Cancer Genetics and Cytogenetics 189 (2009) 132e137

Short communication

BCR/ABL rearrangement with b3a3 fusion transcript in a case ofchildhood acute lymphoblastic leukemia

Juwon Kima, Tae Sung Parka, Chuhl Joo Lyub, Jaewoo Songa, Kyung-A Leea, Sue Jung Kima,Hyeon-Ji Leec, Jong Rak Choia,*

aDepartment of Laboratory Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, KoreabDepartment of Pediatrics, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea

cSeegene Institute of Life Science, 65-5 Bangyi-dong, Songpa-gu, Seoul 138-050, Korea

Received 19 September 2008; received in revised form 29 October 2008; accepted 10 November 2008

Abstract The role of BCR/ABL isoforms and their relati

* Corresponding

0956.

E-mail address: c

0165-4608/09/$ e see

doi:10.1016/j.cancerg

onship to leukemia phenotype have been of majorconcern. Atypical BCR/ABL mRNA transcripts lacking exon a2 have been reported in 12 casesof acute lymphoblastic leukemia (ALL) to date; among them, a b3a3 type transcript has beenreported only once in the childhood ALL. Reported here is the case of a patient with Philadel-phia-positive (Phþ) ALL expressing a b3a3 type transcript, a rare type of BCR/ABL mRNA lackingABL exon a2 sequences. Bone marrow showed a hypercellular marrow with leukemic blasts positivefor CD10, CD19, CD79a, and cytoplasmic m, which is consistent with pre-B ALL. The G-bandingand fluorescence in situ hybridization analyses indicated Phþ. After the patient was diagnosed withALL-L2, induction chemotherapy was performed and imatinib mesylate was thereafter given as themaintenance therapy. Sequencing analysis showed deletion of ABL a2 in the polymerase chain reac-tion product, which corresponded to a b3a3 fusion transcript. To our knowledge, this is the secondreport of an aberrant BCR/ABL product lacking ABL exon a2 in childhood ALL. � 2009 ElsevierInc. All rights reserved.

1. Introduction

The reciprocal translocation t(9;22)(q34;q11.2) resultingin the formation of a BCR/ABL fusion gene is a characteristicfeature of most chronic myeloid leukemias (CML) [1] and isconsidered to be responsible for leukemogenesis. Althoughthe Philadelphia chromosome (Ph) is associated mainly withCML (95% of CML patients are Phþ), 20% of patients withadult acute lymphoblastic leukemia (ALL) [2] and 5% ofchildhood ALL patients are also Phþ [3]. The Ph is of interestin ALL, because patients harboring it have shown poor prog-nosis under conventional therapy [4,5] and because the use ofthe ABL tyrosine kinase inhibitors as a treatment agentsignificantly improves their prognosis [6].

The majority of CML cases have been shown to haveeither e13a2 (b2a2) or e14a2 (b3a2) mRNA transcripts,whereas the typical transcript in ALL cases is the e1a2 tran-script [7]. Rare cases have been reported of CML with BCRbreakpoints outside the three defined cluster regions, orwith unusual breakpoints in ABL resulting in BCR/ABL

author. Tel.: þ82-2-2228-2445; fax: þ82-2-313-

jr0606@yuhs.ac (J.R. Choi).

front matter � 2009 Elsevier Inc. All rights reserved.

encyto.2008.11.006

transcripts with b2a3 or b3a3 junctions, or with aberrantfusion transcripts containing variable lengths of intronicsequence inserts [8]. Among those variants, b3a3 tran-scripts are so exceptionally rare that only 8 such cases ofCML and 1 such case of ALL have been described in theliterature [9e14]. Here, we described the case of a 12-year-old boy with Phþ ALL producing a rare b3a3 aberrantBCR/ABL fusion product; we also reviewed the literature,and consider the role of diversity of BCR/ABL fusion tran-scripts and their relationship to the leukemic phenotype.

