Identification of Cryptic Aberrations and Characterization of Translocation Breakpoints Using Array CGH in High Hyperdiploid Childhood Acute Lymphoblastic Leukemia

Identiﬁcation of cryptic aberrations and characterization of translocation breakpoints using array CGH in high hyperdiploid childhood acute lymphoblastic leukemia

K Paulsson1, M Heidenblad1,HMo¨rse2,A˚ Borg3,4, T Fioretos1 and B Johansson1

1Department of Clinical Genetics, Lund University Hospital, Sweden; 2Department of Pediatrics, Lund University Hospital, Sweden; 3Department of Oncology, Lund University Hospital, Sweden and 4Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Sweden

High hyperdiploidy, characterized by non-random trisomies, is example, studies of clonotypic IGH@ rearrangements in the largest cytogenetic subgroup in childhood acute lympho- neonatal blood spots, concordant hyperdiploid ALLs in twins blastic leukemia (ALL). It is not known whether the gained chromosomes are sufficient for leukemogenesis or if additional and the presence of hyperdiploid cells in cord blood have genetic aberrations are necessary. However, the suboptimal shown that the hyperdiploid pattern frequently, possibly always, 6–10 chromosome morphology of hyperdiploid ALLs makes detec- arises before birth. It is, hence, worth noting that the highest tion of structural abnormalities difficult if using cytogenetic incidence of hyperdiploid ALL is found in children aged techniques; alternative methods are, therefore, needed. We between 3 and 5 years, and that the median age of the 12 performed array comparative genome hybridization (CGH) patients in whom a prenatal origin has been demonstrated is 2.4 analyses, with a resolution of 100 kb, of eight cases of high 1,6–10 hyperdiploid childhood ALL to characterize structural abnorm- years, with the oldest being 9 years at the time of diagnosis. alities found with G-banding/multicolor fluorescence in situ The most likely explanation for the latency period is that hybridization (FISH) and to detect novel changes. The non- additional genetic changes must be acquired for overt ALL to centromeric breakpoints of four rearrangements, including occur. Also, it has been shown that hyperdiploidy generally three translocations and one 1q duplication, were narrowed arises by a simultaneous gain of chromosomes in a single down to o0.2 Mb. Furthermore, four submicroscopic imbal- abnormal mitosis.11–13 The exact mechanism is unknown, but ances involving 0.6–2.7 Mb were detected, comprising two segmental duplications involving 1q22 and 12q24.31 in one one may assume that this cell division is highly abnormal, case and two hemizygous deletions in 12p13.2–31 – including indicating the presence of genetic aberrations in the cell before ETV6 – and in 13q32.3–33.1 in another case. Notably, FISH the hyperdiploidy. analysis of the latter revealed an associated reciprocal Considering that additional abnormalities hence may be t(3;13)(q?;32.2–33.1). In conclusion, the array CGH analyses involved in inducing the hyperdiploid pattern as well as being revealed putative leukemia-associated submicroscopic imbal- required for overt leukemia, remarkably little is known about the ances and rearrangements in 2/8 (25%) hyperdiploid ALLs. The detection and characterization of these additional genetic frequencies and pathogenetic impact of such changes in aberrations will most likely increase our understanding of the hyperdiploid childhood ALL. Gene point mutations, for example 14,15 pathogenesis of high hyperdiploid childhood ALL. in FLT3, seem to be quite common, whereas only a few Leukemia (2006) 20, 2002–2007. doi:10.1038/sj.leu.2404372; recurrent structural rearrangements, such as dup(1q), i(7q) and published online 14 September 2006 i(17q), have been identified; no translocation specific for this Keywords: high hyperdiploidy; childhood acute lymphoblastic cytogenetic subgroup has been described to date.16 This could leukemia; array-based comparative genome hybridization possibly be explained by the often poor chromosome morphology in hyperdiploid cases, leading to difficulties in identifying abnormalities with G-banding as well as with multicolor fluorescence in situ hybridization (M-FISH).13,17,18 Therefore, methods not dependent on the quality of the metaphase plates Introduction may yield novel genetic aberrations. However, studies using conventional comparative genome hybridization (CGH) on 4 High hyperdiploidy ( 50 chromosomes) in childhood acute metaphase chromosomes also have failed in general to detect lymphoblastic leukemia (ALL) is characterized by non-random cryptic abnormalities in hyperdiploid cases.19,20 trisomies and tetrasomies, frequently involving chromosomes X, In the present study, we utilized high-resolution, genome- 4, 6, 8, 10, 14, 17, 18 and 21. It is the most common cytogenetic wide array-based CGH (array CGH) to investigate high pattern in pediatric ALL, occurring in approximately 30% of all 1 hyperdiploid pediatric ALLs. With this technique, imbalances cases. This subgroup is associated with a B-cell precursor involving as little as 100 kb, that is, far below the detection limit phenotype, low white blood cell count and a median age of 1,2 of G-banding, M-FISH and conventional CGH, are seen, 4 years, and has an overall survival of approximately 90%. enabling the identification of hidden deletions and amplifica- The pathogenetic consequences of the supernumerary chromo- tions.21,22 Furthermore, as many leukemia-associated transloca- somes are unknown, but dosage effects are likely to play an 3–5 tions are associated with small deletions close to the important role. chromosomal breakpoints (BPs),23 such rearrangements may Recent data suggest that genetic changes in addition to the be detected with array CGH. However, to the best of our high hyperdiploidy are required for leukemia to develop. For knowledge, no translocation or inversion has as yet been found in neoplasia using this method. The present array CGH analysis, Correspondence: Dr K Paulsson, Department of Clinical Genetics, the first to be reported in high hyperdiploid childhood ALL, University Hospital, SE-221 85, Lund, Sweden. E-mail: [email protected] identified one cytogenetically cryptic translocation, detected Received 5 May 2006; revised 13 July 2006; accepted 18 July 2006; novel submicroscopic abnormalities and enabled precise published online 14 September 2006 characterization of previously described rearrangement BPs. Array CGH analysis of high hyperdiploid ALL K Paulsson et al 2003 Patients, materials and methods applied. In addition, probes for the ETV6 and RUNX1 genes (Vysis, Downers Grove, IL, USA) were used. Hyperdiploid cells Patients were identified with a centromeric probe for chromosome 10 The study comprised eight hyperdiploid pre-B childhood ALLs (Vysis). A probe for the subtelomeric region of 3q and a whole (Table 1) successfully analyzed with array CGH. All cases had chromosome paint (WCP) probe for chromosome 3 (Vysis) were been previously investigated with G-banding and M-FISH.13 The utilized to screen for the t(3;13)(q?;q32.2–33.1) found in the investigation was approved by the Research Ethics Committee of present study (see below). All cases in the investigation were Lund University. analyzed, except case 3, in which no material was available.

