Complex Karyotype Acute Myeloid Leukemia Research Articles

Very Poor Outcome and Chemoresistance of Acute Myeloid Leukemia Patients with TP53 Mutations: Correlation with Complex Karyotype and Clinical Outcome

Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated.Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of TOT/886 AML pts treated at the Serˆgnoli Institute of Bologna between 2002 and 2013.Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation Deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Numberª v7.5 (BioDiscovery).Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc).Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype.Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious..Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively).Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p<0.0001) and relapse free-survival (RFS) (p < 0.0001). (Figure 1)Figure 1: Overall Survival curve of 172 AML patients with (red) or without (blue) TP53 mutations (p< 0.0001).Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p< 0.0001–Fisher’s exact test). They are mostly deleterious mutations and are significantly correlated with worst prognosis, fail to respond to therapy and rapidly progress. We recommend TP53 mutation screening at least in AML pts carrying either complex karyotype or chr. 8 gain.Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Universitˆ 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic cell transplantation in patients with intermediate and high-risk AML: results from the randomized Study Alliance Leukemia (SAL) AML 2003 trial.

The optimal timing of allogeneic hematopoietic stem cell transplantation (HCT) in acute myeloid leukemia (AML) is controversial. We report on 1179 patients with a median age of 48 years who were randomized upfront. In the control arm, sibling HCT was scheduled in the first complete remission for intermediate-risk or high-risk AML and matched unrelated HCT in complex karyotype AML. In the experimental arm, matched unrelated HCT in first remission was offered also to patients with an FLT3-ITD (FMS-like tyrosine kinase 3-internal tandem duplication) allelic ratio >0.8, poor day +15 marrow blast clearance and adverse karyotypes. Further, allogeneic HCT was recommended in high-risk AML to be performed in aplasia after induction chemotherapy. In the intent-to-treat (ITT) analysis, superiority of the experimental transplant strategy could not be shown with respect to overall survival (OS) or event-free survival. As-treated analyses suggest a profound effect of allogeneic HCT on OS (HR 0.73; P=0.002) and event-free survival (HR 0.67; P<0.001). In high-risk patients, OS was significantly improved after allogeneic HCT in aplasia (HR 0.64; P=0.046) and after HCT in remission (HR 0.74; P=0.03). Although superiority of one study arm could not be demonstrated in the ITT analysis, secondary analyses suggest that early allogeneic HCT is a promising strategy for patients with high-risk AML.

Whole-genome bisulfite sequencing of a complex karyotype AML and identification of regulatory aberrations distinct from normal karyotype AML.

11075 Background: We characterized the methylome and transcriptome of blasts from a 20 year old man with a metastatic mediastinal immature teratoma who developed a complex karyotype acute myeloid l...

Whole Genome Sequencing On Donor Cell Leukemia in a Patient with Multiple Myeloma Identified Gene Mutations That May Provide Insights to Leukemogenesis.

AbstractAbstract 2414Donor cell leukemia (DCL) is a rare occurrence and refers to leukemia of donor origin in patients who have received allogeneic hematopoietic stem cell transplantation (HSCT). We have previously described a male patient with IgG-κ myeloma who received non-myeloablative allogeneic HSCT from a HLA-matched brother and developed complex karyotype acute myeloid leukemia (AML) of donor origin 10 years after transplantation. He achieved complete remission (CR) with standard induction and consolidation chemotherapy but relapsed one year afterwards. We hypothesized that a comparison of the donor HSC before transplantation (pre-leukemic) and the subsequent AML at whole genome level will provide a unique dataset that may shed light on the pathogenesis of leukemia.DNA was extracted from an aliquot of donor mobilized peripheral blood mononuclear cells (mPBMNC) frozen before transplantation as well as unfractionated and CD34+ myeloblasts of the patient's bone marrow at diagnosis and subsequent relapse of AML. The complete donor origin of the AML was confirmed by PCR based on polymorphic STRs. Whole-genome sequencing (WGS) was performed to sequence paired-end reads generated by Illumina HiSeq 2000. Reads were aligned to the human referecne genome (hg19, NCBI37) by SOAP3 and analysed to detect single nucleotide variants (SNVs), small insertion and deletion (indels) and copy number variations (CNVs). Selected genes after filtering were independently validated by Sanger sequencing.There were 835M and 810M 100bp paried-end reads with insert distance of 500bp generated from donor mPBMNC and CD34+ myeloblasts of the relapsed DCL with respective mean depths of 43.2X and 42.6X after alignment. The digital karytoyping based on the read depth was consistent with that by conventional cytogenetic study. 3,979,582 and 1,020,717 SNVs and indels were detectable from both samples. Based on the Catalog of Somatic Mutations in Cancer (COSMIC) and excluding those asian specific wildtypes annotated in 1000 genome project, 11 SNVs and 15 indels within coding sequence with potential roles as tumor suppressors or oncogenes were identified. On the other hand, there were 128,752 and 56,330 SNVs and indels detected exclusively in DCL. Those putative non-pathogenic SNP and those changes locating outside the gene regions were filtered. Within the gene region, SNVs in introns and synonymous mutations were also filtered. 142 non-synonymous SNVs (139 missense and 3 nonsense mutations) were identified of which 25 were considered as statistically highly confident and 17 of them could be confirmed by Sanger Sequencing. Twelve of these were also identified from the whole BM sample of DCL at diagnosis. These candidates include transcription factor (SALL1), metabolic enzymes (UGT1A5, SPEG), membrane protein (TMC6, SCN3A), cytoskeleton protein (MYH10), ribonucleoprotein (RAVER1), secreted protein (WNT7A), protein involved in DNA damage repair (APLF) and others (PRPF8, ZNF518B and MKRN3). 26 indels were indentified in the coding region of which 5 were considered as statistically highly confident, however, only one indel could be confirmed by Sanger Sequencing in the relapse sample and was not present in the diagnostic sample.The WGS performed in paired pre-leukemic (donor HSC) and leukemic (DCL) human samples has provided us with unique opportunities to dissect the genetic changes in HSC that may contribute to the initiation of AML with complex karyotype. The potential impacts of bone marrow microenvironment in this patient with myeloma in inducing DCL are also being evaluated. Disclosures:No relevant conflicts of interest to declare.