2. Case report

A 12-year-old boy was admitted to Severance Hospitalof Yonsei University for right inguinal pain in July 2008.The peripheral blood count showed a hemoglobin level of6.2 g/dL, and a platelet count of 73 � 109/L, with a whiteblood cell count of 190 � 109/L (8% segmented neutro-phils, 19% lymphocytes, and 73% blast cells). The bonemarrow aspirate showed a hypercellular marrow withleukemic blasts of variable size (L2 in the FrencheAmeri-caneBritish classification). The blasts were positive forCD10, CD19, CD79a, and cytoplasmic m and were negative

133J. Kim et al. / Cancer Genetics and Cytogenetics 189 (2009) 132e137

for CD2, CD3, CD7, CD13, CD14, CD33, myeloperoxi-dase, surface immunoglobulin, and TdT, consistent withpre-B ALL.

Cytogenetic study on bone marrow cells revealed thet(9;22)(q34;q11.2) translocation. Induction chemotherapyconsisted of daunorubicin, vincristine, L-asparaginase,dexamethasone, methotrexate, cytarabine, and predniso-lone; thereafter, imatinib mesylate was given as the mainte-nance therapy. Follow-up bone marrow biopsy specimenobtained after 11 days exhibited a hypocellular marrowfollowing chemotherapy with 2% lymphoblasts in allnucleated cells. After a consolidation therapy including im-atinib mesylate, routine bone marrow biopsy after 2 monthsof treatment revealed a hypocellular marrow without anyobservable leukemic blasts, and the BCR/ABL fusion tran-script was not detected by real-time quantitative poly-merase chain reaction (RQ-PCR) or fluorescence in situhybridization (FISH) analyses. As of writing, the patienthad achieved complete remission and had been maintainedwell on imatinib mesylate for 5 months.

3. Materials and methods

3.1. Chromosome analysis and fluorescence in situhybridization (FISH)

The G-banding analysis were performed using standardprocedures [15,16]. Interphase FISH analysis using dual-color, dual-fusion, locus-specific identifier probes forBCR/ABL was performed according to the manufacturer’srecommendations (Vysis; Abbott Molecular, Des Plaines,IL). FISH analysis was also performed with Vysis IGH/

Fig. 1. Giemsa-banding karyogram of bone marrow sample in a case of childhoo

the derivative chromosomes 9 and 22.

MYC, p16, MLL, and TEL/AML1 probes. Karyotypesare described according to ISCN 2005 [17].

3.2. Molecular study

Multiplex reverse transcriptaseepolymerase chain reac-tion (RT-PCR) assay was performed with a Seeplexleukemia BCR/ABL kit (Seegene, Seoul, Korea), which isdesigned to detect eight common BCR/ABL transcriptsincluding major, minor, and micro breakpoint clusterregions (M-bcr, m-bcr, and m-bcr), according to the manu-facturer’s instructions. Cycling conditions were as follows:94�C for 15 minutes (1 cycle); 94�C for 30 seconds, 60�Cfor 1 minute 30 seconds, 72�C for 1 minute 30 seconds (37cycles); and 72�C for 10 minutes (1 cycle). The PCR prod-ucts were analyzed with 2% agarose gel electrophoresis at100 V for 30 minutes.

The purified PCR product was inserted into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) according to themanufacturer’s specifications. Plasmid DNA was preparedusing a plasmid mini kit (Qiagen, Hilden, Germany).Sequencing was performed using a BigDye terminatorv.3.1 cycle sequencing kit (Applied Biosystems [ABI],Foster City, CA) and the M13F/M13R primer on a DNAsequencer (ABI model 3730XL). Sequence analysis wasdone using the Ensembl database (http://www.ensem-bl.org/index.html).

4. Results

Cytogenetic analysis showed a 46,XY,t(9;22)(q34;q11.2)karyotype in 2 of the 20 cells analyzed (Fig. 1). The BCR/

d acute lymphoblastic leukemia: 46,XY,t(9;22)(q34;q11.2). Arrows indicate

134 J. Kim et al. / Cancer Genetics and Cytogenetics 189 (2009) 132e137

ABL probe showed nuc ish(ABL�2),(BCR�2),(ABL conBCR�1)[49/316]; that is, the abnormal signal patterns wereseen in 15.5% of the nuclei examined. None of the otherprobes (IGH/MYC, p16, MLL, and TEL/AML1) showedany abnormal findings.

