transient response to imatinib in a chronic eosinophilic leukemia associated with...

Transient Response to Imatinib in a ChronicEosinophilic Leukemia Associated withins(9;4)(q33;q12q25) and a CDK5RAP2-PDGFRAFusion Gene

ChristophWalz,1 Claire Curtis,2,3 Susanne Schnittger,4 Beate Schultheis,1 Georgia Metzgeroth,1

Claudia Schoch,4 Eva Lengfelder,1 Philipp Erben,1 Martin C. Muller,1 Torsten Haferlach,4

Andreas Hochhaus,1 Rudiger Hehlmann,1 Nicholas C. P. Cross,2,3 and Andreas Reiter1*

1III.Medizinische Universit�tsklinik,Fakult�t fˇr Klinische Medizin Mannheimder Universit�t Heidelberg,Mannheim,Germany2Wessex Regional Genetics Laboratory,Salisbury District Hospital,Salisbury,UK3HumanGenetics Division,Universityof Southampton,Southampton,UK4Mˇnchner Leuk�mie-Labor,Munich,Germany

Chronic myeloproliferative disorders with rearrangements of the platelet-derived growth factor receptor A (PDGFRA) gene at

chromosome band 4q12 have shown excellent responses to targeted therapy with imatinib. Here we report a female patient

who presented with advanced phase of a chronic eosinophilic leukemia. Cytogenetic analysis revealed an ins(9;4)(q33;q12q25)

in 5 of 21 metaphases. FISH analysis with flanking BAC probes indicated that PDGFRA was disrupted. A novel mRNA in-frame

fusion between exon 13 of the CDK5 regulatory subunit associated protein 2 (CDK5RAP2) gene, a 40-bp insert that was par-

tially derived from an inverted sequence stretch of PDGFRA intron 9, and a truncated PDGFRA exon 12 was identified by 50-RACE-PCR. CDK5RAP2 encodes a protein that is believed to be involved in centrosomal regulation. The predicted

CDK5RAP2-PDGFRA protein consists of 1,003 amino acids and retains both tyrosine kinase domains of PDGFRA and several

potential dimerization domains of CDK5RAP2. Despite achieving complete cytogenetic and molecular remission on imatinib,

the patient relapsed with imatinib-resistant acute myeloid leukemia that was characterized by a normal karyotype, absence of de-

tectable CDK5RAP2-PDGFRA mRNA, and a newly acquired G12D NRASmutation. VVC 2006Wiley-Liss, Inc.

INTRODUCTION

Patients with clinical characteristics of chronic

myeloid leukemia (CML) who lack the Ph-chro-

mosome and/or the BCR-ABL1 fusion gene are

usually referred to as having atypical CML (aCML).

In many cases the hematological features overlap

with other recognized subtypes of chronic myelo-

proliferative disorders (CMPD) or myelodysplas-

tic/myeloproliferative disorders (MDS/MPD), par-

ticularly chronic eosinophilic leukemia (CEL) and

chronic myelomonocytic leukemia (CMML) (Var-

diman et al., 2002). In the majority of patients, the

molecular pathogenesis of these diseases is poorly

understood, but a minority of patients present with

acquired chromosomal aberrations. The molecular

cloning of these aberrations has revealed diverse

tyrosine kinase fusion genes that most commonly

involve PDGFRA, PDGFRB, FGFR1, or JAK2 (Crossand Reiter, 2002; Macdonald et al., 2002; Krause

and Van Etten, 2005; De Keersmaecker and Cools,

2006). The recurrent involvement of tyrosine ki-

nase-encoding genes at translocation breakpoints

has led to searches for other, cryptic abnormalities

of these genes, and currently the most commonly

known abnormality in aCML is the V617F JAK2mutation (Jones et al., 2005).

The most common abnormalities in CEL are

FIP1L1-PDGFRA, which results from a cytogeneti-

cally invisible deletion on 4q12 (Cools et al., 2003),

and the ETV6-PDGFRB fusion gene in CMML

with a t(5;12)(p12;q31-33) (Golub et al., 1994).

