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Leukemia (2003) 17, 547–553 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Amplification of AML1 on a duplicated chromosome 21 in acute lymphoblastic leukemia: a study of 20 cases L Harewood1,4, H Robinson1, R Harris1, M Jabbar Al-Obaidi1, GR Jalali1, M Martineau1, AV Moorman1, N Sumption1, S Richards2, C Mitchell3 and CJ Harrison1on behalf of the Medical Research Council Childhood and Adult Leukaemia Working Parties 1 Leukaemia 2 Research Fund Cytogenetics Group, Cancer Sciences Division, University of Southampton, Southampton, UK; Clinical Trial Service Unit, Radcliffe Infirmary, Oxford, UK; and 3Paediatric Oncology, John Radcliffe Hospital, Oxford, UK This study identifies multiple copies of the AML1 gene on a duplicated chromosome 21, dup(21), as a recurrent abnormality in acute lymphoblastic leukemia (ALL). Clusters of AML1 signals were visible at interphase by fluorescence in situ hybridization (FISH). In metaphase, they appeared tandemly duplicated on marker chromosomes of five distinct morphological types: large or small acrocentrics, metacentrics, submetacentrics or rings. The markers comprised only chromosome 21 material. Karyotypes were near-diploid and, besides dup(21), no other established chromosomal changes were observed. A total of 20 patients, 1.5 and o0.5% among consecutive series of childhood and adult ALL respectively, showed this phenomenon. Their median age was 9 years, white cell counts were low and all had a pre-B/common immunophenotype. Although this series is not the first report of this abnormality, it is the largest, permitting a detailed description of the variety of morphological forms that duplicated chromosome 21 can assume. Leukemia (2003) 17, 547–553. doi:10.1038/sj.leu.2402849 Keywords: amplified AML1; acute lymphoblastic leukemia; duplicated chromosome 21 Introduction The AML1 (CBFA2) gene, located in the chromosomal band 21q22, has recently attracted a lot of interest in terms of its role in leukemogenesis. AML1 is involved in a number of chromosomal translocations in leukemia. Of particular note in acute lymphoblastic leukemia (ALL) is the t(12;21)(p13;q22), in which AML1 fuses to the TEL (ETV6) gene. This translocation accounts for approximately 25% of B-lineage ALL in children.1–4 Additional copies of AML1 have often been observed in acute leukemia. Acquired trisomy 21, a frequent finding in childhood ALL, itself produces one extra copy of the gene. Structural rearrangements involving duplication of the long arm of chromosome 21, dup(21), have also been described.5–14 Fluorescence in situ hybridization (FISH) techniques showed that extra copies of the AML1 gene were present on these abnormal chromosomes.6,9–14 In one of two patients, for whom the amplified regions were shown to extend beyond the AML1 gene in both centromeric and telomeric directions, the amplification included the ERG and ETS2 genes.10 Amplification of AML1 has been reported in acute myeloid leukemia (AML), where structural rearrangements resulted in partial gains of chromosome 21. Among seven cases, two had evolved from a prior myelodysplasia.15–18 Point mutations19–22 and cryptic chromosome 21 rearrangements associated with Correspondence: CJ Harrison, Leukaemia Research Fund Cytogenetics Group, Cancer Sciences Division, University of Southampton, MP 822 Duthie Building, Southampton General Hospital, Southampton SO16 6YD, UK; Fax: 44 (0)23 8079 6432 4 Current address: Medical Research Council Human Genetics Unit, Edinburgh, UK Received 12 September 2002; accepted 12 November 2002 deletions of AML123,24 have also been reported in myeloid disorders. Collectively, these observations point to possible dosage effects of AML1 in the pathogenesis of leukemia. Further studies in larger groups of patients are needed in order to elucidate the role of AML1 and to help identify the