MLL Partial Tandem Duplication Research Articles

Common Leukemia- and Lymphoma-Associated Genetic Aberrations in Healthy Individuals

Leukemia- and lymphoma-associated (LLA) chromosomal rearrangements are critical in the process of tumorigenesis. These genetic alterations are also important biological markers in the diagnosis, prognosis, and treatment of hematopoietic malignant diseases. To detect the presence or absence of these genetic alterations in healthy individuals, sensitive nested RT-PCR analyses were performed on a large number of peripheral blood samples for selected markers including MLL partial tandem duplications (PTDs), BCR-ABL p190, BCR-ABL p210, MLL-AF4, AML1-ETO, PML-RARA, and CBFB-MYH11. Using nested RT-PCR, the presence of all of these selected markers was detected in healthy individuals at various prevalence rates. No correlation was observed between incidence and age except for BCR-ABL p210 fusion, the incidence of which rises with increasing age. In addition, nested RT-PCR was performed on a large cohort of umbilical cord blood samples for MLL PTD, BCR-ABL p190 and BCR-ABL p210. The results demonstrated the presence of these aberrations in cord blood from healthy neonates. To our knowledge, the presence of PML-RARA and CBFB-MYH11 in healthy individuals has not been previously described. The present study provides further evidence for the presence of LLA genetic alterations in healthy individuals and suggests that these mutations are not themselves sufficient for malignant transformation.

RUNX1 Mutations in Acute Myeloid Leukemia: Results From a Comprehensive Genetic and Clinical Analysis From the AML Study Group

To evaluate frequency, biologic features, and clinical relevance of RUNX1 mutations in acute myeloid leukemia (AML). Diagnostic samples from 945 patients (age 18 to 60 years) were analyzed for RUNX1 mutations. In a subset of cases (n = 269), microarray gene expression analysis was performed. Fifty-nine RUNX1 mutations were identified in 53 (5.6%) of 945 cases, predominantly in exons 3 (n = 11), 4 (n = 10), and 8 (n = 23). RUNX1 mutations clustered in the intermediate-risk cytogenetic group (46 of 640, 7.2%; cytogenetically normal, 34 of 538, 6.3%), whereas they were less frequent in adverse-risk cytogenetics (five of 109, 4.6%) and absent in core-binding-factor AML (0 of 77) and acute promyelocytic leukemia (0 of 61). RUNX1 mutations were associated with MLL-partial tandem duplications (P = .0007) and IDH1/IDH2 mutations (P = .03), inversely correlated with NPM1 (P < .0001), and in trend with CEBPA (P = .10) mutations. RUNX1 mutations were characterized by a distinct gene expression pattern; this RUNX1 mutation-derived signature was not exclusive for the mutation, but also included mostly adverse-risk AML [eg, 7q-, -7, inv(3), or t(3;3)]. RUNX1 mutations predicted for resistance to chemotherapy (rates of refractory disease 30% and 19%, P = .047, for RUNX1-mutated and wild-type patients, respectively), as well as inferior event-free survival (EFS; P < .0001), relapse-free survival (RFS, P = .022), and overall survival (P = .051). In multivariable analysis, RUNX1 mutations were an independent prognostic marker for shorter EFS (P = .007). Explorative subgroup analysis revealed that allogeneic hematopoietic stem-cell transplantation had a favorable impact on RFS in RUNX1-mutated patients (P < .0001). AML with RUNX1 mutations are characterized by distinct genetic properties and are associated with resistance to therapy and inferior outcome.

Targeted Next-Generation Sequencing (NGS) Enables a Broad Screening of 95 Molecular Mutations In a One-Step Approach and Detects Recurrent Mutations In AML with a Normal Karyotype In MYH11 and NOTCH1

