Hepatocellular Carcinoma in Ten Children Under FiveYears of Age With Bile Salt Export Pump Deficiency

A. S. Knisely,1 Sandra S. Strautnieks,2 Yvonne Meier,3 Bruno Stieger,3 Jane A. Byrne,2 Bernard C. Portmann,1

Laura N. Bull,4 Ludmila Pawlikowska,4 Banu Bilezikci,5 Figen Ozcay,6 Aranka Laszlo,7 Laszlo Tiszlavicz,8

Lynette Moore,9 Jeremy Raftos,10 Henrik Arnell,11 Bjorn Fischler,11 Antal Nemeth,11 Nikos Papadogiannakis,12

Joanna Cielecka-Kuszyk,13 Irena Jankowska,14 Joanna Pawłowska,14 Hector Melın-Aldana,15 Karan M. Emerick,16

Peter F. Whitington,16 Giorgina Mieli-Vergani,2 and Richard J. Thompson2

Hepatocellular carcinoma (HCC) is rare in young children. We attempted to see if immu-nohistochemical and mutational-analysis studies could demonstrate that deficiency of thecanalicular bile acid transporter bile salt export pump (BSEP) and mutation in ABCB11,encoding BSEP, underlay progressive familial intrahepatic cholestasis (PFIC)—or “neona-tal hepatitis” suggesting PFIC—that was associated with HCC in young children. We stud-ied 11 cases of pediatric HCC in the setting of PFIC or “neonatal hepatitis” suggesting PFIC.Archival liver were retrieved and immunostained for BSEP. Mutational analysis of ABCB11was performed in leukocyte DNA from available patients and parents. Among the 11 non-related children studied aged 13-52 months at diagnosis of HCC, 9 (and a full sibling, withneonatal hepatitis suggesting PFIC, of a tenth from whom liver was not available) hadimmunohistochemical evidence of BSEP deficiency; the eleventh child did not. Mutations inABCB11 were demonstrated in all patients with BSEP deficiency in whom leukocyte DNAcould be studied (n � 7). These mutations were confirmed in the parents (n � 14). Withrespect to the other 3 children with BSEP deficiency, mutations in ABCB11 were demon-strated in all 5 parents in whom leukocyte DNA could be studied. Thirteen different muta-tions were found. In conclusion, PFIC associated with BSEP deficiency represents apreviously unrecognized risk for HCC in young children. Immunohistochemical evidence ofBSEP deficiency correlates well with demonstrable mutation in ABCB11. (HEPATOLOGY 2006;44:478-486.)

Abbreviations: HCC, hepatocellular carcinoma; BSEP, bile salt export pump; PFIC, progressive familial intrahepatic cholestasis; GGT, �-glutamyltranspeptidase;PEBD, partial external biliary diversion; FIC1, familial intrahepatic cholestasis 1; MRP2, multidrug resistance-associated protein 2; ATP, adenosine triphosphate; LT,liver transplantation; AFP, �-fetoprotein; TTI, tyrosinemia type I; BRIC, “benign” recurrent intrahepatic cholestasis.

From the 1Institute of Liver Studies, King’s College Hospital, London, UK; the 2Division of Gene and Cell Based Therapy, Department of Liver Studies andTransplantation, King’s College London School of Medicine, London, UK; the 3Division of Clinical Pharmacology and Toxicology, Department of Medicine, UniversityHospital, Zurich, Switzerland; the 4University of California–San Francisco Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, SanFrancisco, CA; the Departments of 5Pathology and 6Pediatric Gastroenterology, Hepatology, and Nutrition, Baskent University Hospital, Ankara, Turkey; the 7Departmentof Pediatrics, Albert Szent-Gyorgyi Medical Centre and University of Szeged, Szeged, Hungary; the 8Department of Pathology, University of Szeged, Szeged, Hungary; the9Department of Histopathology and the 10Paediatric Emergency Department, Women’s and Children’s Hospital, North Adelaide, South Australia, Australia; the11Department of Pediatrics, Karolinska University Hospital, Huddinge and Solna, Stockholm, Sweden; the 12Section of Perinatal Pathology, Department of Pathology,Karolinska University Hospital, Huddinge, Stockholm, Sweden; the Departments of 13Pathology and 14Pediatric Gastroenterology, Hepatology, and Immunology, TheChildren’s Memorial Health Institute, Warsaw, Poland; and the Departments of 15Pathology and 16Pediatrics, Northwestern University Feinberg School of Medicine andChildren’s Memorial Hospital, Chicago, IL.

