Cholestatic diseases of Infancy – a liver biopsy pattern-based approach

Basel Seminars in Paediatric Pathology and Genetics

Pierre Russo, MD Director, Division of Anatomic Pathology The Children’s Hospital of Philadelphia Professor, Department of Pathology and Laboratory Perelman School of Medicine at the University of Pennsylvania

Objectives

• To define neonatal cholestasis and neonatal • To outline the evaluation of neonatal cholestasis • To review the potentially fatal / treatable etiologies of neonatal cholestasis where the correlation between clinical features and histopathology is critical • To illustrate various histologic patterns in liver biopsies from cholestatic and their differential diagnosis

Pediatric

• Children are not little adults • However, many adult liver diseases are also seen in pediatric patients – Hepatitis B – Hepatitis C – Autoimmune hepatitis • Unique to pediatric – Neonatal cholestasis – Emphasis on metabolic and genetic disorders

Definitions

• Cholestasis – impairment of formation or reduction of bile flow from the liver. – Bile acids – – Cholesterol – Organic anions, drugs and toxins • Hepatitis - inflammation of the liver • – discoloration of skin, sclera or mucous membranes resulting from elevated unconjugated or conjugated bilirubin • Conjugated hyperbilirubinemia > 2.0 mg/dl or >15% of total bilirubin

15% Of Neonates Develop Jaundice

Physiologic vs pathologic jaundice • Physiologic jaundice – essentially means unconjugated hyperbilirubinemia - common and nearly never dangerous • Neonatal cholestasis is always pathologic, and essentially means conjugated hyperbilirubinemia (with a few exceptions) • Neonatal cholestasis - an emergency with potential for fatal outcome and chronic disease. It is never normal. • Any with jaundice after 2 weeks of age should have a fractionated bilirubin to assess for conjugated hyperbilirubinemia

Differential Diagnosis Of Neonatal Cholestasis

Neonatal Hepatitis Cholestatic syndromes • Peroxisomal disorders • Idiopathic NH • PFIC – Zellweger • Viral NH – Type 1 Byler P-type ATPase – Infantile Refsum – CMV – Type 2 Canalicular Bile Acid Tx – Other enzymopathies – Herpes – Type 3 MDR3 deficiency • Bile acid synthetic disorders – Rubella • Aagenaes cholestasis lymphedema – 3b-hydroxysteroid dehydrogenase/i – Reovirus – Adenovirus • N. Am. Indian Cholestasis – D4-3-oxosteroid 5b-reductase – Enteroviruses • Nielsen Greenland Eskimo cholestasis – Oxosterol 7a-hydroxylase – Parvovirus B19 • Benign Recurrent Intrahepatic cholestasis Toxic – Paramyxovirus • Dubin Johnson MRP2 cMOAT deficiency • Drugs – Hepatitis B • Rotor syndrome • Parenteral alimentation – HIV Metabolic disorders • Aluminum • Bacterial and parasitic • a1-antitrypsin deficiency Miscellaneous associations – bacterial • • Shock/hypoperfusion – UTI • Neonatal iron storage disease • Histiocytosis X – Syphilis • Endocrinopathies • Neonatal lupus erythematosus – Listeriosis – Hypopituitarism • Indian childhood – Toxoplasmosis – Tuberculosis – Hypothyroidism • Autosomal trisomies 17, 18, 21 – Malaria • Amino acid disorders • Graft v host disease obstruction – Tyrosinemia • Erythrophagocytic lymphohistiocytosis • Cholangiopathies – Hypermethionemia • ECMO – – Mevalonate kinase deficiency • Veno-occlusive disease – Choledochal • Lipid disorders • Donahue leprechaunism – Nonsyndromic Paucity – Niemann-Pick A, B • Arthrogryposis cholestasis – – Niemann-Pick C • Erythroblastosis fetalis – Sclerosing Cholangitis – Gaucher – Spontaneous duct perforation – Wolman – – Cholesterol ester storage ds – Congenital hepatic fibrosis – Bile duct stenosis • Urea cycle disorders • Other – Arginase deficiency – Inspissated bile/mucus • Carbohydrate disorders – Cholelithiasis – – Tumors – Fructosemia – Masses – Glycogen storage IV • Mitochondrial disorders – Oxidative phosphorylation

