To identify novel genomic regions of interest in acute myeloid leukemia (AML) with complex karyotypes, we applied comparative genomic hybridization to microarrays (array-CGH), allowing high-resolution genome-wide screening of genomic imbalances.

Sixty AML cases with complex karyotypes were analyzed using array-CGH; parallel analysis of gene expression was performed in a subset of cases.

Genomic losses were found more frequently than gains. The most frequent losses affected 5q (77%), 17p (55%), and 7q (45%), and the most frequent genomic gains 11q (40%) and 8q (38%). Critical segments could be delineated to genomic fragments of only 0.8 to a few megabase-pairs of DNA. In lost/gained regions, gene expression profiling detected a gene dosage effect with significant lower/higher average gene expression levels across the genes located in the respective regions. Furthermore, high-level DNA amplifications were identified in several regions: 11q23.3-q24.1 (n = 7), 21q22 (n = 6), 11q23.3 (n = 5), 13q12 (n = 3), 8q24 (n = 3), 9p24 (n = 2), 12p13 (n = 2), and 20q11 (n = 2). Parallel analysis of gene expression in critical amplicons displayed overexpressed candidate genes (eg, C8FW and MYC in 8q24).

In conclusion, a large spectrum of genomic imbalances, including novel recurring changes in AML with complex karyotypes, was identified using array-CGH. In addition, the combined analysis of array-CGH data with gene expression profiles allowed the detection of candidate genes involved in the pathogenesis of AML.

Nonrandom, clonal chromosome aberrations are detected in approximately 50% of adult patients with acute myeloid leukemia (AML), and it is well established that karyotype provides important prognostic information that influences therapy and outcome of this disease.1-3 The cloning and molecular characterization of balanced translocations and inversions, such as t(8;21)(q22;q22), inv(16)(p13q22), and t(15;17)(q22;q11∼21), has resulted in the identification of cellular pathways that are causally implicated in the pathogenesis of distinct subtypes of AML.

In contrast, little is known about the molecular pathogenesis of AML with complex karyotypes, commonly defined as the presence of three or more chromosome abnormalities in the absence of t(8;21), inv(16)/t(16;16), t(15;17), and t(11q23). Complex karyotypes are found in approximately 10% to 15% of adult AML cases.4,5 They are often associated with prior myelodysplastic phase or prior exposure to toxic agents4,6 and almost invariably with a dismal outcome.1,2,4,5,7 Cytogenetic analysis has been hampered by the difficulty of dissecting the complex changes by conventional chromosome banding techniques alone. In particular, marker chromosomes, or the cytogenetic hallmarks of DNA amplifications, double minutes and homogeneously staining regions, cannot always be precisely localized to specific chromosomal bands. Previous cytogenetic and molecular cytogenetic studies have shown that genomic losses or gains are much more frequent than balanced rearrangements in this subset of AML.8-10 Among the imbalances most frequently found are losses affecting 5/5q (−5/5q–), −7/7q–, −17/17p–, −18/18q–, 12p–, and −16/16q–; and gains affecting 8/8q (+8/+8q), +21/+21q, +11q, and +22q.

Array-based comparative genomic hybridization (array-CGH) represents a novel technique that allows high-throughput, genome-wide screening of genomic imbalances with a 20- to 100-fold higher spatial resolution than conventional CGH.11-13 In hematologic research, array-CGH has so far mainly been applied to the analysis of lymphoproliferative disorders.14-16 The potential of this technology for the detection of genomic amplifications was also demonstrated in a recent study of AML cases with complex karyotypes that were analyzed using a chromosome 21q–specific microarray.17 In addition, parallel analysis of gene expression using microarray technology has also been shown to provide useful information regarding the delineation of candidate genes in genomic regions characterized by copy number alterations (CNAs).17-19

To identify novel candidate regions and disease-related genes, we studied 60 AML cases with complex karyotypes using genome-wide high resolution array-CGH in combination with global gene-expression profiling in a subset of cases.

