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: Ricardo_Aguiar@dfci.harvard.edu

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.

Ricardo_Aguiar@dfci.harvard.edu

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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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REFERENCES

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11. Darbyshire PJ, Shortland D, Swansbury GJ, Sadler J, Lawler SD, Chessells JM. A

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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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