Hepatobiliary disease in cystic fibrosis patients with pancreatic sufficiency

Hepatobiliary Disease in Cystic Fibrosis Patients With Pancreatic Sufficiency DONNA L. WATERS,1 STUART F. A. DORNEY,1 MARGIE A. GRUCA,1 HUGH C. O. MARTIN,2 ROBERT HOWMAN-GILES,3 ALEX E. KAN,4 MERE DE SILVA,5 AND KEVIN J. GASKIN1

F o c a l a n d m u l t i l o b u l a r biliary cirrhosis are considered p a t h o g n o m o n i c o f cystic fibrosis (CF) a n d a l m o s t invariably h a v e b e e n r e p o r t e d in p a t i e n t s w i t h steatorrhea. In contrast, p a t i e n t s w i t h p a n c r e a t i c sufficiency a n d n o r m a l a b s o r p t i o n are c o n s i d e r e d less likely to dev e l o p liver or biliary tract problems. The a u t h o r s report t h r e e p a t i e n t s w i t h CF a n d p a n c r e a t i c sufficiency, pres e n t i n g w i t h r e c u r r e n t a b d o m i n a l pain (unrelated to pancreatitis). All h a d c o m m o n bile duct disease, o n e w i t h m u l t i l o b u l a r cirrhosis a n d portal h y p e r t e n s i o n . P a n c r e a t i c sufficiency w a s p r o v e n by quantitative pancreatic s t i m u l a t i o n tests, 3-day fecal fat analyses, a n d s e r u m p a n c r e a t i c i s o a m y l a s e s . All t h r e e p a t i e n t s h a d mild l u n g disease. T w o w e r e h o m o z y g o u s for t h e comm o n A F 5 0 8 m u t a t i o n , a n d t h e other, a AF508 c o m p o u n d h e t e r o z y g o t e . H e p a t o b i l i a r y structure a n d f u n c t i o n w e r e d e t e r m i n e d by serial h e p a t o b i l i a r y scintigraphy, p e r c u t a n e o u s t r a n s h e p a t i c c h o l e c y s t o g r a p h y , a n d bioc h e m i c a l liver f u n c t i o n tests. P a t i e n t s 1 a n d 3 h a d mild h e p a t o m e g a l y , n o r m a l liver b i o c h e m i s t r y , a n d distal c o m m o n bile duct strictures. P a t i e n t 2 h a d a firm nodular liver w i t h s p l e n o m e g a l y , a b n o r m a l liver b i o c h e m i s try, a n d a c h o l a n g i o g r a p h i c a p p e a r a n c e of s c l e r o s i n g cholangitis. All h a v e u n d e r g o n e o p e r a t i v e t r e a t m e n t for p e r s i s t e n t a b d o m i n a l pain. T h e s e c a s e s c o n f i r m the occ u r r e n c e of c o m m o n bile duct p a t h o l o g y a n d liver dise a s e in p a t i e n t s w i t h CF a n d p a n c r e a t i c sufficiency. T h e y d e m o n s t r a t e that liver and biliary tract disease c a n o c c u r i n d e p e n d e n t l y o f t h e u n d e r l y i n g disease severity a n d t h e p r e s e n c e of steatorrhea. Further, t h e y s u g g e s t that o b s t r u c t i o n o f t h e biliary tract m a y be an a d d i t i o n a l factor in t h e e v o l u t i o n o f liver disease in CF. (HEPATOLOGY 1995;21:963-969.)

Abbreviations: CF, cystic fibrosis; PS, pancreatic sufficiency; PI, pancreatic insufficiency; CFTR, cystic fibrosis transmembrane conductance regulator. From the Departments of 1Gastroenterology, 2Surgery, 3Nuclear Medicine, 4Histopathology, and 5Radiology, Royal Alexandra Hospital for Children, Camperdown, Sydney, Australia. Received June 3, 1994; accepted October 25, 1994. Supported by grant (90/0206) from the National Health and Medical Research Council of Australia and The James Fairfax Institute of Paediatric Clinical Nutrition. Presented in part at the 24th Annual Meeting of the Paediatric Research Society of Australia, Newcastle, N.S.W., September 26-27, 1991. Address reprint requests to Kevin J. Gaskin, MD, James Fairfax Institute, Royal Alexandra Hospital for Children, P.O. Box 34, Camperdown, NSW 2050, Australia. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2104-001153.00/0