Patients

Frozen cells (−70°C) from peripheral blood (PB; n = 20) or bone marrow (n = 40) from 60 adult patients with newly diagnosed AML and complex karyotypes (de novo AML, n = 35; secondary AML after myelodysplastic syndrome [MDS], n = 16; therapy-induced AML, n = 4; unknown, n = 5) were analyzed. Complex karyotypes were defined as the presence of at least three chromosome abnormalities in the absence of t(8;21), inv(16)/t(16;16), t(15;17), and t(11q23). The diagnosis of AML was made according to the French-American-British Cooperative Group criteria.20 The median age of the patients was 62 years (range, 22 to 81 years), the median blast count in PB 40% (range, 10% to 89%), and in bone marrow 69% (range, 30% to 91%). All patients had given informed consent for cryopreservation and molecular analyses.

Cytogenetic and Molecular Genetic Analysis

All samples were studied by chromosome banding analysis using standard techniques, and chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature.21

Validation of array-CGH was performed by fluorescence in situ hybridization (FISH). The corresponding bacterial artificial (BAC) or P1-derived artificial chromosome (PAC) clones were hybridized to metaphase and interphase cells of healthy volunteers to verify localization and hybridization specificity. Using the validated clones patients' metaphase and interphase cells were then hybridized as previously described.22

Array-CGH

We designed a microarray consisting of 2,799 different BAC or PAC clones which were selected according to the NCBI database information (www.ncbi.nlm.nih.gov/mapview) released in March 2003. For detailed description of array design, hybridization conditions, image analysis, and identification of CNAs see the Appendix (online only). In brief, cutoff levels were determined for each individual experiment. After computing the ratios from dye-swap hybridization and subsequent normalization, an individual set of balanced clones for each experiment was used to calculate the mean and standard deviation. The cutoff levels were defined as mean ± 3× the standard deviation. Frequently affected regions recently detected as copy number polymorphisms (5q11, 7q22, 7q35, 14q32, and 15q11) were excluded from data analysis.23,24 For further refinement of genomic aberrations array-CGH using the previously described 6.0-k microarray was performed as recently published.25

Gene Expression Analyses

On the basis of the availability of material (blast count > 50% before enrichment of malignant cells by Ficoll gradient density centrifugation and good RNA quality), for a subset of 24 cases, global gene expression profiling (GEP) analyses were performed as previously described.26 Seven of these cases have already been published,26 whereas 17 cases were newly analyzed. Following normalization of fluorescence ratios by mean-centering genes for each array and then for each gene across all arrays26 map positions for arrayed cDNA clones were assigned using the NCBI May 2005 genome assembly (accessed through the UCSC genome browser). In this way, approximately 35,000 arrayed clones representing 18,000 unique genes could be assigned map positions. The complete gene expression microarray data set is available at the Stanford Microarray Database (http://smd.stanford.edu).27

Statistical Analyses

For hierarchical clustering, we applied average linkage hierarchical clustering and visualized results using TreeView.28 For two-class supervised analyses, we used the SAM (significance analysis of microarrays) method29 which utilizes a modified t-test statistic, with sample-label permutations to evaluate statistical significance.

Frequency of Genomic Imbalances

Genomic imbalances were detected in all 60 cases. Losses were more frequent than gains (Fig 1). The most frequent losses affected 5q (77%), 17p (55%), 7q, (45%); followed by losses of 16q (32%), 18q (30%), 17q (22%), 3p and 12q (20% each), 20q and 12p (18% each), 18p (15%), and 13q and 11q (12% each). The most frequent genomic gains affected 11q (40%) and 8q (38%); followed by gains of 21q and 1p (25% each), 9p (18%), 22q (17%), 13q (15%), and 6p and 19p (12% each).

Delineation of Copy Number Alterations

The significant genomic losses and gains are summarized in Table 1, and the 41 DNA amplifications are summarized in Table 2. Some important findings are described in detail according to their frequency:

Chromosome 5.

Fluorescence signal ratios indicated loss of 5q in 46 (77%) of the 60 cases. All losses were large, encompassing the commonly deleted regions (CDRs) previously described in 5q31 and 5q32.30,31

Chromosome 17.

Thirty-seven cases (62%) exhibited losses of chromosome 17. Two CDRs were identified: one in 17p13 between 7.3 Mb and 8.1 Mb containing the TP53 gene (33 cases); and a second in 17p11-17q11 between 23.1 Mb and 29.5 Mb containing the NF1 gene (18 cases). Seventeen of the latter cases had concomitant loss of the TP53 region; in nine of these 17 cases, the losses were noncontiguous; in one case, there was a small loss on 17q11 encompassing NF1 without loss of TP53.