specific chromosomal regions and interacting genes involved in this process. The series of patients described here revealed the existence of a distinct subgroup of older children with ALL and amplification of the AML1 gene, arising from partial duplication of the long arm of chromosome 21, including the 21q22 region. Materials and methods Patients The 20 patients in this study emerged from a series of children and adults with a diagnosis of ALL entered to one of the Medical Research Council (MRC) ALL treatment trials, UKALL XI or ALL 97 for children aged 1–18 years inclusive, or UKALL XII for adults aged 15–55 years. White blood cell count (WBC), immunophenotyping and morphological classification were carried out in the local referral centers and these data, including follow-up information, were collected by the Clinical Trial Service Unit (CTSU), Oxford. In the current childhood trial, ALL 97, no distinction was made between pre-B and common ALL. Cytogenetics Cytogenetic analysis of diagnostic bone marrow or peripheral blood samples was carried out in the regional cytogenetics laboratories. The G-banded slides were reviewed at the Leukaemia Research Fund UK Cancer Cytogenetics Group (UKCCG) Karyotype Database in Acute Leukaemia (Database).25 Karyograms were generated using MacKtype software (Applied Imaging International, UK) and the karyotypes were described according to the International System of Human Cytogenetic Nomenclature.26 FISH Interphase FISH screening is routinely carried out on all trial patients for the prognostically significant abnormalities, TEL/ AML1 or BCR/ABL fusion and rearrangements of the MLL gene. This is performed on the fixed cell suspensions used for cytogenetic analysis. The dual color probe kit, LSIs TEL/AML1 ES (Vysis, UK), designed to detect the translocation, t(12;21)(p13;q22), was hybridized to cell suspensions according to the manufacturer’s instructions. This kit contains a probe spanning the AML1 gene at 21q22, labeled with Spectrum Amplification of AML1 on duplicated chromosome 21 L Harewood et al 548 Orange, and a second probe covering exons 1–4 of the TEL gene, labeled with Spectrum Green. To confirm the presence of the AML1 gene within the duplicated chromosome 21 regions, two exon-specific cosmid probes together covering the entire gene were hybridized individually onto interphase and, wherever possible, metaphase cells from each patient. Cosmid ICRF c103 CO664 contains exons 1–5 of AML1 and cosmid H11086 contains exons 6 and 7. These probes were directly labeled with Spectrum Red using a Nick Translation kit (Vysis, UK). Slides with selected metaphases were co-denatured with 0.04 mg of labeled probe in 10 ml of Hybrisol VII solution (Appligene-Oncor, Qbiogene, UK) at 721C for 2 min and hybridized at 371C overnight. They were then washed in 0.4x SSC with 0.3% IGEPAL (Sigma, UK), followed by 2 SSC with 0.1% IGEPAL at room temperature. Cells were examined using a Zeiss Axioskop fluorescence microscope fitted with appropriate filters (Karl Zeiss, Germany). Whole chromosome paints (wcp) (STAR*FISH, Cambio, UK) were applied to metaphases where possible to resolve the Gbanded karyotypes. Multiplex-FISH (M-FISH) was performed on patients 3956, 4178, 5607 and 5754 using the SpectraVysion 24 color chromosome painting kit (Vysis, UK). These probes were hybridized according to the manufacturers’ instructions. FISH images from interphase and metaphase cells were captured using an Olympus BX microscope with a cooled coupled device (CCD) camera (Photometrics, USA) and analyzed using MacProbe and M-FISH software (Applied Imaging International Ltd, UK) as appropriate. Results A total of 20 patients with multiple copies of the AML1 signal were identified during a routine interphase FISH screening program using the LSIs TEL/AML1 ES probe (Figures 1a and b). In all, 13 patients were part of a consecutive series of 870 