Hepatocellular Carcinoma in Ten Children Under Five
Years of Age With Bile Salt Export Pump Deficiency
A. S. Knisely,1Sandra S. Strautnieks,2Yvonne Meier,3Bruno Stieger,3Jane A. Byrne,2Bernard C. Portmann,1
Laura N. Bull,4Ludmila Pawlikowska,4Banu Bilezikc ¸i,5Figen O¨zc ¸ay,6Aranka L´ aszl´ o,7L´ aszl´ o Tiszlavicz,8
Lynette Moore,9Jeremy Raftos,10Henrik Arnell,11Bj¨ orn Fischler,11Antal N´ emeth,11Nikos Papadogiannakis,12
Joanna Cielecka-Kuszyk,13Irena Jankowska,14Joanna Pawłowska,14Hector Melı ´n-Aldana,15Karan M. Emerick,16
Peter F. Whitington,16Giorgina Mieli-Vergani,2and 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 the
canalicular 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-
Archival liver were retrieved and immunostained for BSEP. Mutational analysis of ABCB11
was 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, with
neonatal hepatitis suggesting PFIC, of a tenth from whom liver was not available) had
ABCB11 were demonstrated in all patients with BSEP deficiency in whom leukocyte DNA
could be studied (n ? 7). These mutations were confirmed in the parents (n ? 14). With
respect 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 a
BSEP deficiency correlates well with demonstrable mutation in ABCB11. (HEPATOLOGY 2006;
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 the1Institute of Liver Studies, King’s College Hospital, London, UK; the2Division of Gene and Cell Based Therapy, Department of Liver Studies and
Transplantation, King’s College London School of Medicine, London, UK; the3Division of Clinical Pharmacology and Toxicology, Department of Medicine, University
Hospital, Zu ¨rich, Switzerland; the4University of California–San Francisco Liver Center Laboratory and Department of Medicine, San Francisco General Hospital, San
of Pediatrics, Albert Szent-Gyo ¨rgyi Medical Centre and University of Szeged, Szeged, Hungary; the8Department of Pathology, University of Szeged, Szeged, Hungary; the
9Department of Histopathology and the10Paediatric Emergency Department, Women’s and Children’s Hospital, North Adelaide, South Australia, Australia; the
11Department of Pediatrics, Karolinska University Hospital, Huddinge and Solna, Stockholm, Sweden; the12Section of Perinatal Pathology, Department of Pathology,
Karolinska University Hospital, Huddinge, Stockholm, Sweden; the Departments of13Pathology and14Pediatric Gastroenterology, Hepatology, and Immunology, The
Children’s Memorial Health Institute, Warsaw, Poland; and the Departments of15Pathology and16Pediatrics, Northwestern University Feinberg School of Medicine and
Children’s Memorial Hospital, Chicago, IL.
Received February 16, 2006; accepted May 24, 2006.
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:
email@example.com; 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).
Potential conflict of interest: Nothing to report.
activity, or low-GGT PFIC, once known as “Byler dis-
ease,” encompasses several disorders.1These are defined
by persistent nonremitting conjugated hyperbiliru-
binemia with elevated serum concentrations of bile acids
(hypercholanemia), lack of abnormal bile acid species in
serum and urine, patency of a normally formed biliary
or ileal exclusion is undertaken,2-4evolution into end-
(FIC1) deficiency and bile salt export pump (BSEP) defi-
ciency. The former is caused by mutation in ATP8B1,
which encodes FIC1.5,6FIC1 is a putative aminophos-
pholipid flippase.7The latter is caused by mutation in
ABCB11,8which encodes BSEP.8,9BSEP is the principal
canaliculus.10,11At biopsy on presentation in infancy,
patients with PFIC due to FIC1 deficiency generally have
bland canalicular cholestasis, whereas patients with PFIC
due to BSEP deficiency generally have “neonatal hepati-
Hepatocellular carcinoma (HCC) is rare in children.13
We identified 11 unrelated children with clinically diag-
nosed PFIC in whom HCC was diagnosed between the
ages of 13 and 52 months. In 10 of these children, BSEP
deficiency was demonstrated immunohistochemically
and mutation in ABCB11 was demonstrated via molecu-
ily members. We describe clinical and histopathological
findings, attempt correlation with results of mutational
analysis and processes of carcinogenesis, and suggest im-
plications for management.