Chromosome 7.

Twenty-seven cases (45%) had 7q losses. These were large encompassing known CDRs on 7q (ie, 7q22-q31.1, and 7q35-q36).32-34

Chromosome 20.

Eleven cases (18%) had 20q losses encompassing the known approximately 2.6-Mb CDR in 20q12-q13.1.35

Chromosome 11.

Genomic gains and amplifications of 11q were found in 24 cases (40%). Three commonly gained regions were identified: the first region was located between 59.1 Mb and 79.6 Mb in 11q12-q14 (15 cases). The second region mapped to 11q23.3 (21 cases), extended from 116.3 Mb to 119.6 Mb containing the MLL and DDX6 genes; five of these 21 cases showed high-level DNA amplifications within this region. The third region between 127.1 Mb and 129.3 Mb on 11q23.3.-q24.1 (20 cases), including the ETS1 and FLI1 genes, was highlighted by seven cases exhibiting high-level DNA amplifications. These findings were confirmed by FISH using corresponding clones.

Chromosome 8.

Chromosome 8 gains were detected in 23 cases (38%). Three cases exhibited an approximately 10.2-Mb-sized amplified segment between 122.9 Mb and 133.1 Mb on 8q24.12-q24.13 encompassing the C8FW and MYC genes (Fig 2).

Correlation of Array-CGH and Gene Expression Profiling Findings

CNAs are characteristic for malignancies36 and might therefore lead to altered gene expression in terms of gene dosage effects. In consistency with this hypothesis, we, for example, discovered a gene dosage effect in cases with 5q–, 17p–, and 7q–detecting significant lower average gene expression levels across the genes located in the respective regions as compared to unaltered cases (Table 3). A similar effect was demonstrated in cases with trisomy 11q and 8q displaying significant higher average gene expression levels across the gained regions. These observations are in concordance with previous microarray findings in AML.37

In addition to the reported gene dosage effects, supervised analyses using the SAM method29 did not reveal significant gene expression signatures correlating with loss of 5q, 17p, and 7q, or gain of 11q and 8q, respectively (data not shown).

Delineation of Potential Candidate Genes in Amplified Genomic Regions

To delineate potential candidate genes of pathogenetic relevance, we correlated our high resolution array-CGH findings of amplified regions with our gene expression data. Thereby, we were able to disclose highly expressed genes in critical regions of amplicons, narrowing down the number of relevant candidates within these regions. For example, in cases displaying amplicons in 8q24, we found not only high expression levels of the well-known oncogene MYC, but also high expression of NSE2, FLJ32440, and C8FW, also known as TRIB1 (Fig 2). In 9p24 amplified cases, we identified highly expressed JAK2, in 13q14 FOXO1A, and in 13q12 FLT3, CDX2, and PAN3 (Fig 3).

Array-CGH allows comprehensive genome-wide screening of genomic imbalances at high resolution.11-13 There are only few limitations kept in mind interpreting the data: (i) array-CGH does not allow detecting balanced translocations, inversions, or insertions; identified in AMLs with complex karyotypes, albeit with much lower frequency than unbalanced rearrangements8-10; (ii) the assay used is unable to detect imbalances of sex chromosomes reported in this AML subgroup8-10; and (iii) imbalances present in small subclones will not be identified.

With the platforms used in this study, the resolutions were on average 2 Mb down to 0.1 Mb for the 2.8-k platform and 1 Mb down to 0.1 Mb for the 6.0-k platform, respectively. Using these platforms in 60 AMLs with complex karyotypes, we were able to identify novel recurring imbalances that likely pinpoint to regions harboring genes contributing to leukemogenesis.

Genomic losses were more frequent than gains. The most frequent aberrations were losses affecting 5q, 17p, and 7q, known recurring abnormalities in myeloid leukemias, with significant lower average gene expression levels across the genes located in the respective regions as compared to unaltered cases. Due to the heterogeneity of our sample set, known commonly deleted regions on 5q and 7q associated with MDS and AML could not be further refined.30-34

Genomic losses affecting 17p including the TP53 locus were found in 55% of the cases. Thus, the frequency was higher than reported in studies using conventional cytogenetic techniques, and comparable to studies using FISH with a locus-specific probe.8-10,38 Mutations and/or deletions of TP53 have been associated with genetic instability and complex genetic changes.39,40 Thus, the frequent loss of TP53 in our cases, likely leading to dysfunction of the TP53 protein, represents one possible pathway for genetic instability in AML with complex karyotypes.

