acute myeloid leukemia - Nature

Leukemia (2002) 16, 1752–1754  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu

Large deletions 5⬘ to the ETO breakpoint are recurrent events in patients with t(8;21) acute myeloid leukemia C Godon1, J Proffitt2, N Dastugue3, M Lafage-Pochitaloff4, M-J Mozziconacci4, P Talmant1, M Hackbarth2, R Bataille1 and H Avet-Loiseau1 1

Hematology Laboratory, University Hospital, Nantes, France; 2Vysis Inc., Downers Grove, IL, USA; 3Cytogenetic Unit, University Hospital, Toulouse, France; and 4Cytogenetic Laboratory, Institut Paoli-Calmette, Marseille, France

Recurrent chromosomal rearrangements are observed in many leukemia subtypes. Recently, it has been shown that several of these translocations/inversions were associated with the loss of sequences located in the vicinity of the chromosomal breakpoints. So far, such deletions have not been described for the t(8;21) translocation. We have analyzed a series of 65 patients with t(8;21) using several probes specific for the ETO and AML1 regions. We have found six patients (9%) with deletion of the region 5⬘ to ETO. In all six patients, the deletion encompassed at least 260 kb, and was even larger in two patients (up to 2 Mb). A similar analysis of the 21q22 region did not reveal any deletion of the 3⬘AML1 region. In conclusion, cytogenetically undetectable small deletions located immediately 5⬘ to the ETO breakpoint were found to accompany the t(8;21) translocation in a significant percentage of cases. The clinical significance, if any, of these deletions remains to be determined. Leukemia (2002) 16, 1752–1754. doi:10.1038/sj.leu.2402585 Keywords: acute myeloid leukemia; cytogenetics; ETO; fluorescence in situ hybridization; deletion

Introduction The translocation t(8;21)(q22;q22) is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML), especially in the M2 FAB subtype. The overall incidence of the translocation is between 6% and 8%,1,2 although conflicting data have been reported with the use of RT-PCR (reporting a higher incidence).3,4 The t(8;21) translocation is more frequent in younger patients (and especially in children), and is associated with a high complete remission rate. This higher complete remission rate usually translates into longer overall survival, and most therapeutic trials now exclude these patients from front-line allogeneic bone marrow transplant programs.1,5,6 Because of the specificity of these characteristics, AML with either t(8;21) or AML1-ETO fusion transcripts has been identified as a specific entity in the recent WHO classification.7 Many recurrent translocations have been described in hematopoietic malignancies. Most of them are specific for one or a small number of well-defined disease types, such as t(9;22) in chronic myeloid leukemia (CML) and some acute lymphoid leukemia (ALL), t(15;17) in acute promyelocytic leukemia, inv(16) in AML with abnormal eosinophils, or translocations involving the MLL gene at 11q23 in different AML and ALL subtypes. Although these chromosomal rearrangements appear as reciprocal translocations, it has been reported that some of them were associated with submicroscopic deletions in a significant number of cases.8–15 Moreover, at least in the CML model, these deletions may be associated with a specific poorer prognosis.9,15 So far, such deletions have not been

Correspondence: H Avet-Loiseau, Laboratoire d’He´matologie, Institut de Biologie, 9 quai Moncousu, 44093 Nantes Cedex 1, France; Fax: 33 240 08 41 14 Received 16 October 2001; accepted 10 April 2002

described in AML with t(8;21). In order to search for submicroscopic deletions in patients with t(8;21), we performed a fluorescence in situ hybridization (FISH) analysis in a series of 65 patients with t(8;21). Materials and methods

Patients Sixty-five patients (46 males, 19 females) diagnosed with AML and t(8;21)(q22;q22) between 1988 and 2000 in three French cytogenetic laboratories and for whom frozen material was available were entered into this study. Sixty-three were true AML (according to the FAB classification), including 42 of the M2 subtype (65%), and two were MDS, and are now considered as AML according to the WHO criteria.7 In 21 cases (32%), t(8;21)(q22;q22) was the sole abnormality. In 28 cases (61% of the male population), t(8;21) was associated with the loss of chromosome Y, in 11 cases, it was associated with trisomy 8, and in nine patients, t(8;21) was associated with del(9q). Of note, two patients did not display t(8;21) on cytogenetics (complex karytotype), and the ETO-AML1 fusion was detected by RT-PCR.

