Short communication

A case of chronic myelogenous leukemia with e8a2 fusion transcript

Il Joong Parka, Young Ae Lima, Wee Gyo Leea, Joon Seong Parkb, Hugh Chul Kimb,Hyeon-Ji Leec, Sung Ran Choa,*

aDepartment of Laboratory Medicine , Ajou University School of Medicine, San 5 Wonchun-dong, Yeongtong-gu, Suwon 443-721, Republic of KoreabDepartment of Hematology-Oncology , Ajou University School of Medicine, Suwon, Republic of Korea

cSeegene Institute of Life Science, Seoul, Republic of Korea

Received 17 April 2008; received in revised form 30 May 2008; accepted 5 June 2008

Abstract The Philadelphia chromosome and its corresponding fusion gene, BCReABL, is one of the best-known genetic abnormalities in hematological malignancies. Major BCReABL translocation ismuch more common in chronic myelogenous leukemia (CML) and minor BCReABL in acute lym-phoblastic leukemia. We experienced an extraordinarily rare case of CML with an e8a2 variant. Anunusual band, other than the common transcripts, was observed in reverse transcriptionepolymer-ase chain reaction (RT-PCR) for the BCReABL gene rearrangement. Sequence analysis of the PCRproduct revealed an 1172-bp e8a2 fusion with a 14-bp insertion of ABL intron Ia. The patientachieved a complete hematological response 3 months after imatinib treatment. It is necessary tokeep in mind that an unexpected band revealed with RT-PCR may mean the presence of unusualfusion gene. � 2008 Elsevier Inc. All rights reserved.

Cancer Genetics and Cytogenetics 185 (2008) 106e108

1. Introduction

Philadelphia chromosome (Ph), characterized by thet(9;22)(q34;q11.2) translocation, is observed in O95% ofchronic myelogenous leukemia (CML) cases [1,2]. In gen-eral, the BCReABL fusion gene is transcribed as a largechimeric RNA, which typically results in either or both ofb2a2 (e13a2) and b3a2 (e14a2) fusion mRNAs, both orwhich are translated into p210 BCR-ABL protein. The pro-tein plays a crucial role in the pathogenesis of CML in thechronic phase [3]. A minor proportion of CML patients ex-press unusual types of BCReABL transcripts, althoughmost CML patients have b2a2 or b3a2. Recently, severalpatients with unusual e8a2 BCReABL transcripts have beenreported and characterized [4e7]. Here, we report a case ofCML with a novel e8a2 BCReABL transcript with a 14-bpfragment insertion of the ABL intron Ia.

2. Materials and methods

2.1. Case report

A 46-year-old man, who had been referred from asecondary-care hospital because of high white blood cell(WBC) count with a left shift and palpable spleen, was

* Corresponding author. Tel.: þ82-31-219-5780; fax: þ82-31-219-

5778.

E-mail address: sungran@ajou.ac.kr (S.R. Cho).

0165-4608/08/$ e see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.cancergencyto.2008.06.001

admitted to Ajou University Hospital and diagnosed witha chronic phase of CML. On admission, his complete bloodcell count revealed white blood cells 46,200/mL (normal,3,900e9,690) with 4% of blast cells, hemoglobin 13.0 g/dL (normal, 11.7e17.1), and platelets 663,000/mL (normal,134,000e387,000). Peripheral blood and marrow findingswere compatible with CML. Conventional cytogeneticanalysis was not performed, because of a missing order. In-stead, dual-color, dual-fusion fluorescence in situ hybrid-ization (FISH) on peripheral blood was done to test forthe presence of BCReABL rearrangement or t(9;22). Mul-tiplex reverse transcriptionepolymerase chain reaction(RT-PCR) for BCReABL gene rearrangement showed anunexpected band, in addition to the typical fusion tran-scripts (Fig. 1A), whereas FISH analysis showed a typicaltranslocation pattern consistent with CML. Sequence anal-ysis of the PCR product revealed an 1172-bp e8a2 fusionwith a 14-bp insertion of ABL intron Ia (Fig. 1B). Westarted administration of imatinib mesylate at a daily doseof 400 mg, and a complete hematological response wasachieved by 3 months. The patient has been maintainedwell on imatinib at a daily dose of 300 mg.

2.2. FISH analysis

Interphase FISH analysis using Vysis dual-color, dual-fusion, locus-specific identifiers (LSI) BCR and ABLprobes (Abbott Molecular/Vysis, Des Plaines, IL) was

AAGCCCTTCA...

...tacttg tttggttttatcag gtggtt...

...CCCGACGGCA GTCCATGAC...

BCR exon 8

…cttccagABL intron Ia

ABL intron Ia

CPM

Internal control

b2a/2 (major)

600

400

900

1200

...CCCGACGGCAtttggttttatcagAAGCCCTTCA..e1a/2 (minor)

ABL exon 2

u

A B

Fig. 1. (A) Detection of BCReABL transcript by multiplex reverse transcriptaseepolymerase chain reaction. An unusually large band (u), ~1200 bp, was

observed in the patient. Lane M: size marker (100-bp ladder); lane P: patient sample; lane C: positive control with b2a2 (401 bp) and e1a2 (348 bp) fusions

and internal control (600 bp). (B) Sequence analysis of the atypical band revealed an 1172-bp e8a2 fusion with a breakpoint within e8 at position 2686 (Gen-

Bank Y00661) and a 14-bp insertion of Ia intron between e8 and a2.