Further recurring genomic losses were mapped to 18q, 16q, and 17q (35% each). Frequencies of losses of 18q and 16q resulting from deletions or unbalanced structural aberrations were higher than previously reported.8-10,41,42 More importantly, we were able to delineate CDRs. On the basis of two cases, an approximately 2.5-Mb-sized CDR located in 18q21 was delineated including the transcription factor 4 (TCF4) gene. TCF4, which has not yet been associated with leukemogenesis, encodes a transcription factor with homology to other transcription factors, such as TEL and TAL1, that are known to be involved in leukemogenesis. On 16q, an approximately 8.5-Mb-sized CDR encompassing, for example, CBFB and TRADD could be delineated. The smallest lost region in 17q11 was approximately 6.4 Mb in size and contained the NF1 gene. NF1 negatively regulates RAS signaling, and loss of NF1 can lead to a progressive myeloproliferative disorder, but for inducing AML, additional events are required.43,44 Therefore, loss of NF1 might be a cooperating genetic event in leukemogenesis in AML with complex karyotypes.

An enormous spectrum of other recurring losses was identified in a substantial proportion of cases (Fig 1). In part, these aberrations included known recurring losses (eg, 20q–, 12p–, 11q–), in part novel regions of interest. The most frequent genomic gains affected 11q, 8q, 21q, 1p, and 9p. Thereby, cases with trisomy 11q and 8q displayed significant higher average gene expression levels across the gained regions. On the basis of overlapping 11q gains and amplifications, three critical segments were delineated mapping to 11q12-q13, 11q23.3, and 11q23.3-q24.1. Furthermore, our data provided evidence for at least two distinct amplicon clusters in 11q23-q24: one containing MLL and DDX6 (approximately 3.3 Mb in 11q23.3), and another encompassing ETS1 and FLI1 (approximately 2.2 Mb in 11q23.3-q24.1). DDX6, ETS1, FLI1, and most commonly MLL have previously been recognized as targets of 11q23 amplifications.45,46 MLL amplifications have been associated with karyotype complexity, extremely poor survival, and mutations in TP53.45,46 Similarily, in our series, four of five cases with MLL amplifications exhibited TP53 deletion; however, given the high frequency of TP53 loss in our series, this is likely a more general association with karyotype complexity and not restricted to MLL amplifications. Recently, coamplified sequences on 11q13.5 and 11q23-q24 in MLL amplified AML/MDS cases have been described.47 Except for one single case, this was not observed in our study.

The 11q23.3-q24.1 amplicons identified in our study included ETS1 and FLI1, members of the ETS gene family. The ETS gene family includes numerous genes playing important roles in regulating hematopoiesis, proliferation, differentiation, and apoptosis.48-50 Interestingly, six cases in our study without 11q23.3-q24.1 gains exhibited high-level DNA amplifications of ERG and ETS2, two other members of the ETS gene family localized in 21q22. Thus, 13 of the 60 AML cases exhibited high-level DNA amplification of ETS gene family members suggesting a pathogenetic role in AML with complex karyotypes. In accordance with our findings, in a recent study, the ERG and ETS2 loci were also frequently amplified in complex karyotype AML with abnormal chromosome 21.17

In addition, three cases exhibited an amplified region in 8q24 containing among others the MYC gene, a known amplification target in AML.51,52 Surprisingly, further refinement of the 8q24 amplicon using the 6.0-k platform and correlation with GEP revealed overexpression of MYC, NSE2, and FLJ32440. However, the highest expression levels were identified for C8FW, also known as TRIB1, encoding a phosphoprotein regulated by mitogenic pathways. Little is known about C8FW, but it binds to 12-lipoxygenase, which has been shown to be overexpressed in human cancer tissues.53 Moreover, C8FW is homologous to SKIP3, a protein kinase overexpressed in several tumor types assuming a role in tumor progression.54 In accordance with a recently published study, these findings suggest that C8FW might be one candidate target gene of 8q24 amplifications.55