Fluorescence in situ hybridization FISH was performed on cytogenetic preparations conserved in fixative at −20°C, according to the manufacturer’s instructions. Probes were provided by Vysis (Downers Grove, IL, USA). We first tested a dual-fusion probe (LSI AML1/ETO Dual Color, Dual Fusion Translocation Probe), containing a SpectrumGreenlabeled AML1-specific probe (1.3 Mb, spanning the AML1 locus, Figure 1a), and a SpectrumOrange-labeled ETO-specific probe spanning about 480 kb (Figure 1a). In a second step, we selected smaller probes: three partially overlapping BAC probes located on the 5⬘ side of ETO and labeled with SpectrumOrange (5⬘ETO-1 to -3), and one 3⬘AML1 BAC probe labeled with SpectrumGreen (Figure 1a). These probes were obtained from either Research Genetics (Huntsville, AL, USA) for the RPCI clones, or Genome Systems (St Louis, MO, USA) for the GS clones. The 5⬘ETO-1 to -3 probes corresponded to the RPCI11-66D2, GS-55E16 and GS-210E12 clones, respectively, whereas the 3⬘AML1 BAC probe corresponded to the RPCI11–1006L1 clone. We also utilized two additional BAC probes located telomeric to 5⬘ETO region BAC contig. One of these BACs (RPCI11–23B3) contains the AFMA081XC9 marker and is located 500 kb telomeric to ETO. The other BAC (RPCI11–388K12) contains the WI-7945 and AFM074WD11 markers and is located 1.2 Mb telomeric to ETO. Analyses were performed using a Zeiss (Jena, Germany) Axioplan 2 microscope equipped with a CCD (chargecoupled device) camera (Sensys; Photometrics, Tucson,

Deletions 5⬘ to the ETO breakpoint in patients with t(8;21) AML C Godon et al

Figure 1 (a) A schematic representation of the ETO- and AML1specific probes used in this study. The arrows show the location of the breakpoints on each locus. (b) A typical metaphase cell of a patient with a common form of the t(8;21) and hybridized with the LSI AML1/ETO Dual Color, Dual Fusion Translocation Probe. Fusion signals are observed on both derivative chromosomes. (c) A metaphase cell of a patient with t(8;21) and deletion of sequences located on the 5⬘ side of ETO, hybridized with the LSI AML1/ETO Dual Color, Dual Fusion Translocation Probe. In this case, only one fusion signal is observed on the derivative chromosome 8, but not on the derivative chromosome 21.

AZ, USA), connected to an image analysis system (SmartCaptureVP; Vysis). Images were selected and captured with selective filters for SpectrumOrange, FITC and DAPI. For each probe, 100 to 200 interphase cells were analyzed, and metaphase cells were systematically searched for. Using the dual-fusion probe, a normal sample displayed two separate orange and two separate green signals. A typical t(8;21)-positive case displayed one orange (normal chromosome 8), one green (normal chromosome 21) and two fusion signals, one each on the abnormal chromosomes 8 and 21 (Figure 1b). Orange and green signals were considered as fused in interphase cells when they completely or partially overlapped. On metaphases, signals were always fused. The cut-off value was based on the analysis of 2000 cells from 10 normal bone marrow specimens. Results We first determined the cut-off values (mean + 3 standard deviations) of the different probes used in this study. Regarding the LSI AML1/ETO Dual Color probe, the cut-off value for assessment of a translocation (ie, one green, one orange, two fusions; or two green, one orange, one fusion in cases with 5⬘ETO deletion, see Results) was 0.82%. Regarding the values

to assess a BAC probe deletion, they ranged between 4.2% and 6.1%. When the LSI AML1/ETO Dual Color probe was employed, 47 patients displayed the typical common configuration, ie one green, one orange and two fusion signals, in 48% to 99% of the interphase cells (Figure 1b). Eleven patients displayed one extra orange signal, in full agreement with trisomy 8 at karyotype. In one patient, only one fusion was observed, with two orange and two green separated signals. Metaphase analysis of this specimen actually revealed a classical configuration with one green, one orange and two fusion signals, probably reflecting an unidentified complex translocation, with unknown genomic material inserted between the ETO- and AML1-specific sequences. Six patients presented an interphase FISH pattern with only one fusion, two green (AML1) signals and one orange (ETO) signal. In these six cases, metaphase analysis showed that the fusion was always located on the der(8) (the characteristics of these six patients are summarized in Table 1). Of note, in two of these six patients, the t(8;21) was not detected on karyotype, but by RT-PCR. Using smaller 5⬘ETO probes, we first confirmed the deletion of the 5⬘ETO region. All six patients displayed a loss of a minimal commonly deleted region extending from the ETO breakpoint to a region located 260 kb more telomeric (ie the three 5⬘ETO BAC probes were lost). Two patients presented even larger deletions: one patient (patient 2) displayed a loss of the AFM A081XC9 marker (approximately 500 kb telomeric to ETO), whereas a second one (patient 3) presented a large deletion extending at least to the WI-7945 locus (about 1.3 Mb telomeric to ETO). We then tested all the patients lacking deletion with the three 5⬘ETO BAC probes, in order to check possible smaller undetected deletions. In all these patients, all three probe targets were retained. We also tested all the patients with a probe specific for the 3⬘AML1 region, but no deletion was observed.