Liver and biliary tract disease are well-recognized complications of cystic fibrosis (CF). Focal biliary cirrhosis and the more severe multilobular biliary cirrhosis are two forms of cirrhotic liver disease considered pathognomonic of CF 1'2 and in clinical series, have been reported in up to 40% and 13% of patients, respectively. 3'4 An even higher incidence of 72% and 24%, respectively, has been reported in an adult autopsy study, 5 thus alluding to an increasing incidence of liver disease with age in CF populations. Gallbladder disease is also common, 6 with gallstones reported in up to 30% of a group comprising mainly adolescents and young adults, 7 and microgallbladders noted in a similar p r o p o r t i o n Y Less frequently described are common bile duct stenoses and sclerosing cholangitis, with a radiologically proven incidence in two series of 16% and 4%, respectively. 4'9 The cause ofhepatobiliary disease in CF has not been completely elucidated. Fat malabsorption has been incriminated in the development of gallstones by its contribution to increased fecal bile acid losses and the development of lithogenic bile. 6 Although inspissation of secretions caused by the underlying epithelial secretion anomaly m a y aid nucleation, lithogenesis in relation to the severity of malabsorption is a more compelling theory, considering gallstones have not been described in those patients with pancreatic sufficiency (PS) and normal absorption. 1°'11 Fibrotic liver disease also has been considered to occur exclusively in patients with malabsorption or pancreatic insufficiency (PI). 12 This could relate to the malabsorption per se, or to the severity of the underlying lesion, because patients with PI represent a group characterized by the more severe genetic mutations. 13,14However, recent data suggest no genotypic distinction among patients with liver disease, 15 and thus, malabsorption continues to be regarded as a significant contributor to the development of liver disease in CF. The older literature suggests, however, that CF patients with PS can develop fibrotic liver disease or even frank cirrhosis. One report 16 described two previously undiagnosed adolescent patients with no prior history of gastrointestinal disease who presented with cirrhosis and portal hypertension. Another described two cases with liver disease who demonstrated residual tryptic activity in duodenal secretions. 17 Many studies

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964 WATERSET AL have also included older children or adults in their series, whose initial presentation of CF was through hepatic complications, ls2° alluding to the possibility of a previously mild expression of lung and pancreatic disease. The absence of fat balance studies or quantitative pancreatic stimulation testing in these cases questions the preservation of their pancreatic function, but the findings do suggest t h a t some CF patients with severe liver disease m a y have PS. The current report presents three patients with CF and hepatobiliary disease who clearly have PS, as documented by both fat balance studies and pancreatic stimulation tests.