Received February 16, 2006; accepted May 24, 2006.Supported in part by Swiss National Science Foundation grant 31-64140.00 (B. S.); the Liver Foundation for Kids, Lemont, IL (P. F. W.); the Samariten, Sven Jerring,

and Wera Ekstrom Foundations, Stockholm, Sweden (B. F.); National Institutes of Health Grant R01 DK50697 (L. N. B.); and Guy’s and St. Thomas’ Charity, London,and Children’s Liver Disease Foundation, Birmingham, UK (R. J. T., S. S. S.).

A. S. Knisely and Sandra S. Strautnieks contributed equally to this study.Address reprint requests to: A. S. Knisely, Institute of Liver Studies, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom. E-mail:

alex.knisely@kcl.ac.uk; fax: (44) 20-3299-3125.Copyright © 2006 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.21287Potential conflict of interest: Nothing to report.

478

Progressive familial intrahepatic cholestasis (PFIC)with normal or only slightly elevated serum con-centrations of �-glutamyltranspeptidase (GGT)

activity, or low-GGT PFIC, once known as “Byler dis-ease,” encompasses several disorders.1 These are definedby persistent nonremitting conjugated hyperbiliru-binemia with elevated serum concentrations of bile acids(hypercholanemia), lack of abnormal bile acid species inserum and urine, patency of a normally formed biliarytract, and, unless partial external biliary diversion (PEBD)or ileal exclusion is undertaken,2-4 evolution into end-stage liver disease. Two principal categories are recognizedwithin low-GGT PFIC: familial intrahepatic cholestasis 1(FIC1) deficiency and bile salt export pump (BSEP) defi-ciency. The former is caused by mutation in ATP8B1,which encodes FIC1.5,6 FIC1 is a putative aminophos-pholipid flippase.7 The latter is caused by mutation inABCB11,8 which encodes BSEP.8,9 BSEP is the principalconveyor of bile acids from hepatocyte cytoplasm into bilecanaliculus.10,11 At biopsy on presentation in infancy,patients with PFIC due to FIC1 deficiency generally havebland canalicular cholestasis, whereas patients with PFICdue to BSEP deficiency generally have “neonatal hepati-tis.”1,12

Hepatocellular carcinoma (HCC) is rare in children.13

We identified 11 unrelated children with clinically diag-nosed PFIC in whom HCC was diagnosed between theages of 13 and 52 months. In 10 of these children, BSEPdeficiency was demonstrated immunohistochemicallyand mutation in ABCB11 was demonstrated via molecu-lar analysis using materials from the children or their fam-ily members. We describe clinical and histopathologicalfindings, attempt correlation with results of mutationalanalysis and processes of carcinogenesis, and suggest im-plications for management.

PatientsHCC was found in the explanted liver of a boy with

intrahepatic cholestasis (patient A; Table 1) and BSEPdeficiency. The boy’s disorder, which manifested at 3weeks of age, was characterized by failure to thrive andicterus. Clinical and laboratory studies revealed hyper-cholanemia, conjugated hyperbilirubinemia, and lowGGT. Urine screening did not identify abnormal bile acidspecies. Imaging studies did not suggest bile duct obstruc-tion or malformation. Microscopy of a liver biopsy spec-imen obtained at 35 days of age identified “neonatalhepatitis” with intralobular cholestasis and anisocytosis,rosetting, edema, multinucleation, and individual cell ne-crosis of hepatocytes; portal-tract cholestasis was notfound (Fig. 1A). Immunohistochemical studies demon-strated normal expression of multidrug resistance-associ-

ated protein 2 (MRP2) (Fig 1B), like BSEP an adenosinetriphosphate (ATP)-binding cassette protein involved incanalicular transport; MRP2 here served as a functionalcontrol useful in assessing tissue preservation.9 Markingfor BSEP (see Methods section) was entirely absent (Fig.1C). Allogeneic hepatocytes were infused14 in hopes ofaverting orthotopic liver transplantation (LT), and ta-crolimus was given. No clinical effect was apparent, al-though GGT rose slightly, consonant with greater accessof bile acids to the canalicular lumen.1,15 The child waslisted for LT. The serum concentration of �-fetoprotein(AFP) had been 2 IU/L as a neonate (nl � 7). This levelhad risen to 34 IU/L before hepatocyte transplantationand reached 199 IU/L by LT (21 months), after which itfell immediately. The deep green-brown explanted livercontained a pink-white mass 0.5 cm in diameter. Micros-copy of the mass revealed HCC. Abnormalities in nontu-moral liver included cirrhosis, mild inflammation, andhepatocellular and canalicular cholestasis, with hepatocel-lular edema (Fig. 1D). The tumor exhibited clear-cellchange, with intracellular inclusions consistent with gly-coprotein (Fig. 1E). On immunostaining, tumor-cell cy-toplasm and inclusions marked for AFP and tumor nucleimarked for p53 but not for �-catenin. Typing of micro-satellite marker loci on 6 different chromosomes usingDNA from lesion and from patient and donor leukocytesfound the tumor to be of native liver rather than alloge-neic-hepatocyte origin. Mutational analysis of ABCB11using leukocyte DNA revealed compound heterozygosityfor IVS16-8T�G, which induces direct splicing of exon16 to exon 18, and 1939delA, which introduces a frame-shift, with subsequent termination codon, in exon 16.The boy was healthy 32 months after LT.