Characterization of Mesenchymal Stromal Cells (MSCs) Derived From Acute Myeloid Leukemia (AML) Bone Marrow

Abstract 2558 INTRODUCTIONRecent evidence has implicated the bone marrow microenvironment directly in the pathogenesis of preleukemic bone marrow disorders (Raaijmakers MHGP et al, Nature 2010) with potential to transform to AML. Moreover, the bone marrow microenvironment is critical to AML survival (Garrido et al 2001, Meads et al 2008). We sought to investigate aspects of the bone marrow microenvironment which may contribute to the pathogenesis and persistence of AML by direct analysis of primary bone marrow MSCs isolated from AML patients in comparison with primary bone marrow MSCs from normal subjects. Our analyses included (1) a comparison of cytokine elaboration between normal and AML bone marrow MSCs (2) immunophenotyping of normal and AML bone marrow MSCs (3) characterisation of binding by AML cells to their autologous stroma and (4) gene expression profiling of normal and AML bone marrow MSCs and (5) cytogenetic analysis AML bone marrow MSCs. METHODSWe have been able to derive confluent cultures of mesenchymal stromal cells from 80% of AML patient marrow samples. Fresh or cryopreserved bone marrow samples were plated in non-hematopoietic expansion media (Miltenyi) under reduced oxygen conditions. After 48 hours of culture, nonadherent cells are removed, and over a period of 1–2 weeks, cultures of spindle shaped cells are derived, that can be sustained in culture for up to 5 passages. Similar cultures were derived from normal bone marrow. Gene expression profiling of bone marrow MSCs was performed by whole genome analysis using Illumina’s BeadChip microarray platform. Samples included mRNAs isolated from confluent cultures of AML bone marrow MSCs, normal bone marrow MSCs, and the normal bone marrow stromal cell lines HS27a and HS5. RESULTSComparison of cytokine elaboration showed a statistically significant (p = 0.037) 5-fold decrease in stromal MCP-1 production by AML bone marrow MSCs compared with normal bone marrow MSCs (327 ± 169 vs. 1669 ± 570 pg/mL, mean ± SE). Normal and AML MSCs showed no statistically significant differences in the production of G-CSF, GM-CSF, M-CSF, IL6, IL12, SCF, TNFα, MCP1 and SDF1β. Like their normal counterparts, AML bone marrow MSCs strongly express CD90, CD29 (β1 integrin), CD73, CD105, CD146, and CD44. The normal bone marrow derived stromal cell lines HS27a and HS5 demonstrated moderate expression of CD324/E-cadherin (28.4% and 37.9% respectively). E-cadherin expression proved highly variable among normal bone marrow MSCs (1.9%-54.9%) and similarly variable in AML bone marrow MSCs (7.8%–56.5%). AML binding to autologous MSCs primarily dependent on β1 integrin, L-selectin and VCAM-1. In contrast, prior data (Basu et al., ASH 2010 Abstract 2756) demonstrated AML binding to the normal bone marrow stromal cell line HS27a as primarily dependent on β1 integrin, CXCR4, and E-cadherin. Gene expression profiling demonstrated no significant differences between 6 AML and 5 normal bone marrow MSCs. AML bone marrow MSCs, as expected, demonstrated marked differences from AML bone marrow mononuclear cells, expressing higher levels of connective tissue growth factor (128 fold), tropomyosin 1 (84.4 fold), collagen type 1 α1 (194 fold), collagen type 1 α2 (137 fold), collagen type 4 α1 (128 fold), collagen type 5 α1 (119 fold), transgelin (111 fold), cadherin 11 (21 fold), biglycan (137 fold), IGF binding protein 6 (18 fold), and procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (74 fold). In our cytogenetic analyses of MSCs to date, bone marrow MSCs derived from one of the complex karyotype AML patients demonstrated normal cytogenetics. In contrast, bone marrow MSCs derived from a second AML patient shared a common t(2;11) translocation present in the AML cells but demonstrated an abnormal clone with del(4q) which lacked the t(6;9) also present in the AML cells [i.e.-MSC karyotype: t(2;11), del(4q); AML karyotype: t(2;11), t(6;9)]. These results suggest that in some patients AML cells and their autologous MSCs may share the same clonal origin, while in other cases, the MSCs may have a distinct origin. CONCLUSIONAML and normal bone marrow MSCs demonstrate only subtle differences, providing an explanation of the ability of AML bone marrow MSCs to support normal hematopoiesis after leukemic debulking (e.g. via induction chemotherapy or allogeneic stem cell transplant). Disclosures:No relevant conflicts of interest to declare.

Integrative Genomics Approaches Identify Novel Disease-Related Genetic Aberrations in Acute Myeloid Leukemia

