doi:10.1182/blood-2003-04-1150Prepublished online August 7, 2003;
Ricardo C AguiarKathryn Wilkinson, Elvira R Velloso, Luiz F Lopes, Charles Lee, Jon C Aster, Margaret A Shipp and with eosinophilia: involvement of PDGFRB and response to imatinibCloning of the t(1;5)(q23;q33) in a myeloproliferative disorder associated
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Cloning of the t(1;5)(q23;q33) in a Myeloproliferative Disorder associated with Eosinophilia: Involvement of PDGFRB and
Response to Imatinib
Short Title: Imatinib in MPD with t(1;5) and novel PDGFRB fusion
Kathryn Wilkinson, Elvira R P Velloso, Luiz Fernando Lopes, Charles Lee, Jon C Aster, Margaret A Shipp and Ricardo C T
Aguiar
Affiliations:Dana Farber Cancer Institute and Harvard Medical School, Boston (K.W.,
M.A.S., R.C.T.A.); Brigham and Women’s Hospital and Harvard Medical School, Boston (C.L., J.C.A.); Hospital A.C. Camargo, São Paulo, Brazil (L.F.L.); Hospital das Clinicas, University of São Paulo, Brazil (E.R.P.V.).
Support:This study was partially funded by the Leukemia and Lymphoma Society of
America (RCTA). Ricardo C T Aguiar is a V Foundation Scholar.
Correspondence to:Ricardo C. T. Aguiar, MD, PhDDana Farber Cancer Institute44 Binney Street, M514Boston MA 02115Phone: 617-6323881Fax: 617-6324734Email: [email protected]
Word count: 1185; abstract: 150
Scientific section: Neoplasia
Copyright (c) 2003 American Society of Hematology
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ABSTRACT:
Eosinophilia is common in myeloproliferative disorders (MPD) with abnormalities of
chromosome band 5q31-33, including those that present with t(1;5)(q23;q33). With the
development of rational drug therapy, characterization of the molecular targets for these
translocations could guide treatment and impact on patient survival. We cloned the
t(1;5)(q23;q33) and showed that it fuses PDGFRB to the coiled-coil domains of a novel
partner protein, Myomegalin. Using two-color interphase FISH, we also demonstrated
that the eosinophils are clonal in these disorders. Imatinib mesylate has recently been
shown to be efficacious in MPDs with PDGFR activation. Therefore, following our
molecular studies, we were able to redirect this patient’s treatment. Although she had
refractory and progressive disease, once imatinib was started complete clinical and
hematological remission, and major cytogenetic response was achieved. Given the
therapeutic implications, our findings stress the need to aggressively investigate the
molecular basis of these diseases, with emphasis on the PDGFR family.
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INTRODUCTION:
The cloning of genes affected by recurrent chromosomal translocations has
furthered our understanding of the molecular basis of hematological malignancies, and
allowed the development of molecular assays that have improved diagnosis and
monitoring of therapy. It has also led to the development of rational targeted drug
therapy, including the specific tyrosine kinase inhibitor, imatinib mesylate1 (Gleevec,
Novartis).
Eosinophilia is common in several myeloproliferative disorders (MPD), notably
chronic myeloid leukemia (CML) and the 8p11 myeloproliferative syndrome2,3. Less
common causes of eosinophilia include MPDs associated with translocations involving
chromosome band 5q31-334. Cloning of these translocations4-9 showed that they
consistently target the PDGFRB gene. In all cases, partner genes contribute
oligomerization domains that constitutively activate PDGFRB’s tyrosine kinase. Recently,
cryptic deletions of chromosome 4q12 resulting in activation of PDGFRA were found in
cases of hypereosinophilic syndrome (HES)10, suggesting that activation of the PDGFR
receptor tyrosine kinase family is a common theme in this phenotypically similar
collection of disorders.
Of the MPDs with eosinophilia, one unique sub-group presents with a
t(1;5)(q23;q33)11,12. Here, we describe the molecular cloning of this translocation and the
clinical/translational consequences of this finding.
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CASE REPORT AND METHODS:
An 11-month-old girl presented with malaise, poor feeding and
hepatosplenomegaly. The initial blood count was Hb 6.0g/dl, WBC 43.9 x 109/L with a
neutrophilic left shift, marked eosinophilia, monocytosis and thrombocytopenia. After a
bone marrow examination, the diagnosis of an MDS/MPD syndrome with eosinophilia
was established. Karyotype revealed a t(1;5)(q23;q33) in 100% of the bone marrow
metaphases. The patient was refractory to conventional chemotherapy including
etoposide, cytarabine and interferon. At the start of imatinib, one year after the diagnosis,
the patient had progressive disease, and a new cytogenetic study confirmed the presence
of the t(1;5) in 100% of metaphases.