Patients with these fusion genes generally exhibit

an excellent response to treatment with imatinib,

with frequent complete hematologic and complete

cytogenetic responses and even high rates of com-

plete molecular responses in FIP1L1-PDGFRA

*Correspondence to: Andreas Reiter MD, III. Medizinische Uni-versitatsklinik, Fakultat fur Klinische Medizin der Universitat Hei-delberg, Wiesbadener Str. 7-11, 68305 Mannheim, Germany.E-mail: [email protected]

Supported by: German Bundesministerium fur Bildung und For-schung; Grant number: DLR e.V.-01GI9980/6; European Leuke-miaNet, Germany; Leukaemia Research Fund, UK.

Received 2 March 2006; Accepted 6 June 2006

DOI 10.1002/gcc.20359

Published online 14 July 2006 inWiley InterScience (www.interscience.wiley.com).

VVC 2006 Wiley-Liss, Inc.

GENES, CHROMOSOMES & CANCER 45:950–956 (2006)

positive CEL (Pardanani et al., 2006). In contrast,

CMPD-associated fusion genes with involvement

of JAK2, e.g., PCM1-JAK2 resulting from a t(8;9)

(p21;p24), or FGFR1, e.g., ZNF198-FGFR1 result-

ing from a t(8;13)(p11;q13), are resistant to imatinib.

We report here a female patient with a novel

CDK5RAP2-PDGFRA fusion gene as the result of

an ins(9;4)(q33;q12q25) in an advanced phase of a

chronic eosinophilic leukemia. This is the fourth

fusion gene with involvement of PDGFRA besides

BCR-PDGFRA, FIP1L1-PDGFRA, and KIF5B-PDGFRA (Baxter et al., 2002; Cools et al., 2003;

Score et al., 2006).

MATERIALS ANDMETHODS

Case Report

A 71-year-old female patient was admitted

because of splenomegaly, spontaneous hematomas,

and constitutional symptoms. The peripheral

blood revealed a leukocytosis of 125 3 109/l, mild

anemia (Hb 10.2 g/dl), and thrombocytopenia (46 3109/l). The differential blood count showed a left-

shifted leukocytosis with 13% blasts, 14% promye-

locytes, and 11% myelocytes in addition to marked

eosinophilia (19%). Bone marrow morphology

revealed a hypercellular marrow with 5% blasts

(CD34þ, MPOþ, TdT�, CD68�), increased pro-

myelocytes (25%), and a significant increase of

eosinophilic precursors and mature eosinophils

(30%). An ins(9;4)(q33;q12q25)[5]/46,XX [16] was

demonstrated by conventional karyotyping. A diag-

nosis of an accelerated phase of chronic eosino-

philic leukemia was made. The patient was not

eligible for intensive chemotherapy because of

comorbidity. After obtaining written informed con-

sent, treatment with 400 mg imatinib daily was ini-

tiated based on the morphological characteristics

and the cytogenetic involvement of the long arm

of chromosome 4. Molecular cloning revealed a

CDK5RAP2-PDGFRA fusion gene. The patient

achieved a complete cytogenetic and molecular

remission as measured by RT-PCR; however, he-

matologic response was only partial with residual

blasts repeatedly detectable in the blood and mar-

row. Three months after start of treatment with

imatinib, the patient rapidly developed imatinib-

resistant AML, while cytogenetic analysis demon-

strated a normal karyotype in 20 metaphases and

the CDK5RAP2-PDGFRA fusion gene was unde-

tectable by interphase-FISH and RT-PCR (Fig. 1).

In due course an acute appendicitis was diagnosed,

and the patient died soon after surgery because of

rapid increase of blasts and septic shock syndrome.

50-Rapid Amplification of cDNA Ends

Polymerase Chain Reaction

50-rapid amplification of cDNA ends polymerase

chain reaction (RACE-PCR) was essentially per-

formed according to the manufacturers’ instruc-

tions (50/30RACE Kit, Roche Diagnostics, Mann-

heim, Germany). Briefly, 1 lg RNA extracted from

peripheral blood leukocytes using the RNeasy sys-

tem (Qiagen, Hilden, Germany) was reverse tran-

scribed using primer A-R1: 50-TCCAGTGAAAAA-

CAAGCTCTCA-30. Nested PCR was performed

with primer A-R2: 50-GGGACCGGCTTAATC-

CATAG-30 in the first step and primer A-R3: 50-GGCTGTTCCTTCAACCACCT-30 in the second

step in conjunction with RACE anchor primers

supplied by the manufacturer. Products were

cloned using the TOPO cloning kit (Invitrogen,

Leek, The Netherlands) and sequenced.