Abstract 402Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of acquired somatic genetic alterations in hematopoietic progenitor cells that alter mechanisms of self-renewal, proliferation and differentiation. While recently numerous aberrations have been identified, many more mutations involved in the multistep pathogenesis of AML still remain to be discovered.Based on critical regions and differentially expressed genes identified by our SNP/aCGH and microarray-based gene expression profiling analysis of 320 AML cases, we designed a custom capture microarray (Nimblegen, Roche) to enrich the genomic DNA of the entire coding region of 1000 genes of potential leukemia-relevance. In total, we captured and sequenced the respective genes in paired diagnosis and remission samples of 50 AML cases using Next Generation Sequencing (NGS) technology (Illumina GAIIx). We analyzed a representative AML sample cohort (n=50), which included 19 cytogenetically normal (CN) AML cases already known routinely determined mutations (CEBPA, NPM1, FLT3, and WT1), 10 CN-AML cases without known mutations, 7 complex karyotype AML cases, 13 core-binding-factor (CBF) AML cases with t(8;21) or (inv16), and one case with translocation t(4;11).On average, we generated 9.5 million reads per sample, with 28.7% of the reads uniquely mapping to the target regions. The mean target coverage per base analyzed was 58.5-fold. For the analysis of our NGS data, we established a pipeline that allows the detection of single nucleotide variations (SNVs) and insertion/deletions (indels). A first analysis using this pipeline already provided novel insights. We detected on average 3 somatic protein altering SNVs previously not reported as SNPs and 1.3 indels per sample, which affected mostly histone-modifying enzymes, chromatin-organizing molecules or transcriptional factors, suggesting importance and at the same time variability of epigenetic changes underlying AML. We found somatic mutations in genes already known to be involved in epigenetic deregulation of leukemias (TET2, TET1), but also identified novel mutations in genes involved in the regulation of the chromatin structure (such as SATB1 or HIST1H2AA). Notably, overrepresentation analysis of the genes affected by missense SNVs revealed the enrichment of gene sets involved in the chromosome organization, DNA repair, and chromatin modification.In conclusion, while targeted NGS identified many aberrations in AML, further analysis will identify recurrent “driver” mutations that play a pivotal role in the pathomechanism of AML and, thus, might enable better targeted therapeutic approaches, especially with regard to the guidance of epigenetic therapies. Disclosures:No relevant conflicts of interest to declare.

Early allo-SCT for AML with a complex aberrant karyotype—results from a prospective pilot study

In AML, a complex aberrant karyotype is associated with poor response to chemotherapy and dismal prognosis. We prospectively studied the concept of allogeneic haematopoietic SCT (HSCT), performed early and regardless of response to induction treatment in patients with complex karyotype AML (CK-AML). The preparative regimen consisted of fludarabine, Ara-C and amsacrine (FLAMSA) chemotherapy, followed by reduced intensity conditioning (RIC) 3 days later. In vivo T-cell depletion by anti-thymocyte globulin was used to protect from early GvHD, and prophylactic donor lymphocyte transfusion was given from day+120 to augment the GvL effect, once tolerance was established. Eighteen consecutive patients with CK-AML (median age: 53 years) received HSCT from related (n=7) or unrelated (n=11) donors. Before FLAMSA-RIC, nine patients each had received one and two induction courses. Stage at start of FLAMSA-RIC was CR/CRi (n=8) or persistent disease (n=10). Following HSCT, 16 patients achieved CR. After a follow-up of 51 months, 11 patients are alive in CR, whereas seven have died in remission (n=3), or from leukaemia (n=4). Cumulative incidence of relapse, non-relapse mortality, acute GvHD≥II and chronic GvHD were 0.222±0.098, 0.235±0.104, 0.367±0.120 and 0.481±0.123, respectively. Four-year survival from HSCT is 61%. Early HSCT following FLAMSA-RIC may improve the outcome of this unfavourable AML subgroup.

Aberrant Expression of Hepatocyte Growth Factor Induces Autocrine MET Activation Providing a Novel Therapeutic Target In Acute Myeloid Leukemia.

AbstractAbstract 1042Despite improvements in the treatment of acute myeloid leukemia (AML), high risk disease such as complex aberrant karyotype AML remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. Thus, we carried out a genome-wide functional screen in complex karyotype AML using a retroviral library of short hairpin RNAs (shRNAs), and discovered that shRNA-mediated depletion of hepatocyte growth factor (HGF) specifically inhibits growth of AML cells but not a panel of lymphoid cancer cells. HGF was to found to be aberrantly expressed in about 15% of patients with AML, including most patients with complex karyotype disease. In contrast to normal CD34+ cells that express MET (but not HGF), 5 of 7 cell lines derived from patients with complex karyotype AML exhibited aberrant expression of HGF that was associated with autocrine activation of its receptor MET. Depletion of HGF or MET using multiple independent shRNAs profoundly reduced proliferation and induced cell death in AML cells lines that express HGF but not those that lack HGF expression. Inhibition of MET using the tyrosine kinase inhibitor (SU11274) or HGF using neutralizing anti-HGF antibody (R&D Systems) also inhibited growth and induced apoptosis in AML cell lines dependent on HGF/MET activation but not those that lack HGF expression. Thus, aberrant HGF expression causes autocrine MET activation and oncogene dependence in a subset of patients with AML, confers sensitivity to HGF/MET inhibition, and provides a novel therapeutic target for this otherwise lethal disease. Disclosures:No relevant conflicts of interest to declare.

IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study.

PURPOSE To analyze the frequency and associations with prognostic markers and outcome of mutations in IDH genes encoding isocitrate dehydrogenases in adult de novo cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS Diagnostic bone marrow or blood samples from 358 patients were analyzed for IDH1 and IDH2 mutations by DNA polymerase chain reaction amplification/sequencing. FLT3, NPM1, CEBPA, WT1, and MLL mutational analyses and gene- and microRNA-expression profiling were performed centrally. Results IDH mutations were found in 33% of the patients. IDH1 mutations were detected in 49 patients (14%; 47 with R132). IDH2 mutations, previously unreported in AML, were detected in 69 patients (19%; 13 with R172 and 56 with R140). R172 IDH2 mutations were mutually exclusive with all other prognostic mutations analyzed. Younger age (< 60 years), molecular low-risk (NPM1-mutated/FLT3-internal tandem duplication-negative) IDH1-mutated patients had shorter disease-free survival than molecular low-risk IDH1/IDH2-wild-type (wt) patients (P = .046). R172 IDH2-mutated patients had lower complete remission rates than IDH1/IDH2wt patients (P = .007). Distinctive microarray gene- and microRNA-expression profiles accurately predicted R172 IDH2 mutations. The highest expressed gene and microRNAs in R172 IDH2-mutated patients compared with the IDH1/IDH2wt patients were APP (previously associated with complex karyotype AML) and miR-1 and miR-133 (involved in embryonal stem-cell differentiation), respectively. CONCLUSION IDH1 and IDH2 mutations are recurrent in CN-AML and have an unfavorable impact on outcome. The R172 IDH2 mutations, previously unreported in AML, characterize a novel subset of CN-AML patients lacking other prognostic mutations and associate with unique gene- and microRNA-expression profiles that may lead to the discovery of novel, therapeutically targetable leukemogenic mechanisms.