This experience prompted a review of cases of HCCassociated with PFIC at King’s College Hospital and else-where. Ten additional young children with HCC associ-ated with intrahepatic cholestasis were identified; all 11children were unrelated (Table 1). Three of the childrenhad been subjects of previous reports (patients B, F, andK).13,16-20 Each patient exhibited conjugated hyperbiliru-binemia without biliary tract obstruction in infancy. Liverbiopsy in 9 patients revealed “neonatal hepatitis”; in 2patients, each of whom had 3 affected siblings with bi-opsy-documented “neonatal hepatitis,” the parents re-fused biopsy. In the 9 children specifically assessed, GGTwas low. These children also had no evidence of bile acidsynthesis disorder on urine screening. Infection with hep-atitis B virus or hepatitis C virus was not demonstrated inany of the patients. Tyrosinemia type I (TTI) and othermetabolic disorders were excluded in all 11 patients. Se-rum concentrations of AFP were elevated in the 8 chil-dren specifically assessed. Five of the children died from

HEPATOLOGY, Vol. 44, No. 2, 2006 KNISELY, STRAUTNIEKS ET AL. 479

Table 1. Patients, Clinical Courses, and ABCB11 / BSEP Mutations

Patient/Gender;Origins

Sibling(s)With PFIC

Age, PFICManifest Intervention(s)

Age, HCCDiagnosed

Outcome toDate Nucleotide Changes

PredictedConsequences

A/Male;NorthernEuropeanCaucasian

0 Cholestasisfrom 3 wk

Hepatocyte infusion,16 mo

21 mo (incidentalin explant; AFP199, nl � 7),at LT

Healthy(2 y, 8 moafter LT)

1939delA/IVS16-8T�G(compoundheterozygote)

647K then VFTSLX/splice sitedisruption

B/Female;NorthernEuropeanCaucasian

0 Cholestasisfrom 2 wk,hospitalizedfor evaluationaged 12 wk

None 28 mo, at openbiopsy; AFP notdetermined

Palliative careonly; deathaged 33 mo

IVS18�1G�A/74C�A(compoundheterozygote)

Splice site disruption/S25X

C/Male;NorthernEuropeanCaucasian

0 Cholestasisfrom birth

None 23 mo (AFP 30k,nl � 5; livermass); histologicdiagnosis atnecropsy, 24 mo

Palliative careonly; deathaged 24 mo

1445A�G/3691C�T(compoundheterozygote)

D482G/R1231W

D/Male;NorthernEuropeanCaucasian

1 Cholestasisfrom 3 wk

Partial external biliarydiversion (PEBD),11 mo; decreasedpruritustemporarily,clinical-laboratorytest results andgrowth no better

22 mo (AFP 158k,nl � 15); livermass; lung andbone lesions;chemotherapygiven; histologicdiagnosis at LT,25 mo

Death, 6 dafter LT(sepsis; noHCC atnecropsy)

890A�G/890A�G(homozygote)

E297G/E297G

E/Male;NorthernEuropeanCaucasian

1 Growth failurefrom 6 mo;diagnosed9.5 mo

PEBD, 10 mo;no response

29 mo (incidentalin explant; AFP6.4k, nl � 9),at LT

Healthy(5 y, 10 moafter LT)

IVS7�1G�A/890A�G(compoundheterozygote)

Splice site disruption/E297G

F/Male;NorthernEuropeanCaucasian

1 Cholestasisfrom 6 wk

None 16 mo (clinicallyunsuspected),at necropsy;AFP notdetermined

Death withmetastaticHCC inlungs

IVS9�1G�T/notknown

Splice site disruption/not known

G/Female;Arabic

3 Cholestasisfrom 6 wk

None 15 mo (AFP 11k,nl � 7; livermass);histologicdiagnosis at LT,16 mo

Healthy(1 y, 7 moafter LT)

1416T�A/1416T�A(homozygote)

Y472X/Y472X

H/Male;NorthernEuropeanCaucasian

0 Evaluation at6 mo forjaundice andgrowth failure

PEBD, 32 mo;excellentresponse(pruritus resolved,bile salts inserum normalrange, growthresumed)