Classification of Neonatal Cholestasis

Extrahepatic Intrahepatic Biliary atresia Neonatal hepatitis Choledochal Alagille syndrome Bile duct stenosis Galactosemia Stones CMV Tumors TPN Α1- antitrypsin deficiency

There is overlap in clinical presentation, laboratory abnormalities, and histology.

Guiding Principles for Evaluation of the cholestatic infant • There are over 100 etiologies • Many complex, rare metabolic diseases which have new and effective • Design initial evaluation according to: – What is dangerous and treatable? – What is common? – What patterns provide clues for specific etiologies?

Etiologies Of Neonatal Conjugated Hyperbilirubinemia

n= %age Biliary atresia 377 34.7% Idiopathic neon. hepatitis 331 30.5% a1-antitrypsin deficiency 189 17.4% Other hepatitis 94 8.7% Alagille syndrome 61 5.6% Choledochal cyst 34 3.1%

Mieli-Vergani G, et al. Lancet 1989. King’s College Hospital

Important Critical Etiologies: Early Treatment Improves Outcome

• Biliary atresia Kasai portoenterostomy • Extrahepatic obstruction • Galactosemia Lactose restriction • Tyrosinemia NTBC • Hypopituitarism Cortisol, thyroxine • Bile acid synthetic defects Cholic acid • Sepsis and urinary tract infection Antibiotics • Syphilis Antibiotics

Russo P, Rand EB, Loomes KM. Chapter 10 Diseases of the Biliary Tree in

Pathology of Pediatric Gastrointestinal and Liver Disease, Springer, 2014

Percutaneous Liver Biopsy

A useful, commonly essential, and many times mandatory step in the evaluation of neonatal and infantile liver disease prior to OR cholangiogram.

Collaboration – communication Amount of tissue needed Special studies or assays PCR or cultures Special stains Electron microscopy

The Clues to Diagnosis

Jaundice Physiologic --- Pathologic Bilirubin Unconjugated --- Conjugated Site Intrahepatic --- Extrahepatic Type Metabolic --- Anatomic Histology Hepatitis,Paucity--- Proliferation GGT Low --- High Systemic Abnormalities --- Isolated Synthesis Normal --- Abnormal

The liver biopsy – major histologic patterns in neonatal cholestatic disorders

• Obstructive pattern • Giant cell Hepatitis • Intrahepatic bile duct paucity Liver biopsy “obstructive pattern”

2 month-old Biliary Atresia Biliary Atresia • Most severe chronic liver disease of infancy • Fibro-inflammatory obliteration of the biliary tree • Etiology unknown (viral, autoimmune, toxic) • 1/8,000 live births in US (400 cases/yr); higher in Asia • Untreated, death from cirrhosis < 1 year of age • Most common cause of pediatric worldwide

Diagnosis Of Biliary Atresia

Typical scenario Well appearing child Days to weeks old Acholic stools, dark urine Mild icterus Hepatosplenomegaly

Conjugated bilirubin Mildly elevated ALT Elevated GGT

Operative and Endoscopic

Operative cholangiogram Gold standard Difficult in Alagille syndrome

Endoscopic retrograde cholangiogram Not widely available Technically difficult Expensive Rarely therapeutic Risks unknown What does it add? Kasai

Morio Kasai Biliary Atresia – the remnant

Biliary Atresia – remnant histology

Hepatic ducts

Normal, 40 weeks BA, 2 months Biliary Atresia – the explant

80% - 85% of children will require liver transplantation 40% have failure of HPE without achieving good biliary drainage, requiring liver transplantation < 2 yrs Bile duct loss Differential Diagnosis of Obstructive Cholestasis on Liver Biopsy

