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MYELOID NEOPLASIA
Brief report
A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling acute
promyelocytic leukemia
Esperanza Such,1 José Cervera,1 Ana Valencia,1 Eva Barragán,2 Mariam Ibañez,1 Irene Luna,1 Óscar Fuster,2
Maria Luz Perez-Sirvent,1 Leonor Senent,1 Amparo Sempere,1 Jesús Martinez,1 Guillermo Martín-Aragonés,1 and
Miguel A. Sanz1
Departments of 1Hematology and 2Medical Pathology, Hospital Universitario La Fe, Valencia, Spain
Chromosomal translocations in hematological malignancies often result in novel
fusion chimeric genes. We report a case
of acute myeloid leukemia with a clonal
translocation t(11;12)(p15;q13) displaying morphologic and immunophenotypic
features resembling the classical hypergranular subtype of acute promyelocytic
leukemia. The gene fused to NUP98
(nucleoporin 98) was detected by comparative genomic hybridization array as
the retinoid acid receptor gamma gene
(RARG). The involvement of RARG in a
chimeric fusion transcript has not been
reported previously in human leukemia.
(Blood. 2011;117(1):242-245)
Introduction
Chromosomal translocations in hematological malignancies often
result in the generation of novel chimeric genes. The nucleoporin
98 gene (NUP98) located at chromosome 11p15 is recurrently
involved in a variety of rearrangements in both myeloid and
lymphoid malignancies.1,2 After the first NUP98 rearrangement,
Homeobox A9 gene (NUP98/HOXA9), was discovered, more than
20 different fusion partners were reported.1 Among these partners,
other homeobox and non-homeobox genes have been identified as
fusion partners of NUP98. We report here a novel NUP98
rearrangement in a patient with acute myeloid leukemia displaying
morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. This
case harbored a novel fusion gene as a result of a chromosomal
break point at 12q13 detected in a new 11p15 rearrangement. The
gene fused to NUP98 was identified by comparative genomic
hybridization (CGH) array as the retinoid acid receptor gamma
gene (RARG). RARG is a member of the nuclear receptor
superfamily and shares high homology (90%) with RARA and
RARB, the other retinoic acid receptors that are involved in
retinoid signaling. While an artificial construct has been reported in
which RARG is fused to the promyelocytic leukemia gene product
(PML), resulting in an oncogenic protein,3 no cases of human
leukemia–bearing RARG fusions have been described.
Methods
Case reports
This study was approved by the institutional review board of the Hospital
Universitario La Fe. A 35-year-old man was referred to our department with
asthenia, mucosal bleeding, spontaneous ecchymoses, and fever. Blood
tests showed a hemoglobin level of 6 g/dL, a platelet count of 8 ⫻ 109/L,
and a white blood cell count of 12 ⫻ 109/L with 82% blasts and atypical
promyelocytes. Although increased levels of D-dimer were present, the
patient did not fulfill the criteria for coagulopathy.4 Morphologic examination of bone marrow (BM) smears disclosed a monomorphic infiltration by
blast cells, 80% hypergranular (atypical promyelocytes, which frequently
displayed Auer rods) resembling the hypergranular subtype of acute
promyelocytic leukemia (M3) of the French-American-British classification (Figure 1A-B). The immunophenotype of peripheral blood blasts was
positive for CD13, CD33, CD45, CD117, and cMPO, weakly positive for
CD34, and negative for HLA-DR and B-cell and T-cell markers. The
immunofluorescence staining with the anti-PML monoclonal antibody
(PG-M3)5 was negative in both BM and peripheral blood samples.
Moreover, reverse transcription polymerase chain reaction (RT-PCR) to
amplify the PML/RARA fusion gene and conventional karyotyping to
search for the t(15;17) were also negative. Due to the clinical suspicion of
acute promyelocytic leukemia, the patient was started on all-trans retinoic
acid (ATRA) treatment. One day after ATRA initiation, due to the absence
of a PML/RARA rearrangement, ATRA was discontinued and treatment was
switched to a standard 7 ⫹ 3 schedule with cytarabine and idarubicin as
induction therapy. After the achievement of complete remission, the patient
underwent consolidation chemotherapy followed by autologous peripheral
blood stem-cell transplant. Eight months after transplantation, he remains
alive and in complete hematologic remission.