Multiplex RT-PCR assay for BCR/ABL rearrangementin bone marrow specimen revealed PCR products of sizes299 and 600 bp; the product sized 299 bp represents theb3a3 transcript, with an internal control of 600 bp(Fig. 2). Cloning and sequencing confirmed an aberrantfusion product sized 299 bp derived from the joiningof BCR (ENSG00000186716) exon 14 (b3) and ABL exon3 (a3), lacking ABL (ENSG00000097007) exon 2 (a2)(Fig. 3). The other samples were confirmed to haveproducts of expected sizes of e1a2, b3a2, and b2a2/e1a2and were used as positive controls.

5. Discussion

Including the present case, 13 cases with BCR/ABL genelacking ABL exon a2 in ALL have been reported (Table 1).

The predominant BCR/ABL transcripts found in Phþ

CML are b2a2 and b3a2 transcripts [21], whereas thee1a2 transcript is more often encountered in Phþ ALLpatients [8]. Other BCR/ABL variants, however, such ase19a2 (c3a2), e1a3, e6a2, b2a3 (e13a3), b1a1 (e12a1),b3a3 (e14a3), have also been reported by different groups.Some of them are extremely rare, and atypical b3a3 varianthas been reported only once in a Phþ childhood ALL [9]. Ina study conducted by Goh et al. [22] in Korea, among 538of 548 CML patients screened (98.2%) were found to haveb3a2 or b2a2, and the total frequency of other minor

Fig. 2. Multiplex reverse transcriptaseepolymerase chain reaction (RT-PCR) a

lymphoblastic leukemia. A band slightly smaller than the frequently observed b

are listed at the right (as provided by Seegene, Seoul, Korea). Lane M, left: si

ABL transcript (b3a3). Lane 2: RT-PCR product of e1a2 as a positive control. La

5: RT-PCR product of e13a2 (b2a2)/e1a2 as a positive control. Lane 6: blank. L

transcripts was !2%. Nonetheless, the b3a3 transcriptwas not found in any of the patients.

Although more cases are needed to draw a definiteconclusion, BCR/ABL transcripts lacking ABL exon a2 aremore frequently found in patients diagnosed with commonALL (cALL) as shown in table 1. There was no correlationbetween the prognosis or leukemic characteristics and theWBC count, which ranged from 5.0 to 209 � 109/L. Thepresent case showed a pre-B immunophenotype with highWBC count at diagnosis. Although the clear relationshipbetween the molecular defect and the hematologic and clin-ical manifestation is yet to be elucidated, the ABL exon a2has been known to code for a part of the SH3 region of ablprotein, which is believed to have a negative regulatoryeffect on the kinase domain of the abl protein, and thereforethe absence of ABL exon a2 should result in increasedkinase activity [8]. On the other hand, many other reportspointed out that the transcription of ABL exon a2 may notbe required for leukemogenesis or may have no effect onthe severity of the disease [12,13,23], and that the SH3domain is required for STAT5 activation, possibly trig-gering the leukemogenesis [24]. Consequently, it is ex-pected that those who have BCR/ABL transcript withoutABL exon a2, leading to deletion of the SH3 domain, wouldshow a better clinical course [13], as in the present case.Nonetheless, other cases without ABL exon a2 (Table 1)do not seem to follow that rule.

Several studies have indicated that the theoretical inci-dence of BCR/ABL gene lacking ABL exon a2 is less thanthe actual incidence reported, and that this disparity iscaused by some cases that are missed by conventionalRT-PCR using a primer corresponding to ABL exon a2sequences [25]. In the present case, when we identifiedthe Ph through cytogenetic studies, we expected to find

nalysis for detection of BCR/ABL transcripts in a case of childhood acute

and representing e1a2 transcript was detected. Target and amplicon sizes

ze marker 100-bp ladder. Lane 1: RT-PCR product of the patient’s BCR/

nes 3 and 4: RT-PCR product of e14a2 (b3a2) as a positive control. Lane

ane M, right end: size marker 50-bp ladder.