Furthermore, a large number of other high-level DNA amplifications were identified in our study, suggesting a more general role of proto-oncogene activation in AML pathogenesis (Table 2). These included a recurring amplicon mapping to 13q12 (Fig 3) encompassing, for example, FLT3, FLT1, PAN3, and the caudal-type homeobox transcription factor 2 gene (CDX2). ETV6/CDX2 fusion genes have been reported in three AML cases with t(12;13)(p13;q12).56 In a murine model of t(12;13)(p13;q12), it has been recently shown that the ectopic overexpression of Cdx2 was the key transforming event.57 Cdx genes have has a key role as upstream regulator of several Hox genes,58,59 and upregulation of HOX genes has been associated with AML pathogenesis.26,60 Interestingly, parallel analysis displayed the highest count ratios in array-CGH and GEP for FLT3 and PAN3. PAN3, encodes for a regulatory subunit of the polyadenylate-binding protein dependent poly(A) nuclease complex being involved in mRNA deadenylation, a mechanism greatly affecting mRNA stability. Furthermore, a recent study in yeast demonstrated a role in post-transcriptional regulation of DNA repair genes.61,62 Moreover, in seven cases, GEP disclosed overexpression of the forkhead box 01A gene (FOXO1A, also known as FKHR) as involved in cell cycle arrest and the regulation of apoptosis.63 As recently shown, a synthetic fusion of FOXO1A and MLL transform hematopoietic progenitor cells in vitro.64

By using high-resolution genome-wide screening tools such as array-CGH and GEP, we are beginning to unravel the enormous genetic diversity of AML cases with complex karyotypes. Further correlation of high-resolution genomic profiling with global gene expression studies may help to better characterize this disease, and should be followed by subsequent functional analyses to identify disease relevant genes in the critical regions.

Clone selection, preparation and spotting. For the DNA microarray, only clones with corresponding Mb localization and FISH mapping information were selected. The clones were ordered from the libraries RPCIB753 or RPCIP704 of the German Resource Center for Genome Research (RZPD, Berlin, Germany). A set of 1,500 of the 2,799 clones covers the whole human genome in a physical distance of approximately 2 Mb (part of the golden Path clone set). The remaining 1,299 clones either contiguously span genomic regions known to be frequently involved in hematologic malignancies (eg, 1p, 2p, 3q, 7q, 9p, 11q, 12q, 13q, 17p, 18q; n = 600) or contain oncogenes or tumor suppressor genes known to be of pathogenetic relevance in hematologic malignancies (n = 699). In consideration of continuous updating of database information (eg, physical mapping, sequencing, and marker information), critical regions discussed in this study were carefully reviewed. Positions are referred to the first base of each chromosome according to the NCBI database information. Nevertheless, particular genomic information may still vary as a result of the latest database releases.

Preparation of the DNA was performed as previously described (Fiegler H, Carr P, Douglas EJ, et al. Genes Chromosomes Cancer 36:361-374, 2003. Erratum in Genes Chromosomes Cancer 37:223, 2003). The DNA isolated from cultured BAC/PAC clones by alkaline lysis was amplified using three different DOP primers (DOP1, DOP2, DOP3). Because of the high specificity for amplification of human DNA and low specificity for E coli DNA, these primers allow the specific amplification of the human inserts. The obtained polymerase chain reaction (PCR) products representing more than 90% of the initial human DNA insert were pooled and thereafter reamplified using a consensus DOP 4 primer.

Three minor modifications were performed: (i) primer concentrations in all PCR reactions were 10 mM, (ii) the consensus DOP4 primers were not amine modified, and (iii) after adding of 2 μl of 20 × saline sodium citrate to 10 μl of the PCR product, the reamplified PCR products were directly used for spotting.

The PCR products were spotted in quadruplicate onto Corning CMT-Gaps II glass slides (Corning, Corning, NY) using the Omnigrid microarrayer (Gene Machines, San Carlos, CA). The slides were then baked at 80°C for 10 minutes and exposed to ultraviolet light for cross linking of DNA fragments (254 nm/2,400 μJ). The arrays were stored at room temperature.