children, aged between 1 and 18 years, from which we estimated the incidence of this abnormality to be 1.5%. Among adults over the age of 19 years, the incidence was o0.5% (one out of 229 patients). Patients with one or two additional signals corresponding to extra copies of chromosome 21 were excluded. The karyotypes, clinical details and follow-up data for all 20 patients are shown in Table 1. All patients were negative for TEL/AML1 fusion, for BCR/ABL fusion and for rearrangements of the MLL gene. Since the AML1 probe of the LSIs TEL/AML1 ES kit is B500 kb size and extends beyond the 50 and 30 ends of the AML1 gene, exon-specific probes to AML1 were applied in order to verify that the gene itself was amplified. Although these cosmids spanned more than one exon of AML1 (ICRF c103 Figure 1 (a) LSIs TEL/AML1 ES probe on interphase nuclei from patient 2776. The AML1 signals are in red and the TEL signals are green. (b) LSIs TEL/AML1 ES probe on a metaphase from patient 3368. The normal chromosome 21 has a single red signal and the dup(21) has tandemly repeated signals along part of its length. The normal and dup(21) are painted with wcp 21 (yellow). (c) Cosmid ICRF c103 CO664 covering exons 1–5 and (d) cosmid H11086 covering exons 6 and 7 of AML1 hybridized to metaphases from patient 4623. Leukemia Table 1 Patient no. Sex/age (years) WBC 109/l EFS (months) OS (months) AML1 signals/cell Form of dup(21) Karyotype M/10 F/7 M/11 M/20 F/13 F/7 M/14 M/7 F/12 M/8 F/14 F/14 F/13 M/5 M/8 M/9 F/6 F/14 M/8 M/9 2 17 11 6 3 15 19 3 2 4 1 3 3 8 3 2 14 1 4 2 36 19 27 3+ 26+ 27+ 28+ 4 19+ 17 18+ 17+ 1+ 13+ 13 10+ 7+ 2+ 2+ 1+ 60+ 51+ 32 3+ 26+ 27+ 28+ 18 19+ 20+ 18+ 17+ 1+ 13+ 13+ 10+ 7+ 2+ 2+ 1+ 6+ 4–6 6+ 5+ 5+ 4–7 6+ 6–10 5+ 6+ 7+ 6–8 4–9 5–8 6+ 4–7 4–6 4–6 4–6 5–7 M R M AL AS R SM AL R AL SM AL SM SM R R AL AS R M 48,XY,+X,+14,ider(21)(q10)dup(21)(q?) 47,XX,+X,der(21)r(21)(q?)dup(21)(q?) 46,XY,t(1;16)(q23;p13),ider(21)(q10)dup(21)(q?)/51,idem,+X,+3,+10,+14,+21 46,XY,del(7)(p1?5),t(8;22)(q1?1;q13),dup(21)(q?) 45,XX,7,del(12)(p12),dup(21)(q?) 45,XX,dup(8)(p?),11,der(15)t(11;15)(?;q24),der(21)r(21)(q?)dup(21)(q?) 46,XY,der(21)dup(21)(q?)/46,idem,del(5)(q?),der(15)t(5;15)(q?;q?) 45,XY,dic(8;16)(p1;p1),del(13)(q1?4),dup(21)(q?)/46,idem,t(Y;13)(q1;q1?4),+dic(8;16)(p1;p1)b 47,XX,add(7)(q2),+10,der(21)r(21)(q?)dup(21)(q?)/47,idem,del(12)(p13) 46,XY,dup(21)(q?) 46,XX,t(12;16)(q24;p11),del(15)(q24q26),t(17;20)(p1?3;q11),der(21)dup(21)(q?) 46,XX,del(7)(q22),t(14;22)(q32;q11),dup(21)(q?)b 46,XX,der(21)dup(21)(q?)/46,idem,del(16)(q1) 47,XY,12,der(21)dup(21)(q?),+mar1,+mar2 45,Y,t(X;15)(q2?1;q2?4),dic(12;17)(p1;p1),der(21)r(21)(q?)dup(21)(q?) 46,XY,der(21)r(21)(q?)dup(21)(q?) 46,XX,dup(21)(q?)/47,idem,+X 46,XX,del(9)(p22),dup(21)(q?) 46,XY,t(8;11)(p2?1;q21),del(11)(q21),der(21)r(21)(q?)dup(21)(q?)/47,idem,+Xb 46,XY,ider(21)(q10)dup(21)(q?)b Amplification of AML1 on duplicated chromosome 21 L Harewood et al 2423 2776 3131 3368a 3527 3743 3745 3956 3970 4134 4135 4178 4237 4279 4405 4444 4623 5601 5607 5754 Karyotypes and Clinical Details of Patients with dup(21) Previously reported as patient 38.28 Confirmed by M-FISH. M: metacentric; SM: submetacentric; AL: large acrocentric; AS: small acrocentric; R: ring chromosome. a b 549 Leukemia Amplification of AML1 on duplicated chromosome 21 L Harewood et al 550 Figure 2 Partial G-banded karyograms showing examples of the different forms of dup(21). Normal chromosome 21 on the left, dup(21) on the right: (a) a large acrocentric (patient 3956), (b) a small acrocentric (patient 5601), (c) a metacentric (patient 3131), (d) a submetacentric (patient 3745), and (e) a ring chromosome (patient 2776). CO664 contains exons 1–5 and H11086 contains exons 6 and 7), the finding of discrete signals with both probes in each patient suggested that the entire gene was amplified (Figures 1c and d). The approximate number of copies of AML1 signals