AbstractAbstract 175Acute myeloid leukemia (AML) is a heterogeneous disease and based on cytogenetic stratification AML with a normal karyotype (AML-NK) is an intermediate-risk group. In recent years the detection of novel molecular mutations allowed to further stratify AML-NK into different prognostic subgroups. However, thus far still in a subset of AML-NK no recurrent mutations have been identified. In order to detect novel recurrent mutations we performed DNA sequence enrichment from complex genomic samples using microarrays to enable a targeted Next-Generation Sequencing (NGS) analysis. We combined 454 PicoTiterPlate (PTP) pyrosequencing with long-oligonucleotide sequence capture arrays to apply this technique for the comprehensive molecular genetic characterization of AML-NK. 6 bone marrow specimens from untreated de novo AML patients at diagnosis were analyzed (n=4 AML-NOS; n=2 AML-MRC according to WHO classification). All cases were shown to be negative for the most frequent mutations in FLT3 (both internal tandem duplication and tyrosine kinase domain), MLL partial tandem duplication, NPM1, and CEBPA. A custom 1.91 Mb microarray was designed to contain capture probes for all coding regions of 95 genes, in total 1608 exons (NimbleGen 385K format; Madison, WI). These 95 target genes had been selected according to their relevance in leukemogenic pathways, i.e. cell cycle control, cell proliferation and differentiation, multidrug resistance, growth factor receptors, oncogenes, tumor suppressors, and homeobox genes. Starting with 20 μ g of genomic DNA, this array design allowed a median 294-fold DNA enrichment of these targeted genomic loci, as assessed by ligation-mediated LM-PCR. Three patients per lane were sequenced on a 2-lane PTP using the large volume Titanium chemistry assay (454 Life Sciences, Branford, CT). Each case was tagged by a molecular 10-base barcode. Overall, 1,070,724 reads with a median length of 352 bp were generated in the two lanes (GS Reference Mapper software version 2.3). In median, 72.3% on-target bases were derived from the capture array probes, resulting in a 16-fold median coverage. In total, in this proof-of-principle cohort of 6 patients, in median 1534 variants per case were detected. After excluding single nucleotide polymorphisms and noncoding aberrations, 13 nonsynonymous mutations were found in 11/95 genes analyzed (1-4 mutations per case). Single missense mutations were found for transcriptional repressor SPEN (Q340R), histone acetyltransferase and transcriptional coactivator CREBBP (K622R), multi-drug resistance transporter ABCC1 (R633Q), epidermal growth factor EGF (A995P), runt-related transcription factor RUNX1 (G138S) and mixed-lineage leukemia gene MLL (D2890G). A TET2 nonsense mutation was observed in exon 5 (E1178X), being located in a conserved domain as described by Delhommeau et al. (N Engl J Med. 2009 360:2289-301). In addition, in two patients two genes were recurrently hit by a mutation: MYH11 and NOTCH1. MYH11 missense mutations were detected in exons 36 and 39 (E1840D; M1661V). We further observed two missense NOTCH1 mutations in exons 2 and 13 (P22S; E694K), located in the extracellular epidermal growth factor-like repeats domain which is required for ligand interaction. Thus far, activating NOTCH1 mutations have been reported in the context of T cell acute lymphoblastic leukemia (heterodimerization and PEST domains). In conclusion, we demonstrated that the combination of a targeted DNA sequence enrichment assay followed by NGS technology enabled a molecular characterization of 95 genes in AML-NK in a one-step approach. New recurrent aberrations such as NOTCH1 mutations are interesting targets for a broader screening in AML subtypes. In particular, methods like this will enable an unbiased comprehensive genetic characterization of leukemias and other malignancies and are suitable to identify markers to further stratify AML-NK into different risk groups. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Roche Diagnostics GmbH: Research Funding.

Mutations In the Tet Oncogene Family Member 2 (TET2) Gene Refine the New European LeukemiaNet Risk Classification of Primary, Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) In Adults: A Cancer and Leukemia Group B (CALGB) Study