RT-PCR Analysis

RNA was reverse transcribed with random hex-

amers, using standard techniques. Primers used to

detect CDK5RAP2-PDGFRA cDNA were CDK/f:

50-CAAAGGAGACTGCACCATCCGT-30 and PDG-

FRA/r: 50-CGACCAAGCACTAGTCCATCTC-30.Reciprocal PDGFRA-CDK5RAP2 fusion transcripts

were checked by the combination of primers PDG-

FRA/f: 50-AGTCCTGGTGCTGTTGGTGATT-

30 and CDK/r: 50-CTGAAGCAACACGTCCTT-

CTGAT-30. All amplifications were performed for

32 cycles at 608C annealing temperature.

Fluorescence In Situ Hybridization

Bacterial artificial chromosome (BAC) clones

were selected from sequences flanking PDGFRAand CDK5RAP2 from www.ensembl.org or http://

genome.ucsc.edu and obtained from the Sanger

Institute (Cambridge, United Kingdom). The fol-

lowing BACs were used: RP11-98G22 (upstream of

PDGFRA); RP11-24O10 (downstream of PDG-FRA); and RP11-88M19 (upstream of CDK5RAP2).Fluorescence in situ hybridization (FISH) was per-

formed according to standard procedures (Reiter

et al., 1998; Demiroglu et al., 2001).

Mutation Analysis

Screening for point mutations of NRAS using a

melting curve based LightCycler assay was per-

formed at codons 12, 13, and 61 (Nakao et al.,

2000b). Mutation analyses of FLT3 (length muta-

tions and point mutations within the tyrosine ki-

nase domain), NPM1, and MLL (partial tandem

duplications) were performed as previously de-

Genes, Chromosomes & Cancer DOI 10.1002/gcc

951CDK5RAP2-PDGFRA POSITIVE CEL

scribed (Nakao et al., 2000a; Schnittger et al., 2000,

2002, 2005). Samples tested positive were con-

firmed by sequence analysis. Primer sequences are

available on request.

RESULTS

The morphological characteristics of CEL and

the rearrangement of parts of the long arm of chro-

mosome 4 suggested a possible involvement of

PDGFRA. FISH experiments with BACs that

flanked PDGFRA indicated that this gene was

indeed disrupted (not shown). 50-RACE-PCR was

therefore used using reverse primers derived from

sequences located shortly downstream of known

PDGFRA breakpoint locations, e.g., FIP1L1-PDGFRA or BCR-PDGFRA, respectively. The

resulting PCR products consisted of sequences

derived from a known gene, CDK5 regulatory sub-

Figure 1. (A) RT-PCR for the detection of the CDK5RAP2-PDGFRA fusion transcript at different timepoints before (lane 1) and during treatment with imatinib (lanes 2–4). (B) Amplification of BCR as internalcontrol for adequate cDNA quality.

Figure 2. The in frame CDK5RAP2-PDGFRA fusion junction with the corresponding amino acids.CDK5RAP2 sequences are in plain type and PDGFRA sequences in italics. The 40-bp insert is shown in lowercases. The insert partly corresponds to an inverted sequence stretch derived from PDGFRA intron 9.


952 WALZ ET AL.

unit associated protein 2 (CDK5RAP2: REFSEQ:

NM_018249.4), fused to PDGFRA. Chimeric

CDK5RAP2-PDGFRA transcripts were confirmed

using RT-PCR (Fig. 1) and indicated an in-frame

fusion (Fig. 2) between CDK5RAP2 exon 13, a 40-

bp insert, and the truncated PDGFRA exon 12

(88 bp downstream the intron 11/exon 12 boun-

dary). Twenty-two basepairs of the insert could be

matched to an inverted sequence stretch derived

from PDGFRA intron 9, and 18 bp could not be

matched to any known sequence of the human

genome. FISH analysis confirmed the colocaliza-

tion of 50-CDK5RAP2 and 30-PDGFRA sequences

in patient, but not in control cells (not shown).

Reciprocal PDGFRA-CDK5RAP2 transcripts were

not detected by RT-PCR analysis. CDK5RAP2-PDGFRA is predicted to encode a protein of 1,003

amino acids (Fig. 3) consisting of more than 49%

of CDK5RAP2 (494 amino acids) and the entire

tyrosine kinase domain of PDGFRA (509 amino

acids).