52 mo (markedincrease inabdominal size;tumormetastasized atdiagnosis; AFP2x106, nl � 9),at open biopsy

Palliative careonly; death3 wk afterdiagnosis

890 A�G/IVS13del-13 -8(compoundheterozygote)

E297G/splice sitedisruption

I/Male;Central AsianCaucasian

0 Cholestasisfrom birth

None 13 mo (incidentalin explant; AFP831, nl � 23),at LT

Healthy(1 y, 11 moafter LT)

IVS19�2T�C(homozygote)

Splice site disruption

J/Male;Central AsianCaucasian

0 Cholestasisfrom 1 wk

None 14 mo (AFP 4k,nl � 23;liver mass),at biopsy;confirmed atLT, 15 mo

Healthy(1 y, 2 moafter LT)

2316T�G(homozygote)

Y772X

K/Male;NorthernEuropeanCaucasian

3 Cholestasisfrom 3 mo

None 26 mo (markedincrease inabdominal size;tumormetastasized atdiagnosis; AFPnot measured),at open biopsy

Death withmetastaticHCC inlungs, aged27 mo

None sought None predicted

480 KNISELY, STRAUTNIEKS ET AL. HEPATOLOGY, August 2006

Fig. 1. Features of BSEP deficiency with cholestatic “neonatal hepatitis” eventuating in HCC; material from livers of 2 patients (panels A-E, patientA; panel F, patient G). (A) “Neonatal hepatitis,” age 35 days; intralobular cholestasis with edema and multinucleation of hepatocytes. Centrilobularvenule, left, and portal tract, right. (Hematoxylin-eosin; original magnification �200.) (B) Same material as panel A immunostained for MRP2 withhematoxylin counterstain. The arrow marks a canaliculus with reaction product. (Original magnification �200.) (C) Same material as panels A andB immunostained for BSEP with hematoxylin counterstain. Canalicular reactivity is not found. (Original magnification �200.) (D) Nontumoral liver,hepatectomy specimen, age 21 months; hepatocellular edema with intracytoplasmic and canalicular cholestasis. (Hematoxylin-eosin; originalmagnification �200.) (E) HCC with clear-cell features and prominent inclusion bodies, histochemically consistent with glycoprotein; hepatectomyspecimen, age 21 months. (Main image, hematoxylin-eosin; inset, diastase/periodic acid — Schiff technique; original magnification �400.) (F)Hepatocellular carcinoma with trabecular features; hepatectomy specimen, age 16 months. (Hematoxylin-eosin; original magnification �200.)

HEPATOLOGY, Vol. 44, No. 2, 2006 KNISELY, STRAUTNIEKS ET AL. 481

HCC; the other 6 underwent LT. Of these 6, one died ofsepsis, without tumor, shortly after LT, and 5 are well.

Paraffin-wax blocks containing tumor and nontumoralliver were retrieved; sections were stained and immuno-stained (see Methods section). Genomic DNA was obtainedfrom patients’ peripheral blood in 7 instances (patients A, D,E, G, H, I, and J) and from parents’ peripheral blood in 10instances (patients A-J). Mutational analysis of ABCB11 wasundertaken (see Methods section). All studies were consid-ered routine diagnostic assessment.

Materials and Methods

Histological Studies. Tumor was available from all11 patients with HCC. Nontumoral liver was availablefrom 10 patients. In the exception (patient F),16,17 whosesister also had intrahepatic cholestasis manifesting as“neonatal hepatitis”,18,19 liver could not be retrieved fromarchives. The patient’s lung, with metastatic tumor, andliver from his sister were used. Tissue sections cut at 4 �mwere stained with hematoxylin-eosin and, after diastasedigestion, with periodic acid — Schiff technique. Parallelsections were immunostained (DAKO Chem-Mate;DAKO, Ely, UK) with antibodies raised in rabbit againstBSEP21 and raised in mouse against an ATP-binding cas-sette protein, MRP2 (Signet/Bioquote, York, UK). Sec-tions containing tumor were similarly stained withantibodies against AFP (DAKO), p53 (DAKO), and�-catenin (Novocastra, Newcastle-upon-Tyne, UK),markers of stages in or routes toward malignant change inhepatocytes.22 The anti-BSEP antibody was raised, as de-scribed,11,23 against an oligopeptide of the C-terminal 13amino acids of BSEP (Neosystems, Strasbourg, France)and was affinity-purified (AminoLink Kit; Pierce Bio-technology, Boston, MA) against the same oligopeptide.Archival liver from 2 adults with Dubin-Johnson syn-drome, in which MRP2 generally is lacking, and 30 chil-dren with cholestatic disease of known etiology (ATP8B1mutation, n � 10; ABCB11 mutation, n � 5; ABCB4mutation, n � 3; bile acid synthesis disorder, idiopathicacute liver failure, extrahepatic biliary atresia, �1-antitryp-sin storage disorder, Alagille syndrome, and TTI, n � 2each) also were evaluated for MRP2 and BSEP expression.(All commercial products were used according to themanufacturers’ instructions.)