Array CGH Results DNA was extracted from bone marrow or peripheral blood cells, obtained at the time of diagnosis or relapse. Slides containing The array CGH analysis confirmed all numerical changes the ‘32k’ array set, consisting of 32 433 bacterial artificial previously detected with G-banding/M-FISH (Table 1). Four chromosome (BAC) clones (BACPAC Resources, Oakland, CA, structural abnormalities resulting in large-scale imbalances were USA) and covering at least 98% of the human genome,21,22 present. The array CGH revealed that the der(5)t(1;5)(p?;q?) in were produced at the SWEGENE DNA microarray resource case 2 was a der(5)t(1;5)(q11–21.1;q21.3) (chr 1: gain at least center at Lund University, Sweden. Male reference genomic from 142 to 245 Mb; chr 5: gain 0–106.8 Mb; positions DNA was used in all hybridizations (Promega, Madison, WI, according to www.ensembl.org), der(11)t(6;11)(?;q23) in case USA). Labeling, slide preparation and hybridization were 4 was a der(11)t(6;11)(p21.1;q14.1) (chr 6: gain 0–44.5 Mb; chr performed as described previously.26 11: gain 0–80.5 Mb), dup(1)(q?) in case 5 was a dup(1)(q11– The analyses of microarray images were performed with the 21.1q41) (chr 1: gain at least from 142 to 216 Mb), and that the GenePix Pro 4.0 software (Axon Instruments, Foster City, CA, der(8)t(8;14)(p?;q?) in case 8 was a der(8)t(8;14)(p12;q?) (chr 8: USA). For each spot, the median pixel intensity minus the loss 0–37.5 Mb) (Table 1). Two 1q BPs were within the median local background for both dyes was used to obtain a test pericentric heterochromatin of chromosome 1, not represented over reference gene copy number ratio. Data normalization was in the ‘32k’ array set, and they could hence not be precisely performed per array subgrid using lowess curve fitting with a mapped. The 14q BP in case 8 could not be determined because smoothing factor of 0.33.27 To identify imbalances, the MATLAB of the concurrent clone with tetrasomy 14 (Table 1), which toolbox CGH plotter was applied, using moving mean average made it impossible to investigate the partial 14q gain in the over three clones and limits of log 2 40.2.28 Classification as subclone with der(8). All translocation BPs seen with array CGH gain or loss was based on (1) identification as such by the CGH were confirmed with locus-specific metaphase FISH. plotter and (2) visual inspection of the log 2 ratios. In general, Ten submicroscopic imbalances were found with array CGH log 2 ratios 40.5 in at least four adjacent clones were (Table 2). Five were copy number gains, displaying log 2 ratios considered to be deviating. Ratios of 0.5–1.0 were classified corresponding to duplications and involving 0.2–1.9 Mb (med- as duplications/hemizygous deletions; ratios 41.0 were classi- ian 0.5 Mb), whereas five were copy number losses, showing fied as amplifications/homozygous deletions. All normalizations log 2 ratios corresponding to hemizygous deletions and invol- and analyses were carried out in the BioArray Software ving 0.4–2.7 Mb (median 0.5 Mb). All these imbalances were Environment database.29 present in one case each, except the 7q22.1 gain and the 0.5 Mb loss at 15q11.2 (Table 2). Six of them overlapped with copy number polymorphisms (CNPs) previously reported in at least FISH two different studies (the Database of Genomic Variants; To confirm the array CGH findings, metaphase FISH was http://projects.tcag.ca/variation/). In addition, losses involving performed according to standard methods. Signals were 0.2 Mb were detected in the IGK@ and IGH@ loci at 2p11.2 and detected using an Axioplan 2 fluorescence microscope (Carl 14q32.33, respectively, in three of the cases (Table 2). Zeiss, Jena, Germany) and analyzed with the CytoVision Ultra Metaphase FISH was used to investigate two of the detected system (Applied Imaging, Newcastle Upon Tyne, UK). BACs and gains: the 0.2 Mb gain at 7q22.1 and the 1.9 Mb gain at P1 artificial chromosomes (Table 2; BACPAC Resources) were 12q24.31 (Table 2). The FISH analyses revealed signals only at