Alpha-1 antitrypsin deficiency low or absent serum alpha-1 antitrypsin level, PiZZ phenotype

Choledochal cyst Ultrasonographic and cholangiographic findings Alagille syndrome (early) Associated malformations, mutation in JAG1 or Notch2 gene Cystic fibrosis History of meconium ileus, failure to thrive, positive sweat test TPN hepatopathy History of prematurity and TPN use

Neonatal sclerosing cholangitis Ichthyosis, scarring alopecia, dysmorphism, Claudin-1 mutations Progressive familial cholestasis Usually older infants at presentation; absence type 3, ABCB4 disease of immunohistochemical staining for MDR3 along canaliculi Galactosemia Extensive fatty change

Tumor, stone or other mechanical obstruction

a1 ANTITRYPSIN DEFICIENCY

Emphysema Neonatal hepatitis Cirrhosis

Disease due to mutations of protease inhibitor (Pi) gene (chr 14) a1-Antitrypsin Deficiency

7 week-old with a1-AT deficiency α1 Antitrypsin Deficiency Choledochal cyst

•1/15,000 livebirths (1/1000 in Japan)

•4:1 F:M

•50% dx’d < 10yrs of age; 25% < 20 yrs of age

•10% lifetime risk of carcinoma

Obstructive cholestasis - Parenteral Nutrition

3 months Parenteral Nutrition

• 2 month-old ex 32 week premie with jejunal atresia s/p resection and reanastomosis, on TPN majority of his life • Now off TPN but still cholestatic, please evaluate for BA Obstructive pattern- cystic fibrosis

2 month-old boy with cholestasis Obstructive pattern - Galactosemia

• Autosomal Recessive • FTT, vomiting, diarrhea • E. coli septicemia • Hemolytic anemia • Progression to cirrhosis in 6 months if untreated • Treatment is dietary removal of lactose • Neonatal screening Obstructive pattern with fat in liver available

Neonatal sclerosing cholangitis

• Neonatal ichthyosis- sclerosing cholangitis syndrome (NISCH) • Scalp hypotrichosis • Scarring alopecia • Leukocyte vacuolization • Claudin-1 gene, 3q27

Claudin-1 staining, Grosse, Hepatol 2012 5 months Obstructive pattern - Langerhans cell

histiocytosis

8 month-old girl with cholestasis and skin rash CD1a LCH - Liver explant

CD1a

The liver biopsy – major histologic patterns in neonatal cholestatic disorders

• Obstructive pattern • Giant cell Hepatitis • Intrahepatic bile duct paucity Neonatal Giant Cell Hepatitis

Disorders with a Neonatal Giant Cell Hepatitis phenotype • Idiopathic • Infections (TORCH) • Progressive familial cholestasis • Bile acid synthetic disorders • Metabolic disease • Hypopituitarism • Biliary Atresia

Torbenson, AJSP 2010 Familial Cholestatic Disorders

BAAT UGT1A1 ABCB4 MDR3 CFTR ATP8B1 PS? PS? - Cl ATP8B1 PC Bile PL BA SLC10A2 TJ 2 OA flow

ABCB11 BA BCS1L BSEP CIRHA1 ABCC2 Cholangiocyte

Hepatocyte Progressive Familial Intrahepatic

Choestasis type 2 (PFIC 2, ABCB 11)

BSEPBSEP

2 months, Giant cell hepatitis and low serum GGT cholestasis

BSEP control

J Peds 2007, 150:556 Bile Acid Synthesis Defects

• Nine different defects in primary synthetic pathway identified • Defects presenting with neonatal hepatitis with low GGT cholestasis and abnormal bile acids in blood, bile or urine • Severe disease result from enzyme defects acting on sterol nucleus and present early in life