Mutational Analysis. DNA was extracted from pe-ripheral-blood leukocytes using the QIAamp blood DNAmini-kit (QIAGEN, Crawley, UK). ABCB11 was ana-lyzed by sequencing of PCR products of all 28 coding andnoncoding exons. Primer sequences for exonic amplifica-

tion, available on request, included up to 50 bp of in-tronic flanking sequence and hence all sequences criticalfor mRNA splicing.

PCR amplification and product purification were per-formed using Roche FastTaq amplification and HighPure PCR purification systems (Roche Diagnostics Ltd,Lewes, UK). Bidirectional sequencing was performed us-ing the 3.1 Dye Terminator Cycle Sequencing kit (Ap-plied Biosystems, Warrington, UK), followed by ethanolprecipitation and capillary gel electrophoresis on a 3100-Avant Genetic Analyzer (Applied Biosystems). Sequenceanalysis was performed using Sequencher software (GeneCodes, Ann Arbor, MI).

Microsatellite Typing (Patient A). DNA was ex-tracted from paraffin-embedded tumor using the QIAgenDNeasy Tissue kit (QIAGEN) as well as from leukocytesof both patient A and the hepatocyte donor. Fluorescentlylabeled primers were used to amplify informative micro-satellite marker loci on 6 chromosomes. PCR productswere separated on a 373 Automated DNA Sequencer andanalyzed using Genescan and Genotyper software (all Ap-plied Biosystems). Haplotypes for tumor and for patientand hepatocyte-donor leukocytes were constructed andcompared.

Results

Histological Studies. In 6 patients with HCC, liverand tumor were sampled at LT (patients A, D, E, G, I,and J). In 3 patients, liver and tumor were sampled atnecropsy (patients C, F, and K). In 2 patients with wide-spread disease, nontumoral liver was obtained at pre-sentation and follow-up biopsies, whereas tumor wasobtained at diagnostic laparotomy (patients B and H).All 11 patients had cirrhosis when tumor was diag-nosed.

Tumors in the 6 explanted livers consisted of a singlenodule in 4 patients (patients A, D, E, and I), 2 nodules in1 patient (patient J), and 3 nodules in 1 patient (patientG). Tumor in 1 patient (patient A) lacked bile pigmentand consisted of cells with cleared cytoplasm containingglobules of eosinophilic material (Fig. 1E). Tumors werewell-differentiated in 9 patients (Fig. 1F) and occasionallyexhibited bile pigment at the centers of rosettes of cellsresembling hepatocytes. In the patient with 3 tumor nod-ules (patient G), 2 of the nodules were well-differentiated,with bile production, and 1 was composed of clear cells.Neither mucin nor mucus was identified in any tumor.Although occasional hemopoietic cells were found withintumor, no lesion had the zonally biphasic smaller-cell(embryonal)/larger-cell (fetal) appearance of hepatoblas-

482 KNISELY, STRAUTNIEKS ET AL. HEPATOLOGY, August 2006

toma, and heterologous elements (osteoid, squamous ep-ithelium, melanin) were found in none of the patients.

In 8 of the patients with HCC from whom nontu-moral liver was available and in liver from the sister (whoalso had severe intrahepatic cholestasis) of the patientwith HCC from whom nontumoral liver was not avail-able (patient F), BSEP was not detected immunohisto-chemically at any site in nontumoral liver. In nontumoralliver from another patient with HCC (patient H), scantstaining for BSEP was seen along occasional canaliculi. Inyet another patient (patient K) and his brother, BSEPexpression was intact. MRP2 was well-expressed at cana-licular margins in all nontumoral liver. BSEP expressionwas not found in any tumor except in patient K.

All tumors, however, expressed MRP2 at margins ofrosettes or, at cell borders, in linear patterns consistentwith canalicular margins. Canaliculi in all samples fromthe panel of comparison materials but those with Dubin-Johnson syndrome expressed MRP2. Canaliculi in allsamples from the panel but those with known ABCB11mutation expressed BSEP. AFP was demonstrated in cy-toplasm in all tumors. Nuclei in all tumors but 3 (patientsB, J, and K) stained for p53; �-catenin accumulation innuclei was not detected in any of the tumors.