AbstractAbstract 98Mutations in the TET2 gene were recently identified in a variety of myeloid neoplasms including AML. However, the frequency and clinical relevance of TET2 mutations in CN-AML, the largest cytogenetic subgroup of adult AML, have not been well defined. We report here the frequency and spectrum of TET2 mutations, and their associations with clinical and molecular characteristics, treatment outcomes and genome-wide gene- and microRNA (miR)-expression signatures in a relatively large cohort of 427 patients (pts) with primary CN-AML. The pts, aged 18–83 years, were intensively treated on CALGB frontline protocols, and were analyzed centrally for TET2 mutations by PCR and direct sequencing, and for other prognostic gene mutations (FLT3 internal tandem duplications [ITD] and tyrosine kinase domain mutations, MLL partial tandem duplications, and mutations in NPM1, CEBPA, WT1 and IDH1&IDH2). If available, buccal swabs or remission marrow samples were used to determine TET2 germline status. Gene- and miR-expression profiles were derived using microarrays (Affymetrix HG-U133 plus 2.0 and OSUCCC custom miR array v4.0). At least 1 sequence variation in TET2 was found in 104 pts. Frameshift (n=59) and nonsense (n=34) variations were distributed throughout all coding exons, while missense changes (n=37) clustered mainly (28/37) in 2 evolutionarily conserved domains of TET2. The remaining missense variations in 9 pts were located outside the conserved domains, and analysis of available buccal swabs or remission samples showed that these sequence changes were present in the germline. Since it is unclear whether they represent innocent polymorphisms or disease-relevant mutations, these 9 pts were excluded from further analyses. TET2-mutated (TET2-mut) pts were older (P<.001), had higher white blood counts (P=.04), a lower frequency of IDH1 and IDH2 mutations (P<.001), and showed a trend towards higher frequency of CEBPA mutations (P=.07) compared with TET2 wild-type (TET2-wt) pts. The European LeukemiaNet (ELN) recently proposed a standardized reporting system for AML, in which CN-AML pts are assigned to Favorable-risk (Fav; pts with mutated CEBPA and/or mutated NPM1 without FLT3-ITD) or Intermediate-I-risk (Int-I; all remaining CN-AML pts) categories. We assessed the prognostic relevance of TET2 mutations in the context of the Fav (n=199 pts) and Int-I (n=219) ELN categories. TET2 mutations tended to be more frequent in Fav than in Int-I CN-AML pts (27% v 19%, P=.08), even though types and location of mutations were similar in both groups. Within the Fav category, TET2-mut pts had shorter event-free survival (EFS; P<.001), a lower complete remission (CR) rate (P=.007) and shorter disease-free survival (DFS; P=.003; Fig 1), and shorter overall survival (OS; P=.001; Fig 2) compared with TET2-wt pts. In contrast, in the Int-I category, no difference in EFS (P=.45), CR rates (P=1.0), DFS (P=.36; Fig 1) or OS (P=.72; Fig 2) was found between TET2-mut and TET2-wt pts. In multivariable models, TET2 mutations associated with shorter EFS (P=.004; hazard ratio [HR], 1.71), lower CR rate (P=.03; odds ratio, 0.62) and shorter DFS (P=.049; HR, 1.54) only among Fav, but not among Int-I, CN-AML pts. A TET2 mutation-associated gene-expression signature consisting of 213 probe sets (136 named genes) was identified in ELN Fav CN-AML pts and included genes previously implicated in AML pathogenesis, e.g., upregulated CEBPA, APP, NCAM1 and IDH1, and downregulated MLL. In contrast, no signature of differentially expressed genes was identified in Int-I pts. miR profiling revealed distinct TET2 mutation-associated miR-expression signatures in the ELN Fav and Int-I risk groups. Among miRs upregulated in ELN Fav/TET2-mut pts were miR-148a (targeting DNA methyltransferases, highly expressed in refractory chronic lymphocytic leukemia) and miR-24 (stimulating myeloid cell proliferation, blocking granulocytic and erythroid differentiation). In Int-I/TET2-mut pts, one of the upregulated miRs was miR-204 (targeting HOXA10 and MEIS1, downregulated in NPM1-mut AML). We conclude that TET2 mutations are associated with lower remission rates and inferior survival in the ELN Fav category of CN-AML, and may be useful to refine the ELN molecular classification. TET2 mutation-associated gene- and miR-expression signatures, first identified here, may contribute to our understanding of the biology of TET2-mutated CN-AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Low frequency of MLL-partial tandem duplications in paediatric acute myeloid leukaemia using MLPA as a novel DNA screenings technique

Mixed-lineage leukaemia ( MLL)–partial tandem duplications (PTDs) are found in 3–5% of adult acute myeloid leukaemia (AML), and are associated with poor prognosis. In adult AML, MLL–PTD is only detected in patients with trisomy 11 or internal tandem duplications of FLT3 ( FLT3-ITD). To date, studies in paediatric AML are scarce, and reported large differences in the frequency of MLL–PTD, frequently utilising mRNA RT-PCR only to detect MLL–PTDs. We studied the frequency of MLL–PTD in a large cohort of paediatric AML ( n = 276) and the results from two different methods, i.e. mRNA RT-PCR, and multiplex ligation-dependent probe amplification (MLPA), a method designed to detect copy number differences of specific DNA sequences. In some patients with an MLL-rearrangement, MLL–PTD transcripts were detected, but were not confirmed by DNA-MLPA, indicating that DNA-MLPA can more accurately detect MLL–PTD compared to mRNA RT-PCR. In paediatric AML, MLL–PTD was detected in 7/276 patients (2.5%). One case had a trisomy 11, while the others had normal cytogenetics. Furthermore 4 of the 7 patients revealed a FLT3-ITD, which was significantly higher compared with the other AML cases ( p = 0.016). In conclusion, using DNA-MLPA as a novel screenings technique in combination with mRNA RT-PCR a low frequency of MLL–PTD in paediatric AML was found. Larger prospective studies are needed to further define the prognostic relevance of MLL–PTD in paediatric AML.