Array-CGH. Genomic DNA of the tumor specimens was isolated using standard protocols. Pooled PB DNA from five female or five male healthy volunteers was used as common reference in each experiment. In total, 300 ng of tumor DNA and reference DNA were differentially labeled with Cy3- and Cy5-conjugated dCTP (2′-deoxycytidine 5′-triphosphate) by random primed labeling. Microcon membrane column centrifugation was used to remove unincorporated nucleotides. To account for different incorporation of fluorochrome-labeled nucleotides, each experiment was performed in duplicate labeling tumor DNA either with Cy3 or Cy5 and reference DNA with Cy5 or Cy3, respectively (dye-swap). Because labeled male tumor DNA was cohybridized with differentially labeled female DNA as an internal control, alterations affecting the sex chromosomes were not detectable. Labeled DNA, 75 μg of human Cot-1 DNA, and 75 μg of E coli DNA were precipitated and resolved in 130 μl of prewarmed Ultrahyb buffer (Ambion, Austin, TX) at 37°C for 30 minutes. After consecutive denaturation at 75°C for 10 minutes and preannealing at 37°C for 60 minutes, hybridization was performed for 36 hours at 37°C using a GeneTac hybridization chamber (Genomic Solutions, Cambridgeshire, United Kingdom). Slides were washed three times in 2x saline sodium citrate, 50% formamide and 0.1% Tween20, pH 7.0 at 45°C, followed by washing for 2 minutes in 1× phosphate buffered saline, 0.05% Tween20, pH 7.0 at 25°C. Afterwards, slides were dried by spinning for 5 minutes at 2,500 rpm (Kohlhammer H, Schwaenen C, Wessendorf S, et al. Blood 795-801, 2004. Schwaenen C, Nessling M, Wessendorf S, et al. Proc Natl Acad Sci U S A 101:1039-1044, 2004).

Image analysis and identification of CNAs. Microarrays were imaged using a dual laser scanner and the GenePix Pro 6.0 imaging software (GenePix 4000B, Axon Instruments, Union City, CA). Raw data were analyzed as previously described (Schwaenen C, Nessling M, Wessendorf S, et al. Proc Natl Acad Sci U S A 101:1039-1044, 2004). Fluorescence ratios were normalized using the median of the fluorescence ratios computed as log2 values from the 1,503 DNA control fragments spanning the whole genome. The cutoff level was determined for each individual experiment. After computing the ratios from the dye-swap hybridization and subsequent normalization, an individual set of balanced clones for each experiment was used to calculate the mean and standard deviations. The cutoff levels were defined as mean plus/minus three times the standard deviation. Frequently affected regions recently detected as copy number polymorphisms (5q11, 7q22, 7q35, 14q32, and 15q11) were excluded from data analysis (Sebat J, Lakshmi B, Troge J, et al. Science 305:525-528, 2004. Iafrate AJ, Feuk L, Rivera MN, et al. Nat Genet 36:949-951, 2004).

The authors indicated no potential conflicts of interest.

Conception and design: Frank G. Rücker, Lars Bullinger, Konstanze Döhner, Hartmut Döhner

Administrative support: Peter Lichter, Konstanze Döhner, Hartmut Döhner

Provision of study materials or patients: Carsten Schwaenen, Swen Wessendorf, Martin Bentz, Richard F. Schlenk, Bernhard Radlwimmer, Peter Lichter

Collection and assembly of data: Frank G. Rücker, Lars Bullinger, Daniel B. Lipka, Simone Miller

Data analysis and interpretation: Frank G. Rücker, Lars Bullinger, Swen Wessendorf, Stefan Fröhling, Claudia Scholl, Bernhard Radlwimmer, Hans A. Kestler, Jonathan R. Pollack, Hartmut Döhner

Manuscript writing: Frank G. Rücker, Lars Bullinger, Carsten Schwaenen, Stefan Fröhling, Jonathan R. Pollack, Peter Lichter, Konstanze Döhner, Hartmut Döhner

Final approval of manuscript: Frank G. Rücker, Hartmut Döhner

Table

Table 1. Delineation of Genomic Losses and Gains in 60 Acute Myeloid Leukemia Cases With Complex Karyotypes Using the 2.8-k Platform

Table 1. Delineation of Genomic Losses and Gains in 60 Acute Myeloid Leukemia Cases With Complex Karyotypes Using the 2.8-k Platform