Mutational Analysis. Mutation in ABCB11 wasfound on both alleles in 9 families and on 1 allele in thefamily of patient F, from which only maternal leukocyteswere available. In 7 patients, mutations were found onanalysis of proband leukocyte DNA; these were con-firmed in parental material for each. With respect to theother 4 patients, from whom no peripheral blood leuko-cyte DNA was available (patients B, C, F, and K), muta-tion in ABCB11 was found in material from the 5 parentswho consented to genetic studies (both parents of patientB, both parents of patient C, and only one parent ofpatient F). The parents of patient K could not be traced,and mutational analysis was not attempted.

A total of 13 different mutations, 10 novel, was iden-tified (Table 1). The common Polish mutation1445A�G (D482G)8 was present in one parent of a pa-tient from whom leukocytes were not available (patientC). The common European missense mutation 890A�G(E297G)8 was present in 3 patients and their parents (pa-tients D, E, and H). Except for 890A�G (E297G), noneof the changes found was present in 500 control individ-uals representative of all major populations (University ofCalifornia San Francisco Pharmacogenetics Project24;http://pharmacogenetics.ucsf.edu).

Microsatellite Typing (Patient A). At all marker locistudied, haplotypes constructed for tumor matched thosefor patient DNA rather than for hepatocyte-donor DNA.

DiscussionSeveral descriptions exist of HCC in early childhood

associated with “giant-cell hepatitis” or “neonatal hepati-tis”.13,16-20,25,26 Two such children had one sibling withfatal intrahepatic cholestasis,16-19,26 and another had 3 sib-lings with intrahepatic cholestasis, one of whom diedfrom HCC in adolescence.20 HCC also has been de-scribed in older children or children of unstated age, ad-olescents, or young adults with “neonatal hepatitis”,familial cholestatic cirrhosis of childhood, Byler disease,or PFIC.20,27-32 What causes giant-cell hepatitis or “neo-natal hepatitis” in these children, or what sort of PFICaffects them, is a matter of debate.

In a search of published instances of HCC in earlychildhood and in a review of materials at five pediatrichepatology centers, we identified 11 children, 3 of whomhad been subjects of the case reports cited above,13,16-20 inwhom “neonatal hepatitis” and persistent cholestasis wereassociated with development of HCC at less than 52months of age and from whom (and whose families) ar-chival materials or blood samples were available for study.Archival materials for 2 other similar children25,26 weresought but were unavailable (T. Higgins and S. Falkmer,personal communications). Attempts to gain access tomaterials from a child with PFIC and with HCC manifestat an unstated age32 were unsuccessful.

Liver disease in 9 of the 11 patients met the clinico-pathological criteria set forth for the diagnosis of PFIC. Inthe other 2 patients (patients F and K),16-20 GGT was notmeasured, and primary disorders of bile acid synthesiswere not excluded. In all 11, clinicopathological findingssuggested BSEP deficiency. In 9, BSEP could not be dem-onstrated immunohistochemically at canalicular margins(8 patients) or was present but very scant (patient H). Inpatient F,16,17 nontumoral liver was not available for as-sessment of BSEP expression. The liver of a sister withcholestatic liver disease fatal in childhood and initiallymanifest as “neonatal hepatitis”,18,19 however, entirelylacked immunohistochemically demonstrable BSEP.Liver disease in this sibling pair thus was assessed as likelydue to BSEP deficiency. In 10 of 11 instances of HCC inearly childhood associated with clinically diagnosedPFIC, deficiency of BSEP expression was directly demon-strated or could be reasonably inferred. The exception waspatient K20 (see below).

Lesions in ABCB11 predicted to disrupt synthesis offunctional BSEP were identified in all 10 families studiedby mutational analysis (Table 1). Mutation was found onboth alleles of ABCB11 in 9; paternal DNA was not avail-able for the exception (patient F), and only a maternalchange could be identified. Two ABCB11 mutations were

HEPATOLOGY, Vol. 44, No. 2, 2006 KNISELY, STRAUTNIEKS ET AL. 483

found in each of the 7 children with deficiency of BSEPexpression from whom peripheral blood leukocytes wereavailable. With respect to the other 3 children withproven or likely deficiency of BSEP expression, each ofthe 5 parents whose DNA could be studied proved tocarry a single ABCB11 change. The lack of immunohis-tochemically detectable BSEP in liver tissue from childrenB and C and from the sibling of child F argues stronglythat B, C, and F each carried 2 defective ABCB11 alleles.In all 10 of the patients whose liver disease was associatedwith BSEP deficiency, then, mutation in ABCB11 wasdirectly demonstrated or could be reasonably inferred.