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sis, only Patient 1 has required a re-admission for the t r e a t m e n t of chest disease. All were referred for assessment of recurrent right upper q u a d r a n t abdominal pain in the absence of clinical and biochemical evidence of pancreatitis or distal intestinal obstruction syndrome. All had biliary tract anomalies with mild to severe liver disease and underwent cholecystojejunostomy for the relief of abdominal pain. The pain was episodic, varying in frequency from once per week to daily, was severe enough to awaken the children from sleep, could be associated with certain (fatty)foods, and caused school avoidance. For Patient 1, the pain was exacerbated by right-sided chest physiotherapy. The PATIENTS AND METHODS mechanism producing abdominal pain in these patients Three patients attending the Cystic Fibrosis Clinic at The is unclear, but the exclusion of pancreatitis and relief of Royal Alexandra Hospital for Children, Sydney, Australia, are described. The diagnosis of CF was suggested in patients pain after surgical bypass suggest t h a t this presenting 1 and 3, currently aged 7 and 8 years, respectively, by eleva- feature may be caused by distension or inflammation of tion of immunoreactive trypsin on newborn screening. Pa- the biliary tree or gallbladder as a result of the partial tient 2 presented with pneumonia at 6 weeks of age. He is common-duct obstruction. Patients i and 2 are homozycurrently 12 years of age and was born before the commence- gous for the AF508 mutation, and Patient 3 is a AF508 ment of the screening program. Diagnosis was confirmed by compound heterozygote. elevation of sweat chloride (>60 mmol/L) in all patients. Patient 1 (female) had a normal fecal fat excretion Investigations. Assessment of pancreatic function was by (8%) at i month of age, and colipase secretion was 23% quantitative pancreatic stimulation testing, 3-day fecal fat of normal (2,934 U/kg/hr) by pancreatic stimulation analysis, and measurement of serum pancreatic isoamylase, test performed at 5 months of age. Fecal fat studies using methods described previouslyY Pancreatic function was defined as sufficient to prevent the malabsorption of fat repeated on two occasions over the next year indicated if colipase output was greater than 120 units per kilogram maintenance of pancreatic function (5% and 8% fat exof body weight per hour (U/kg/hr) i.e., greater than 1% of cretion), and a serum pancreatic isoamylase at 6 years mean normal levels for age; fecal fat excretion was less than was 73 U/L. This patient has never received pancreatic 8% of measured fat intake without pancreatic enzyme supple- enzyme supplements, and height and weight standard mentation, and serum pancreatic isoamylase was greater deviation (Z) scores and lung function results were conthan or equal to 15 U/L in a child older than 2 years of age. sistently within the normal range (height Z score: -0.6; Physical examination and measurement of height and weight Z score: 0.3; and forced expiratory volume in 1 weight were performed at routine 3-monthly (Patients i and second 103% of predicted normal at 5½ years of age 3) and 6-monthly (Patient 2) clinic visits. Each 6 months, when referred). Because she was experiencing abdomilung function was tested by standard spirometry, and liver biochemistry was performed. Total serum bile acids were col- nal pain two to three times weekly and regularly misslected after an overnight fast and measured by an enzymatic, ing school, a hepatobiliary scan was performed before colorimetric method (Enzabile, Nycomed Pharma AS, Oslo, 6 years of age. This study showed normal hepatocyte Norway) (normal, <7 #mol/L). Normal ranges for liver func- function and clearance and no functional obstruction tion tests in children as measured by the Beckmann CX-5 of bile flow. Results of biochemical liver function tests Analyser are: total bilirubin, <10 #mol/L; alanine amino- and serum bile acids were normal. Because of further transferase <50 U/L; gamma glutamyl transpeptidase, <40 liver enlargement and continuing abdominal pain, she U/L; and serum alkaline phosphatase, <300 U/L. DNA analy- underwent percutaneous transhepatic cholecystogram sis for the AF508 mutation was performed, but we have yet examination 8 months later. This showed a large gallto screen clinic patients for other CF gene mutations. bladder and a long segment of irregularly narrowed Liver and biliary tract disease were also investigated by serial hepatobiliary scintigraphy. This technique and the cri- common bile duct, which had a spiral appearance on teria for partial common bile duct obstruction have been de- two-dimensional view. Liver histology showed some scribed elsewhere.4 Definitive testing for biliary tract obstruc- fatty change and cholangiolitis. Because abdominal tion by percutaneous transhepatic cholecystogram has also pain had increased to two to three times daily and was been described previously. 4 Percutaneous needle biopsies of restricting physical therapy and food intake, Patient 1 the liver using a 16-gauge Biopty instrument (C. R. Bard underwent cholecystojejunostomy. Focal cirrhosis was Inc., Covington, GA) were performed at the time of cholecys- apparent at laparotomy, and liver histology showed octography with the patient under general anesthesia. All pa- casional mucus-plugged small ducts and acute and tients subsequently underwent cholecystojejunostomy and chronic cholangitis (Fig. 1A and 1B). open liver biopsy. Informed parental consent was obtained Patient 2 (male) is from a rural area. He had not for all procedures, which were approved by the hospital's undergone formal pancreatic function testing at diagEthics Committee. nosis and was treated with pancreatic enzyme suppleRESULTS ments. A serum pancreatic isoamylase at 3½ years of All three patients had mild lung disease and were age was mildly elevated (93 U/L), and after referral for pancreatic sufficient. Since hospitalization at diagno- investigation of his abdominal pain at 5½years, a fecal