rogressive familial intrahepatic cholestasis (PFIC)
with normal or only slightly elevated serum con-
centrations of ?-glutamyltranspeptidase (GGT)
HCC was found in the explanted liver of a boy with
intrahepatic cholestasis (patient A; Table 1) and BSEP
deficiency. The boy’s disorder, which manifested at 3
weeks of age, was characterized by failure to thrive and
icterus. Clinical and laboratory studies revealed hyper-
cholanemia, conjugated hyperbilirubinemia, and low
tion or malformation. Microscopy of a liver biopsy spec-
imen obtained at 35 days of age identified “neonatal
hepatitis” with intralobular cholestasis and anisocytosis,
crosis of hepatocytes; portal-tract cholestasis was not
found (Fig. 1A). Immunohistochemical studies demon-
strated normal expression of multidrug resistance-associ-
ated protein 2 (MRP2) (Fig 1B), like BSEP an adenosine
triphosphate (ATP)-binding cassette protein involved in
canalicular transport; MRP2 here served as a functional
control useful in assessing tissue preservation.9Marking
for BSEP (see Methods section) was entirely absent (Fig.
1C). Allogeneic hepatocytes were infused14in hopes of
averting orthotopic liver transplantation (LT), and ta-
crolimus was given. No clinical effect was apparent, al-
though GGT rose slightly, consonant with greater access
of bile acids to the canalicular lumen.1,15The child was
listed for LT. The serum concentration of ?-fetoprotein
(AFP) had been 2 IU/L as a neonate (nl ? 7). This level
had risen to 34 IU/L before hepatocyte transplantation
and reached 199 IU/L by LT (21 months), after which it
fell immediately. The deep green-brown explanted liver
contained 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, and
lular edema (Fig. 1D). The tumor exhibited clear-cell
change, with intracellular inclusions consistent with gly-
coprotein (Fig. 1E). On immunostaining, tumor-cell cy-
marked for p53 but not for ?-catenin. Typing of micro-
satellite marker loci on 6 different chromosomes using
DNA from lesion and from patient and donor leukocytes
found the tumor to be of native liver rather than alloge-
neic-hepatocyte origin. Mutational analysis of ABCB11
using leukocyte DNA revealed compound heterozygosity
for IVS16-8T?G, which induces direct splicing of exon
16 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 HCC
associated with PFIC at King’s College Hospital and else-
where. Ten additional young children with HCC associ-
ated with intrahepatic cholestasis were identified; all 11
children were unrelated (Table 1). Three of the children
had been subjects of previous reports (patients B, F, and
K).13,16-20Each patient exhibited conjugated hyperbiliru-
biopsy in 9 patients revealed “neonatal hepatitis”; in 2
patients, each of whom had 3 affected siblings with bi-
opsy-documented “neonatal hepatitis,” the parents re-
fused biopsy. In the 9 children specifically assessed, GGT
was low. These children also had no evidence of bile acid
atitis B virus or hepatitis C virus was not demonstrated in
any of the patients. Tyrosinemia type I (TTI) and other
metabolic 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, 2006KNISELY, STRAUTNIEKS ET AL.479
Table 1. Patients, Clinical Courses, and ABCB11 / BSEP Mutations
from 3 wk
21 mo (incidental
in explant; AFP
199, nl ? 7),
28 mo, at open
biopsy; AFP not
(2 y, 8 mo
647K then VFTSLX/
from 2 wk,
aged 12 wk
None Palliative care
aged 33 mo
Splice site disruption/
0 None 23 mo (AFP 30k,
nl ? 5; liver
necropsy, 24 mo
22 mo (AFP 158k,
nl ? 15); liver
mass; lung and
diagnosis at LT,
29 mo (incidental
in explant; AFP
6.4k, nl ? 9),
16 mo (clinically
15 mo (AFP 11k,