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Discussion Several studies have described large submicroscopic deletions (up to several Mb) adjacent to the breakpoints of different recurrent chromosomal rearrangements.8–15 So far, no deletion has been reported in patients with t(15;17) or t(8;21)AML, but only 14 patients with t(8;21) had been tested.15 Using a dual-fusion probe, we have analyzed 65 patients with AML and t(8;21), and found deletions 5⬘ to the ETO breakpoint in 9% (6/65) of them. This deleted segment varied in size among patients. Nonetheless, a common region extending from the ETO breakpoint region to at least 260 kb towards the telomere was found in all six patients. In two patients, the deleted region was even larger, encompassing up to 1.3 Mb. In contrast, we did not identify any patient with either a deletion of the 3⬘ part of AML1, or a deletion smaller than 260 kb of the 5⬘ETO region. The mechanism and the significance of these ETO region deletions are unknown. Deletions 5⬘ of ABL and 3⬘ of BCR have been associated with a worse prognosis.9,15 For some translocation-associated deletions, a high density of Alu repeats, which are known to facilitate illegitimate recombination, have been described in the vicinity of deletion breakpoints. Thus, Alu-mediated recombination is one possible deletion mechanism.15 However, this mechanism remains a hypothetical model, and there may be several possible mechanisms causing deletions to occur near translocation breakpoints. Different deletion mechanisms may be associated with different translocations. Leukemia

Deletions 5⬘ to the ETO breakpoint in patients with t(8;21) AML C Godon et al

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Table 1

Main clinico–biological characteristics of the six patients with t(8;21) and deletion of sequences located on the 5⬘ side of ETO

Patients Sex/ Age

WBC FAB (% blast cells)a typeb

Karyotype

Size of deletion

Treatmentc Relapse

OS (months)d

1

M/47

3 G/L (4%)

M2

45,X,⫺Y,t(8;21)(q22;q2),del(9)(q11q22)[3]

苲260 kb

CT

No

39+

2

F/21

1.5 G.L (0%)

M2

46,XX,t(4;16)(q1?3;p13),t(8;21)(q22;q22), del(13)(q13q14)[12]/46,XX[10]

苲500 kb

CT

Yes (10 months)

12

3

F/49

10 G/L (70%)

M2

45,X,-X,t(8;21)(q22;q22)[8]/46,XX[1]

苲1300 kb CT

No

83+

4

M/53

6 G/L (15%)

M2

45,X-Y,t(2;20)(p21;q12),?inv(8)(p22q21), 苲260 kb add(21)(q21)[3]/idem,add(11)(p15)[17] 46,XX,add(5)(q35),t(8;9)(q22;q34)[19]/46,XX[1] 苲260 kb

CT

No

28+

Allo BMT

No

63+

45,X,⫺Y,t(8;21)(q22;q22)[7]/idem,del(9)(q12q21) 苲260 kb [5]/idem,del(9)del(9)(q12q21)del(9)(q31q33)[9]

CT

Yes (15 months)

19

5

F/31

2 G/L (0%)

M2

6

M/73

7.9 G/L (59%)

M4Eo

a

WBC, white blood cell counts at diagnosis. FAB, leukemia has been classified according to the FAB classification. c Treatment: CT, chemotherapy; alloBMT, allogeneic bone marrow transplantation. d OS, overall survival; + means that the patient is still alive and in complete remission. b

In our study, we have included patients from three institutions, with a large age range (3–72 years), treated with different therapeutic programs, over a period of more than 12 years. Consequently, no prognostic information can be obtained from this series. The only possible comment is that 4/6 patients with deletions remain disease free with a median follow-up for these 6 patients of 4 years (range 28–83 months). The analysis of larger prospective trials will be necessary in order to determine if such deletions have a clinical significance. In conclusion, t(8;21)-associated submicroscopic deletions appear to be a recurrent event in AML. This finding extends the scope of the phenomenon of translocationassociated deletions observed in hematopoietic malignancies.

7

8

9

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