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Cancer Genet Cytogenet 121:52–55 (2000)

2000 Elsevier Science Inc. All rights reserved.655 Avenue of the Americas, New York, NY 10010

A Unique Structural Abnormality of Chromosome 16

Resulting in a CBF

b

-MYH11 Fusion Transcript in a Patient with Acute Myeloid Leukemia, FAB M4

John O’Reilly, Lucia Chipper, Francisca Springall,and Richard Herrmann

ABSTRACT:

A 43-year-old female with a peripheral white cell count of 118.0

3

10

9

/L and 96% blastswas diagnosed with acute myeloid leukemia (AML), FAB M4. Cytogenetics, performed on a bone mar-row sample, revealed the following abnormal karyotype: 46,XX,ins(16)(q22p13.1p13.3). Fluorescence insitu hybridization (FISH) confirmed the inter-arm insertion using a probe for 16p. The result of this

structural rearrangement was the fusion of CBF

b

to

MYH11 seen commonly in inv(16)(p13q22). Thepatient commenced high-dose intensive combination chemotherapy (big ICE; Idarubicin, Cytarabine,and Etopiside). Five days post chemotherapy, she developed febrile neutropenia. Despite broad spec-trum intravenous antibiotics and antifungal therapy, the patient died at day nine post chemotherapy.This case demonstrates a previously unreported structural abnormality of chromosome 16 in a patientwith AML M4, which represents a third mechanism to inv(16)(p13q22) and t(16;16)(p13q22) in produc-ing the CBF

b

-MYH11 fusion. CBF

b

-MYH11 fusions masked by cryptic translocations at the cytogeneticlevel have been detected by FISH and PCR techniques. Due to the improved prognosis associated withCBF

b

-MYH11 fusions compared to the standard risk group for AML, its detection remainsimportant. © 2000 Elsevier Science Inc. All rights reserved.

INTRODUCTION

Structural abnormalities of chromosome 16 in associationwith acute myeloid leukemia and elevated levels of bonemarrow eosinophilia (AML M4Eo) were initially de-scribed in 1983 [1]. The abnormality was shown to be apericentric inversion of chromosome 16 with breakpointsat p13 and q22 [2]. This was followed by the discoverythat the translocation between the homologous chromo-some 16 pair at the same breakpoints was also associatedwith AML M4Eo [3].

The inv(16)(p13q22), although predominantly associ-ated with AML M4Eo, has also been reported in up to10% of AML M4 cases without eosinophilia [4–6].

Recent cloning of the breakpoints identified the genes

CBF

b

at 16q22 and

MYH11

at 16p13 [7]. The inv(16)(p13q22) and t(16;16)(p13;q22) result in a transcription-ally active fusion gene,

CBF

b

-MYH11, which can be de-

tected by reverse transcriptase-polymerase chain reaction(RT-PCR) [5]. Initial studies indicate the fusion gene mayalter transcriptional regulation mediated by the core bind-ing complex (CBC) and that the smooth muscle heavychain domain is required for the transforming ability ofthe fusion protein [8].

We describe a patient with AML M4 not having theM4Eo subtype with an abnormal chromosome 16. The ab-normality in this patient represents a unique structuralchange of chromosome 16, which has resulted in the

CBF

b

-MYH11 fusion gene.

CASE HISTORY

A 43-year-old female presented to her local doctor com-plaining of a skin rash on her leg, a sore throat, and cervi-cal lymphadenopathy. A full blood picture was ordered atthis time and revealed: Hb 133 g/L, platelets 123

3

10

9

/L,and WBC 118.0

3

10

9

/L (96% blasts). A subsequent bonemarrow aspirate revealed hypercellular fragments with90% myeloblasts, some containing granules and somewith monocytoid features. These blasts expressed CD13⁄33 and CD 68 antigens and a diagnosis of AML FABM4 was established.

From the Department of Haematology, Royal Perth Hospital(J. O., L. C., R. H.), Perth, Australia; and Kanematsu Laboratories,Royal Prince Alfred Hospital (F. S.), Sydney, Australia.

Address reprint requests to: John O’Reilly, Department of Hae-matology, Royal Perth Hospital, Wellington Street, Perth, WesternAustralia 6001.

Received November 11, 1999; accepted February 10, 2000.

Ins(16) Resulting in a CBF

b

-MYH11 Fusion

53

The patient was started on high-dose intensive com-bination chemotherapy (big ICE). Five days post chemo-therapy, she developed febrile neutropenia. Despitebroad spectrum intravenous antibiotics and antifungaltherapy, the patient succumbed, dying at day nine postchemotherapy.

MATERIALS AND METHODS

Cytogenetics

Cytogenetic studies were performed on bone marrow us-ing short term synchronized cultures in RPMI mediumsupplemented with 20% fetal calf serum. The cultureswere stimulated with interleukin 3 (R&D Systems). Thecells were arrested at metaphase using Colcemid (GIBCOBRL) and treated with a hypotonic solution (0.075M KCl).The cells were then fixed in methanol:acetic acid (3:1)and dropped onto microscope slides. GTG banding wasperformed to detect chromosome abnormalities, whichwere described according to the International System forCytogenetic Nomenclature [9].

