Advisor for Genetic Research Paul Mohabir, M.D. Email: [email protected] University of Michigan National Institute of Diabetes and Digestive Outpatient Director, Cystic Fibrosis 2922 Taubman Center and Kidney Diseases Division of Pulmonary and Critical Debra Kroeker, R.N., C.P.N. 1500 E. Medical Center Drive National Institutes of Health Care Medicine Department of Pulmonology Ann Arbor, MI 48109 Two Democracy Plaza, Room 6103 Stanford University Medical Center Children’s Hospital Central California Telephone: (734) 936-9623 6707 Democracy Boulevard, MSC 5460 300 Pasteur Drive, H3143 9300 Valley Children’s Place Email: [email protected] Bethesda, MD 20892-5460 Stanford, CA 94305-5236 Madera, CA 93636 Telephone: (301) 594-8810 Telephone: (650) 804-4811 Telephone: (559) 353-5550 Bruce Marshall, M.D. Email: [email protected] Email: [email protected] Email: [email protected] Vice President of Clinical Affairs Cystic Fibrosis Foundation 6931 Arlington Road Bethesda, MD 20814 Telephone: (800) 344-4823 Email: [email protected]

56 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 57 Participants List Participants List

Jean Molleston, M.D. Joel Nations, M.D. Keyur Patel, M.D. Elizabeth Reid, M.S. Professor of Clinical Pediatrics Staff Physician Assistant Professor Clinical Nutrition Specialist Division of Pediatric Gastroenterology, Pulmonary Medicine Service Duke Gastroenterology/Hepatology Pediatric Pulmonology Hepatology, and Nutrition National Naval Medical Center Site Coordination Penn State Milton S. Hershey Medical Center Indiana University School of Medicine 8901 Wisconsin Avenue Duke Clinical Research Institute P.O. Box 850, HO 85 Riley Hospital for Children Bethesda, MD 20889 2400 Pratt Street 500 University Drive 702 Barnhill Drive, Room ROC 4210 Telephone: (301) 295-4217 Durham, NC 27705 Hershey, PA 17033 Indianapolis, IN 46202 Email: [email protected] Telephone: (919) 668-7186 Telephone: (717) 531-4679 Telephone: (317) 274-3774 Email: [email protected] Email: [email protected] Email: [email protected] Andrea Nepa, M.S. Clinical Dietitian Thankam Paul, M.D. Patricia Robuck, Ph.D., M.P.H. Veronique Morinville, M.D. Department of Clinical Nutrition Assistant Professor Director, Clinical Trials Program Assistant Professor of Pediatrics The Children’s Hospital of Philadelphia Department of Pediatrics Division of Digestive Diseases Division of Pediatric Gastroenterology 34th Street and Civic Center Boulevard Rhode Island Hospital and Nutrition and Nutrition Philadelphia, PA 19104 593 Eddy Street National Institute of Diabetes and Digestive Montreal Children’s Hospital Telephone: (215) 590-3217 Providence, RI 02903 and Kidney Diseases D-562 Email: [email protected] Telephone: (401) 258-1949 National Institutes of Health 2300 Tupper Street Email: [email protected] Two Democracy Plaza, Room 679 Montreal, QC H3H 1P3 Joe Palermo, M.D., Ph.D. 6707 Democracy Boulevard, MSC 5450 Canada Instructor Elizabeth Perkett, M.D. Bethesda, MD 20892-5450 Telephone: (514) 412-4474 Department of Pediatrics Professor Telephone: (301) 594-7789 Email: [email protected] Washington University in St. Louis Department of Pediatrics Email: [email protected] One Children’s Place Division of Pediatric Allergy, Immunology, Rosemary Nagy, M.B.A. St. Louis, MO 63110 and Pulmonary Medicine Rene Romero, M.D. Clinical Research Coordinator Telephone: (314) 454-6173 Vanderbilt Medical School Associate Professor Department of Pediatrics Email: [email protected] 11215 DOT Department of Pediatrics Washington University School of Medicine 2200 Children’s Way Emory University School of Medicine CB 8208 Shruti Paranjape, M.D. Nashville, TN 37232-9500 2015 Uppergate Drive 660 S. Euclid Avenue Assistant Professor of Pediatrics Telephone: (615) 343-7617 Atlanta, GA 30322 Saint Louis, MO 63110 Eudowood Division of Pediatric Respiratory Email: [email protected] Telephone: (404) 785-1832 Telephone: (314) 454-2295 Sciences Email: [email protected] Email: [email protected] Johns Hopkins University Peggy Radford, M.D. DM Rubenstein Child Health Building Director of Pulmonary Research Carl Rountree, M.D. Michael Narkewicz, M.D. 200 N. Wolfe Street Pediatric Pulmonary Assistant Professor Professor of Pediatrics Baltimore, MD 21287 Phoenix Children’s Hospital Department of Pediatrics and Division of Pediatric Gastroenterology, Telephone: (410) 502-5792 1919 E. Thomas Road Pharmacology Hepatology, and Nutrition Email: [email protected] Phoenix, AZ 85016 Penn State College of Medicine University of Colorado Denver School of Telephone: (602) 546-0985 500 University Drive, H085 Medicine and The Children’s Hospital Email: [email protected] Hershey, PA 17033 13123 E. 16th Avenue Telephone: (717) 531-5901 Aurora, CO 80045 Email: [email protected] Telephone: (720) 777-6669 Email: [email protected]

58 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 59 Participants List Participants List