is shown in Table 1. This number varied between patients and also from cell to cell within the same sample, the close association of signals in many nuclei making accurate enumeration impossible. In metaphase cells, the AML1 signals, either discrete or in clusters, were observed in tandem duplication on a distinctive marker chromosome (Figures 1b, c and d). The marker was proved to be composed entirely of chromosome 21 material in the 14 cases tested with wcp 21 (Figure 1b) and the four tested by M-FISH, indicating that at least part of it had been duplicated. The results from the AML1-specific probes confirmed that the duplication clearly included the chromosomal region 21q22, where the AML1 gene is located. Since the extent of the duplication and the number of copies of the AML1 signal were difficult to determine, the abnormalities were described in the karyotypes as dup(21)(q?) (Table 1). The marker assumed one of five distinct morphological forms in G-banded cells (Table 1 and Figure 2): a small acrocentric F dup(21)(q?) (two patients); a large acrocentric F dup(21)(q?) (five patients); a metacentric resembling an isochromosome Fider(21)(q10)dup(21)(q?) (three patients); a submetacentric F der(21)dup(21)(q?) (four patients); or a ring chromosome F der(21)r(21)(q?)dup(21)(q?) (six patients), each with a variable number of dark staining Giemsa-positive bands along its length. From microscopic observation of the G-banded metaphases, it was sometimes difficult to determine the true form of the marker between metaphases from an individual patient, raising the possibility that it may change from one type to another. The karyotypes of all 20 patients were abnormal with some similar features. The stemlines were all near-diploid with 45–48 chromosomes, 12 of them being pseudodiploid. In all patients, the dup(21) replaced one copy of the normal chromosome 21, being the sole karyotypic change in the stemline in six of them. In association with the interphase FISH observations described above, there was no cytogenetic evidence of any other established chromosomal changes. Five patients showed the gain of an X chromosome as a secondary change. Chromosomes 7, 8, 12, 15 and 16 (p arm) were involved in accompanying structural abnormalities in three or more patients. The children in this series were older, being outside the usual distribution for childhood ALL of 2–5 years. Their median age was 9 years with a range from 5 to 14 years, with nine of them being more than 10 years old. The only adult patient with dup(21) was aged 20 years. The patients all had a low WBC, pre-B or common immunophenotypes and L1 morphology. As far as prognosis is concerned, follow-up time was too short for conclusions to be drawn. Of the six patients who relapsed, one had an event-free survival (EFS) of o4 months and an overall Leukemia survival of only 18 months, while the remainder relapsed between 13 and 36 months. Discussion This study presents a series of 20 patients with ALL, each with an abnormal marker chromosome of variable morphology, replacing one copy of a normal chromosome 21. The markers were composed entirely of chromosome 21 material, with multiple copies of the AML1 gene duplicated in tandem along their length. Previous reports have described similar markers, and those in which the same or comparable techniques were used in their identification, are shown in Table 2 (patients 1–11). Probes to the AML1 gene were used in all 11 cases, while the involvement of chromosome 21 was proved by whole chromosome painting for patients 1–8, by spectral karyotyping (SKY) for patient 9 and by comparative genomic hybridization for patients 10 and 11.6,9–12,14 Two earlier reports described markers that were defined by cytogenetics as triplication or quadruplication of chromosome 21.5,7 They resembled the large acrocentrics of our own and other series.9,11,12,14 Since wcp 