Since acquired, activating mutations are fre-

quent events in AML, we checked samples at two

different time points (diagnosis and first detection

of imatinib-resistant AML) for known mutations of

NPM1, FLT3, NRAS, and MLL. We identified a

G12D NRAS mutation in the AML sample, but not

in the sample from diagnosis. The presence of the

mutation was confirmed by sequence analysis. No

mutations were detected in codon 13 and 61 of

NRAS or any of the other genes.

DISCUSSION

The karyotype in this case was not clearly sug-

gestive for a rearrangement of PDGFRA. The pres-

ence of a CDK5RAP2-PDGFRA fusion and the lack

of a reciprocal PDGFRA-CDK2RAP2 fusion there-

fore indicated a more complex rearrangement.

This hypothesis was supported by the insertion of

an inverted 22-bp sequence stretch derived from

PDGFRA intron 9 and additional insertion of a

20-bp sequence stretch of unknown origin be-

tween the two genes. Complex rearrangements in

obviously simple chromosomal rearrangements

have been confirmed by FISH analysis in several

other MPDs that are associated with tyrosine kinase

fusion genes, e.g., the t(8;13), t(5;10) and t(8;17),

resulting in the generation of the corresponding

fusion genes ZNF198-FGFR1, H4-PDGFRB, and

MYO18A-FGFR1, respectively (Reiter et al., 1998;

Kulkarni et al., 2000; Walz et al., 2005). The pre-

sence of complex aberrations may explain, at least

in part, why these fusions are so rare or even

unique.

The clinical characteristics at presentation and

the clinical course of our case were unusual.

Recent analyses of imatinib treated CML patients

have demonstrated the risks of point mutations in

the tyrosine kinase domain of ABL1 (Gorre et al.,

2001; Hochhaus et al., 2002; Shah et al., 2002;

Branford et al., 2003) or alternative genetic abnor-

malities, e.g., amplification of BCR-ABL1, ulti-

Figure 3. Diagrammatic representation of normal PDGFRA, normal CDK5RAP2, and the CDK5RAP2-PDGFRA fusion protein.



mately leading to imatinib-resistance (Gorre et al.,

2001; Hochhaus et al., 2002). In the present case,

imatinib failure was not due to known mechanisms

of imatinib-resistance. It was the consequence of

the outgrowth of a second imatinib-resistant leuke-

mic clone with a normal karyotype that had

acquired a G12D NRAS mutation. Although this

mutation was not detectable in a sample from diag-

nosis, this clone may already have coexisted with,

and possibly preceded the ins(9;4) clone at diagno-

sis. The hypothesis that CDK5RAP2-PDGFRA was

a secondary abnormality is supported by the pres-

ence of the ins(9;4) in only 5 of 21 metaphases at

diagnosis although features of the peripheral and

the marrow clearly indicated a CEL in accelerated

phase. A similar secondary fusion of CEV14 to

PDGFRB was reported in a patient with relapsed

AML (Abe et al., 1997). It can only be speculated

about the clinical course of the patient if treatment

with imatinib had not been initiated. It is conceiva-

ble that treatment and response to imatinib accel-

erated the outgrowth of the more malignant AML.

Mutations of NRAS, which is involved in regula-

tory processes of proliferation, differentiation, and

apoptosis, have been described in various solid

tumors and hematologic malignancies (Beaupre

and Kurzrock, 1999). They seem to be rare in

BCR-ABL1 negative MPDs, as no NRAS mutations

were found in a mixed series of 64 patients with

polycythemia vera, essential thrombocythemia,

and idiopathic myelofibrosis (Tsurumi et al., 2002).

However, the same G12D NRAS mutation as iden-

tified in our patient could be found in about 50%

in patients with CMML (Hirsch-Ginsberg et al.,

1990). It was speculated whether the occurrence of

additional events like NRAS mutations might con-

tribute to transformation into acute leukemia in

MPDs, but the actual rate of newly acquired NRASmutations in CML blast crisis compared with that

in CML chronic phase is low (Ahuja et al., 1991;

Watzinger et al., 1994). In addition, it has been

demonstrated that mutations of NRAS, FLT3, andTP53 are not involved in the transformation of

V617F JAK2 mutation positive MPDs (Suzuki

et al., 2006). The impact of the additional mutation

for disease progression in our case therefore

remains unclear.

