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Cancer Genet Cytogenet 107:48–50 (1998)

Elsevier Science Inc., 1998655 Avenue of the Americas, New York, NY 10010

A Philadelphia-Negative Chronic Myeloid Leukemia with a

BCR/ABL

Fusion Gene on Chromosome 9

K. S. Reddy and B. Grove

ABSTRACT:

A 40-year-old man had chronic myeloid leukemia (CML) and an apparently normal kary-otype. Fluorescence in situ hybridization with a BCR/ABL1-S probe, which is formatted to display a

BCR/ABL

fusion signal on chromosome 22, gave a positive fusion signal on a chromosome 9. Thereforethis patient has a

BCR/ABL

fusion gene on chromosome 9. The BCR/ABL1-D probe, formatted to dis-play a fluorescent signal for both the reciprocal products of a 9/22 rearrangement, gave a positive fusionsignal on the derivatives 9 and 22. These findings favor either a cryptic reciprocal exchange between

BCR

and

ABL

loci or the reversal of a Philadelphia translocation. An insertion of

BCR

next to

ABL

isruled out. The reverse-transcriptase polymerase chain reaction provided molecular evidence that atypical CML chimeric product resulting from a fusion of

BCR

exon 2 with

C-ABL

exon II, a

2

b

2

, ispresent. © Elsevier Science Inc., 1998

INTRODUCTION

Between 5 and 10% of chronic myeloid leukemia (CML)cases are Philadelphia (Ph) chromosome negative. Ofthem, about 5% are Ph negative and

BCR/ABL

fusion genepositive [1]. In most cases, the

BCR/ABL

fusion gene is onchromosome 22. There are few reports of the

BCR/ABL

fu-sion signal on chromosome 9 [2–8]. Of such cases, fourhave apparently normal karyotypes and the

BCR/ABL

fu-sion signal is on chromosome 9 [2–5]. The patient in thisreport is the fifth case. The mechanism of origin of the

BCR/ABL

fusion signal on 9q34 is discussed.

CASE REPORT

A 40-year-old man presented with left-leg swelling, pain,and ecchymosis. He had no organomegaly or lymphaden-opathy. His white blood cell (WBC) count was 59,000/mm

3

and his platelet count was 768,000/mm

3

with a hem-ocrit of 42%. The differential was 53.3% neutrophils,18.3% bands, 7.3% lymphocytes, 2% monocytes, 1.7%eosinophils, 2.3% basophils, 11.3% metamyelocytes, 2.7%myelocytes, 1% blasts, and 1.7 nucleated red blood cells/100 WBCs. The basophils were abnormal in being poorly

granulated. The bone marrow was 100% cellular with 4%myeloblasts and an 8:1 myeloid:rubroid ratio.

He had been treated since September 1997 with hy-droxyurea, 1.5 g daily. His WBC count was 31,700/mm

3

inDecember and his treatment was changed to 3 millionunits of intravenous

a

-interferon 6 days a week. The WBCcount in February of 1998 was 15,400/mm

3

.

MATERIALS AND METHODS

The bone marrow specimen was cultured for 24 hours and48 hours. The cultures were harvested and the slides GTGbanded.

Fluorescence in situ hybridization (FISH) was performedwith the use of the mbcr/abl, bcr/abl1-D, bcr/abl1-S, andD9Z5 (9

b

-satellite, which maps to the pericentric hetero-chromatin) probes according to the manufacturer’s in-structions (Oncor Inc.) with minor modifications. The bcrprobe is direct labeled with a red fluorochrome and gives ared signal, the abl probe is direct labeled with a green flu-orochrome and gives a green signal. The biotin-labeledD9Z5 probe is detected by using fluorescein-labeled avidin,which gives a green signal. The slides were denatured at70

8

C for 2 minutes. After overnight hybridization withbcr/abl1-D plus 9

b

-satellite and bcr/abl-S plus 9

b

-satellite,the slides were washed in 0.5

3

SSc at 72

8

C for 5 minutes.RNA was extracted from the bone marrow specimen by

using RNAzol (Tel-Test Inc). The extracted RNA was re-verse transcribed by using MoMuLV reverse transcriptase(RT; BRL 8025 SA, 200 U/

m

L). The polymerase chain reac-tions (PCRs) for amplifying P1, P2, and P3 rearrangementswere set up [9] and amplified for 35 cycles. The PCR reac-

From Quest Diagnostics Inc. (K. S. R.), San Juan Capistrano,California, USA; and the Santa Clara Valley Health and HospitalSystem, (B. G.), San Jose, California, USA.

Address reprint requests to: Dr. Kavita S. Reddy, Cytogenetics,Quest Diagnostics Inc., 33608 Ortega Highway, San Juan Capist-rano CA 92690.

Received November 21, 1997; accepted March 24, 1998.

Ph-Negative CML with

BCR/ABL

on Chromosome 9

49

tion (250

m

L) was dot blotted. The filter strips were probedwith

32

P 5

9

-end-labeled P1, P2, and P3, washed, and ex-posed to X-ray film overnight.