Cytogenetic analysis and FISH
A BM sample was processed after short-term culture (24 hours) following
standard procedures. The chromosomes were stained by G-banding and the
karyotype reported according to International System for Human Cytogenetic Nomenclature recommendations.6 Fluorescent in situ hybridization
(FISH) was performed on 200 interphase cells using the dual-color
translocation probes Vysis LSI PML/RARA and the Vysis LSI RARA dual
color break apart (both from Abbott Laboratories) to discard variant
translocations of RARA.
RT-PCR
Molecular analysis by reverse transcription polymerase chain reaction
(RT-PCR) for different PML/RARA isoforms was performed as described
elsewhere.7 The presence of NUP98 rearrangements with the genes
Submitted June 23, 2010; accepted September 19, 2010. Prepublished online
as Blood First Edition paper, October 8, 2010; DOI 10.1182/blood-2010-06-291658.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2011 by The American Society of Hematology
242
BLOOD, 6 JANUARY 2011 䡠 VOLUME 117, NUMBER 1
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BLOOD, 6 JANUARY 2011 䡠 VOLUME 117, NUMBER 1
A NOVEL NUP98/RARG GENE FUSION IN ACUTE MYELOID LEUKEMIA
243
Figure 1. Morphologic features and results of CGH array of an acute myeloid leukemia patient with a clonal translocation t(11;12)(p15;q13) resembling the classical
hypergranular subtype of acute promyelocytic leukemia. May-Grümwald-Giemsa–stained bone marrow smear (1000⫻, Nikon Eclipse 80i microscope and Nikon DS-Fi 1
camera) showing (A) promyelocytes with hypergranulated cytoplasm; several nuclei are invaginated and (B) promyelocyte with bundles of Auer rods (faggot cell).
(C) Representative karyotype of BM cells with t(11;12)(p15;q13). (D) Results of CGH array. Top, microdeletions in chromosome 11p15 involving the NUP98 gene. Bottom,
microdeletions in chromosome 12q13 involving the RARG gene.
described in 12q13, HOXC11 and HOXC13, was also determined using
previously reported assays.8,9 The NUP98/RARG mRNA was reversetranscribed into cDNA using random hexamers, and PCR was performed
using the following primers: NUP98 exon 10 NUP98F: 5⬘-GGG CTT GGT
GCA GGA TTT GG-3⬘, and RARG exon 7 RARGR: 5⬘-TGG GTC CGG
TTC AGG GTC AGC-3⬘. These primers were also used to amplify the
fusion transcript break points (GenBank accession number: HQ229990).
CGH array
Genomic DNA was quantified and its purity confirmed by spectrometry
(ND1000, NanoDrop Technologies). Non-amplification labeling of DNA
(direct method) was obtained following the Agilent Oligonucleotide
Array-Based CGH for Genomic DNA Analysis protocol (Version 5.0;
Agilent Technologies). DNA (2000 ng) from patient leukemic cells was
fragmented in a restriction-digestion step. Digestion was confirmed and
evaluated by the DNA 7500 Bioanalyzer assay. Cyanine 3-dUTP and
cyanine 5-dUTP were used for fluorescent labeling of, respectively, the BM
sample and the patient DNA control sample for the patient digested gDNA
using a genomic DNA enzymatic labeling kit according to the manufacturer’s instructions. Labeled DNA was hybridized with Human Genome CGH
Microarray 244K containing 236,000⫹ coding and noncoding human
sequences represented. Arrays were scanned in a microarray scanner
(Agilent) according to the manufacturer’s protocol, and data were extracted
using feature extraction software Version 9.5.3.1; Agilent). All microarray
data are available at ArrayExpress (http://www.ebi.ac.uk/arrayexpress)
under accession number E-MEXP-2928.
