CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis

Abstract The recent discovery of acquired uniparental disomy (aUPD) in acute myeloid leukemia (AML) has been linked to homozygosity for mutations in certain genes (Raghavan et al, Cancer Res. 2005, Fitzgibbon et al, Cancer Res. 2005). Although this phenomenon, which is undetectable by conventional cytogenetics, has been confirmed in subsequent small-scale studies, its extent and frequency remains uncertain. To determine the frequency and distribution of aUPD, DNA samples from 455 young adult AML patients entered in the UK Medical Research Council AML10 trial were analyzed using Mapping 10K 2.0 single nucleotide polymorphism (SNP) arrays (Affymetrix Inc.). Genomic DNA from blood samples of ten non-leukemic individuals was used as control to estimate the copy number values (control set I). We defined aUPD as 50 consecutive homozygous markers but allowed 2 heterozygous calls to accommodate contaminating normal tissue. Using this criterion a false positive rate of 3.3% was calculated from an available data of 90 independent controls (control set I). Overall, 120 regions of UPD were observed in 79 AML cases (17%), 87% of which involved at least one breakpoint, i.e. resulted from mitotic recombination, and 13% were whole chromosome aUPDs arising from chromosomal non-disjunction. They were the sole aberration, as detected by SNP arrays, in 61 samples (13%), and 84% of these had only a single region of aUPD. There was a non-random distribution across chromosomes; 13q (n=18 cases), 11p (n=8) and 11q (n=9) were the most frequently affected. Other chromosomes with regions of recurrent aUPD were 2p (n=7), 2q (n=6), 1p (n=5), 19q (n=4), 17q12–q21.2 (n=4), 21q (n=4), 9p (n=3), Xq (n=3), 6p (n=2), and 17p (n=2). Acquired UPDs were observed across all cytogenetic risk groups: in 25% of adverse risk patients, 11% of favorable risk, 19% of normal karyotype and 10% of the remaining intermediate risk patients. Samples with aUPD13q (5% of samples) belonged exclusively to the intermediate risk group. Chromosome 13 was the only chromosome to show recurrent whole chromosome aUPD. Fifteen samples with aUPD13q covered the region of the FLT3 gene at 13q12.2; all 15 had a FLT3-internal tandem duplication (ITD) and all cases with a high FLT3-ITD mutant level > 50% of total had 13q aUPD. Gains and losses were observed in 12% and 14% of the samples respectively. As expected, gains on chromosome 8 and losses on chromosomes 5 and 7 were common, confirming the general utility of this approach. No homozygous losses were observed. Comparison of arrays with cytogenetic analysis showed that additional information (aUPDs and/or copy number changes) was obtained in 23% of cases with a normal karyotype and 38% of cases without available cytogenetics. This study highlights the importance of aUPD in the development of AML and pinpoints regions that may contain novel mutational targets.

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Chromosome Instability in Acute Myeloid Leukemia Is Associated to increased spontaneous breakage and Copy Number Variations (CNVs).

Blood ◽

10.1182/blood.v114.22.4240.4240 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4240-4240

Author(s):