nl ? 7; liver
diagnosis at LT,
52 mo (marked
2x106, nl ? 9),
at open biopsy
13 mo (incidental
in explant; AFP
831, nl ? 23),
14 mo (AFP 4k,
nl ? 23;
LT, 15 mo
26 mo (marked
at open biopsy
aged 24 mo
from 3 wk
Partial external biliary
11 mo; decreased
test results and
growth no better
PEBD, 10 mo;
Death, 6 d
1 Growth failure
from 6 mo;
from 6 wk
(5 y, 10 mo
Splice site disruption/
1None Death with
Splice site disruption/
from 6 wk
(1 y, 7 mo
0 Evaluation at
6 mo for
PEBD, 32 mo;
bile salts in
3 wk after
(1 y, 11 mo
Splice site disruption
from 1 wk
(1 y, 2 mo
from 3 mo
None Death with
None soughtNone predicted
480KNISELY, 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, patient
A; panel F, patient G). (A) “Neonatal hepatitis,” age 35 days; intralobular cholestasis with edema and multinucleation of hepatocytes. Centrilobular
venule, left, and portal tract, right. (Hematoxylin-eosin; original magnification ?200.) (B) Same material as panel A immunostained for MRP2 with
hematoxylin counterstain. The arrow marks a canaliculus with reaction product. (Original magnification ?200.) (C) Same material as panels A and
B 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; original
magnification ?200.) (E) HCC with clear-cell features and prominent inclusion bodies, histochemically consistent with glycoprotein; hepatectomy
specimen, 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, 2006KNISELY, STRAUTNIEKS ET AL.481
HCC; the other 6 underwent LT. Of these 6, one died of
sepsis, without tumor, shortly after LT, and 5 are well.
Paraffin-wax blocks containing tumor and nontumoral
liver were retrieved; sections were stained and immuno-
E, G, H, I, and J) and from parents’ peripheral blood in 10
undertaken (see Methods section). All studies were consid-
ered routine diagnostic assessment.
Materials and Methods
Histological Studies. Tumor was available from all
11 patients with HCC. Nontumoral liver was available
from 10 patients. In the exception (patient F),16,17whose
sister also had intrahepatic cholestasis manifesting as
archives. The patient’s lung, with metastatic tumor, and
were stained with hematoxylin-eosin and, after diastase
digestion, with periodic acid — Schiff technique. Parallel
sections were immunostained (DAKO Chem-Mate;
DAKO, Ely, UK) with antibodies raised in rabbit against
BSEP21and raised in mouse against an ATP-binding cas-
sette protein, MRP2 (Signet/Bioquote, York, UK). Sec-
tions containing tumor were similarly stained with
antibodies against AFP (DAKO), p53 (DAKO), and
?-catenin (Novocastra, Newcastle-upon-Tyne, UK),
hepatocytes.22The anti-BSEP antibody was raised, as de-
scribed,11,23against an oligopeptide of the C-terminal 13
amino 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 (ATP8B1
mutation, n ? 10; ABCB11 mutation, n ? 5; ABCB4
mutation, n ? 3; bile acid synthesis disorder, idiopathic
sin storage disorder, Alagille syndrome, and TTI, n ? 2
(All commercial products were used according to the
Mutational Analysis. DNA was extracted from pe-
mini-kit (QIAGEN, Crawley, UK). ABCB11 was ana-
noncoding exons. Primer sequences for exonic amplifica-
tion, available on request, included up to 50 bp of in-
tronic flanking sequence and hence all sequences critical
for mRNA splicing.
PCR amplification and product purification were per-
formed using Roche FastTaq amplification and High
Pure 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 ethanol
precipitation and capillary gel electrophoresis on a 3100-
Avant Genetic Analyzer (Applied Biosystems). Sequence
analysis was performed using Sequencher software (Gene
Codes, Ann Arbor, MI).