Fig. 3. (A) Sequence analysis of the PCR product in a case of childhood acute lymphoblastic leukemia. Directly sequenced BCR/ABL cDNA PCR product

revealed a b3a3 junction and the absence of ABL exon a2 derived sequences. (B) Sequencing result of the PCR product. The vertical line indicates the break-

point and BCR/ABL junction of the fusion gene.

135J. Kim et al. / Cancer Genetics and Cytogenetics 189 (2009) 132e137

an e1a2 type transcript, the most frequently encounteredtranscript in ALL patients. We almost failed to detect thevariant fusion transcript in our patient, because the sizedifference of the band between e1a2 and b3a3 is difficultto be noticed unless electrophoresis is done in an optimalrun time (Fig. 1).

Table 1

Published cases of acute lymphoblastic leukemia with BCR/ABL transcripts lack

Patient Age/Sex

BCR/ABL

transcript

Type of

ALL Karyotype

WB

(�1

1 3/F b3a3 cALL Phþ 11.4

2 1/F e1a3 cALL Phþ 95

3 39/F e1a3 pre-pre-B Phþ 8.4

4 61/F b2a3 pre-B Phþ 209

5 Unknown b2a3 Unknown Unknown Unk

6 62/M b2a3 cALL Phþ 10

7 64/F e1a3 cALL 46,XX 1.8

8 31/M e1a3 cALL no metaphase 14

9 61/F e1a3 cALL no metaphase 14

10 48/M e1a3 cALL 46,XY 62

11 37/F b2a3 cALL Phþ 70

12 45/F e1a3 pre-B Phþ 5

13 12/M b3a3 pre-B Phþ 190

Abbreviations: ALL, acute lymphoblastic leukemia; cALL, common acute ly

Philadelphia chromosome positive; WBC, white blood cells.

When we realized that the barely distinguishableproduct of smaller size could actually be a variant formof BCR/ABL transcript, we decided to use cloning andsequencing to confirm the exact type of fusion productand its breakpoint. The directly sequenced BCR/ABL cDNAPCR product revealed a b3a3 junction and the absence of

ing ABL exon a2

C count

09/L)

Clinical

outcome

Duration of

follow-up Reference

Dead 41 mo Inukai et al., 1993 [9]

Dead 54 mo Iwata et al., 1994 [11]

Dead 9 mo Soekarman et al., 1990 [18]

Dead 14 mo Soekarman et al., 1990 [18]

nown Unknown Unknown Tuszynski et al., 1993 [19]

Dead 18 mo Burmeister et al., 2007 [20]

Dead 16 mo Burmeister et al., 2007 [20]

CR 13 mo Burmeister et al., 2007 [20]

CR 18 mo Burmeister et al., 2007 [20]

Dead 6 mo Burmeister et al., 2007 [20]

CR 140 d Burmeister et al., 2007 [20]

CR 130 d Burmeister et al., 2007 [20]

.9 alive 5 mo present case

mphoblastic leukemia; CR, complete remission; F, female; M, Male; Phþ,

136 J. Kim et al. / Cancer Genetics and Cytogenetics 189 (2009) 132e137

ABL exon a2 derived sequences. If the expected product isnot detected or appears in different size by PCR method,sequencing should be used to detect some of the rarelyencountered variant fusion transcripts of BCR/ABL. Thisalso applies to other types of fusion transcripts. Previouscase reports from our group emphasize the need forsequencing when a rare type of transcript is encountered[26,27]. For instance, a new variant form of CBFBeMYH11rearrangement (K-type), which could have been missed bythe conventional RT-PCR method, was detected by use ofmultiplex RT-PCR; it was later confirmed by sequencing[27].

In conclusion, we report a rare b3a3 transcript in a Phþ

childhood ALL. To ensure the detection of the rare BCR/ABL fusion transcripts, different approaches should beattempted, especially if the product is of an unusual sizeor if a slightly different size is noticed. More cases shouldbe accumulated to disclose the mechanism of leukemogen-esis in terms of fusion transcripts, and further evaluationshould be conducted to elucidate possible clinical and prog-nostic implications of the findings.

Acknowledgments

The authors wish to thank Ms. Kyung-Ran Hong,Mr. Ho-Young Chung, and Seegene Company (Seoul,Korea) for their excellent technical support in the analysisof BCR/ABL fusion transcripts.

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