Trisomy 11 in myelodysplastic syndromes defines a unique group of disease with aggressive clinicopathologic features.

Trisomy 11 in myelodysplastic syndromes (MDS) is rare, with undefined clinical significance and is currently assigned to the International Prognostic Scoring System (IPSS) intermediate-risk group. Over a 15-year period, we identified 17 MDS patients with trisomy 11 either as a sole abnormality (n=10) or associated with one or two additional alterations (n=7), comprising 0.3% of all MDS cases reviewed. Of 16 patients with Bone Marrow material available for review, 14 (88%) patients presented with excess blasts, 69% patients evolved to acute myeloid leukemia (AML) in a 5-month median interval and the median survival was 14 months. For comparison, we studied 19 AML patients with trisomy 11 in a noncomplex karyotype, of which, a substantial subset of patients had morphologic dysplasia, and/or preexisting cytopenia(s)/MDS. Genomic DNA PCR showed MLL partial tandem duplication in 5 of 10 MDS and 7 of 11 AML patients. A review of literature identified 17 additional cases of MDS with trisomy 11, showing similar clinicopathologic features to our patients. Compared with our historical data comprising 1165 MDS patients, MDS patients with trisomy 11 had a significantly inferior survival to patients in the IPSS intermediate-risk cytogenetic group (P=0.0002), but comparable to the poor-risk group (P=0.97). We conclude that trisomy 11 in MDS correlates with clinical aggressiveness, may suggest an early/evolving AML with myelodysplasia-related changes and is best considered a high-risk cytogenetic abnormality in MDS prognostication.

FLT3, NPM1 and MLL Mutations Help Risk Stratification in Pediatric Acute Myeloid Leukemia.

Abstract 1574▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-600 IntroductionAcute myeloid leukemia (AML) is a heterogeneous disease which harbors various genetic alterations. Among theses genetic events, Mutations of FLT3, NPM1, MLL and other genes often predict prognosis, particularly in cases cytogenetic normal (CN-AML). Could these be criteria for risk stratification in Pediatric AML ? Patients and Methods155 cases of de novo AML were diagnosed routinely according to morphology, immunology, cytogenetics, and molecular biology examination on bone marrow (BM) aspirates between Jan. 2002 and Dec. 2008. All patients received chemotherapy according to the AML-XH-99 protocol, which consist of Daunorubicin, Cytosine arabinoside, Etoposide, Homoharringtonine. For acute promyelocytic leukemia, all-trans retinoic acid and Arsenic trioxide were also included. Meanwhile, total RNA of leukemic cells form all diagnostic BM samples were extracted, and then reverse transcribed. MLL partial tandem duplication (MLL/PTD) fusion transcripts were screened by real-time quantitative polymerase chain reaction. FLT3 internal tandem duplication (FLT3/ITD), FLT3 tyrosine kinase domain mutation (FLT3/TKD) and NPM1 mutation were examined by High resolution melting analysis. ResultsOf the 155 children with de novo AML, 121(78.1%) had received chemotherapy for more than one week with data available for analysis. Among them, 55(45.5%) was cytogenetically normal (CN-AML). In this total cohort of patients 49(27.09%) had FLT3/ITD (32.70% in CN-AML), 14 (9.03%) had FLT3/TKD (7.30% in CN-AML), 62 (40%) had NPM1 mutation (49% in CN-AML), and additional 8 (5.16%) had MLL/PTD (5.50% in CN-AML). In this cohort of patients 98 (63.22%) had at least one mutation. The clinical outcomes were listed in table 1. Generally, patients with FLT3 mutation (ITD or TKD mutation) usually have worse results after chemotherapy, as reported previously by other researchers. Meanwhile, NPM1 mutations usually predict better prognosis in our cohort of AML patients. MLL/PTD always predicts the worst outcome in AML as other MLL rearrangements in leukemia. Among CN-AML patients, 5-year EFS and OS were similar to whole cohort of patients according to those mutations. Cox regression analysis in a univariate model revealed that the presence of FLT3/ITD and NPM1 was significant prognostic factor of EFS, (P<0.05). We therefore proposed a molecular-risk classification of pediatric AML patients based on the data we got in this study. For the newly classified groups of low, medium and high risk groups, EFS rate was 62.03%±8.42%, 45.42%±4.52%, and 14.85%±2.99%, respectively, P=0.00. CRD for the 3 groups was 27.69±21.34 months, 22.62±19.64 months, 13.26±11.95 months, respectively, p=.022. Our results indicate that combinations of these couple of molecular events may be the useful tool for further classify AML in children.Table 1The clinical outcomes of pediatric AML patients with different gene mutationsMutations/wildFirst course CR (%)5-year EFS (%)5-year OS (%)CR Duration (Month)FLT3/ITD50/76.5 (p = 0.004)24.65 ± 3.93/55.01 ± 5.09 (p = 0.007)49.13 ± 7.08/62.24 ± 4.96 (p = 0.173)18.58 ± 17.37/23.71 ± 20.09 (p = 0.185)FLT3/TKD72.7/68.2 (p = 0.527)43.68 ± 9.79/50.48 ± 4.319 (p = 0.373)49.21 ± 9.25/60.595 ± 4.31 (p = 0.233)38.45 ± 29.84/20.55 ± 17.05 (p = 0.003)NPM1 Mutation72.3/66.2 (p = 0.308)61.41 ± 6.26/36.30 ± 4.24 (p = 0.014)75.45 ± 5.29/47.12 ± 5.18 (p = 0.001)27.15 ± 20.93/19.03 ± 17.78 (p = 0.024)MLL/PTD25/70.1 (p = 0.091)8.50 ± 3.10/50.98 ± 4.18 (p = 0.04)14.33 ± 1.76/60.39 ± 4.19 (p = 0.091)8.5 ± 6.19/55.65 ± 19.53 (p = 0.024) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Overexpression of BAALC and MN1 Predict Worse Outcome in De Novo AML with Partial Tandem Duplication of MLL.