21 was only used on one of them7 and they had not been tested with probes to AML1, they were not included in Table 2. Ours is clearly the largest series so far reported and is the first to identify the incidence of dup(21) at approximately 1.5% among children. In two smaller series, the incidence appeared to be slightly higher. Two patients (6 and 7, Table 2) were found in a consecutive series of 107 patients14 and a further two patients (10 and 11, Table 2) were observed in a series of 109.9 There is no mention of this latter series being consecutive, but the majority of patients came from a single center. It is however noteworthy that they were collected over a 25-year period, whereas our cases, which were UK wide in origin, had been collected in less than 6 years. Abnormal numbers of AML1 signals, but on an apparently normal chromosome 21, have been reported before.27–29 Patients 12–15 (Table 2) were from a recent study in which four of 41 childhood ALL patients were reported to have AML1 amplification with signals repeated in a tandem fashion on a chromosome 21, which showed no evidence of duplication. Patient 16, a young adult, showed the same phenomenon, with five AML1 signals per interphase nucleus and tandem copies of AML1 also on an apparently normal chromosome 21. No details are provided of the five patients with amplified AML1 and a normal karyotype from the series of Najfeld;27 therefore they have not been included in Table 2. The four patients reported by Mikhail et al 29 formed a group that appeared clinically distinct from our series in that they were all 6 years old or less and three had WBC in excess of 50 109/l. Karyotypes were normal in three of them, although one harbored a t(12;21) translocation. Although these observations may indicate that this group of cases are distinct from those with an obviously abnormal Table 2 Patient no. 1 2 Patient no. in paper Sex/age (years) WBC 109/l EFS (months) OS (months) Diagnosis AML1 Signals/cell 110 210 M/15 M/6 4 5 33+ NA 33+ NA L1/pre-B L2/common/pre-B 5 4–5 36,10 111 211 114 214 113 512 19 29 129 2329 3129 4129 3628 F/11 F/10 M/11 M/17 F/19 F/8 ?/12 M/12 F/6 M/3 F/2 F/6 F/6 F/17 23 1 6 1 10 1 2 4 26 100 78 2 56 2 65 14+ 7+ 10+ 12+ NA NA NA NA NA NA NA NA NA NA 14+ 7+ 10+ 12+ NA NA NA NA NA NA NA NA NA B-ALL Common Common Pre-B Pre-B Pre-B B-lineage Pre-B Pre-B Pre-B Pre-B Pre-B Early pre-B Common 5 6 7 8 6–8 5–10 6+ 10–15 6 3 or 4 3 or 4c 3 or 4 4 5 Karyotype 46,X,Y,add(21)(q22),+mar 53,XY,+X,+Y,inv(3)(p14q21),add(4)(q35),+9,+17,+21,+21,+add(21)(q22)/54,idem, +add(21)(q22)/53,idem,inv(3)(p14q21),+mar1,+mar2/54,idem,+21a 46,XX,21,+mar 46,XX,der(21) 46,X,+X,inv(Y)(p11.2q12),+10,20,der(21) 46,XY,add(1)(q25),add(21)(q21) 46,XX,del(7)(p14p21),21,+mar 47,XX,+X,del(21)(q22),der(21) +X,+10,del(11)(q23),qdp(21)(q11.1q22)b 46,XY,del(18)(p11),der(21) 48,XX,20,+der(21),+2mar 46,XY 46,XX 45,XX,19 46,XX 46,XX a Has only two copies of AML1 on the add(21); the other additional signals correspond to extra copies of chromosome 21. Complete karyotype not provided. c Extra copies of AML1 associated with TEL/AML1 fusion. ? indicates gender of patient not provided; superscripts represent reference numbers; NA: not available. b Amplification of AML1 on duplicated chromosome 21 L Harewood et al 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Previously Published Cases with dup(21) and/or extra copies of AML1 551 Leukemia Amplification of AML1 on duplicated chromosome 21 L Harewood et al 552 marker, it is perhaps more realistic to think of them as different ends of a spectrum that encompasses all degrees of duplication of segments of chromosome 21, the end result of which, among others, is amplification of at least the AML1 gene. In our series, we only used probes to the AML1 gene. Other studies have however shown amplification of regions involving genes both centromeric and telomeric to AML1 on