107I.J. Park et al. / Cancer Genetics and Cytogenetics 185 (2008) 106e108

performed according to the manufacturer’s recommenda-tions. The slides were freshly prepared from the patient’speripheral blood, and 200 interphase nuclei were counted.

2.3. Molecular studies

2.3.1. Multiplex RT-PCRThe multiplex RT-PCR assay was performed according

to the manufacturer’s instructions with a Seeplex leukemiaBCReABL kit (Seegene, Seoul, Korea), which is designedto detect eight common BCReABL transcripts, includingthe major breakpoint cluster region (M-bcr), minor bcr(m-bcr), and micro bcr (m-bcr). Cycling conditions wereas follows: 94�C for 15 minutes (1 cycle); 94�C for 30 sec-onds, 60�C for 1 minute 30 seconds, 72�C for 1 minute 30seconds (37 cycles); and 72�C for 10 minutes (1 cycle). ThePCR products were analyzed by 2% agarose gel electropho-resis at 100 V for 30 minutes.

2.3.2. Cloning and sequencingThe purified PCR product (~1200 bp) was inserted into

the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) ac-cording to the manufacturer’s specifications. PlasmidDNA was prepared using a Qiagen plasmid mini-kit (Hil-den, Germany). Sequencing was performed using an ABIBigDye terminator version 3.1 cycle sequencing kit (PEApplied Biosystems, Foster City, CA) and the M13F/M13R primer on an ABI 310 DNA sequencer. Sequenceanalysis was done using the basic local alignment searchtool BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi)and Ensembl (http://www.ensembl.org/index.html).

3. Results

Interphase FISH showed a typical translocation pattern(i.e., 2 yellow BCReABL fusion signals, 1 green BCR sig-nal, and 1 orange ABL signal) in 65% of nuclei counted.Multiplex RT-PCR for BCReABL showed an unusuallylarge band (~1200 bp), in addition to the internal control

band (Fig. 1A). Sequencing revealed that this band corre-sponded to a junction between BCR exon e8 and ABL exona2 including a 14-nucleotide fragment insertion from ABLintron Ia at the fusion point (nt 88286e88299, GenBankNW_924573 [http://www.ncbi.nlm.nih.gov]) (Fig. 1B).

4. Discussion

Several unusual BCReABL transcripts are uncommon inboth CML and acute lymphoblastic leukemia, includinge2a2 [8], e3a2 [9], e6a2 [3], e8a2 [4,6,7], e15a2 [10],e19a2 [11,12], e1a3 [13], e13a3 [14], and e14a3 [15]. In-cluding the present case, 13 CML cases with e8a2 junctionmore frequent than other variants have been reported todate. The e8a2 is an example of transcripts with fusion ofout-of-frame ABL and BCR exons [16]. The direct junctionbetween BCR exon e8 and ABL exon a2 would not producean oncogenic BCReABL protein due to a generation ofa premature stop codon (UAG) at position 7 after the fusion[5]. Therefore, either the chromosomal breakpoint must oc-cur inside exons or intronic sequences must be interposedto restore the reading frame. In previously reported cases,e8a2 mRNA revealed various sequences by an insertionof intronic sequences generally derived from the ABL intronIb, by breakpoints within the BCR exon 8 or the ABL exona2, or by both mechanisms [4e7].

The most frequent rearrangement in patients with e8a2revealed by molecular study is an identical 55-bp invertedfragment insertion from ABL intron Ib; this accounts forO50% of the e8a2 patients described to date. Demehriet al. [5] reported a case of CML with e8a2 having an151-bp insertion of ABL intron Ia and breakpoint withinthe exon a2. The present case showed a breakpoint withine8 exon and a 14-bp fragment insertion from ABL intronIa. The present case thus appears to be the second case withe8a2 transcript showing an ABL intron Ia insertion.

For the present it seems that e8a2-positive CML patientshave no distinctive clinical features except for good re-sponse to imatinib. Because the number of cases hitherto

108 I.J. Park et al. / Cancer Genetics and Cytogenetics 185 (2008) 106e108

reported is small, collecting more cases is necessary tocharacterize e8a2-positive CML more definitively. Patientswith e8a2 transcripts were reported to be resistant to inter-feron-a treatment, suggesting a worse prognosis of CMLwith e8a2 fusion gene [5]. After the introduction of imati-nib mesylate, however, a few investigators have reportedthat e8a2 CML might be sensitive to the ABL tyrosine ki-nase inhibitor, imatinib [7,17]. The present case showeda good response to imatinib, too.

A variety of multiplex RT-PCR assays are now availablefor BCReABL fusion transcripts, to detect not only thecommon BCReABL fusion (M-bcr, m-bcr) but also unusualvariants. In clinical laboratories using optimal reagents andprocedures, an aberrant band in multiplex RT-PCR forBCReABL may indicate the presence of unusual fusiongene, and the identification of the atypical transcript byDNA sequencing may be necessary to further validate thefinding.

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