In Acute Myeloid Leukemia with Complex Karyotype TP53 Alterations Are Associated with Specific Genomic Aberrations and Predict Inferior Survival.

Abstract 2632Poster Board II-608In acute myeloid leukemia (AML) complex karyotype is defined as the presence of ≥3 chromosome abnormalities in the absence of one of the chromosomal rearrangements listed in the category “AML with recurrent genetic abnormalities” (WHO 2008). This subset of AML accounts for approximately 10 to 15 % of all AML and it is characterized by a very poor prognosis. To identify novel genomic regions of interest in this AML subgroup, we recently applied microarray-based techniques (array-comparative genomic hybridization [CGH] and single nucleotide polymorphism [SNP] analysis) allowing high-resolution genome-wide screening of genomic imbalances in 245 patients; 171 of the 245 (70%) patients were enrolled in clinical protocols using intensive chemotherapy. Applying these techniques, we identified deletions of 17p13 encompassing the TP53 locus in 94 of 245 (38%) cases. Based on these findings and the fact that TP53 is a frequently affected gene in numerous malignancies, we aimed to investigate its role in complex karyotype AML. Therefore, we screened 188 of the 245 patients forTP53 mutations either by direct sequencing of exon 5 to 9 or by denaturating high-performance liquid chromatography of exon 4 to 10 on a WAVE® system followed by direct sequencing of the positive cases. In total, we identified 137 mutations (exon 4 [e4] n=2; e5 n=41; e6 n=26; e7 n=35; e8 n=26; e10 n=1; splice sites n=6) in 106 patients (56%) with a maximum of four mutations per case. Of these 137 mutations, 108 were missense mutations, 15 frame shift mutations, 8 nonsense mutations, and 6 were located at intronic splice sites.Combining the findings from array-based and mutation analyses, TP53 gene alterations were identified in 129 of 188 (69%) patients. Of note, 29% of these cases showed biallelic TP53 gene alterations with deletion of one allele and at least one mutation in the remaining allele.When correlating TP53 alterations (mutations and/or deletions) with genomic data from array-based analyses, we found positive correlations with the presence of 5q and 7q losses (Chi-square test P < .001 and P = .001, respectively), total number of deletions (P < .001), gains (P = .002), high-level DNA amplifications (P < .001), and genomic complexity as measured by total number of aberrations per case (P < .001). Correlation of TP53 alterations with clinical characteristics revealed that patients with TP53 alterations were older (median age 60 versus 55 years, P = .01) and showed in trend a lower WBC (median, 6.6 versus 14.2 × 109/L, P = .14); there was no difference in bone marrow and blood blast counts, in platelet counts, or in type of AML (de novo vs. secondary vs. treatment-related). Clinical outcome analyses were restricted to patients enrolled in prospective clinical trials (n=171). AML exhibiting a TP53 alteration had significantly lower complete remission (CR) rates [25% (26/104) versus 56% (24/43), P < .001], inferior event-free survival (EFS, P = .009), relapse-free survival (RFS, P = .02), and overall survival (OS, P < .001) compared with TP53 wild type AML. In multivariate models for achievement of CR, RFS, and OS, TP53 alteration retained its significance as an independent risk factor (CR: OR 0.38, P < .001; RFS: HR 1.65, P = .02; OS: HR: 2.15, P = .002).In conclusion, TP53 is the most frequently known affected gene in complex karyotype AML. We found that the TP53 status is associated with specific genomic aberrations as well as the degree of genomic complexity, a finding that fits well into the TP53 pathomechanism of genomic instability. Importantly, among AML with complex karyotype TP53 alterations significantly predicted inferior outcome allowing for a further refinement of this AML subgroup. Disclosures:No relevant conflicts of interest to declare.

Genome-Wide Analysis of Alternative Splicing Points to Novel Leukemia Relevant Genes in Acute Myeloid Leukemia.

Abstract 2391Poster Board II-368Alternative mRNA splicing represents an effective mechanism of regulating gene function as well as a key element to increase the coding capacity of the human genome. Today, an increasing number of reports illustrates that aberrant splicing events can contribute to human disease and that alterations in the splicing machinery are common and functionally important for cancer development. Aberrant splice forms can for example have genome-wide effects by deregulating key signaling pathways. However, for most of the aberrant mRNA transcripts detected it remains unclear whether they directly contribute to the malignant phenotype or just represent a by-product of cellular transformation. Thus, more comprehensive analyses of the transcriptome splicing are warranted in order to get novel insights into the biology underlying malignancies like, e.g., acute myeloid leukemia (AML).Here, we performed a genome-wide screening of splicing events in AML using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). We analyzed forty AML cases with complex karyotypes and twenty Core Binding Factor (CBF) AML cases (entered on a multicenter trial for patients <60 years, AMLSG 07-04) using this microarray approach allowing the detection of splice variants. In order to detect alternative splicing events distinguishing different leukemia subgroups we applied a commercial and an open source software tool: XRAY version 3.9 (Biotique Systems) and the OneChannelGUI package for R (version 1.10.7 available at http://www.bioinformatica.unito.it/oneChannelGUI/). Using XRAY supervised analysis comparing subgroups of CBF and complex karyotype AML we identified 1120 transcripts to be potentially alternatively spliced. In parallel, the analysis of the same AML subgroups using the OneChannelGUI package in R revealed 1439 candidates with an overlap of only 211 genes. Of these transcripts, that have been indicated by both programs as potentially alternatively spliced, selected candidates were further investigated by RT-PCR, quantitative RT-PCR and sequence analysis for the presence of splice-variants. Of 26 candidate genes studied, we could confirm alternative splice forms for 5 genes that might potentially be involved in driving leukemogenesis, such as the protein coding gene arginine methyltransferase 1 (PRMT1), which regulates transcription through histone methylation and participates in DNA damage response. Furthermore, we could confirm differential exon usage in the protein tyrosine phosphatase non-receptor type (PTPN6) transcript, which encodes for a negative regulator of numerous signaling pathways involved in cell cycle control and apoptosis. Similarly, the mRNA of the protein Rho GTPase activating protein 4 (ARHGAP4), which has been shown to regulate cell motility, was alternatively spliced between CBF and complex karyotype subgroups.In summary, these first gene expression data demonstrate that the attempt to elucidate the splicing of transcriptome in AML by applying Exon microarray technology is challenging in particular with regard to the currently available software solutions. Nevertheless, our results show that this approach offers the ability to detect novel alternatively spliced candidate genes. Being involved in cell cycle control, regulation of transcription or remodeling of the cytoskeleton, alternative splicing of these genes might play a potential role in the pathomechanism of distinct AML subgroups. Thus, in the future more extensive Exon array profiling with more sophisticated software solutions at hand is likely to provide additional insights into the molecular mechanisms of leukemogenesis and might reveal novel targets for refined therapeutic strategies in AML. Disclosures:No relevant conflicts of interest to declare.