Ariela Freya Fundia ◽

Irene Beatriz Larripa ◽

Susana Hayde Acevedo

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Copy Number Variations ◽

Fragile Sites ◽

Bone Marrow Cultures ◽

Acute Myeloid ◽

Marrow Cultures

Abstract Abstract 4240 Acute Myeloid Leukemia (AML) is a genetically pleiomorphic disease characterized by multiple genetic lesions including structural and numerical chromosome aberrations, gene mutations and changes in gene expression. The underlying mechanism behind the acquisition of these genetic abnormalities is not known, but it is likely that are facilitated by factors that increase chromosomal and genetic instability. The aim of this work was to explore chromosomal instability (CIN) in de novo AML patients by evaluating chromosome fragility and acquired DNA copy number variations (CNVs). Leukemic karyotypes were scored on bone marrow cultures (24-48 hs, without mitogens) from 24 AML patients using conventional cytogenetic and FISH. Spontaneous and FUdR -induced chromosomal fragility was studied on PHA-stimulated lymphocytes cultures (72 hs) from 10 patients and 10 healthy individuals, with and without FUdR (10mg/ml). One hundred metaphases were analyzed blind using conventional cytogenetic with sequential GTG banding. CNVs were scored on bone marrow cultures from 14 AML patients using fluorescence in situ hybridization (FISH) with two probes targeting specific regions on chromosomes 5q31 (LSI EGR/D5S721:D5S23) and 7q31 (LSI D7S522/CEP7), loci reported to be critical hotspots involved in AML. The cut-off value for FISH scoring was calculated after analyzing 250 cells from each of 10 normal bone marrow samples. The cutoff for allelic losses was equal to 0.8% for either 5q or 7q probe. Nine out of 24 AML cases presented normal karyotypes in bone marrow samples while the following abnormalities: t(3;8), t(3;11), t(4;11), t(15;17), t(9;22) and inv(16) were detected in the remaining patients. Significantly increased frequencies of spontaneous chromosome breakage, scored on untreated cultures, were detected in patients (0.22±0.03) respect to controls (0.07±0.03) (p<0.05), showing a random pattern of distribution. No differences were observed between patients and controls with FUdR treatment. Statistical analysis with Ch2 test considering data of FUdR breakpoint distribution over all individuals, identified 21 common fragile sites (c-fra) in AML cases (p<0.005). The most common sites were located at 1p32, 1p22, 1q21, 3p14, 3q27, 4q31, 5q31, 6p21 and 9q13. A high inter-individual variation in the pattern of expression was observed. Using FISH, we obtained the DNA copy numbers at 5q and 7q in all AML samples. Only losses of DNA were found with both probes. The 5q signal count in patients with normal karyotypes was on average 6.4% (range 0.5-30%), while it was 24.9% (range 0.8-90%) for patients with abnormal cytogenetic. The 7q signal count was on average 3.9% (range 0.5-10.3%) and 20.6% (range 2-63%) for cases with normal and abnormal karyotypes, respectively. Particularly, we showed that 12/14 (85.7%) AML cases, either with normal or abnormal karyotypes, exhibited copy number losses at both regions, showing different values between each patient. These findings showed that leukemic patients exhibit a CIN phenotype, providing an unstable background and facilitating the acquisition of additional genetic changes, such as CNVs, which could play an important role in disease progression. Disclosures: No relevant conflicts of interest to declare.

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Genomic Copy Number Variations in the Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients with del(5q) and/or -7/del(7q)

International Journal of Medical Sciences ◽

10.7150/ijms.12612 ◽

2015 ◽

Vol 12 (9) ◽

pp. 719-726 ◽

Cited By ~ 9

Author(s):

Rui Zhang ◽

Young-Mi Kim ◽

Xianfu Wang ◽

Yan Li ◽

Xianglan Lu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myelodysplastic Syndrome ◽

Copy Number ◽

Myeloid Leukemia ◽

Copy Number Variations ◽

Genomic Copy Number ◽

Genomic Copy ◽

Acute Myeloid

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Genome-Wide Micro-Deletions and Amplifications Acquired at Relapse in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.166.166 ◽

2009 ◽

Vol 114 (22) ◽

pp. 166-166 ◽

Cited By ~ 2

Author(s):