Results and discussion
G-banding karyotype analysis in BM cells revealed a clonal
translocation t(11;12)(p15;q13) in 16 of 20 metaphases analyzed
(Figure 1C). FISH analysis using the PML/RARA and RARA probes
hybridized to the normal copies of chromosomes 15 and 17, indicating
the absence of cryptic PML/RARA fusion or RARA rearrangements. Two
previous rearrangements in apparently similar cases bearing t(11;12)
aberrations have been described,8,9 and these involved the NUP98
gene in 11p15 and HOXC11 or HOXC13 genes in 12q13. These
fusion transcripts were ruled out by specific RT-PCR analyses.
A CGH array was performed to better map the rearrangement
region and to search for minor or cryptic genomic imbalances
flanking the break-point translocation. A 1.0-Mb microdeletion in
11p15 and a 2.5-Mb microdeletion in 12q13, involving the NUP98
and RARG genes, respectively, were identified. No other copy
number variations were found (Figure 1D). To confirm a NUP98/
RARG fusion, an 881-bp product was specifically amplified from
the patient’s cDNA but not from the control (Figure 2A). The
transcript fusion product was coamplified in the same reaction and
sequenced. Sequence analysis of the NUP98/RARG fusion transcript revealed that NUP98 exon 12 was fused in-frame to RARG
exon 4 (Figure 2B). The reciprocal RARG/NUP98 fusion product
was not detected with either single-step or nested PCR (Figure 2A).
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244
SUCH et al
BLOOD, 6 JANUARY 2011 䡠 VOLUME 117, NUMBER 1
Figure 2. Electrophoresis agarose gel and chromatogram showing the NUP98/RARG rearrangement, and schematic representation of the NUP98/RARG fusion
protein. (A) NUP98/RARG fusion. The reciprocal RARG/NUP98 fusion product was not detected. (B) Nucleotide and protein sequences surrounding the NUP98/RARG fusion
region. The fusion was in-frame, so that the open reading frames of both genes in the fusion transcript were retained. The arrows indicate the break-point and fusion sites of the
NUP98/RARG gene. (C) Schematic representation of the NUP98/RARG fusion proteins and its wild-type counterparts. The locations of the primers used for RT-PCR are
indicated by horizontal arrows. GLFG repeats have been shown to be docking sites for transport receptors. P indicates patient cDNA (881bp); C1 and C2, PML/RARA patient’s
cDNA; M6, molecular weight; GLEBS, motif of nuclear export of poly(A)⫹ RNA and nuclear pore complex (NPC) structure; RBD, RNA-binding domain; DBD, In the
abbreviations for Figure 2, “DBD, DNA-binding domain and distribution and arrows the joining site” has been rephrased as “DBD, DNA-binding domain. Arrows indicate the
joining sites.” Please confirm or correct. DNA-binding domain. Arrows indicate the joining sites.
This latter finding might be related to the demonstrated microdeletions in both genes. The NUP98/RARG fusion mRNA (Figure 2C)
is predicted to encode an 862–amino acid protein. The NUP98
5⬘-region encoding the GLFG-repeat and the GLEBS-like motifs
were fused to the 3⬘-region of RARG, which included the
DNA- and ligand-binding domains of the gene. We hypothesize
that, as was observed with RARA fusion genes in acute promyelocytic leukemia, this newly described chimeric fusion can disrupt
normal retinoid signaling by acting as an aberrant RARG receptor
(Figure 2C).
The NUP98 gene encodes a protein component of the nuclear
pore complex that regulates the nucleocytoplasmic transport of
proteins and mRNAs. NUP98 contains a domain with a GLFG
repeat that has been shown to activate transcription, providing
docking sites for nuclear transport to conduct RNA and protein
traffic between the nucleus and cytoplasm. Chimeric transcripts
formed by the NUP98 N-terminal GLFG repeats fused to the
C-terminus of the partner proteins are expressed in all NUP98
fusions reported, suggesting that the NUP98 N-terminus may be
important for leukemogenesis.10,11 Here we identified RARG as a
new NUP98 partner.