Table 1 Cytogenetic features of eight high hyperdiploid childhood ALL cases

Case Sex/Age Karyotypea

1 M/2 57,XY,+X,+Y,+4,+6,+10,+14,+17,+18,+21,+21,+mar 2b M/2 61–63,XY,+X,+Y,+4,+der(5)t(1;5)(q11–21.1;q21.3),+6,+8,+9,+10,+11,+12,+14,+14,+17,+18,+21,+21,+22 3c M/2 54,XY,+X,+4,+6,+10,+14,+17,+18,+21/55,idem,+21 4b M/3 61,XY,+X,+4,+5,+7,+8,+8,+10,+der(11)t(6;11)(p21.1;q14.1),+12,+14,+14,+17,+18,+21,+22/63,idem,+18,+21 5 F/3 56,XX,+X,dup(1)(q11-21.1q41),+4,+6,+8,+10,+14,+14,+18,+21,+21 6 F/4 54,XX,+X,+4,+6,+14,+17,+18,+21,+21 7 M/4 56,XY,+X,+4,+6,+8,+10,+14,+17,+21,+21,+21/57,idem,+15 8 F/15 57,XX,+X,+X,+4,+6,+10,+14,+14,+17,+18,+21,+21/56,idem,der(8)t(8;14)(p12;q?),À14/57,idem,+8,der(8) Â 2,À14 Abbreviations: ALL, acute lymphoblastic leukemia; M, male; F, female. Abnormalities further characterized with array CGH are indicated in bold type. aAll cases had been investigated using G-banding and M-FISH. All cases except no. 3 have previously been published in Paulsson et al.13 bNote that, from a formal point of view, the karyotypes of these cases are hypotriploid and should be written accordingly.24 cPreviously published in Davidsson et al.25

Leukemia Leukemia 2004

Table 2 Submicroscopic imbalances detected with array CGH in eight high hyperdiploid childhood ALLs