Wael Sayej, M.D. Leonard Seeff, M.D. Claude Sirlin, M.D. Jeffrey Teckman, M.D. Fellow Senior Scientist Department of Radiology Associate Professor Pediatric Gastroenterology Liver Disease Research Branch University of California, San Diego Department of Pediatrics Digestive Diseases and Nutrition Center National Institute of Diabetes and Digestive 408 Dickinson Street, MC 8226 Division of Gastroenterology Women and Children’s Hospital of Buffalo and Kidney Diseases San Diego, CA 92103 and Hepatology 219 Bryant Street, Second Floor National Institutes of Health Telephone: (619) 471-0513 Cardinal Glennon Children’s Medical Center Buffalo, NY 14222 Building 31, Room 9A27 Email: [email protected] Saint Louis University Telephone: (201) 921-1312 31 Center Drive, MSC 2560 1465 S. Grand Boulevard Email: [email protected] Bethesda, MD 20892-2560 Ronald Sokol, M.D. St. Louis, MO 63104 Telephone: (301) 435-3338 Vice Chair of Pediatrics and Chief Telephone: (314) 577-5647 Detlef Schuppan, M.D., Ph.D. Email: [email protected] Gastroenterologist Email: [email protected] Professor Department of Gastroenterology Department of Medicine Girish Sharma, M.D. The Children’s Hospital/University Yumi Turmelle, M.D. Division of Gastroenterology Cystic Fibrosis Center Director of Colorado Denver Transplant Hepatologist Beth Israel Deaconess Medical Center Department of Pediatrics 13123 E. 16th Avenue, B290 Division of Pediatric Gastroenterology 330 Brookline Avenue Rush University Medical Center Aurora, CO 80045 and Nutrition Boston, MA 02215 1653 W. Congress Parkway, Suite 452 Telephone: (720) 777-6669 St. Louis Children’s Hospital Telephone: (617) 667-2371 Chicago, IL 60612 Email: [email protected] Washington University School of Medicine Email: [email protected] Telephone: (312) 563-2291 Campus Box 8116 Email: [email protected] Sean Sullivan, M.D. One Children’s Place Kathleen Schwarz, M.D. Pediatric Gastroenterologist St. Louis, MO 63110 Professor Ross Shepherd, M.D. Department of Pediatrics Telephone: (314) 454-6173 Department of Pediatrics Professor Naval Medical Center Portsmouth Email: [email protected] Johns Hopkins University School Department of Pediatrics 620 John Paul Jones Circle of Medicine Washington University School of Medicine Portsmouth, VA 23708 Hubert van der Doef, M.D. Brady 320 Queensland Institute for Medical Research Telephone: (757) 953-2645 Pediatric Gastroenterology 600 N. Wolfe Street One Children’s Place Email: [email protected] University Medical Center Utrecht Baltimore, MD 21287 St Louis, MO 63110 Postbox 85090, KE 04.133.1 Telephone: (410) 955-8769 Telephone: (314) 420-2553 Horst-Dietmar Tauschel, Ph.D. Utrecht 3508 AB Email: [email protected] Email: [email protected] Medical Sciences The Netherlands Dr. Falk Pharma GmbH Telephone: (31) 887555294 Sarah Jane Schwarzenberg, M.D. Marilyn Siegel, M.D. Leinenweberstrasse 5, PO Box 6529 Email: [email protected] Associate Professor Professor of Radiology and Pediatrics Freiburg D-79041 Department of Pediatrics Mallinckrodt Institute of Radiology Germany Alexandra Vasilescu, D.O. University of Minnesota Washington University Medical Center Telephone: 49-761-1514-127 Pediatric Gastroenterology Fellow MMC 185 510 S. Kingshighway Boulevard Email: [email protected] Pediatric Gastroenterology 420 Delaware Street, S.E. St. Louis, MO 63110 Johns Hopkins University Minneapolis, MN 55455 Telephone: (314) 454-6229 900 N. Wolfe Street Telephone: (612) 624-4669 Email: [email protected] Baltimore, MD 21287 Email: [email protected] Telephone: (410) 614-4621 Email: [email protected]

60 Cystic Fibrosis Liver Disease | Clinical Research Workshop Cystic Fibrosis Liver Disease | Clinical Research Workshop 61 Participants List

Xavier Villa, M.D., M.S.P.H. Patti Yanchuk Director Adult Cystic Fibrosis Program Coordinator Division of Gastroenterology, Hepatology, Pulmonary Medicine and Nutrition Penn State Milton S. Hershey Medical Center Department of Pediatrics Box 850, Mailcode H041 University of Texas Medical Branch 500 University Drive at Galveston Hershey, PA 17033 301 University Boulevard Telephone: (717) 531-6525 Houston, TX 77555-0352 Email: [email protected] Telephone: (713) 838-9687 Email: [email protected] Patrick Young, M.D. Director of Clinical Research Diana Wetmore, Ph.D. Department of Gastroenterology Vice President National Naval Medical Center Alliance Management Building 9, First Floor Cystic Fibrosis Foundation Therapeutics 8901 Wisconsin Avenue 6931 Arlington Road, Suite 200 Bethesda, MD 20889 Bethesda, MD 20814 Telephone: (301) 295-6044 Telephone: (301) 907-2535 Email: [email protected] Email: [email protected] ConTrACTor SupporT: notes Ronni Wetmore, M.S. Coordinator, Adult Cystic Fibrosis Center Denise Hoffman, CMP Pulmonary and Critical Care Associates The Scientific Consulting Group, Inc. LaVilla Medical Building, Suite 202 656 Quince Orchard Road, Suite 210 425 N. Lee Street Gaithersburg, MD 20878 Jacksonville, FL 32204 Telephone: (301) 670-4990 Telephone: (904) 318-8807 Email: [email protected] 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]

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