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FIO. 1. (A) Open liver biopsy showing focal cirrhosis (patient no. 1). The hepatic lobules are irregularly replaced by large tracts of fibrous tissue containing increased bile ducts and ductules with an inflammatory infiltrate. (Hematoxylin-eosin stain; original magnification ×7.) (B) Acute inflammatory cells in fibrous tissue, bile ducts, and ductules around a small island of disrupted liver parenchyma. One small bile duct is plugged by inspissated secretion. (Hematoxylin-eosin stain; original magnification ×37.)

fat analysis was performed while not taking enzymes and found to be clearly within the normal range (5% fat excretion). Quantitative pancreatic stimulation test showed colipase secretion was 6,147 U/kg/hr or 48% of mean normal, and therefore pancreatic enzyme therapy was ceased. Height and weight Z scores and lung function tests were within the normal range at referral (height Z score: 0.6; weight Z score: 0.6; and forced expiratory volume in 1 second 107% of predicted normal). He was experiencing nocturnal abdominal pain, and initial abdominal examination showed a firm liver edge 3 cm below the costal margin. A hepatobiliary scan was performed and showed a large gallbladder with partial obstruction to bile flow at the distal end of the common bile duct; tracer distribution was patchy and there was mild cholestasis (Fig. 2A). Serum bilirubin was raised (25 #mol/L), but other biochemical parameters, including serum bile acids, were normal. The

patient underwent percutaneous transhepatic cholecystogram, which showed a large, distended gallbladder with an irregularly narrowed common bile duct (similar to sclerosing cholangitis) and a distal common bile duct stricture (Fig. 2B). Liver biopsy showed increased fibrous tissue in portal tracts, proliferation of bile ductules, and acute cholangitis. Five measurements of serum pancreatic isoamylase over 2½ years were normal or mildly elevated (range, 32 to 58 U/L). Annual hepatobiliary scans suggested progression of liver disease and cirrhosis. The persistence of recurrent abdominal pain and hepatomegaly with a firm, irregular liver edge plus the development of splenomegaly and abnormal liver biochemistry (total bilirubin = 20 #mol/L, alanine aminotransferase = 111 U/L, gamma glutamyl transpeptidase = 97 U/L) necessitated surgical referral. A cholecystojejunostomy was performed at 8 years of age, showing macronodular

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A

FIG. 2. (A) Hepatobiliary scan with Tc99m DISIDA (patient no. 2). The image at 42 minutes shows the liver to be moderately enlarged with a patchy distribution of tracer. There is narrowing of the distal common bile duct, with mild proximal dilatation and mild dilatation of the left hepatic duct. There is patchy retention of tracer in smaller bile ducts, including tertiary ducts. A large gallbladder is seen. The h a l f clearance time was 45 minutes (normal, < 3 8 minutes). (B) Percutaneous t r a n s h e p a t i c cholecystogram (patient no. 2), showing a large, distended gallbladder with irregularly narrowed common bile duct and distal common bile duct stricture. (C) Hepatobiliary scan with Tc99m DISIDA (patient no. 3). The frame at 24 minutes shows a mild to moderate dilatation of the m a i n hepatic ducts and tertiary bile ducts. There is distal common bile duct narrowing and mild dilatation proximal to this segment. A gallbladder was visualized; it was larger on delayed images. The h a l f clearance time was 32 minutes (normal, <38 minutes). (D) Percutaneous t r a n s h e p a t i c cholecystogram (patient no. 3) illustrates narrowing of the distal one quarter of the common bile duct and minimal dilatation of the left hepatic biliary tree.

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FIG. 3. Macronodular cirrhosis (patient 2) at laparotomy.