Chromosome Position Cloneb Size (Mb) Gain/loss Gene(s) in regiona Reported Case(s) FISH ﬁndings Comment band (Mb)a CNPc ra G nlsso ihhprili ALL hyperdiploid high of analysis CGH Array

1p36.13 16.4–16.9 RP11–45I3 0.5 G SPATA21, NECAP2, NBPF1, MSTP2, Yes 5 ND F EIF1AP1,MSTP9 1q22 151.5–152.1 RP11–540E15 0.6 G KCNN3, PMVK, PBXIP1, PYGO2, SHC1, CKS1B, No 7 ND F FLAD1, LENEP, ZBTB7B, DCST2, DCST1, ADAM15, EFNA4, EFNA3, EFNA1, RAG1AP1, DPM3, KRTCAP2, TRIM46, MUC1, THBS3, MTX1, GBA, SCAMP3, CLK2, HCN3, PKLR, FDPS, RUSC1 2p11.2 89 RP11–15J7 0.2 L IGK@ F 4, 5 ND Somatic immunoglobulin Paulsson K rearrangement 7q22.1 101.7–101.9 RP13–771B7 0.2 G PRKRIP1, POLR2J, POLR2J3, RASA4 Yes 3, 5 Segmental F

duplication al et 8q21.2 86.7–87.0 RP11–574H12 0.3 G GOR Yes 7 ND F 12p13.2–31 9.6–12.3 RP11–711K1 2.7 L KLRB1, CLEC2D, CD69, KLRF1, CLEC2A, CLEC2B, No 8 Hemizygous F CLEC12A, CLEC1B, CLEC9A, CLEC1A, CLEC7A, deletion OLR1, GABARAPL1, KLRD1, KLRC4, KLRC1, KLRA1, STYK1, CSDA, TAS2, T2R55, PRB4, ETV6, BCL2L14, LRP6 12q24.31 122.6–124.5 RP11–665C13 1.9 G DDX55, EIF2B2, GTF2H3, TECT2, ATP6V0A2, No 7 Segmental F DNAH10, NCOR2, SCARB1, UBC, DHX37, BRI3BP, duplication AACS 13q32.3–33.1 98.7–100.9 RP11–44I7 2.2 L PHGDHL1, EBI2, GPR18, TM9SF2, CLYBL, ZIC5, No 8 Hemizygous Cryptic t(3;13)(q?; ZIC2, PCCA, VGCNL1, ITGBL1 deletion/t(3;13) q32.2–33.1) 14q32.33 105 RP11–284A8 0.2 L IGH@ F 4, 6 ND Somatic immunoglobulin rearrangement 15q11.2 19.7–20.1 RP11–585E3 0.4 L OR4N4 Yes 6 ND F 15q11.2 21.9–22.4 RP11–710L6 0.5 L No genes Yes 2, 3 Normal F 16p11.2 32.6–33.0 RP11–261A7 0.4 L No genes Yes 4 ND F Abbreviations: ALL, acute lymphoblastic leukemia; array CGH, array-based comparative genome hybridization; CNP, copy number polymorphism; FISH, fluorescence in situ hybridization; G, gain; ND, not done; L, loss; F, not applicable. aAccording to www.ensembl.org. bRepresentative clone involved in imbalance. cAt least two different studies have reported an overlapping CNP (The Database of Genomic Variants; http://projects.tcag.ca/variation. Array CGH analysis of high hyperdiploid ALL K Paulsson et al 2005 the expected loci, suggesting segmental duplications. Three of the losses seen with array CGH were analyzed with metaphase FISH. These included the 2.7 Mb loss at 12p13.2–31, the 2.2 Mb loss at 13q32.3–33.1 and the 0.5 Mb loss at 15q11.2 (Table 2). The first two of these were confirmed to be hemizygous deletions, whereas the latter yielded signals in both homologs. This discrepancy between the array CGH and the FISH results for the 15q11.2 region may be due to the presence of different clones with and without the deletion.26 FISH utilizing probes localized telomeric to the 13q32.3–33.1 deletion revealed that the distal part of 13q had been translocated to 3q (Figure 1). Hence, this deletion was associated with a cryptic reciprocal t(3;13)(q?;q32.2–33.1). Screening for this translocation in six of the remaining high hyperdiploid ALLs did not reveal any additional case harboring the t(3;13).