the abnormal chromosomes.6,10,27 The size of the duplicated segments suggests that this is probably true for the majority of patients with dup(21). Although with hindsight it is easy to suspect the presence of a duplicated chromosome 21 in a karyogram that contains a distinctive marker and from which one copy of a normal chromosome 21 is missing, the abnormality was in fact discovered by chance at interphase, while screening for the TEL/AML1 fusion. Abnormal numbers of AML1 signals in interphase nuclei led to the metaphase investigations, which located them on the marker. This was also true for patients 6, 7, 10 and 11 (Table 2).9,14 Patient 9 (Table 2) is an exception. The dup(21) was identified firstly by SKY and the presence of AML1 was confirmed subsequently.12 The markers in the 20 patients reported here could be classified into five distinct types with some overlapping features. Five of our patients had large acrocentric dup(21), with signal numbers for AML1 in interphase cells, varying from five to 10 between them. The markers in patients 4, 5, 6, 9 and 11 (Table 2) also appeared as large acrocentrics with between 4 and 8 copies of AML1 in interphase cells.9,11,12,14 In two of our patients, the markers were small acrocentrics, each having 4–6 AML1 interphase signals. They appear to compare with patient 2 in Table 2. Seven cases in our series appeared to be either large metacentric or submetacentric chromosomes with from 4 to 9 AML1 interphase FISH signals. Patients 3, 7 and 10 (Table 2) also had metacentric markers. Patient 3 had five AML1 signals in interphase, two of which were sited on each arm of the marker in metaphase. Patient 10 had the highest number of signals reported for this or any other series with from 10 to 15 signals at interphase that were ‘tandemly placed in two sites’ on the ‘der(21)’. Patient 8 was different from the other reported cases. A single AML1 signal was observed on one arm of the marker, with duplicated signals on the other arm. In addition, the AML1 signal was deleted from the other chromosome 21. This is the only patient described in which both chromosomes 21 are abnormal.13 Our report is the first to describe the dup(21) in the form of a ring. These chromosomes, which were observed in six patients, had from 4 to 7 AML1 signals at interphase. Patient 1 (Table 2) also appeared to have a marker of a unique form, not identified in our series. It was described as ‘dicentric’ in four cells and as ‘telomeric’ in four others, while the pattern of uneven hybridization of certain probes along its length was said to confirm its dicentric origin.10 The marker in patient 3 (Table 2) was described in more detail in an earlier publication.6 Here also there was uneven hybridization of probes, but this time on the two arms of a metacentric marker, causing the authors to suggest that it may have arisen from secondary breakage of a chromosome 21 that originated as a ring. In our own series, the difficulties we encountered in classifying the markers of any one patient into a single cytogenetic type may be an indication that they have the inherent instability ascribed to ring chromosomes and may indeed have evolved from them. This could explain the variation in size between the different forms of the markers, although, interestingly, the relative size of the marker was not universally paralleled by an increase in the number of AML1 signals. Leukemia Mikhail et al 29 demonstrated that the detection of extra AML1 signals by FISH was associated with overexpression of AML1 measured by real-time quantitative RT-PCR, both in the cases listed in Table 2 and those with additional signals resulting from trisomy 21. It is of interest to consider whether amplification within a single chromosome, either visible at the cytogenetic level or not, has the same effect as an acquired gain of chromosome 21, and to what extent the structure