RARG is a member of the retinoid acid receptor (RAR) family,
along with RARA, which is known to be involved in the PML/RARA
fusion of acute promyelocytic leukemia, and RARB. RARs are
nuclear hormone receptors functioning as ligand-activated transcription factors that interact specifically to modulate transcription of
DNA elements.12 They function as heterodimers with retinoid X
receptors. Specifically, heterodimers formed by RXRA-RARG are
necessary for growth arrest and visceral and primitive endodermal
differentiation.3,12
RARG rearrangements in human leukemia have not been
previously described. Recently, La Starza et al1 reported a case of
an adult patient with acute myeloid leukemia of the M2 subtype
carrying a t(11;12)(p15;q13) translocation that involved a NUP98
rearrangement with 1 of the following 3 putative candidate genes
localized at the translocation break point: RARG, MFSD5, or
ESPL1. Although, in the above case the partner gene of NUP98 was
not identified, the authors speculated that RARG was probably not
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BLOOD, 6 JANUARY 2011 䡠 VOLUME 117, NUMBER 1
A NOVEL NUP98/RARG GENE FUSION IN ACUTE MYELOID LEUKEMIA
implicated because it was incorrectly oriented. This hypothesis is
reinforced by the fact that, unlike our case, the patient reported by
La Starza et al1 did not share any morphologic features characteristic of acute promyelocytic leukemia. It should be noted that an
artificial PML/RARG fusion protein is able to produce the same
biological effects mediated in the cells by PML/RARA and also to
confer responsiveness to differentiation treatment with retinoid
acid.3 In addition, inoculated transduced cells expressing PML/
RARG were able to trigger leukemia in vivo in a murine model,
displaying morphologic features resembling those obtained with
transduction of PML/RARA.12
In conclusion, we describe here a novel NUP98/RARG gene
rearrangement that conferred to leukemic cells acute promyelocytic
leukemia–like morphologic and immunophenotypic features. The
favorable outcome with a standard 7 ⫹ 3 chemotherapy approach,
followed by consolidation and intensification with autologous
stem-cell transplantation, does not allow us ascertain the sensitivity
to ATRA, due to the early discontinuation of the drug in this patient.
Further studies are needed to better investigate the biological
properties, in particular sensitivity to retinoids, of leukemias
bearing this new NUP98/RARG fusion protein.
Acknowledgments
The authors thank Federico Gomis for the morphological photographs, Beatriz Costán for her daily support in performing molecular and cytogenetic studies, and the Microarray Analysis Service of
245
the Centro de Investigación Principe Felipe of Valencia for CGH
arrays. We also thank Dr Francesco Lo Coco for his invaluable help
and critical review of the manuscript.
This work was supported in part by grant R06/0020/0031 from
Red Temática de Investigación Cooperativa en Cancer, grant
BES2008-008053 from the Ministerio de Ciencia e Innovación,
and grants CA08/00141, CM09/00038, PI06/0657, and 09/01828
from Instituto de Salud Carlos III.
Authorship
Contribution: E.S. designed molecular studies and wrote the paper;
M.L.P.-S. and L.S. performed morphologic analysis; E.S. and A.V.
performed cytogenetic analysis and FISH experiments; A.S. performed PG-M3 immunofluorescence study; M.I. and O.F. performed molecular studies; E.B. supervised molecular studies; J.C.
supervised clinical and experimental findings; J.M. and G.M.-A.
were involved in the management of the patient; I.L. provided
clinical data; and M.A.S. was responsible for the conception of the
study and final approval of the draft. All authors reviewed the
manuscript and contributed to the final draft.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Miguel A. Sanz, Hospital Universitario La Fe,
Avenida Campanar 21, 46009 Valencia, Spain; e-mail:
[email protected].
References
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109-111.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2011 117: 242-245
doi:10.1182/blood-2010-06-291658 originally published
online October 8, 2010
A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling
acute promyelocytic leukemia
Esperanza Such, José Cervera, Ana Valencia, Eva Barragán, Mariam Ibañez, Irene Luna, Óscar
Fuster, Maria Luz Perez-Sirvent, Leonor Senent, Amparo Sempere, Jesús Martinez, Guillermo
Martín-Aragonés and Miguel A. Sanz
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