Cholestatic liver diseases of Infancy – a liver

biopsy pattern-based approach

Basel Seminars in Pathology Paediatric Pathology

and Genetics

Pierre Russo, MD

Director, Division of Anatomic Pathology

The Children’s Hospital of Philadelphia

Professor, Department of Pathology and Laboratory Medicine

Perelman School of Medicine at the University of Pennsylvania

Objectives

• To define neonatal cholestasis and neonatal hepatitis

• 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 infants and their differential diagnosis

Pediatric Liver Disease

• 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 hepatology

– Neonatal cholestasis

– Emphasis on metabolic and genetic disorders

Definitions

• Cholestasis – impairment of bile formation or reduction of bile

flow from the liver.

– Bile acids

– Bilirubin

– Cholesterol

– Organic anions, drugs and toxins

• Hepatitis - inflammation of the liver

• Jaundice – 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 infant with jaundice after 2 weeks of age should have a fractionated bilirubin to assess for conjugated hyperbilirubinemia

Differential Diagnosis Of Neonatal Cholestasis

Neonatal Hepatitis

• Idiopathic NH

• Viral NH

– CMV

– Herpes

– Rubella

– Reovirus

– Adenovirus

– Enteroviruses

– Parvovirus B19

– Paramyxovirus

– Hepatitis B

– HIV

• Bacterial and parasitic

– bacterial sepsis

– UTI

– Syphilis

– Listeriosis

– Toxoplasmosis

– Tuberculosis

– Malaria

Bile duct obstruction

• Cholangiopathies

– Biliary atresia

– Choledochal cysts

– Nonsyndromic Paucity

– Alagille syndrome

– Sclerosing Cholangitis

– Spontaneous duct perforation

– Caroli disease

– Congenital hepatic fibrosis

– Bile duct stenosis

• Other

– Inspissated bile/mucus

– Cholelithiasis

– Tumors

– Masses

Cholestatic syndromes

• PFIC

– Type 1 Byler P-type ATPase

– Type 2 Canalicular Bile Acid Tx

– Type 3 MDR3 deficiency

• Aagenaes cholestasis lymphedema

• N. Am. Indian Cholestasis

• Nielsen Greenland Eskimo cholestasis

• Benign Recurrent Intrahepatic cholestasis

• Dubin Johnson MRP2 cMOAT deficiency

• Rotor syndrome

Metabolic disorders

• a1-antitrypsin deficiency

• Cystic fibrosis

• Neonatal iron storage disease

• Endocrinopathies

– Hypopituitarism

– Hypothyroidism

• Amino acid disorders

– Tyrosinemia

– Hypermethionemia

– Mevalonate kinase deficiency

• Lipid disorders

– Niemann-Pick A, B

– Niemann-Pick C

– Gaucher

– Wolman

– Cholesterol ester storage ds

• Urea cycle disorders

– Arginase deficiency

• Carbohydrate disorders

– Galactosemia

– Fructosemia

– Glycogen storage IV

• Mitochondrial disorders

– Oxidative phosphorylation

• Peroxisomal disorders

– Zellweger

– Infantile Refsum

– Other enzymopathies

• Bile acid synthetic disorders

– 3b-hydroxysteroid dehydrogenase/i

– D4-3-oxosteroid 5b-reductase

– Oxosterol 7a-hydroxylase

Toxic

• Drugs

• Parenteral alimentation

• Aluminum

Miscellaneous associations

• Shock/hypoperfusion

• Histiocytosis X

• Neonatal lupus erythematosus

• Indian childhood cirrhosis

• Autosomal trisomies 17, 18, 21

• Graft v host disease

• Erythrophagocytic lymphohistiocytosis

• ECMO

• Veno-occlusive disease

• Donahue leprechaunism

• Arthrogryposis cholestasis

• Erythroblastosis fetalis

Classification of Neonatal Cholestasis

Extrahepatic Intrahepatic

Biliary atresia

Choledochal cyst

Bile duct stenosis

Stones

Tumors

Neonatal hepatitis

Alagille syndrome

Galactosemia

CMV

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 therapies

• 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 Surgery

• 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 liver

transplantation 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 Cholangiography

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 Hepatoportoenterostomy

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

type 3, ABCB4 disease

Usually older infants at presentation; absence

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

available

Obstructive pattern with fat in liver

Neonatal sclerosing cholangitis

• Neonatal ichthyosis- sclerosing

cholangitis syndrome (NISCH)

• Scalp hypotrichosis

• Scarring alopecia

• Leukocyte vacuolization

• Claudin-1 gene, 3q27

5 months Claudin-1 staining, Grosse, Hepatol 2012

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

PC

ABCB4

MDR3

OA

ABCC2

ABCB11

BSEP

BA

ATP8B1

PS?

Cholangiocyte

CFTR

TJ 2

Cl -

BA

SLC10A2

Bile

flow

PL

ATP8B1

BCS1L

CIRHA1

PS?

BAAT

UGT1A1

Familial Cholestatic Disorders

Hepatocyte

Progressive Familial Intrahepatic

Choestasis type 2 (PFIC 2, ABCB 11)

2 months, Giant cell hepatitis and

low serum GGT cholestasis

BSEP control

BSEP BSEP

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 ultrasound showed nonspecific hepatosplenomegaly, contracted gallbladder, and trace perihepatic ascites 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

Alagille syndrome

Alpha-1 antitrypsin deficiency

Peroxisomal disorders (Zellweger)

Congenital cytomegalovirus infection

Biliary Atresia (late)

Secondary feature

Graft versus Host disease

Chronic hepatic allograft rejection

Primary biliary cirrhosis

Primary (or 2ary) sclerosing cholangitis

Sarcoidosis

Hodgkin disease (post-treatment)

Drug-associated (antibiotics)

Occasionally documented associations

Cystic fibrosis

HNF-1β mutations (renal cysts, diabetes)

PFIC

Familial hemophagocytic lymphohistiocytosis

Arthrogryposis-renal dysfunction-cholestasis

Niemann-Pick type C

Mitochondrial DNA depletion syndrome

Trisomy 18

Trisomy 21

Prune Belly syndrome

Williams syndrome

Congenital syphilis

Congenital rubella

•Autosomal Dominant

•Mutation in JAG1

(chr. 20p12) •Multi-system

•Liver, heart,

•Skeleton, eye,

•Face, kidney,

•vasculature

Alagille Syndrome (ALGS)

PA/PPS 67%

PVS 3%

TOF 16%

ASD 4%

PDA 5%

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