Manoj Raghavan ◽

Manu Gupta ◽

Tracy Chaplin ◽

Sabah Khalid ◽

T. Andrew Lister ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Snp Array ◽

Uniparental Disomy ◽

Normal Karyotype ◽

P Value ◽

Copy Number Changes ◽

Acute Myeloid

Abstract Abstract 166 Recurrence of acute myeloid leukemia AML has a poor prognosis with only 20% of adults surviving to 5 years. Therefore it is of importance to identify molecular changes that explain the pathogenesis of relapsed AML. Previous studies had not identified consistently acquired cytogenetic changes at relapse. Recently, acquired uniparental disomy due to mitotic recombination was described in 40% of relapsed AML (Raghavan et al 2008). Most of the events lead to homozygosity for FLT3 mutations. This study aimed to discover if there are further genetic abnormalities acquired at disease recurrence that cannot be identified by conventional cytogenetics, i.e. microdeletions or gains. Twenty-one presentation and relapse paired AML patient blood and marrow samples were stored with consent at St Bartholomew's Hospital, London. Eleven patient samples had a normal karyotype at diagnosis, two had favourable prognosis cytogenetics (inv(16) and t(8;21)) and others had varying numerical cytogenetic abnormalities and rearrangements associated with an intermediate prognosis. DNA from the samples was analysed by array based high-resolution single nucleotide polymorphism (SNP) genotyping (Affymetrix Human SNP array 6.0). Data was analysed using Partek Genome Browser (Partek, MO). In all cases, the leukemia infiltrate of the marrow or blood was greater than 60% and most cases were greater than 90% allowing accurate identification of DNA copy number changes. Abnormalities of a size that would be identified by cytogenetics were disregarded. Using segmentation analysis using a p-value less than 0.001, over 400 microdeletions and gains were detected that were acquired at relapse in the 21 pairs. Each of the copy number changes was less than 2 megabases in size. One AML sample with a normal karyotype at diagnosis and trisomy 8 and add(9)(q34) at relapse had not acquired any microdeletions or gains. In contrast, in other samples as many as 69 microdeletions/gains were detected. There was no correlation between increased complexity of the karyotype of the leukemia and the number of microdeletions/gains. Several of the acquired microdeletions/gains were in regions containing genes known to be involved in AML, including a deletion of 234Kb at 13q12.2 involving FLT3 and CDX2, and an acquired deletion at 21p11.2 of 150Kb involving exons encoding the runt domain of RUNX1. Another copy number gain was detected at the MLL locus, suggestive of partial tandem duplication. Other detected locations are in Table 1.Table 1Location by cytobandCopy number changeSize / KbP valueGene13q12.2Deletion23410−33FLT3, CDX221q22.12Deletion15010−13RUNX111q23.3Gain5.10.0099MLL11p15.4Gain830.00001NUP9817q21.31Deletion8.00.0007BRCA1The results indicate that recurrent AML may be associated with the deletion or gain of several genes involved in leukaemogenesis. Many other locations are involved throughout the genome, suggesting at least some of these are also involved in the clonal evolution of the leukaemia at recurrence. Further studies should identify novel genes from these regions involved in the pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

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Mlecular Monitoring of Acute Myeloid Leukemia Patients Carrying Nucleophosmin (NPM1) Mutations Undergoing An Autologous Peripheral Blood Stem Cell Transplantation

Blood ◽

10.1182/blood.v112.11.4864.4864 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4864-4864

Author(s):

Valentina Gianfelici ◽

Daniela Diverio ◽

Saveria Capria ◽

Silvia maria Trisolini ◽

Sonia Buffolino ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Peripheral Blood ◽