All of the laboratory investigations and treatment decisions were approved by the
ethics committee of the patient’s institution of origin (Hospital AC Camargo, Brazil).
Written consent was obtained from the patient’s guardians.
Molecular cloning: Southern blot and 5’ RACE PCR were performed as described
previously3,13. Appropriate PCR products were cloned and sequenced.
RT-PCR: Single step RT-PCR was used for detection of the PDE4DIP- PDGFRB and
reciprocal PDGRFB-PDE4DIP fusions . To define the specific PDE4DIP ( Myomegalin)
isoform fused to PDGFRB, a long-range nested RT-PCR was performed. All oligos
sequences are available upon request. All relevant PCR products were sequenced.
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Western blot: Protein was isolated from the patient’s peripheral blood cells at diagnosis
and two months into imatinib treatment. Immunoblotting was performed as previously
described13, with total AKT and phosho-AKT (S473) antibodies used as directed by the
manufacturer (Cell Signaling, MA).
Two-color fluorescence in-situ hybridization: An eosinophil-enriched cell population
was isolated from the patient’s peripheral blood as described previously 14. BAC clones
mapping centromeric (CTC-307M15, CTB-46E9, and CTB- 13H5) and telomeric (CTB-
5M9, CTB-108B20, and CTB-171P15) to the PDGFRB locus were from ResGen
(Carlsbad, CA). BAC DNAs were hybridized to the patient’s slides as described
previously3. After counterstaining with DAPI, images were processed using an Olympus
AX70 fluorescence microscope and Genus imaging software (Applied Imaging). To
assess post-treatment samples for cytogenetic response, cells were spun onto slides, fixed,
and hybridized to PDGFRB probes as described above. Cells were scored for the
presence of PDGFRB rearrangements on the BioView Duet Imaging system (Rehovot,
Israel).
RESULTS AND DISCUSSION
PDGFRB is fused to a novel gene, PDE4DIP, in the t(1;5)(q23;q33). With
Southern blot analysis, we detected rearrangement of the PDGRFB gene in the t(1;5)
DNA (Figure 1A). Subsequently, sequencing of several 5’ RACE-PCR clones
consistently showed the same novel DNA sequence fused in-frame to PDGFRB exon 11.
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Database search confirmed that these sequences mapped to chromosome 1q21-23 and
corresponded to a novel gene, PDE4DIP (phosphodiesterase 4D interacting protein ,
myomegalin)15. Southern blot analysis with a PDE4DIP probe confirmed its disruption in
this translocation (data not shown).
The chimeric PDE4DIP-PDGFRB mRNA was readily detectable in the patient with the
t(1;5)(q23;q33), but not in two normal controls (Figure 1B). The reciprocal PDGFRB-
PDE4DIP fusion is not expressed (Figure 1B). Myomegalin is the protein encoded by
PDE4DIP and it was characterized because of its binding to the phosphodiesterase
PDE4D14. Of relevance for this report, myomegalin encodes several putative
oligomerization domains capable of activating PDGFRB. They include a leucine z ipper
(LZ) domain and several coiled-coil structures (Fig. 1C). In humans, there are at least two
major isoforms of myomegalin (KIAA0454 and KIAA0477)15, encoding unique N and C
termini (Figure 1C). This feature is of significance because one of these isoforms encodes
an N-terminal LZ domain. Surprisingly, we found that in our case PDGFRB is fused to
the PDE4DIP isoform lacking the LZ domain (KIAA0477) (Figure 1C) indirectly
implicating the coiled-coils domains in the constitutive activation of PDGFRB.
In summary, in this fusion protein the first 905 amino acids of myomegalin are
joined in-frame to the transmembrane and tyrosine kinase domains of PDGFRB (Figure
1D). It is highly likely that deregulation of PDGFRB activity is the major pathogenic
defect in this MPD.
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Figure 1.
In MPD with t(1;5)(q23;q33) the eosinophils are part of the malignant clone.
Eosinophilia is a common feature of several hematological malignancies. It is generally
assumed that, when a clonal cytogenetic abnormality has been demonstrated in a
MDS/MPD with eosinophilia, these cells are part of the neoplastic clone. However, this is
not always true16-18, and only rarely has the clonality of the eosinophils been clearly
established19. As seen in figure 2, eosinophils are readily identified based on the auto-
fluorescent of eosinophilic granules, a feature that is unique among cells of the
granulocytic series19-21. These granules were pseudo-colorized white to allow for clear
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visualization of the FISH signals. Using a two-colour interphase FISH assay with probes
flanking the PDGFRB locus, we observed that this patient's eosinophils have
rearrangements of PDGFRB and are therefore components of the malignant clone (Figure
2).