and expression of the gene are altered in these different situations. Although point mutations of the runt domain of the AML1 gene have been observed in myeloid disorders,19–22 there was no evidence of point mutations in exons 3–5 of AML1 (encoding the runt domain) in five ALL patients tested (patients 1–3, 6 and 7).10,14 Although these studies could not rule out point mutations in other exons, it was presumed to be unlikely. This led the authors to suggest that it may be the amplification of wild-type AML1 that is important in leukemogenesis. It could be that additional copies of AML1 result in dysregulation of the normal AML1 gene product. Further studies using DNA microarray techniques will provide information on the expression of AML1 in relation to other leukemia-associated genes both on chromosome 21 and elsewhere within the genome. The karyotypes in our series were near-diploid, with the majority being pseudodiploid, which was consistent with most of the previously reported karyotypes in Table 2. In six patients in our series and in two previously reported, the dup(21) was the sole karyotypic change, at least in the stemline,6,11 suggesting that it may occur as a primary genetic event. Apart from dup(21), no known established numerical or structural chromosomal change was present. Gain of an X chromosome, observed in five cases, was also seen in four reported cases,6,11–13 while chromosome Y was involved in a translocation with chromosome 13 in one of our cases and respectively gained, lost or inverted in three reported cases.10,11 Among the 11 patients previously reported, the clinical features were similar to those observed in our series. Followup was too short to say anything conclusive about the prognostic implications of this abnormality. From the screening of our own adult series, in conjunction with the 42 adult ALL patients in the study of Penther et al,14 there were no patients over the age of 21 years identified with this abnormality. This adds further evidence to that already established for the ETV6/AML1 translocation,28 that abnormalities involving chromosome 21 in ALL appear to be more common in children than in adults. In this report, we have made a significant contribution to the number of cases described with a dup(21) and associated amplification of the AML1 gene, increasing the number of reported cases to 31. We have confirmed that the abnormal chromosome may assume a variety of shapes, including the previously unidentified form of a ring. The important number of cases included in our series has permitted a realistic estimate of the incidence of this abnormality to be 1.5% in childhood ALL. Our observations reinforce the importance of chromosome 21 in leukemogenesis. It seems likely that the amplification of AML1, and possibly of other genes within close proximity, directly contributes to the etiology of acute lymphoblastic leukemia. Acknowledgements We thank the Leukaemia Research Fund for financial support and the UK cytogenetics laboratories that contributed samples to this study: Belfast, Birmingham, Cambridge, Cardiff, Croydon, Dundee, Edinburgh, Great Ormond Street Children’s Hospital, London, Manchester, Newcastle, Norwich, Royal Marsden Amplification of AML1 on duplicated chromosome 21 L Harewood et al 553 Hospital, London, Salisbury, Sheffield, and University College Hospital, London. Cosmids ICRF c103 CO664 and H11086 were kindly donated by Dr Anne Hagermeijer, EU Concerted Action and Dr Roland Berger, Paris, respectively. We are grateful to Dr John Crolla and his team, Wessex Regional Genetics Laboratory, Salisbury for the growing and preparation of the AML1 specific cosmids as part of an ongoing collaboration. We thank Dr Helena Kempski for helpful discussions. References 1 Romana SP, Poirel H, Leconiat M, Flexor MA, Mauchauffe M, Jonveaux P et al. 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