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examination, she had a firm liver edge palpable 3 cm below the costal margin. Height, weight, and lung function results were within the normal range (height Z score: -0.6; weight Z score: 0.0; forced expiratory volume in 1 second: 89% of predicted normal), and biochemical liver function tests and serum bile acids were also normal. A hepatobiliary scan was suggestive of partial common bile duct obstruction, showing mild to moderate dilatation of the hepatobiliary system and common bile duct (Fig. 2C). A percutaneous transhepatic cholecystogram (Fig. 2D) showed a slow and markedly irregular flow of contrast material into the duodenum, with narrowing of the distal one quarter of the common bile duct and minimal dilatation of the left hepatic biliary tree. The liver biopsy showed normal liver tissue with no evidence of fibrosis, inflammation, fatty change, or duct obstruction. Repeat hepatobiliary scan, liver function tests, and liver biopsy performed 2 years after the cholangiogram were unchanged; however, the patient continued to experience abdominal pain with postprandial nausea and vomiting. There was no clinical or biochemical evidence of pancreatitis. Frequent school absence because of these symptoms prompted surgical referral, and cholecystojejunostomy was performed at 8 years of age. The liver surface looked normal at surgery with normal microscopy on needle biopsy.

cirrhosis (Fig. 3). The pancreas was palpably fibrotic, and operative cholangiography confirmed a stricture at the lower end of the common bile duct. A wedge biopsy of the liver showed a spectrum of features, ranging from virtually normal hepatic and parenchymal archiDISCUSSION tecture and morphology to biliary cirrhosis with eosinoThis report documents the occurrence of mild to sephilic plugging and active cholangitis. Two years after surgery, a needle biopsy of the liver showed fibrosis vere cirrhotic liver disease or biliary tract anomalies with proliferated bile ducts, mixed inflammatory infil- in three patients with CF and PS as proven by normal trate, and a small number ofductules containing eosin- absorption on fat balance studies and adequate levels ophilic hyaline plugs and basophilic granular material. of colipase secretion during pancreatic stimulation Although subsequent hepatobiliary scans retained the tests. Two of the patients had cirrhosis at the time of features of macronodular cirrhosis, they demonstrated surgery, one with focal disease, the other with multilobsignificant improvement in tracer excretion and clear- ular cirrhosis and portal hypertension. All three paance from the liver, with free passage into the bowel tients had biliary tract disease, two with distal common through the cholecystojejunostomy. The patient's bile duct stenoses, the other with an irregularly narheight and weight measurements remained consis- rowed bile duct and distal stricture. These cases demtently above the 50th percentile, and lung function con- onstrate that liver and biliary tract disease do occur in tinues within the normal range (forced expiratory vol- CF patients with PS, and are not the exclusive compliume in 1 second: 117%). Serum pancreatic isoamylase cations of patients with malabsorption from pancreatic measurements had remained either normal or mildly insufficiency. The occurrence of cirrhosis in patients with PS queselevated, and he remains off enzyme therapy. His biochemical profile has remained essentially unchanged tions the speculative role of malabsorption in the geneuntil his serum albumin recently decreased to 30 g/L. sis of liver disease in CF. The original histological dePatient 3 (female) is a AF508 compound heterozy- scriptions of CF liver disease 17'22'23 suggested that gote. At diagnosis, her fecal fat excretion was only 3% inspissation and stasis of biliary secretions produced of intake, and colipase secretion was 11% of normal for bile ductular plugging, obstruction, and inflammation, age at 6 months. Repeat pancreatic stimulation test at leading to the characteristic focal fibrotic lesion. Al2½years of age confirmed preservation of function with though the precise role of pancreatic insufficiency and colipase secretion 39% of normal (4,958 U/kg/hr) and malabsorption in this mechanism was unknown, the pancreatic isoamylase estimations consistently within association of malabsorption with increased fecal bile the normal range (25 and 32 U/L at 2½ and 5 years, acid losses and the subsequent development of lithorespectively). This patient has not received pancreatic genic bile has been well documented. 1° The latter, by enzyme supplements. She was referred at 5 years of enhancing the inspissation of biliary secretions, could age complaining of worsening abdominal pain over a explain the predisposition of PI patients to develop 12-month period, and in the intervening 6 months had liver disease. 12 Equally important has been the demonexperienced vomiting and pallor after eating. At initial stration of normal fecal bile acid losses in PS pa-