Analysis of the ETS Family Member Genes ERG, ETS2, ETS1 and FLI1 in Acute Myeloid Leukemia (AML) Patients with Normal Cytogenetics: Expression Levels and Impact On Clinical Outcome. A Study of the AMLSG.

Abstract 1600▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-626 BackgroundThe V-ets erythroblastosis virus E26 (ETS) oncogene family is one of the largest families of transcription factors. ETS transcription factors are characterized by two major functional domains, a transcription domain and an evolutionarily highly conserved DNA-binding domain, also known as ETS domain that mediates binding to purine-rich DNA sequences. Most ETS family proteins are nuclear targets for activation of the Ras-MAP kinase signalling pathway. Therefore, they play a significant role in regulating cellular functions such as cell growth, apoptosis, development and differentiation. ETS transcription factors have been implicated in leukemia by chromosomal rearrangement, and more commonly by gene amplification and/or overexpression. Moreover, overexpression of ERG was shown to be an adverse predictor for clinical outcome in AML with normal cytogenetics (CN). In our recent study on complex karyotype AML, array-CGH (comparative genomic hybridization) analysis identified small genomic amplifications affecting ERG/ETS2 in 21q22 and ETS1/FLI1 in 11q23 in about 10% of the cases. Correlation with global gene expression profiling showed that ERG and ETS2 as well as ETS1 and FLI1 were overexpressed in these cases.Aims: To evaluate expression levels of ERG, ETS2, ETS1 and FLI1 in a large cohort of younger (16 to 60 years of age) adult CN-AML patients (pts) and their impact on clinical outcome. MethodsThe expression of ERG, ETS2, ETS1 and FLI1 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay in 343 CN-AML pts who were entered on 3 AMLSG treatment protocols (AMLHD93, AML HD98-A, AMLSG 07-04). ERG, ETS2, ETS1, and FLI1 were dichotomized into two major groups according to their expression levels. The upper quartile was chosen as the cut point and the set of patients with gene expression above were defined as Q4 group. Univariable as well as multivariable regression models were used to evaluate the influence of ERG, ETS2, ETS1 and FLI1 on induction success, event-free, relapse-free and overall survival. Multivariable analyses were stratified for AMLSG treatment protocols. ResultsPartial correlation analysis revealed positive correlations of expression levels between ETS2 and ERG (ρ=0.45) being the strongest, followed by ERG and FLI1 (ρ=0.4), as well as ETS1 and FLI1 (ρ=0.31). Correlation of ERG, ETS2, ETS1 and FLI1 with white blood count (WBC) revealed a significant association between high gene expression (Q4) and elevated WBC (ERG, p=0.004; ETS2, p=0.002, FLI1 p<0.001), whereas high expression of ETS1 was associated with a significantly lower WBC (p<0.001). Univariable as well as multivariable analyses on induction success revealed high ETS2 as an unfavourable marker (OR, 0.29, p=0.01). In univariable analysis, there was a significantly inferior relapse-free survival (RFS) and overall survival (OS) for high ERG (p=.01; p=.06, respectively) and high ETS2 (p=.002; p=.03, respectively) that was even more pronounced when both ERG Q4 and ETS2 Q4 (ERG Q4 ∩ ETS2 Q4) (p<0.001; p=.001, respectively) were included as one variable and compared with the rest. In multivariable analysis for the endpoints event-free survival (EFS), RFS and OS, a significant effect was found for RFS for ERG Q4 ∩ ETS2 Q4 (p=.002); the only significant variables that consistently appeared in the model were NPM1mut, FLT3-ITDpos and WBC. In subgroup analysis for the genotypes CEBPAmut, NPM1mut/FLT3-ITDneg, and all others (NPM1mut/FLT3-ITDpos, NPM1wt/FLT3-ITDpos, NPM1wt/FLT3-ITDneg) according to the hierarchical model, ERG Q4 was associated with an inferior EFS (p=.04) and OS (p=.03) in the favorable CEBPAmut genotype and became even more significant for the variable ERG Q4 ∩ ETS2 Q4 (EFS, p=.007, RFS, p=.002; OS, p=.06, respectively). For the NPM1mut/FLT3-ITDneg subgroup, again ERG Q4 ∩ ETS2 Q4 was associated with an adverse RFS (p=.04), but not with OS (p=0.07). ConclusionsIn our study on a large cohort of homogenously treated CN-AML patients, ERG and ETS2 expression were highly correlated. Overexpression of both genes had a significant impact on clinical outcome of CN-AML patients. Moreover, adverse effects of high ERG and high ETS2 expression on prognosis were also shown for the genetic AML subgroups CEBPAmut and NPM1mut/FLT3-ITDneg. DisclosuresNo relevant conflicts of interest to declare.