FISH

FISH studies were performed using an inv(16) probe (On-cor) which targeted 16p11 and 16p13. The digoxigenin-

labeled probe was detected with a fluorescein anti-digoxi-genin reporter system (Oncor). Probe and target denatur-ation together with post-hybridization washing in 0.5

3

SSC were performed at 72

8

C for 5 minutes. Hybridizationwas performed at 37

8

C in a humid chamber, overnight.Fluorescent signals were imaged from a Leitz Dialux 22 tothe VySIS FISH imaging system.

RT-PCR

Total RNA was extracted from the bone marrow sampleusing TRI

ZOL

LS reagent (Life Technologies). cDNA wassynthesized from 2

m

g of total RNA in a total reaction vol-ume of 40

m

L containing 1

3

PCR buffer (10mM Tris HCl,50mM KCl), 5mM MgCl

2

, 1mM of each dNTP, 40U/reac-tion RNAsin (Promega), 2.5

m

M random hexamers (Bresatec),and 2.5U/

m

L M-MuLV reverse transcriptase RNase H

2

(Promega). The reaction was incubated at 42

8

C for onehour, then heated to 95

8

C for five minutes.For PCR, replicates of patient cDNA (5

m

L) were ampli-fied in a 50

m

L reaction containing 200

m

M of each dNTP,150

m

M of each primer, 1

3

PCR buffer (10mM Tris HCl,50mM KCl), 1.5 mM MgCl

2

, and 1.25U/reaction of Ampli-taq Gold (Perkin Elmer). Primers for the amplification ofCBF

b

-MYH11: primer 1 is derived from Claxton et al.,1994 [10] and primer 5M is derived from Tobal et al., 1995

Figure 1 G-Banded karyotype from the diagnostic bone marrow sample demonstrating the ins(16)(q22;p13.1p13.3) (arrowed).

54

J. O’Reilly et al.

[4]. A single round of PCR was performed. Amplificationwas for 45 cycles with a three-step PCR: 60

8

C for 30 sec,72

8

VC for 45 sec, 95

8

C for 30sec. Using this protocol, typeA CBF

b

-MYH11 fusion transcripts gave a band size of245 bp.

RESULTS

Cytogenetic analysis of this bone marrow sample gave thefollowing abnormal karyotype: 46,XX,ins(16)(q22p13.1p13.3)(Fig. 1). This abnormality was detected in all 20 metaphasecells analyzed. This was confirmed using FISH withprobes targeted to 16p11 and 16p13 (Fig. 2). The signalnormally present at 16p13 was relocated to 16q on the ab-normal chromosome 16.

The presence of a CBF

b

-MYH11 fusion transcript wasdemonstrated in this abnormal chromosome 16 by RT-PCR (Fig. 3). The patient band seen in Figure 3 corre-sponded to the common type A breakpoint.

DISCUSSION

We have demonstrated in this patient a previously unre-ported structural abnormality of chromosome 16 resultingin a

CBF

b

-

MYH11

fusion gene. This represents a third

mechanism to the inv(16) and t(16;16) for the formation ofthis fusion gene. This abnormality, which is an inter-arminsertion, has two breaks in the short arm of chromosome16 (p13.1 and p13.3) and insertion of this segment into thelong arm at q22, indicating the possible involvement ofthe

CBF

b

and

MYH11

genes. This requires a direct insertfor correct orientation of the

CBF

b

-

MYH11

fusion genes.Consequently, the reciprocal

MYH11-CBF

b

fusion as seenin the standard inv16 would not occur in this rearrange-ment, further emphasizing its lack of a role in leukemo-genesis.

This case highlights how well-resolved banding in amalignant cytogenetic sample can identify structuralchanges to define a target for follow-up molecular studies.The follow-up with FISH and PCR to augment cytogenet-ics has become important. This is especially the casewhere BCR-ABL [11], PML-RAR

a

[12], and CBF

b

-MYH11[5] fusion transcripts have been detected in cytogeneti-cally normal samples with presumed cryptic transloca-tions.

Many chromosome abnormalities have now been de-scribed in AML and have shown to constitute tumormarkers of diagnostic and prognostic value [13]. Theinv(16)(p13q22) abnormality has been shown to be an in-dicator of good prognosis when compared to the standardrisk group for AML [14, 15]. The combination of cytoge-netics, FISH, and PCR would better stratify patients with

Figure 2 Metaphase FISH using probes to p11 and p13 onchromosome 16 (Oncor). Note the split signal on the abnormalchromosome 16 as a result of the inter-arm insertion.

Figure 3 RT-PCR detection of the CBFb-MYH11 fusion tran-script. Abbreviations: MW, 100bp DNA ladder (Pharmacia); P,patient sample; PC, CBFb-MYH11 type A positive control; NC,normal control; WC, water control.

Ins(16) Resulting in a CBF

b

-MYH11 Fusion

55

AML who would benefit from the appropriate clinicalmanagement and counseling. Unfortunately, due to treat-ment-related mortality, no follow-up data will be avail-able on this patient to assess the prognostic impact of thisabnormality. Whether the presence of the

CBF

b

-

MYH11

fusion gene is sufficient to include this abnormality intothe inv(16), t(16;16) prognostic group needs to be assessedon more patients.

This work was supported by the Western Australian Adult Leu-kaemia Foundation.

REFERENCES

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