Microsatellite Typing (Patient A). DNA was ex-
DNeasy Tissue kit (QIAGEN) as well as from leukocytes
labeled primers were used to amplify informative micro-
satellite marker loci on 6 chromosomes. PCR products
were separated on a 373 Automated DNA Sequencer and
plied Biosystems). Haplotypes for tumor and for patient
and hepatocyte-donor leukocytes were constructed and
Histological Studies. In 6 patients with HCC, liver
and tumor were sampled at LT (patients A, D, E, G, I,
and J). In 3 patients, liver and tumor were sampled at
necropsy (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 was
obtained at diagnostic laparotomy (patients B and H).
All 11 patients had cirrhosis when tumor was diag-
Tumors in the 6 explanted livers consisted of a single
1 patient (patient J), and 3 nodules in 1 patient (patient
G). Tumor in 1 patient (patient A) lacked bile pigment
and consisted of cells with cleared cytoplasm containing
globules of eosinophilic material (Fig. 1E). Tumors were
exhibited bile pigment at the centers of rosettes of cells
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 within
tumor, 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 (who
also had severe intrahepatic cholestasis) of the patient
with HCC from whom nontumoral liver was not avail-
able (patient F), BSEP was not detected immunohisto-
liver from another patient with HCC (patient H), scant
staining for BSEP was seen along occasional canaliculi. In
yet another patient (patient K) and his brother, BSEP
expression was intact. MRP2 was well-expressed at cana-
licular margins in all nontumoral liver. BSEP expression
was not found in any tumor except in patient K.
All tumors, however, expressed MRP2 at margins of
rosettes or, at cell borders, in linear patterns consistent
with canalicular margins. Canaliculi in all samples from
the panel of comparison materials but those with Dubin-
Johnson syndrome expressed MRP2. Canaliculi in all
samples from the panel but those with known ABCB11
mutation expressed BSEP. AFP was demonstrated in cy-
B, J, and K) stained for p53; ?-catenin accumulation in
nuclei was not detected in any of the tumors.
Mutational Analysis. Mutation in ABCB11 was
found on both alleles in 9 families and on 1 allele in the
family of patient F, from which only maternal leukocytes
were available. In 7 patients, mutations were found on
analysis of proband leukocyte DNA; these were con-
firmed in parental material for each. With respect to the
other 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 parents
who consented to genetic studies (both parents of patient
B, both parents of patient C, and only one parent of
patient 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 mutation
1445A?G (D482G)8was present in one parent of a pa-
tient from whom leukocytes were not available (patient
(E297G)8was present in 3 patients and their parents (pa-
tients D, E, and H). Except for 890A?G (E297G), none
of the changes found was present in 500 control individ-
uals representative of all major populations (University of
California San Francisco Pharmacogenetics Project24;
Microsatellite Typing (Patient A). At all marker loci
studied, haplotypes constructed for tumor matched those
for patient DNA rather than for hepatocyte-donor DNA.
Several descriptions exist of HCC in early childhood
associated with “giant-cell hepatitis” or “neonatal hepati-
tis”.13,16-20,25,26Two such children had one sibling with
lings with intrahepatic cholestasis, one of whom died
from HCC in adolescence.20HCC 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.20,27-32What causes giant-cell hepatitis or “neo-
natal hepatitis” in these children, or what sort of PFIC
affects them, is a matter of debate.
In a search of published instances of HCC in early
childhood and in a review of materials at five pediatric
hepatology centers, we identified 11 children, 3 of whom
had been subjects of the case reports cited above,13,16-20in
associated with development of HCC at less than 52
months of age and from whom (and whose families) ar-
Archival materials for 2 other similar children25,26were
sought but were unavailable (T. Higgins and S. Falkmer,
personal communications). Attempts to gain access to
at an unstated age32were unsuccessful.
Liver disease in 9 of the 11 patients met the clinico-
the other 2 patients (patients F and K),16-20GGT was not
measured, and primary disorders of bile acid synthesis
were not excluded. In all 11, clinicopathological findings
onstrated immunohistochemically at canalicular margins
(8 patients) or was present but very scant (patient H). In
patient F,16,17nontumoral liver was not available for as-
sessment of BSEP expression. The liver of a sister with
cholestatic liver disease fatal in childhood and initially
manifest as “neonatal hepatitis”,18,19however, entirely
lacked immunohistochemically demonstrable BSEP.