No deficiency of BSEP expression was found in liverfrom either patient K or his older brother, both of whomhad clinically diagnosed PFIC and both of whom died ofHCC.20 Liver disease in this sibling pair thus was assessedas not likely due to BSEP deficiency. The status of patientK with respect to ABCB11 mutation is unknown.Though lesions in ABCB11 impeding BSEP function butnot BSEP expression may have been present, the liverdisease clinically diagnosed as PFIC and manifest as “neo-natal hepatitis” in that child and his siblings20 perhapsrepresented a disease different from BSEP deficiency. Thedefinition of PFIC now generally in use1 did not yet exist30 years ago, when patient K and his siblings were evalu-ated: GGT values and results of bile acid synthesis defectscreening, in particular, were not used to lessen heteroge-neity within a wide mix of cholestatic disorders.

The polyclonal, affinity-purified anti-BSEP antibodythat we employed21 identified appropriately distributedcanalicular BSEP in patients with a variety of liver diseasesof known etiology other than mutation of ABCB11. As ina previous study using snap-frozen tissue,9 we assessednonspecific preservation of canalicular antigens in termsof expression of MRP2, which, like BSEP, is an ATP-binding cassette protein and a canalicular transporter.MRP2 expression was present and unremarkable in allmaterials except, as expected, those from patients withDubin-Johnson syndrome.

In all 10 children with HCC and deficiency in expres-sion of BSEP, mutational analysis in ABCB11 found le-sions predicted to abrogate synthesis of functional BSEP.Failure to express immunohistochemically identifiableBSEP, or severe deficiency in BSEP expression (patientH), thus was paired with mutation in ABCB11 in everyinstance studied. All patients with mutation in ABCB11expressed immunohistochemically identifiable MRP2.We conclude that MRP2 served as an appropriate techni-cal control. We also conclude that lack of expression ofBSEP, when assessed immunohistochemically, correlatedwell with demonstration of mutation in ABCB11.

We could not associate progression to HCC with anyparticular kind of mutation in ABCB11, although splice-site mutations formed a plurality among the 13 mutationsidentified in the 10 families genetically studied. The var-ious predicted effects (Table 1) include splicing defects(patients A, B, E, F, H, and I), a frameshift (patient A),missense changes at conserved amino acid residues (pa-tients C, D, E, and H), and direct introduction of prema-ture termination codons (patient B, exon 2; patient G,exon 13; patient J, exon 19). The frameshift, which wasfound in exon 16, generates 6 altered amino acid residuesfollowed by premature termination. Among the missensechanges is 1 in exon 27 (patient C); those in patients D, E,and H (890A�G [E297G], exon 9) and the other muta-tion in patient C (1445A�G [D482G], exon 14) havebeen described previously.8 The 890A�G (E297G) and1445A�G (D482G) mutations reportedly affect BSEPtransport activity,21,33 with altered trafficking of BSEP tothe canalicular membrane.33-35 The mutation in patientC, 3691C�T (R1231W), is predicted to alter an aminoacid residue between the ATP-binding cassette signaturemotif and the Walker B motif of BSEP. This residue isconserved in all MDR subfamily members and in therelated transporters MRP2 and CFTR.

One of the mutations predicted to alter splicing hasbeen described. IVS18�1G�A, found in patient B, was(in combination with 3148C�T [R1050C]) associatedwith “benign” recurrent intrahepatic cholestasis(BRIC)—namely, intrahepatic cholestasis with intermit-tent clinical manifestations (published as IVS19�1G�Athrough typographical error [L. Klomp, personal commu-nication]).36 IVS16-8T�G, found in patient A, has beenassociated with PFIC in several other patients. This mu-tation leads to skipping of exon 17 with an associatedframeshift and introduction of 5 amino acid residues fol-lowed by protein truncation (unpublished data).IVS13del-13ˆ-8 removes 6 bases of the 3� splice site andchanges the location of the branch point sequence.IVS18�1G�A and the other 3 splice site changes (Table1) affect the invariant GT 5� donor splice site consensussequences and are predicted to lead to exon skipping andassociated frameshifts or to cryptic splice site activation.

Of interest is the difference in phenotype between pa-tient B, with PFIC in association with the genotypeIVS18�1G�A / 74C�A (S25X), and a patient withBRIC whose less severe disease was associated with theIVS18�1G�A / 3148C�T (R1050C) genotype.24 Fac-tors modulating penetrance of ABCB11 mutation haveyet to be defined. Also of interest is the presence amongour patients of the 890A�G (E297G) mutation (patientD, homozygous; patients E and H, heterozygous) and the1445A�G (D482G) mutation (patient C, heterozy-

484 KNISELY, STRAUTNIEKS ET AL. HEPATOLOGY, August 2006

gous). The former can be associated with either PFIC orBRIC8,24; preliminary observations suggest association ofboth with favorable response to PEBD.