Copy Number ◽

Myeloid Leukemia ◽

Peripheral Blood Stem Cell ◽

Normal Karyotype ◽

Blood Stem Cell Transplantation ◽

Acute Myeloid

Abstract Nucleophosmin (NPM1) gene mutations represent the most common genetic alterations in adult acute myeloid leukemia (AML), accounting for about 30% of cases and 50–60% of AML with normal karyotype. In addition to their recognized prognostic value (when combined with analysis of the FLT3 gene), NPM1 mutations represent an ideal marker for monitoring of minimal residual disease (MRD) since they are very stable during the course of the disease. PCR quantitative monitoring of NPM1 mutant copies has been mainly restricted to patients treated with conventional chemotherapy. In our study, we retrospectively analyzed, by RQ-PCR, MRD in 12 consecutive NPM1-mutated AML patients who underwent an autologous peripheral blood stem cell transplantation (PBSCT) between September 2000 and March 2008 at the Division of Hematology, “Sapienza” University of Rome. By sequencing of the region encompassing NPM1 exon 12, we demonstrated the presence of a type A mutation in 10/12 patients and of a type B in the remaining 2. Moreover, at diagnosis molecular analysis of the FLT3 gene revealed an internal tandem duplication (ITD) in 7/12 patients. Cytogenetic analysis showed a normal karyotype in 11/12, whereas 1 patient presented a minor karyotype aberration (trisomy 4). RQ-PCR was retrospectively performed on bone marrow samples in all patients at diagnosis, in 11/12 patients before the transplant and in 9/12 after the graft. Six of the 12 patients are at present in continuous complete remission (CCR) with a median follow-up of 54 months (range 4–89), 1 patient has died in CCR 55 months after PBSCT due to a pancreatic carcinoma and 5/12 have relapsed. Four of the latter 5 patients relapsed shortly after the transplant (range 3–10 months); 2 of them died of disease progression and 2 of salvage chemotherapy toxicity. One patient, who relapsed 92 months from PBSCT, is alive in second hematological remission following re-induction and consolidation chemotherapy. No significant differences in median levels of transcript at diagnosis were found between relapsing and non-relapsing patients (93,000 and 68,000; range: 86,000–103,000 and 37,000–116,000, respectively). Nine patients were analyzed after PBSCT: 3 persistently positive relapsed and the copy number of the transcript was always higher than at diagnosis. In the other 6 patients, the transcript was undetectable (3 of them were already in molecular remission pre-transplant): 5 are still in CCR while 1 patient relapsed 92 months after the graft. Regarding the FLT3-ITD status at diagnosis, 3/7 patients are in CCR whereas 4 have relapsed. The retention of NPM1 mutations even at relapse further confirms that NPM1 alterations are highly stable, pointing to this event as critical for leukemia growth and survival. Moreover, in our study long-term survival was always associated with a copy number of NPM1 mutant transcripts below the detectable level of the method, suggesting that disappearance of the transcript should be the primary clinical endpoint. Prospective studies and larger case series are necessary in an attempt to identify a cut-off level during treatment which could be predictive of the clinical outcome of AML patients harboring NPM1 mutations and help in therapeutic choices.

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Adverse Prognostic Role of IDH2 Mutations at Diagnosis and during Follow-up in Patients with Acute Myeloid Leukemia and Normal Karyotype

Blood ◽

10.1182/blood-2021-149719 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2366-2366

Author(s):