In contrast to our case, the vast majority of

patients with PDGFRA fusions are male (Baxter

et al., 2002; Cools et al., 2003; Pardanani et al.,

2003; Trempat et al., 2003; Klion et al., 2004; Safley

et al., 2004; Vandenberghe et al., 2004; Score et al.,

2006), and a similar male predominance has also

been reported for fusion genes with involvement

of PDGFRB and JAK2 (Lacronique et al., 1997;

Peeters et al., 1997; Steer and Cross, 2002; Jones

and Cross, 2004; Bousquet et al., 2005; Murati

et al., 2005; Reiter et al., 2005). The reasons for

this observation remain obscure and it can only be

speculated about the possibility of uncharacterized

gender-dependent genetic alterations or other

mechanisms. Possibly these unknown mechanisms

may not apply to secondary abnormalities, and it is

interesting to note that the secondary CEV14-PDGFRB fusion was also seen in a female.

PDGFRA was initially identified as a fusion part-

ner of BCR in an aCML with t(4;22)(q12;q11)

(Baxter et al., 2002). Meanwhile, four cases have

been identified in which four different BCR exons

(exon 1, 7, 12 and 17, respectively) are fused with a

truncated PDGFRA exon 12 (Trempat et al., 2003;

Safley et al., 2004). In addition to the more fre-

quent FIP1L1-PDGFRA fusion, a KIF5B-PDGFRAfusion gene was identified recently in a male

patient with CEL and a complex rearrangement

involving chromosomes 3, 4, and 10 (Score et al.,

2006). All four known fusions have in common that

unrelated partner genes are fused to a truncated

PDGFRA exon 12 retaining the entire tyrosine ki-

nase domain of PDGFRA. With exception of the

KIF5B-PDGFRA fusion, short intron-derived

sequences are frequently found to be inserted

between the exons of the partner gene and

PDGFRA. Three fusion proteins retain potential

dimerization domains of the partner genes (BCR,KIF5B, and CDK5RAP2). In contrast, no potential

dimerization domains could be identified within

the FIP1L1 gene. By creating a series of FIP1L1deletions fused to PDGFRA, it has been shown that

FIP1L1 may not be essential for the transforming

activity of the truncated PDGFRA protein (Stover

et al., 2004). It was therefore speculated that the

increased tyrosine kinase activity of the FIP1L1-

PDGFRA fusion protein is due to loss of an autoin-

hibitory domain that is predominantly encoded by

PDGFRA exon 11. Why then some PDGFRA part-

ners have dimerization domains and some do not

remains a mystery.

CDK5RAP2 encodes a 24-kDa centrosomal pro-

tein that is widely expressed in the developing

embryo. It contains several functional motifs that

are retained in the CDK5RAP2-PDGFRA fusion

protein including regions that are predicted to form

multiple coiled-coils. In addition, it seems to have

a role as a binding partner for activation proteins of

CDC2-like kinase (NCLK), which are implicated

in differentiation of neuronal cells (Wang et al.,

2000; Bond et al., 2005). It was recently shown that


954 WALZ ET AL.

homozygous mutations in CDK5RAP2 and CENPJare associated with the occurrence of autosomal re-

cessive primary microcephaly (Bond et al., 2005).

During prometaphase and metaphase, CDK5RAP2

is concentrated at the mitotic spindle poles raising

the possibility that CDK5RAP2 may play a regula-

tory role in mitosis (Bond et al., 2005). Of interest,

analysis of the CDK5RAP2 sequence showed

homologies with the intermediate filament asso-

ciated protein (RESTIN) gene, which is strongly

expressed in Reed-Sternberg cells of Hodgkin’s

disease (Ching et al., 2000). Furthermore, CDK5-

RAP2 adds to a growing list of centrosomal partner

proteins in fusion genes involving tyrosine kinases

(Delaval et al., 2005).

In conclusion, we have identified the fourth

PDGFRA fusion gene in a female patient present-

ing with CEL. The patient achieved complete

cytogenetic and molecular remission on treatment

with imatinib. Subsequent failure to treatment

with imatinib was not due to common mechanisms

of resistance, but was due to rapid progression of

an imatinib-resistant normal karyotype AML with

a G12D NRAS mutation.

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transient response to imatinib in a chronic eosinophilic leukemia associated with...

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