– 3β-hydroxy δ5-C27 steroid dehydrogenase – Δ 4-3-oxosteroid 5β-reductase • Disorders of bile acid conjugation present later in life with failure to thrive and fata malabsorption • Mass spectrometry analysis of urine bile acids • Treatment with bile acids to restore physiologic bile acid function and to decrease synthesis of toxic abnormal bile acids Δ 4-3-oxosteroid 5β-reductase Giant cell hepatitis Fibrosis Distorsion of canaliculi on EM 3β-hydroxy δ5-C27 steroid dehydrogenase Pre-treatment Post-treatment

A case of Neonatal Giant cell hepatitis Clinical Vignette • This full-term 2 month-old male was referred to The Children’s Hospital of Philadelphia for jaundice and scleral icterus • At 4 days of life, he was noted to have a total bili 12.8 (direct bili 1.8) He had no further labs until his referral to CHOP • He underwent a work up that revealed hepatosplenomegaly, cholestasis, and elevated transaminases. • Abdominal showed nonspecific hepatosplenomegaly, contracted gallbladder, and trace perihepatic and doppler revealed normal flow. • DISIDA excreted. • Work up included normal urinalysis, negative blood cultures and negative testing for CMV, HSV, adenovirus, enterovirus, and parechovirus. • He underwent a liver biopsy

Diagnosis: Niemann-Pick type C mutation in NPC 1 Biliary atresia with prominent giant cell changes

The liver biopsy – major histologic patterns in neonatal cholestatic disorders

• Obstructive pattern • Giant cell Hepatitis • Bile duct paucity Bile duct paucity

Definition of Bile Duct Paucity

• Duct / portal tract ratio in full-term infants, children and adults is 0.9 to 1.8 • Ratio is lower in premature infants (physiological) • May be transient in some cases • Paucity = duct / portal tract ratio < 0.5 (in children) • Must have adequate # of portal tracts (10) • Comparison with hepatic artery may be more reliable – Ratio of BD/HA = 1 with comparable external diameters

Over-reporting of Bile Duct Paucity

• Non-pathologist authors • Self-perpetuating references • Sampling or observational error – Non-representative biopsy – Sampling of peripheral portal tracts in premature infants and infants < 1month of age (bile ducts develop up to first month of life)

Conditions Associated with Bile Duct Paucity

Well documented association Occasionally documented associations Alagille syndrome Cystic fibrosis Alpha-1 antitrypsin deficiency HNF-1β mutations (renal cysts, diabetes) Peroxisomal disorders (Zellweger) PFIC Congenital cytomegalovirus infection Familial hemophagocytic lymphohistiocytosis Biliary Atresia (late) Arthrogryposis-renal dysfunction-cholestasis Secondary feature Niemann-Pick type C Graft versus Host disease Mitochondrial DNA depletion syndrome Chronic hepatic allograft rejection Trisomy 18 Primary biliary cirrhosis Trisomy 21 Primary (or 2ary) sclerosing cholangitis Prune Belly syndrome Sarcoidosis Williams syndrome Hodgkin disease (post-treatment) Congenital syphilis Drug-associated (antibiotics) Congenital rubella

Alagille Syndrome (ALGS)

•Autosomal Dominant •Mutation in JAG1 (chr. 20p12) •Multi-system •Liver, heart, •Skeleton, eye, •Face, , •vasculature PDA 5% PA/PPS 67%

PVS 3% TOF 16% ASD 4%

VSD +ASD 1% Bile duct paucity – Alpha 1 antitrypsin deficiency

2 month-old with cholestasis Bile duct paucity – Alpha 1 antitrypsin deficiency

Liver explant at 8 months of age

Summary points

• Hepatic histology is an essential component of the diagnostic algorithm of the cholestatic infant • The pathologist must be familiar with the differential diagnosis of various histologic patterns in the liver biopsy and communicate effectively with the clinician • Integration of clinical and laboratory findings with histologic features and close collaboration between hepatologist and pathologist are essential for accurate and timely diagnosis

You find only what you look for, you seek only what you know