Liver disease in this sibling pair thus was assessed as likely
due to BSEP deficiency. In 10 of 11 instances of HCC in
early childhood associated with clinically diagnosed
patient K20(see below).
Lesions in ABCB11 predicted to disrupt synthesis of
functional BSEP were identified in all 10 families studied
by mutational analysis (Table 1). Mutation was found on
able for the exception (patient F), and only a maternal
HEPATOLOGY, Vol. 44, No. 2, 2006KNISELY, STRAUTNIEKS ET AL.483
found in each of the 7 children with deficiency of BSEP
expression from whom peripheral blood leukocytes were
available. With respect to the other 3 children with
proven or likely deficiency of BSEP expression, each of
the 5 parents whose DNA could be studied proved to
carry a single ABCB11 change. The lack of immunohis-
B and C and from the sibling of child F argues strongly
that B, C, and F each carried 2 defective ABCB11 alleles.
In all 10 of the patients whose liver disease was associated
with BSEP deficiency, then, mutation in ABCB11 was
directly demonstrated or could be reasonably inferred.
No deficiency of BSEP expression was found in liver
from either patient K or his older brother, both of whom
had clinically diagnosed PFIC and both of whom died of
HCC.20Liver disease in this sibling pair thus was assessed
K with respect to ABCB11 mutation is unknown.
Though lesions in ABCB11 impeding BSEP function but
not BSEP expression may have been present, the liver
natal hepatitis” in that child and his siblings20perhaps
definition of PFIC now generally in use1did not yet exist
30 years ago, when patient K and his siblings were evalu-
ated: GGT values and results of bile acid synthesis defect
screening, in particular, were not used to lessen heteroge-
neity within a wide mix of cholestatic disorders.
The polyclonal, affinity-purified anti-BSEP antibody
that we employed21identified appropriately distributed
a previous study using snap-frozen tissue,9we assessed
nonspecific preservation of canalicular antigens in terms
of expression of MRP2, which, like BSEP, is an ATP-
binding cassette protein and a canalicular transporter.
MRP2 expression was present and unremarkable in all
materials except, as expected, those from patients with
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 identifiable
BSEP, or severe deficiency in BSEP expression (patient
H), thus was paired with mutation in ABCB11 in every
instance studied. All patients with mutation in ABCB11
expressed immunohistochemically identifiable MRP2.
We conclude that MRP2 served as an appropriate techni-
cal control. We also conclude that lack of expression of
BSEP, when assessed immunohistochemically, correlated
well with demonstration of mutation in ABCB11.
We could not associate progression to HCC with any
particular kind of mutation in ABCB11, although splice-
identified 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 was
found in exon 16, generates 6 altered amino acid residues
followed by premature termination. Among the missense
and H (890A?G [E297G], exon 9) and the other muta-
tion in patient C (1445A?G [D482G], exon 14) have
been described previously.8The 890A?G (E297G) and
1445A?G (D482G) mutations reportedly affect BSEP
transport activity,21,33with altered trafficking of BSEP to
the canalicular membrane.33-35The mutation in patient
C, 3691C?T (R1231W), is predicted to alter an amino
acid residue between the ATP-binding cassette signature
motif and the Walker B motif of BSEP. This residue is
conserved in all MDR subfamily members and in the
related transporters MRP2 and CFTR.
One of the mutations predicted to alter splicing has
been described. IVS18?1G?A, found in patient B, was
(in combination with 3148C?T [R1050C]) associated
(BRIC)—namely, intrahepatic cholestasis with intermit-
tent clinical manifestations (published as IVS19?1G?A
nication]).36IVS16-8T?G, found in patient A, has been
associated with PFIC in several other patients. This mu-
tation leads to skipping of exon 17 with an associated
frameshift 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 and
changes the location of the branch point sequence.
IVS18?1G?A and the other 3 splice site changes (Table
1) affect the invariant GT 5? donor splice site consensus
sequences and are predicted to lead to exon skipping and
associated frameshifts or to cryptic splice site activation.