Loss or GainChromosomeNo. of CasesRegion (approximate Mb)Consensus Region
Candidate Genes
Approximate MbNo. of Cases
Loss3p120.7-88.732.6-38.13MLH1
Loss12p13110.1-32.712.6-15.611CDKN1, CDKN1B, ETV6*
Loss16q221933.7-88.458.8-67.34TRADD, CBFB*
Loss17p330.3-27.77.3-8.133TP53*
Loss17p11-q111823.1-29.5NF1*
Loss18p11.3-p11.290.2-11.09
Loss18q211818.1-74.850.1-52.616TCF4*
Gain6p73.5-63.728.7-33.22TNF, PBX2
Gain9p112.1-68.32.1-9.910JAK2
Gain11q12-q131559.1-79.6
Gain11q23.321116.3-119.6MLL, DDX6
Gain11q23.3-q24.120127.1-129.3ETS1, FLI1*
Gain19p70.7-23.76.7-21.36
Gain22q1020.2-48.820.2-38.33BCR, CHEK2, NF2

*Validated by fluorescence in situ hybridization analyses.

Table

Table 2. Characteristics of 41 High-Level DNA Amplifications in 60 Acute Myeloid Leukemia Cases With Complex Karyotypes Using the 2.8-k Platform

Table 2. Characteristics of 41 High-Level DNA Amplifications in 60 Acute Myeloid Leukemia Cases With Complex Karyotypes Using the 2.8-k Platform

Chromosomal BandAmplification size (approximate Mb)No. of CasesCandidate Genes
4q28132.8-136.71
6q1370.0-76.51
8q24.12-q24.13122.9-133.13FLJ32440, C8FW, MYC*
9p242.1-9.92JAK2
9p21.3-9p21.117.4-24.01IFN, MLLT3, CDKN2A, CDKN2B
10p155.8-14.71IL15RA, PRKCQ
11q1372.2-81.31PDE2A, SERPINH2, WNT11, GARP, RAB30
11q23.3116.3-119.65MLL, DDX6
11q23.3-q24127.1-129.37ETS1, FLI1*
12p130.1-5.91RBBP2, FGF6, CCND2
12p134.1-5.92FGF6, CCND2
13q11-q1219.9-31.73FLT3, PAN3, CDX2*
15q25-q2692.5-100.01IGF1R
19q1349.2-49.91BCL3
19q13.458.3-59.11MYADM, PRKCG
20q1128.2-30.52ID1, BCL2L1
20q1348.7-54.81ZNF217, BCAS1
21q2238.6-43.76ERG, ETS2*
22q11.2-q13.118.6-38.31CHEK2, NF2

*Validated by fluorescence in situ hybridization analyses.

Table

Table 3. Gene Dosage Effect (altered cases versus unaltered cases in the respective genomic regions)

Table 3. Gene Dosage Effect (altered cases versus unaltered cases in the respective genomic regions)

Copy Number AlterationUnaltered Cases (mean)Altered Cases (mean)P
5q−1.022147020.84830826.002
7q−1.007816000.85570572< .001
17p−1.023671050.91129908.006
+8q1.002808141.08271694.038
+11q1.004719611.17009351.005

© 2006 by American Society of Clinical Oncology

published online ahead of print at www.jco.org on July 24, 2006.

Supported by Bundesministerium für Bildung und Forschung (BMBF), Grant No. 01GS0439 NGFN2.

Presented in part at Gemeinsame Jahrestagung der Deutschen, Österreichischen und Schweizerischen Gesellschaft für Hämatologie und Onkologie, Innsbruck, Austria, October 2-6, 2002; Gemeinsame Jahrestagung der Deutschen, Österreichischen und Schweizerischen Gesellschaft für Hämatologie und Onkologie, Hannover, Germany, October 1-5, 2005; 10th Congress of the European Hematology Associate, Stockholm, Sweden, June 2-5, 2005; and the 26th Annual Meeting of the American Society of Hematology, San Diego, CA, December 4-7, 2004.

F.G.R. and L.B. contributed equally to this work.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

We thank the members of the AML Study Group (AMLSG) for providing leukemia specimens. We gratefully acknowledge Stefan Hein, Zoraja Keresman, and Sandra Ruf for technical assistance.

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DOI: 10.1200/JCO.2005.04.5450 Journal of Clinical Oncology 24, no. 24 (August 20, 2006) 3887-3894.

Published online September 21, 2016.

PMID: 16864856

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