Our patients D, E, and H, all with 890A�G (E297G)mutation, came to PEBD. Patients D and E had no im-munohistochemically identifiable BSEP; patient H re-tained some expression, which was very scant. Patient Hresponded well to PEBD, but HCC developed nonethe-less. Close monitoring appears in order for BSEP-defi-cient PFIC even when clinical response to PEBD is good.It may also be in order for BSEP-deficient BRIC.

Our patients’ tumors were not morphologically uni-form. No tumor met criteria for the diagnosis of hepato-blastoma or cholangiocarcinoma. (A hepatocellularmalignancy interpreted as hepatoblastoma has been de-scribed in association with PFIC; we have demonstrateddeficiency of BSEP expression in ambient liver, but con-sider the tumor more likely HCC.37) In 9 of the 10 pa-tients with demonstrated or inferred deficiency of BSEPexpression, the single lesion was a well-differentiatedHCC with bile production. Clear-cell features were foundin the other patient with a single lesion (patient A). In thepatient who had 3 tumors (patient G), 1 had clear-cellfeatures; and 2 were well-differentiated HCC.

Common characteristics of the tumors, however, wereincreased synthesis of AFP and nuclear accumulation ofp53 protein. None of the tumors exhibited nuclear accu-mulation of �-catenin. Although these observations mayimplicate specific pathways toward malignancy,22 theyidentify no particular carcinogenic species or event. Ofinterest is a preliminary report of cholangiocarcinoma in 2older children with PFIC and ABCB11 mutation.38 Thismay indicate mutagenesis affecting cells that can differen-tiate along either hepatocellular or cholangiocytic lines.

The mechanism of hepatocarcinogenesis in BSEP de-ficiency is unclear. Perhaps hepatocarcinogenesis in BSEPdeficiency or malfunction is among nonspecific conse-quences of various mutations in ABCB11. One such con-sequence—increased intracellular concentrations of bileacids—may be mutagenic.39 This, however, fails to ac-count for the dearth of HCC in other cholestatic andhypercholanemic disorders of infancy such as extrahepaticbiliary atresia.13

It is also possible that TTI provides an analogy. In TTI,a wide range of mutations in fumarylacetoacetate hydro-lase can result in inhibition of DNA ligase 1 by succinyl-acetone, leading to accumulation of genetic injury.40 Anyspecific agent predisposing to malignancy in BSEP defi-ciency has yet to be identified.

Of interest in BSEP deficiency, and distinct from TTI,is the speed with which HCC may develop. Althoughpharmacotherapy and intervention with LT have reduced

the proportion of children with TTI who develop HCCto as low as 10%,41,42 HCC historically occurred in 37%of children with TTI aged � 2 years.43 Among the casesreviewed, although dates of diagnosis are not cited, HCCled to death in no patient aged � 4 years.43 Instances ofHCC manifesting before the age of 24 months in TTIappear rare.44,45 By contrast, 7 of our 10 patients withBSEP deficiency and HCC were aged � 24 months atdiagnosis of HCC.

PFIC—whether due to deficiency of FIC1, deficiencyof BSEP, or other causes—is treated principally with sup-portive measures. It can be argued that to identify BSEPdeficiency as underlying PFIC has few practical implica-tions for care. Our findings imply that, on the contrary,identification of BSEP deficiency may be important. Wesuggest that monitoring for development of HCC is in-dicated in BSEP deficiency, perhaps with determinationsof serum concentrations of AFP and with sonography.Whether such monitoring is indicated in FIC1 deficiencyis an open question, though we know of no instance ofHCC associated with that condition.

Our observations also call into question treatment withallograft hepatocyte infusions14 or even gene therapy inPFIC owing to BSEP deficiency. To establish clones ofexogenous or modified hepatocytes that secrete bile acidsmay not only effectively treat cholestasis and pruritus butalso reduce risk of malignancy liver-wide. However, be-cause this approach may leave BSEP-deficient—and pos-sibly premalignant—hepatobiliary cells in place, it isperhaps less desirable than LT.

In conclusion, mutation in ABCB11, with deficiencyof immunohistochemically identifiable BSEP, increasesrisk of HCC in early life. Immunohistochemical defi-ciency of BSEP in PFIC is closely correlated with demon-strable mutation in ABCB11. These findings suggestapproaches to more efficient diagnosis of PFIC associatedwith ABCB11 mutation and warrant particular care in theobservation and management of patients with PFIC.

Note Added in Proof: The liver of yet another childwith PFIC and hepatocellular carcinoma46 (3 years, 5months of age at transplant hepatectomy) does notexpress BSEP; the common Polish mutation1445A�G(D482G)8 is present in one copy of ABCB11.Genetic analysis continues.

Acknowledgment: We thank Anne Rayner for excel-lent technical work.

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