Discussion

We utilized high-resolution, genome-wide array CGH to investigate eight cases of high hyperdiploid childhood ALL. Four structural rearrangements had previously been seen with G-banding and M-FISH (Table 1), and it was possible to analyze seven of their eight BPs with array CGH. All except the proximal 1q BPs were narrowed down to o200 kb, showing that array CGH is an efficient method for mapping BPs in cases with unbalanced aberrations. Gain of parts of 1q is the most frequent structural abnormality in the hyperdiploid subgroup.16 Two of the present cases (2 and 5) displayed such changes; both had BPs within, or in the very close proximity of, the pericentric heterochromatic region, which is not believed to contain any gene. Thus, dosage effects may be the main pathogenetic outcome of the 1q gains. However, the regions close to the heterochromatin are gene rich (www.ensembl.org) and additional 1q duplications need to be investigated before involvement of specific genes can be excluded. Also, the 5q BP in case 2 was in the gene EFNA5; further studies will show if this or other loci are targeted by the translocation. The der(11)t(6;11) in case 4 was shown to involve 6p21.1 and 11q13.1 (Table 1). The 6p BP occurred in a region containing many genes, whereas no known genes are localized in the 11q BP. Hence, like the 1q gains, it is possible that this rearrangement does not target a specific locus, but may instead lead to general dosage effects. However, it is worth noting that RUNX2 Figure 1 FISH findings of a cryptic del(13)(q32.2q33.1) associated with a t(3;13)(q?;32.3–33.1) in case 8. (a) Metaphase FISH analysis is situated in 6p21.1, approximately 1 Mb distal to the BP utilizing a WCP probe for chromosome 13 (green) and a BAC probe suggested by array CGH. We have previously suggested that (red) localized distally to the 13q deletion, showing that the segment homologs to RUNX1 and ETV6 – the genes involved in the telomeric to the deletion had been translocated (arrow). (b) Metaphase t(12;21)(p13;q22) – may be cryptically rearranged in hyper- FISH analysis with WCP probes for chromosomes 3 (red) and 13 diploid cases.25 Unfortunately, no material was available for (green). A reciprocal t(3;13) is detected (arrows). FISH analyses to definitely exclude involvement of RUNX2. The chromosome 8 BP of the der(8)t(8;14) in case 8 was shown to be in 8p12 by array CGH (Table 1). Because the found in the present study involved genes (Table 2), but too few chromosome 14 BP could not be mapped and the BP region in cases were investigated to speculate on their possible role in the 8p contained more than 10 genes, it was not possible to identify etiology of childhood ALL. a possible target. Four of the submicroscopic imbalances detected with array A total of 12 small duplications and hemizygous deletions CGH were putative leukemia-associated genetic changes. These were detected with array CGH, including 0.2 Mb deletions in were segmental duplications at 1q22 and 12q24.31 in case 7 the IGK@ and IGH@ loci at 2p11.2 and 14q32.33, respectively, and hemizygous deletions at 12p13.2–31, involving the ETV6 in some of the cases (Table 2). The latter two most likely resulted gene, and 13q32.3–33.1 in case 8 (Table 2). The deletions were from somatic immunoglobulin rearrangements clonotypic for confirmed with FISH (Table 2) and were only present in the the hyperdiploid blast cells,30 and were thus not involved in hyperdiploid cells. The role of segmental duplications in leukemogenesis. Six of the remaining submicroscopic imbal- leukemogenesis is not known, but we have previously detected ances overlapped with reported CNPs (Table 2), and were hence similar imbalances in myeloid malignancies with trisomy 8.26 probably constitutional. The majority of the possible CNPs Considering that gain of 1q is frequent in hyperdiploid