The Predictive Value of TP53 FISH Analysis for Treatment Response and Survival in Cytogenetic Subgroups of AML Patients.

Abstract 2617▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-593In recent years, a number of studies emphasized AML with complex aberrant karyotype as a distinct biological entity which is characterized by a unique gene expression pattern, with very frequent alteration in TP53 and a median overall survival (OS) of less than 6 months. However, the definition of a complex karyotype (CK) or whether monosomal karyotype (MK) provides a better prognostic prediction than CK is not well established. In the present study, we examined whether a prompt and simple Fluorescence in situ hybridization (FISH) test for TP53 deletion (presence or absence of 17p13) at diagnosis, has a predictive value for response to therapy and overall survival in AML patients (pts) with CK or chromosomal deletions (either complete or partial of any other chromosome). Between 2000 and 2009 we analyzed 38 patients with newly diagnosed AML, with age ranged from 18 to 82 years (median 52 years). On cytogenetic analysis from bone marrow at diagnosis, 16 (42%) pts had a CK (≥3 chromosome abnormalities), 16 pts (42%) had a normal karyotype (NK) and 6 (16%) pts had other monosomy or deletion of any chromosomal region. Seven patients had a post myelodysplastic syndrome AML and 1 post therapy for a prior malignancy. For induction treatment patients received idarubicine, 12 mg/m2/d (age 18– 55y) or mitoxantron 10 mg/m2/d intravenous (IV) on days 1-3 (age 55–68y) and cytarabine, 100mg/m2/d by continuous IV infusion on days 1 through 7. Pts entering complete remission (CR) received three courses of consolidation with high dose cytarabine. Patients with a histocompatibale donor, which did not received CR or relapsed, were allografted. Elderly patients, 69–82 years old, were treated with one induction chemotherapy which included Mylotarg (3mg/m2/2h on day 1) plus cytarabine (100 mg/m2/24h on days 2-8). Patients at this group of age did not received consolidation therapy.Of the16 pts with CK AML, 14 were treated with chemotherapy but only 3 pts (21%) achieved CR, 4 pts died during induction therapy. By comparison, of the 16 pts with NK AML, 15 pts were treated with chemotherapy, 11 pts achieved CR (73%), 3 pts died during induction therapy. Of the 6 AML pts with monosomy or deletion in cytogenetic analysis, 3 pts (50%) achieved CR (p =0.0067). In all NK AML patients examined the TP53 FISH was normal (mean 4% of cells examined), where as in patient with CK 75% of pts had TP53 deletion (mean 51%), and in the group with chromosomal deletion 50% of pts showed a loss of one TP53 (mean 18%) p=0.0016. Of note, in 13 pts (34%) with TP53 deletion by FISH analysis, cytogenetic analysis found no anomaly of 17p chromosome. In a stepwise logistic regression model FISH groups significantly entered the model in first step, and no other variable did (p=0.0056, C=0.833). In pts in which two copies of TP53 were found in FISH analysis ('10%) the median survival time is 440 days, whereas in pts with TP53 deletion was detected the median survival time was 189 days. The OS in pts with deletion of TP53 was significantly shorter as compared to pts that TP53 deletion was not found (p=0.025). However, a multivariate analysis including age, karyotype, deletion of TP53 and allogeneic transplantation showed that only CK and allogeneic transplantation are independent prognostic factors in this analysis.In conclusion, TP53 status by FISH at diagnosis is important due to the following findings:The test is sensitive, rapid and simple.2. TP53 deletions are frequent in AML with CK and tend to segregate with additional chromosomal deletions.TP53 status at diagnosis has a predictive value with respect to chemotherapy response and OS in AML pts with CK and chromosomal deletions. Disclosures:No relevant conflicts of interest to declare.

Heterogeneity of Acute Myeloid Leukemia at the Stem Cell Level.

Leukemias are considered hierarchically organized being maintained by a leukemia stem cell (LSC). Whereas LSCs become the primary focus for targeted therapies, little is known about the pathways regulating LSC fidelity. Using retroviral gene transfer of MN1, NUP98HOXD13 (ND13), or HOXA9 oncogenes and limiting-dilution transplantation we modelled leukemias with different LSC frequencies, and characterized critical signaling pathways by loss-of-function analysis. Here we establish the concept that LSCs are heterogeneous based on the number of activated transcriptional networks, and functionally characterize downstream targets that are critical for LSC activity. Constitutive expression of the very potent myeloid oncogene MN1 with the ND13 fusion gene in murine bone marrow cells results in acute myeloid leukemia (AML) that is phenotypically very similar to MN1-induced AML. However, limiting dilution analysis showed that the LSC frequency was 33 fold higher in MN1+ND13 cells compared to MN1 cells, and disease latency at the limiting dilution was significantly shorter in the combination model (p=.009). Whereas MN1-LSCs expanded 68-fold over a period of 6 days, MN1+ND13-LSCs expanded 131-fold more than MN1-LSCs as determined by the competitive repopulation unit (CRU) assay. To screen for functional differences of the two models we screened for differential cytokine responses in vitro. Interestingly, MN1+ND13 expressing cells proliferated in response to GM-CSF, whereas MN1 cells or ND13 cells did not. This was confirmed as well for MN1+HOXA9 expressing cells and their MN1+CTL or HOXA9+CTL expressing counterparts. We found that Stat1, Stat3, Stat5, and Erk1/2 were selectively phosphorylated upon cytokine stimulation in MN1+ND13 and MN1+HOXA9 cells compared to single-oncogene transduced cells. To test the role of Stat1 and Stat5b for LSC fidelity Stat1 −/− and Stat5b −/− cells were co-transduced with MN1 and HOXA9 and compared to wildtype cells in vitro and in vivo. Stat1 −/− cells transduced with MN1+HOXA9 proliferated slower than wildtype cells in response to GM-CSF but not with IL3/IL6/SCF. Proliferation of Stat5b −/− cells transduced with MN1+HOXA9 proliferated slower than wildtype cells in response to both GM-CSF and IL3/IL6/SCF (p<.05). CRU assays with MN1+HOXA9-transduced Stat1 −/− and Stat5b −/− cells demonstrated that the day 6 CRU was 6 and 77 fold reduced, respectively, compared to wildtype cells. As MN1 and HOXA9 are upregulated in distinct subsets of normal karyotype AML we speculated that their combined overexpression may model subsets of complex karyotype AML. We performed gene set enrichment analysis on cytogenetic subsets of previously published gene expression data from 285 AML patients. 12 of 13 Stat-related pathways were enriched in complex karyotype patients compared to 4 and 8 of 13 Stat-related pathways in inv(16) and normal karyotype AML, respectively, thus supporting a critical role of Stat activation in LSCs of AML with multiple active pathways like complex karyotype AML. In conclusion we demonstrate considerable heterogeneity of LSC fidelity depending on the number of activated oncogenes and establish a critical role of Stat1 and Stat5b in mediating this LSC fidelity. Stat1 and Stat5b may become important therapeutic targets in complex karyotype AML.