RESULTS

The 20 metaphases analyzed were found to have a normal46,XY karyotype. FISH with the use of the mbcr/abl probewas positive for the fusion signal in 200/208 interphasesand 7/7 metaphases. In each metaphase, probe bcr/abl1-Sdisplayed three signals: a fusion red–green signal on chro-mosome 9 with a control D9Z5 green pericentromeric sig-nal, an

ABL

green signal on the normal 9 with a controlD9Z5 signal and a

BCR

red signal on a G-group chromo-some (Fig. 1A.). The bcr/abl1-D probe displayed the recip-rocal fusion signals on a chromosome 9 with a green D9Z5control signal and on a G-group chromosome (Fig. 1B.).The

BCR

and

ABL

signals are on a G-group and a 9 chro-mosome, respectively (Fig. 1B).

The molecular test is positive for the P2 mRNA, whichis produced by the fusion of

BCR

exon 2 to

C-ABL

exon II(a

2

b

2

). This fusion product is specific for chronic myeloidleukemia (CML).

DISCUSSION

The classic Philadelphia translocation (9;22) or a variantPhiladelphia translocation is found in 90–95% of CMLcases. About 5–10% do not have the Philadelphia translo-

cation. A substantial number of Ph-negative cases have amicroscopically invisible insertional change resulting in

BCR/ABL

fusion. The majority of rearrangements result inthe typical

BCR/ABL

fusion on chromosome 22. However,there are a few reports [2–8] of Ph-negative cases with the

BCR/ABL

fusion signal on chromosome 9. The cases re-ported by Inazawa et al. [2], Abeliovich et al. [5], Hage-meijer et al. [3], and Nacheva et al. [4] and the present casehave normal karyotypes with the

BCR/ABL

fusion signalon chromosome 9. The fusion signal is detected by FISHand Southern blot or RT-PCR. In all cases, FISH is themost informative and located the

BCR/ABL

fusion gene tochromosome 9.

There are two mechanisms by which the

BCR/ABL

fu-sion signal can be located on chromosome 9. One is by aninsertion of

BCR

next to

ABL

[10]. The second mechanismincludes the typical Philadelphia rearrangement t(9;22),followed by a reverse translocation. This restores apparentcytogenetic normality but results in a shift of the

BCR/ABL

fusion gene to chromosome 9 [2]. In this study, by using acombination of probes and RT-PCR, we have shown thatthe second mechanism is most probably in operation. ByFISH with the bcr/abl1-S probe, the fusion signal is shownto be on chromosome 9q34, RT-PCR provides the molecu-lar evidence that the typical CML a

2

b

2

chimeric product ispresent. Probe bcr/abl1-D, which detects the fusion on thederivative chromosomes 9 and 22 resulting from a Phila-delphia rearrangement, displayed a signal on 9q34 and an-other on 22. A reciprocal

BCR/ABL

fusion signal on chro-mosome 22 excludes a cryptic insertion of

BCR

next to

Figure 1 FISH results with BCR/ABL1-S, BCR/ABL1-D, and chromosome 9 b-satellite probe D9Z5, which mapsto the centromeric region. (A) FISH with BCR/ABL1-S probe displays the fusion red–green signal on chromosomes9 with b-satellite signal on the pericentric heterochromatin (arrow), a red BCR signal on a G-group chromosome,and a green ABL signal on chromosome 9 with a b-satellite signal. (B) FISH with BCR/ABL1-D probe displays thefusion red–green signal on chromosome 9 with b-satellite sequence signal on the pericentric heterochromatin(arrow) and the reciprocal BCR/ABL fusion product on a G-group chromosome (arrow). Red BCR signal is on aG-group chromosome and green ABL signal on chromosome 9 with a b-satellite signal.

50

K. S. Reddy and B. Grove

ABL

and favors a reversal of a typical Philadelphia rear-rangement or a reverse, cryptic

BCR

and

ABL

exchange.Cases with the “reverse”

BCR/ABL

fusion on chromo-some 9 are postulated to have a worse prognosis thanthose with the typical

BCR/ABL

fusion gene on chromo-some 22 [7, 10]. Why these patients, although they havethe typical

BCR/ABL

fusion seen in CML, should have aworse prognosis is perplexing. Therefore evaluation ofmore cases with

BCR/ABL

on chromosome 9 is needed be-fore a conclusion can be drawn about prognosis.

CML cases that are

BCR

-negative are considered to havea poor prognosis compared with

BCR

-positive cases. There-fore, establishing the presence of the

BCR/ABL

fusion genein a cytogenetically masked case is important for diagnosisand prognosis. Using FISH rather than a molecular test hasthe advantage of detecting and localizing the fusion signalto a particular chromosome. Further, to monitor a Ph-neg-ative and

BCR/ABL

-positive CML, routine cytogenetics isof limited use. It can identify only secondary changes andnot the

BCR/ABL

fusion. FISH in conjunction with routinecytogenetics can give the maximum information. Thecombination can detect both

BCR/ABL

fusion and any sec-ondary change, especially those associated with blast cri-sis. Because FISH is rapid and simple and allows quantita-tion of the

BCR/ABL

fusion signals, it is preferred toRT-PCR, which also is rapid but difficult to quantitate.

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1

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