Of interest is the difference in phenotype between pa-
tient B, with PFIC in association with the genotype
IVS18?1G?A / 74C?A (S25X), and a patient with
BRIC whose less severe disease was associated with the
IVS18?1G?A / 3148C?T (R1050C) genotype.24Fac-
tors modulating penetrance of ABCB11 mutation have
yet to be defined. Also of interest is the presence among
our patients of the 890A?G (E297G) mutation (patient
1445A?G (D482G) mutation (patient C, heterozy-
484KNISELY, STRAUTNIEKS ET AL. HEPATOLOGY, August 2006
gous). The former can be associated with either PFIC or
BRIC8,24; preliminary observations suggest association of
both 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 H
responded 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 hepatocellular
malignancy interpreted as hepatoblastoma has been de-
scribed in association with PFIC; we have demonstrated
deficiency 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 BSEP
expression, the single lesion was a well-differentiated
in the other patient with a single lesion (patient A). In the
patient who had 3 tumors (patient G), 1 had clear-cell
features; and 2 were well-differentiated HCC.
Common characteristics of the tumors, however, were
increased synthesis of AFP and nuclear accumulation of
p53 protein. None of the tumors exhibited nuclear accu-
mulation of ?-catenin. Although these observations may
implicate specific pathways toward malignancy,22they
identify no particular carcinogenic species or event. Of
older children with PFIC and ABCB11 mutation.38This
tiate along either hepatocellular or cholangiocytic lines.
The mechanism of hepatocarcinogenesis in BSEP de-
deficiency or malfunction is among nonspecific conse-
quences of various mutations in ABCB11. One such con-
sequence—increased intracellular concentrations of bile
acids—may be mutagenic.39This, however, fails to ac-
count for the dearth of HCC in other cholestatic and
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.40Any
specific agent predisposing to malignancy in BSEP defi-
ciency has yet to be identified.
is the speed with which HCC may develop. Although
the proportion of children with TTI who develop HCC
to as low as 10%,41,42HCC historically occurred in 37%
of children with TTI aged ? 2 years.43Among the cases
reviewed, although dates of diagnosis are not cited, HCC
led to death in no patient aged ? 4 years.43Instances of
HCC manifesting before the age of 24 months in TTI
appear rare.44,45By contrast, 7 of our 10 patients with
BSEP deficiency and HCC were aged ? 24 months at
diagnosis of HCC.
PFIC—whether due to deficiency of FIC1, deficiency
of BSEP, or other causes—is treated principally with sup-
portive measures. It can be argued that to identify BSEP
deficiency as underlying PFIC has few practical implica-
tions for care. Our findings imply that, on the contrary,
identification of BSEP deficiency may be important. We
suggest that monitoring for development of HCC is in-
dicated in BSEP deficiency, perhaps with determinations
of serum concentrations of AFP and with sonography.
Whether such monitoring is indicated in FIC1 deficiency
is an open question, though we know of no instance of
HCC associated with that condition.
allograft hepatocyte infusions14or even gene therapy in
PFIC owing to BSEP deficiency. To establish clones of
exogenous or modified hepatocytes that secrete bile acids
may not only effectively treat cholestasis and pruritus but
also reduce risk of malignancy liver-wide. However, be-
cause this approach may leave BSEP-deficient—and pos-
sibly premalignant—hepatobiliary cells in place, it is
perhaps less desirable than LT.
In conclusion, mutation in ABCB11, with deficiency
of immunohistochemically identifiable BSEP, increases
risk of HCC in early life. Immunohistochemical defi-
ciency of BSEP in PFIC is closely correlated with demon-
strable mutation in ABCB11. These findings suggest
approaches to more efficient diagnosis of PFIC associated
observation and management of patients with PFIC.
Note Added in Proof:
with PFIC and hepatocellular carcinoma46(3 years, 5
months of age at transplant hepatectomy) does not
1445A?G(D482G)8is present in one copy of ABCB11.
Genetic analysis continues.
The liver of yet another child
lent technical work.
We thank Anne Rayner for excel-
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