The Genotype Consisting of Complex Karyotype and TP53 Gene Mutation Is Specific to Acute Myeloid Leukemia with Multilineage Dysplasia

Acute myeloid leukemia (AML) is a heterogeneous disorder of hematopoietic progenitor cells. The World Health Organization (WHO) classification of the myeloid neoplasm incorporates genetic, immunophenotypic, biologic and clinical features. Although the WHO classification recognizes several chromosomal translocations they are frequently seen in AML patients, for categorizing the AML with recurrent genetic abnormalities, a number of other genetic alterations such as point mutations and gene rearrangements have not been included. To date, several genetic alterations, which are involved in the pathogenesis of AML and associated with prognosis of patients, have been documented. Therefore, it is required to establish the detailed classification of AML according to the genetic status. In this study, we comprehensively analyzed the genetic alterations and in de nove AML patients and investigated the association with mutational status, cytogenetic status and clinical features in comparison with the WHO classification. The study population included 144 newly diagnosed de nove AML patients consisting of 38 recurrent genetic abnormalities (RGA), 34 multilineage dysplasia (MLD) and 72 not otherwise categorized (NOC) according to WHO subcategories. Bone marrow samples were obtained from the patients after obtaining informed consent for banking and molecular analyses. Mutations in FLT3, cKIT, NPM1, N-RAS, TP53, C/EBPA, AML1, WT1 and MLL/PTD were analyzed as previously described. In consistent with previous reports, FLT3 (24%), cKIT (5%), NPM1 (20%), N-RAS (8%), C/EBPA (12%), AML1 (2%), MLL-PTD (9%), WT1 (3%) and TP53 (8%) mutations were frequently observed. No significantly different distribution was found in the prevalence of FLT3, N-RAS, C/EBPA, AML1 and MLL-PTD mutations among the WHO categories. However, the skewed prevalence was found in cKIT, NPM1 and TP53 mutations: cKIT mutation was frequently found in AML-RGA, NPM1 mutation was not found in AML-RGA, and TP53 mutation was preferentially found in AML-MLD. N-RAS, C/EBPA, AML1 and WT1 mutations were not identified in AML-RGA, though their distributions were not statistically significant among the WHO categories. It is notable that 9 of 12 (75%) patients who have TP53 mutation are categorized in AML with MLD and 8 of the 9 have complex-karyotype. Importantly, 8 of 9 (88.9%) showed complex karyotype, and 5 of them deleted chromosome 17. In this study, we found 2 additional patients harboring TP53 mutation in the other categories, while they did not show the complex karyotype and chromosome 17 abnormality. Furthermore, complex karyotype was found in 14 of the 144 entire AML patients, while 10 of them were categorized in AML-MLD. The remaining 4 patients were categorized in AML-NOC. Three of them showed del(17) or del(17p), though TP53 mutation was not identified. The genotype consisting of complex karyotype and TP53 mutation was, therefore, specifically found in AML-MLD. Thus TP53 mutation and complex-karyotype AML are significantly correlated and associated with the presence of multi-lineage dysplasia (P= .000 and P= .000, respectively). Moreover, these patients have significantly inferior induction rate and overall survival in AML with MLD. These results suggest that TP53 mutation and complex-karyotype AML distinguish a disease entity in AML with MLD.

The Level of AKT Phosphorylation on Threonine 308 but Not on Serine 473 Is Associated with High-Risk Cytogenetics and Predicts Poor Overall Survival in Acute Myeloid Leukemia.