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tients, 11'24 because if the above mechanism is relevant, PS patients would be unlikely to develop lithogenic bile or subsequent liver disease. The occurrence of liver disease in the current cases with PS demonstrate that malabsorption or lithogenic bile are not necessarily prerequisites to the development of liver disease, and other factors must prevail. It is not known whether the m u t a n t cystic fibrosis t r a n s m e m b r a n e conductance regulator (CFTR) protein produces similar disturbance to chloride transport in all affected tissues within an individual. 2~ However, a secretion anomaly in bile duct epithelium could explain the development of bile duct plugging and liver disease. 2GAlthough bile duct epithelium from CF patients has not been specifically studied, CFTR has been identiffed and localized to normal h u m a n bile duct epithelial cells in culture. 27'2s These results imply that CFTR mutations in CF could impair biliary secretion through the primary defect in bile duct cells analogous to the effect of abnormal chloride channel regulation in airw a y epithelium. Current studies of the variable mislocalization of CFTR from the apical membrane, both in vitro and in the CF sweat gland, 29'3° m a y partly explain the variation observed in secretion defects and the apparent influence of different mutations in determining the severity of lung and pancreatic disease, and perhaps the variable occurrence of liver disease. Patients homozygous for the common missense mutation (AF508) have been described as more likely to have severe lung disease and PI, whereas those with a small group of non-AF508 mild mutations have near normal lung function and PS. 13'3~ These observations suggest that if specific alterations of CFTR function produce a varying severity of disease, patients with severe lung and pancreatic problems would be more likely to develop liver disease. However, the Cystic Fibrosis Genotype-Phenotype Consortium 32 has recently shown that homozygosity for AF508 does not specifically correlate with severe disease. In addition, liver pathology appears to be distributed among patients with different CF mutations, 15'33 and thus it is unlikely that specific mutations determine the variable occurrence of liver disease. In the current group, two of the three cases developed liver disease, and both were homozygous for AF508. Both have normal lung function and excellent pancreatic function, thus supporting the concept that AF508 homozygosity does not necessarily confer severe disease. Although we have not specifically studied CFTR in the liver and biliary tree, the excellent condition of these patients suggests they have a milder underlying secretion anomaly. In this respect, it is difficult to reconcile this observation with the occurrence of multilobular cirrhosis and portal hypertension. If liver disease is related to genotype, its appearance in these patients could only be explained by specific mutations having a differential effect on secretion within specific organs. An alternative hypothesis is that in addition to the underlying secretion anomaly other factors contribute to the genesis of plugging and stasis in the biliary tree.

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Previously we found that a large proportion of CF patients with liver disease also had common bile duct disease, most with distal common bile duct stenoses and the remainder with an appearance resembling sclerosing cholangitis. 4 These findings occurred even in the absence of changes to liver biochemistry or serum bile acid measurements. The current cases clearly have PS and have few other risk factors for liver disease, but they demonstrate the same spectrum of common bile duct pathology. This suggests that partial obstruction of the biliary tract m a y contribute to the pathogenesis of liver disease by enhancing biliary stasis and bile ductular plugging. Although common bile duct pathology has been infrequently recognized in other series, we would suggest it is another factor that m a y account for the differential occurrence and highly variable progression of liver disease in CF. Distal common duct strictures are likely to result from external compression of the common duct by an enlarged fibrotic or inflamed pancreas, ~4'35but the cause of the irregular narrowing of the duct resembling sclerosing cholangitis as seen in one case is less clear and could result from an intrinsic abnormality of the bile duct epithelium or intramural fibrosis. 36 Thus, although the effect of CFTR mutations on biliary epithelium will clearly be important in defining the pathogenesis of liver and biliary tract disease in CF, a simple genotype-phenotype relationship is not apparent. Further work is required to elucidate other factors that determine the presence or absence and severity of liver disease in some patients regardless of their pancreatic and overall clinical status.

Acknowledgment: We are indebted to Drs J. Brown, P. Van Asperen, M. Gett, and C. Mellis and acknowledge the help and support of the Physicians, staff, and patients of the Camperdown Cystic Fibrosis Clinic. REFERENCES

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