Erika Borlenghi ◽

Diego Bertoli ◽

Chiara Cattaneo ◽

Margherita Sciumé ◽

Elisa Cerqui ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

Prognostic Role ◽

Advisory Committees ◽

Relapse Risk ◽

Acute Myeloid

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogeneous disorder characterized by a wide range of cytogenetic and molecular aberrations, that affect prognosis and guide treatment decisions. However there is a still large group of patients (pts) considered at intermediate risk whose outcome needs to be better defined. Next-generation sequencing (NGS) can simultaneously detect various mutations, leading to better define its prognostic profile. The role of some mutations, including isocitrate dehydrogenase (IDH) mutations (IDHm), is still controversial. Aim: We evaluated by NGS monitoring at different time points the prognostic role of IDH1/2m in AML pts with normal karyotype, both in the subgroup with mutations of NPM1 (NPM1m) or FLT3 (FLT3m) and in the subgroup without detectable mutations (wt-AML). Methods: Using Sophia Myeloid Solution kit (SOPHiA Genetics), we performed targeted NGS, covering 30 gene regions, in 104 bone marrow samples collected at diagnosis (53), after first consolidation (30) and at relapse (21), in 53 pts (M/F: 24/29; median age: 56 y, range 22-74), treated according to NILG-AML00 protocol (NCT00400673). Standard PCR to detect NPM1m and FLT3m was performed and we identified 20 NPM1m, 3 NPM1+FLT3-ITDm, 4 FLT3-ITDm and 26 wt-AML. Results: At diagnosis, among 219 pathogenic mutations detected, IDHm represented 10.5% of them (median VAF: 39.1%; range 6.2-49.6%). IDHm was observed in 23/53 pts (43.4%) (IDH1m in 11 and IDH2m in 12). In these pts , more frequently commutated genes were DNMT3A (28%), NPM1 (13%), FLT3-ITD/TKD (14%), ASXL1 (6%), SRSF2 and NRAS (9% each). Complete remission (CR) was achieved in 49/53 (92.5%) pts without difference in response rate according to IDH status (86% in IDHm vs 94% in IDH wild-type, wt). Relapse occurred in 28/49 (57%) pts after a median of 11 months (mo), range 2-61. The frequency of relapse was not significantly different across all types of mutations identified, except for IDH2m which was associated with a higher risk of relapse (10/11 in IDH2m vs 18/38 in IDH2wt; p: 0.014), without differences between R172K and R140Q. On the contrary, IDH1m, present in 18% of relapsed pts, did not impact on relapse (5/10 vs 23/39, p: 0.7). Particularly, in the wt-AML group, the IDH2m was prevalent in pts developing relapse (6/11, 54.5%) and all pts with IDH2m relapsed, with median of 13 mo, range 6-24 (6/6 in IDH2m vs 5/17 in IDH2wt, p:0.0046). Among the co-occurrence mutations, the IDH2/DNMT3A was associated with higher relapse risk (9/9 vs 19/40; p: 0.0063). DNMT3A associated with other mutations did not impact on relapse risk. At a median follow-up of 23 mo, median relapse free survival (RFS) and overall survival (OS) of whole population were 24 and 53 mo, respectively. The IDH2m impacted on OS: 23.5 mo in IDH2m vs 72 in IDH2wt pts (p:0.0093) (Fig 1a), but not in RFS (13 vs 29 mo in IDH2m and IDH2wt, respectively (p:0.1). Considering the subgroups of wt-AML, the RFS (Fig. 1b) and OS were 13 and 23.5 mo in IDH2m vs undefined in IDH2-wt (p:0.0014 and p:0.1), respectively. In pts with NPM1 or FLT3m, RFS and OS were 9 and 53 mo in IDH2m vs 29 and 73 mo in IDH2-wt (p:0.2 and p:0.15), respectively. We did not find the other genomic pattern predicting relapse in this group. After consolidation, NGS monitoring was performed in 30 pts in CR. Of the 13 IDH AML pts evaluated, no mutations was observed in 4 (28.5%); the persistence of IDHm was not associated with a significantly higher relapse (p:0.5). Among other mutations present at diagnosis, NGS clearance after consolidation occurred in pts with NRAS, KRAS, PTPN11 and FLT3-ITD/TKD. Conversely, it was limited for the following mutations: TET2 (8/11), DNMT3A (7/13), SRSF2 (6/6), IDH2 (4/5), ASXL1 (2/2), IDH1 (2/4) and NPM1 (1/12). Overall, the persistence of any type of gene mutations after consolidation was predictive of relapse (2/9 vs 6/7, p:0.04), only in wt-AML subgroup. At relapse, of the 11 IDHm pts analyzed, 7/7 IDH2m and 3/4 IDH1m showed the reappearance of mutations. Conclusion: In this retrospective monocentric study, the presence at diagnosis of IDH2m correlated with relapse risk and with survival, suggesting that additional treatment with targeted agents and or consolidation with allogeneic transplant should be considered. In addition, in AML without NPM1m or FLT3m, the persistence of genes mutation detected by NGS monitoring after consolidation had a significant prognostic value to predict subsequent relapse. Figure 1 Figure 1. Disclosures Borlenghi: Amgen, Janssen: Consultancy. Rossi: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Tucci: Gentili: Membership on an entity's Board of Directors or advisory committees; janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

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Broad copy neutral-loss of heterozygosity regions and rare recurring copy number abnormalities in normal karyotype-acute myeloid leukemia genomes

Genes Chromosomes and Cancer ◽

10.1002/gcc.20810 ◽

2010 ◽

Vol 49 (11) ◽

pp. 1014-1023 ◽

Cited By ~ 21

Author(s):

Vincenza Barresi ◽

Alessandra Romano ◽

Nicolò Musso ◽

Carmela Capizzi ◽

Carla Consoli ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Loss Of Heterozygosity ◽

Copy Number ◽

Myeloid Leukemia ◽

Neutral Loss ◽

Normal Karyotype ◽

Acute Myeloid

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Heterogeneity of Acute Myeloid Leukemia at the Stem Cell Level.

Blood ◽

10.1182/blood.v112.11.1355.1355 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1355-1355

Author(s):

Michael Heuser ◽

Laura Sly ◽

Courteney Lai ◽

Malina Leung ◽

Grace Lin ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Critical Role ◽

Normal Karyotype ◽

Complex Karyotype ◽

Gm Csf ◽

Limiting Dilution ◽

Acute Myeloid

Abstract 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.

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