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Figure 2
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Excellent response to imatinib in MPD associated with t(1;5)(q23;q33) and
PDGFRB disruption. After documenting the PDGFRB disruption in this patient, she
received a trial of imatinib therapy. All clinical and hematol ogical abnormalities rapidly
resolved. The bone marrow was normal at 5 months. FISH of peripheral blood cells after
seven months of therapy showed the t(1;5) in only 7.1% of the cells (split apart PDGFRB
signals in 8 of 106 cells, versus 0 of 210 cells from a normal control). We also evaluated
the effects of imatinib on known PDGFRB targets in vivo, comparing AKT
phosphorylation in the patient’s primary cells pre- and post-treatment. We demonstrated
that post-treatment, there was a marked decrease in the phosphorylation of AKT (Figure
1E). Since AKT is one of the well-defined targets of PDGFRB mitogenic and
transforming effects in hematopoietic cells, via activation of the PI3K pathway4, these
studies are consistent with imatinib specifically targeting the deregulated PDGFRB in
this patient’s MPD.
In summary, we have shown that the t(1;5)(q23;q33) targets PDGFRB and that
complete remission can be achieved with imatinib in a heavily pre-treated patient with
progressive disease. Of interest, although only a few cases of MPD with t(1;5)(q23;q33)
have been described11,12 this disease appears to be more common in infants; it also lacks
the extreme male bias found in other cases MPD associated with PDGRB activation4. Our
results confirm and extend the clinical relevance of imatinib treatment in cases of MPD
with eosinophilia22,23 .
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FIGURE LEGENDS:
Figure 1. PDGFRB is fused to PDE4DIP in an MPD associated with eosinophilia
and t(1;5)(q23;q33). A) Southern blot analysis of the t(1;5) and control DNA with a
HindIII-XhoI PDGFRB genomic probe spanning exon 11. Rearranged bands are
indicated by a star. Cloning of the genomic breakpoints for this fusion confirmed that the
wild-type PDGFRB and PDE4DIP-PDGFRB HindIII restriction fragments are of very
similar size and therefore co-migrated in the Southern blot. At DNA level, these genes
fusion occurs at IVS 12 -767 (PDE4DIP) and IVS 10 +289 (PDGFRB). B) Single step
RT-PCR confirms the expression of the PDE4DIP-PDGFRB fusion. The reciprocal
fusion is not expressed. PDGFRB RT-PCR confirms the integrity of the cDNAs. C)
Isoform-specific nested RT-PCR showing that the PDE4DIP isoform lacking the LZ
domain (KIAA 0477) is fused to PDGFRB in the t(1;5). Diagram of the primary protein
structure of human myomegalin, putative oligomerization domains, the breakpoint in the
t(1;5), and the location of the primers (horizontal arrows) used in the isoform-specific
nested RT-PCR. D) Diagrammatic representation of the Myomegalin (MM)-PDGFRB
protein fusion and contributing nucleotides, aminoacids and domains. E) Western blot
analysis showing lower levels of phosphorylated AKT (upper panel) in the patient’s
primary cells two months into imatinib treatment. Total AKT is shown in the lower panel.
Densitometry of relevant bands showed a 60% reduction in the expression of phospho-
AKT post-imatinib (right panel)
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Figure 2. The eosinophils have a rearranged PDGFRB locus and are part of the
neoplastic clone. We used a two-color interphase FISH assay in an esosinophil-enriched
preparation to define the clonality of these cells. Differentially labeled BAC clones
located centromeric (red) or telomeric (green) to the PDGFRB breakpoint region in the
t(1;5) were used as probes. Schematic representation of chromosome 5, breakpoint and
probes location is shown on the left side. In normal metaphases and interphases (upper
panel) the red and green signals are juxtaposed (yellow) in both chromosomes 5. In the
patient’s peripheral blood eosinophils (lower panel), one pair of signals is juxtaposed
(normal chromosome 5), whereas the other signals are split, indicating rearrangement of
the PDGFRB locus. The eosinophilic granules were pseudo-colorized in white due to
their auto-fluorescence.
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ACKNOWLEDGEMENTS:
We thank Patricia Dahia, Chuck Stiles and James Griffin (Dana Farber Cancer Institute),
Beatriz Beitler (Hospital das Clinicas, University of São Paulo), Nick Cross ( Wessex
Regional Genetics Laboratory, UK) and David Parkinson (Novartis, USA). Imatinib was
provided on a compassionate basis by Novartis, Brazil.
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