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Acknowledgments: We thank A. Ren for technical help with
data collection and preprocessing. S. Shamma, C. Espy-Wilson,
E. Cibelli, K. Bouchard, and I. Garner provided helpful
comments on the manuscript. E.F.C. was funded by NIH
grants R01-DC012379, R00-NS065120, and DP2-OD00862
and the Ester A. and Joseph Klingenstein Foundation. E.F.C.,
C.C., and N.M. collected the data. N.M. and C.C. performed the
analysis. N.M. and E.F.C. wrote the manuscript. K.J. provided
phonetic consultation. E.F.C. supervised the project.
Materials and Methods
Figs. S1 to S12
16 September 2013; accepted 17 January 2014
Published online 30 January 2014;
Detection of a Recurrent DNAJB1-PRKACA
Chimeric Transcript in Fibrolamellar
Joshua N. Honeyman,1,2 Elana P. Simon,1,3 Nicolas Robine,4 Rachel Chiaroni-Clarke,1
David G. Darcy,1,2 Irene Isabel P. Lim,1,2 Caroline E. Gleason,1 Jennifer M. Murphy,1,2
Brad R. Rosenberg,5 Lydia Teegan,1 Constantin N. Takacs,1 Sergio Botero,1
Rachel Belote,1 Soren Germer,4 Anne-Katrin Emde,4 Vladimir Vacic,4 Umesh Bhanot,6
Michael P. LaQuaglia,2 Sanford M. Simon1†
Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and
young adults with no history of primary liver disease or cirrhosis. We identified a chimeric
transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the
result of a ~400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code
for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular
chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A.
Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed
in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence
supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs
examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.
Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor that was first described in 1956 and that historically
has been considered a variant of hepatocellular
carcinoma (1, 2). It is histologically characterized
by well-differentiated neoplastic hepatocytes and
thick fibrous bands in a noncirrhotic background
(3, 4). FL-HCC has a clinical phenotype distinct
from conventional hepatocellular carcinoma and
usually occurs in adolescents and young adults.
Patients have normal levels of alpha fetoprotein
without underlying liver disease or history of vi-
ral hepatitis (3–6). Little is known of its mo-
lecular pathogenesis. FL-HCC tumors do not
respond well to chemotherapy (7, 8), and surgical
resection remains the mainstay of therapy, with
overall survival reported to be 30 to 45% at
5 years (1, 6, 8, 9).
To investigate the molecular basis of FL-HCC,
we performed whole-transcriptome and whole-
genome sequencing of paired tumor and adjacent
normal liver samples. To determine whether there
were tumor-specific fusion transcripts among the
coding RNA, we ran the program FusionCatcher
(10) on RNA sequencing (RNA-Seq) data from
29 samples, including primary tumors, metastases,
recurrences, and matched normal tissue samples,
derived from a total of 11 patients (table S1).
There was only one recurrent candidate chimeric
transcript detected in every tumor sample. This
candidate transcript is predicted to result from
the in-frame fusion of exon 1 from the DNAJB1
gene, which encodes a member of the heat
shock 40 protein family, with exons 2 to 10 from
PRKACA, the gene encoding the adenosine 3′,5′-
monophosphate (cAMP)–dependent protein ki-
nase A (PKA) catalytic subunit alpha. This fusion
transcript was not detected in any of the available
paired normal tissue samples (n = 9). This fusion
is not found in the COSMIC database (11) and
has not previously been reported in the literature.
To further characterize the candidate fusion
transcript, we directly examined those RNA-Seq
reads that mapped to PRKACA and DNAJB1. We
examined PRKACA transcript levels with DESeq2
(12) and found that they were increased relative
to normal in tumors from all nine patients tested
[P value adjusted for multiple testing (pAdj) < 10−12,
range three- to eightfold]. To determine whether
the increased expression was attributable to a
specific isoform of PRKACA, we quantified reads
mapping to different exons and evaluated differential expression using DEXSeq (13). In all nine
patients, there was an increase in the expression
of exons 2 to 10 of PRKACA in the tumor relative to exon 1 and relative to the expression in normal tissue (Fig. 1A, left). This exon expression
pattern does not correspond to a known isoform
of PRKACA. Rather, it reflects an increase in
PRKACA transcripts lacking the first exon, which
encodes the domain that engages the regulatory
subunits of PKA. All reads mapping to PRKACA
in normal tissue were either contained within
exons or bridged the junctions between adjacent
exons at annotated splicing sites (Fig. 1B, left,
blue). All tumor samples additionally had reads
mapping from the start of the second exon of
1Laboratory of Cellular Biophysics, Rockefeller University, 1230
York Avenue, New York, NY 10065, USA. 2Division of Pediatric
Surgery, Department of Surgery, Memorial Sloan-Kettering
Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
3The Dalton School, 108 East 89th Street, New York, N Y 10128,
USA. 4New York Genome Center, 101 Avenue of the Americas,
New York, NY 10013, USA. 5Whitehead Presidential Fellows
Program, The Rockefeller University, 1230 York Avenue, New
York, NY 10065, USA. 6Pathology Core Facility Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY
*These authors contributed equally to this work.
†Corresponding author. E-mail: firstname.lastname@example.org