The PI3K/Akt pathway is an important signaling pathway governing cell survival, proliferation and drug resistance in AML. However, the prognosis impact of Akt phosphorylation in AML has yielded some controversies as far as the phosphorylation on Ser473 is concerned. Because full activation of Akt requires phosphorylation on both Thr308 and Ser473, the assessment of these two sites would allow a more complete analysis of Akt activation in AML cells. We have studied the level of phosphorylation on both sites in primary AML samples by flow cytometry. We retrospectively assessed samples from 58 AML patients treated between 12/2000 and 02/2005. The clinical characteristics were: median age, 46.5 y (15–61); sex ratio (M/F) 1.24; FAB, 1 M0, 29 M1/M2, 25 M4/M5, 1 M6, 2 unclassified; median WBC, 33.4 G/L (1.2–225); Hb, 9.2 g/dL (5–13.5); platelets, 48 G/L (6–328); % marrow blasts, 78 (20–97); cytogenetic risk group (favorable, 28%; intermediate, 31%; unfavorable, 19%); FLT3-ITD, 10/54 (19%). The complete response rate after induction chemotherapy (anthracyclines + ara-C) was 83%. Patients in CR1 were referred to consolidation chemotherapy, autologous or allogeneic stem-cell transplantion according to the cytogenetic risk. The global overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) were of 22.1, 14.1, and 15.6 months, respectively. The levels of Akt phosphorylation (Thr308 and Ser473) were evaluated on pretreatment marrow samples and expressed by the ratio between mean fluorescence intensity of the stained AML sample and non-relevant IgG control (rMFI). The values of both Thr308 and Ser473 represented a continuum ranging from 0.3 to 5.6 and 0.4 to 2.87, respectively. The median level of phosphorylation was 2.3 (±0.89) and 1.3 (±0.27) for Thr308 and Ser473, respectively. There were no significant correlations between age, gender, FAB, WBC, FLT3-ITD and Akt phosphorylation. However, the level of phosphorylation on Thr308 but not on Ser473 was significantly correlated with the cytogenetic risk group. Indeed, the median rMFIs on Thr308 were 1.95 (± 0.37); 2.2 (±1) and 3.1 (±0.49) for favorable, intermediate and unfavorable karyotype, respectively (p=0.0069). In univariate analysis, unfavorable karyotype, FLT3-ITD and Thr308high correlated with poor OS, EFS, and RFS. Thr308high patients (> median value) had significantly shorter OS (11 vs 47 months; p=0.01), EFS (9 vs 26 months; p=0.005) and RFS (10 months vs not reached; p=0.02) than Thr308low patients. In the Cox model for multivariate analysis, only the cytogenetic risk independently predicted worse RFS, EFS and OS. Neither mutation screening for AKT1 E17K in an independant series of 148 AML cases nor changes in the level of PTEN expression and phosphorylation could explain the increased phosphorylation of Thr308 in high-risk cytogenetics AML cells. However, we found a statistically significant correlation between protein phosphatase 2A (PP2A) activity and complex karyotypes. Indeed, PP2A activity was markedly reduced by 40% in complex karyotypes compared with normal karyotypes. To specifically assess the role of Akt in the survival and proliferation of complex karyotype AML cells, we used the allosteric Akt kinase inhibitor targeting Akt1 and Akt2 (Akt-i). Akt-i inhibited the phosphorylation of Akt on both sites in these cells. Akt-i inhibited the clonogenic activity of AML cells from normal and complex karyotype samples in a dose-dependent manner, but this effect was significantly more potent in complex than in normal karyotype samples. After 24 h incubation with 10 μM Akt-i, features of apoptosis were detected by Annexin V staining (25% ±3,38 in treated vs 14% in untreated cells). Moreover, Akt-i enhanced the toxicity of daunorubicin (37% ±2,9, Akt-i+dnr vs 25% ±3,5, dnr). We also detected Akt phosphorylation in the immature leukemic compartment (CD34+ CD38− CD123+). Accordingly, Akt-i also induced apoptosis and enhanced dnr activity in CD34+ CD38− CD123+ cells from complex karyotype samples. This study shows that AML cells with complex karyotype display high level of phosphorylation of AktThr308 and suggests that this anti-apoptotic pathway may represent a valuable therapeutic target. Thus, it will be important to determine in clinical trials if this aberrant activation of Akt sensitizes AML cells to PI3K/Akt inhibitors specifically in this subgroup of very high-risk patients.

Identification of High-Level DNA Amplifications in AML with Complex Karyotype Using Array-CGH.

Complex karyotype acute myeloid leukemia (AML), commonly defined as the presence of three or more chromosome abnormalities without specific fusion transcripts, is seen in approximately 10–15% of all AML cases. In this subset of cases, genomic losses and gains are much more frequent than balanced translocations, indicating other mechanisms of leukemogenesis. One possible mechanism is the activation of oncogenes through high-level DNA amplifications. To detect high-level DNA amplifications and to identify corresponding candidate genes, we applied comparative genomic hybridization to microarrays (array-CGH) in 100 cases of complex karyotype AML and correlated the findings with gene expression profiling (GEP) data. For array-CGH a custom-made 2.8k-microarray was used consisting of 2799 different BAC- or PAC-vectors with an average resolution of approximately 2 Mb. Hybridization experiments were performed in a dye-swap setting; significant aberrations were defined as mean plus/minus three times the standard deviation of a set of balanced clones for each individual experiment. In selected cases correlation with global gene expression studies was performed to further delineate candidate genes.We identified 50 high-level DNA amplifications in 20 different genomic regions. Amplifications occurring in at least two cases mapped to (candidate genes in the amplicon) 11q23.3-q24.1 (n=10; ETS, FLI1, APLP2); 11q23.3 (n=8;MLL, DDX6, LARG, SC5DL); 21q22 (n=5; ERG, ETS2); 9p24 (n=4; JAK2); 13q12 (n=4; CDX2, FLT3, PAN3); 8q24 (n=3; C8FW, MYC); 12p13 (n=2; FGF6, CCND2); 20q11 (n=2; ID1, BCL2L1); and 11q13 (n=2; STARD10, GARP, RAD30, DLG2). To better characterize the genomic architecture of the amplicons, we applied array-CGH using an 8.0k-microarray with an average resolution of approximately 1 Mb. Using this approach highly complex amplicon structures with several distinct amplicon peaks were identified for e.g. the amplified regions in 8q24, 11q23, and 13q12. In addition, parallel analysis of GEP in a subset of 43 of 100 cases displayed overexpressed candidate genes in the critical amplified regions; for some of the genes an oncogenic role has been implicated e.g. C8FW and MYC in 8q24, ETS1, FLI1 and APLP2 in 11q24.1, as well as FLT3 and CDX2 in 13q12. In conclusion, using high-resolution genome-wide screening tools such as array-CGH, a large number of high-level DNA amplifications were identified in AML with complex karyotype suggesting a more general role for protooncogene activation in this AML subset. This high-resolution technique allows the detection of complex amplicon structures with several distinct amplicon peaks pinpointing to selective candidate genes. In addition, correlation with GEP studies facilitates the delineation of overexpressed candidate genes within the amplified regions.