Leukemia Array CGH analysis of high hyperdiploid ALL K Paulsson et al 2006 childhood ALLs,16 it is worth noting that one of the segmental childhood acute lymphoblastic leukemia. Genes Chromosomes duplications involved 1q22; it is tempting to speculate that Cancer 2004; 41: 191–202. genes in this small region may be the targets for larger 1q 5 Andersson A, Olofsson T, Lindgren D, Nilsson B, Ritz C, EdeńP duplications in ALL. Deletion of one copy of ETV6 is a common et al. Molecular signatures in childhood acute leukemia and 31 their correlations to expression patterns in normal hemato- secondary aberration in childhood ALL with t(12;21), but little poietic subpopulations. Proc Natl Acad Sci USA 2005; 102: is known about such deletions in other subgroups of ALL. 19069–19074. However, FISH investigations have revealed ETV6 deletions 6 Yagi T, Hibi S, Tabata Y, Kuriyama K, Teramura T, Hashida T without cytogenetically visible 12p alterations in 3–6% of et al. Detection of clonotypic IGH and TCR rearrangements t(12;21)-negative childhood ALLs, including high hyperdiploid in the neonatal blood spots of infants and children with cases.31–33 B-cell precursor acute lymphoblastic leukemia. Blood 2000; 96: 264–268. Interestingly, FISH analysis of the 13q deletion in case 8 7 Panzer-Gru¨mayer ER, Fasching K, Panzer S, Hettinger K, Schmitt K, showed that it was associated with a t(3;13)(q?;32.3–33.1) Sto¨ckler-Ipsiroglu S et al. Nondisjunction of chromosomes leading (Table 2; Figure 1). The t(3;13) was cytogenetically cryptic and to hyperdiploid childhood B-cell precursor acute lymphoblastic had not been detected in the previous G-banding/M-FISH leukemia is an early event during leukemogenesis. Blood 2002; analyses,13 most likely because the involved chromosomal 100: 347–349. regions were too small to be visible with these rather low- 8 Taub JW, Konrad MA, Ge Y, Naber JM, Scott JS, Matherly LH et al. resolution techniques. Deletions in BP regions have previously High frequency of leukemic clones in newborn screening blood samples of children with B-precursor acute lymphoblastic leuke- been described in several fusion gene-forming rearrange- 23 mia. Blood 2002; 99: 2992–2996. ments. As the t(3;13) was reciprocal and the 13q BPs were 9 Maia AT, van der Velden VHJ, Harrison CJ, Szczepanski T, in, or very close to, several genes, PHGDHL1, GPR18, EBI2, Williams MD, Griffiths MJ et al. Prenatal origin of hyperdiploid VGCNL1 and ITGBL1, it is possible that this rearrangement may acute lymphoblastic leukemia in identical twins. Leukemia 2003; have resulted in a fusion gene. 17: 2202–2206. In conclusion, using array CGH, we identified previously 10 Maia AT, Tussiwand R, Cazzaniga G, Rebulla P, Colman S, Biondi A et al. Identification of preleukemic precursors of hyperdiploid undetected putative leukemia-associated chromosome abnorm- acute lymphoblastic leukemia in cord blood. Genes Chromosomes alities in 2/8 (25%) of the investigated hyperdiploid cases, Cancer 2004; 40: 38–43. including one novel reciprocal translocation. Interestingly, none 11 Onodera N, McCabe NR, Rubin CM. Formation of a hyperdiploid of these submicroscopic aberrations involved any of the karyotype in childhood acute lymphoblastic leukemia. Blood chromosomes that are frequently gained in hyperdiploid ALL. 1992; 80: 203–208. Although segmental duplications would be difficult to see with 12 Paulsson K, Panagopoulos I, Knuutila S, Jee KJ, Garwicz S, Fioretos T et al. Formation of trisomies and their parental origin in array CGH analysis in a chromosome that is present in more hyperdiploid childhood acute lymphoblastic leukemia. Blood than two copies, deletions should be detectable, at least those 2003; 102: 3010–3015. involving two or more homologues. The fact that we did not find 13 Paulsson K, Mo¨rse H, Fioretos T, Behrendtz M, Stro¨mbeck B, any cryptic changes in the commonly supernumerary chromo- Johansson B. Evidence for a single-step mechanism in the origin of somes supports the hypothesis that the pathogenetic outcome of hyperdiploid childhood acute lymphoblastic leukemia. Genes the tri- and tetrasomies in high hyperdiploid pediatric ALL is not Chromosomes Cancer 2005; 44: 113–122. simply duplication of submicroscopic genetic abnormalities in 14 Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA et al. FLT3 mutations in childhood acute lymphoblastic leukemia. the extra chromosome, as has previously been suggested for 34,35 Blood 2004; 103: 3544–3546. some neoplasia-associated trisomies. Finally, the array 15 Taketani T, Taki T, Sugita K, Furuichi Y, Ishii E, Hanada R CGH analysis narrowed down several BPs of structural et al. FLT3 mutations in the activation loop of tyrosine kinase aberrations to o200 kb. 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