Abstract 1002Poster Board I-24 Background and purpose:Overexpression of BAALC, MN1, or ERG gene has been described to have adverse impact on the outcome of acute myeloid leukemia (AML) with normal karyotype. The majority of patients with partial tandem duplication of MLL gene (MLL-PTD) had normal karyotypes. The prognostic relevance of overexpression of these genes in MLL-PTD AML was not clear. Aims:We aimed to (1) measure the mRNA expression levels of FLT3, BAALC, FHIT, MN1, and ERG genes in AML patients with MLL-PTD (2) compare the expression levels of these genes with normal controls, and (3) determine their prognostic significance. Patients and methods:Bone marrow samples from 93 de novo AML patients with MLL-PTD at diagnosis were analyzed. MLL-PTD was screened by Southern blot analysis or reverse transcriptase polymerase chain reaction (RT-PCR) then confirmed by real-time quantitative PCR (RQ-PCR). RQ-PCR assay with TaqMan probe was performed to measure the expression of FLT3, BAALC, FHIT, MN1, and ERG genes in MLL-PTD AML as well as in 34 normal controls for comparison. The expression levels of target genes were calculated as the copy number of each gene normalized to the copy number of ABL control gene (NCN). Positive and negative controls as well as standard curve constructs were included in each assay. Mutational analysis was performed by DNA/cDNA PCR followed by GeneScan analysis for detection of internal tandem duplication of FLT3 gene (FLT3/ITD). The expression levels of each target genes were dichotomized at the median value to low and high expression groups. The event-free and overall survivals (EFS and OS) were compared between the 2 groups. Results:The expression levels of mRNAs for FLT3, BAALC, FHIT, MN1, and ERG genes at diagnosis in MLL-PTD AML and 34 normal controls are shown in Table.GeneMLL-PTDNCN/100 ABL(Mean±SE)ControlsNCN/100 ABL(Mean±SE)PFLT3703.4±59.663.6±6.9<0.001BAALC377.2±81.927.2±4.8<0.001FHIT36.5±4.426.9±3.50.089MN122.5±2.95.3±0.8<0.001ERG251.1±23.6147.2±22.90.012MLL-PTD patients had significantly higher expression levels of FLT3, BAALC, MN1, and ERG compared to normal controls. The expression of FHIT was also higher than that of controls, but did not reach statistic significance. FLT3/ITD was present in 26 of 52 patients (50%). The expression levels of the above genes were not different between patients with FLT3/ITD and those without. The median age of the entire cohort was 56 years. The median EFS and OS of the 52 patients who received standard induction chemotherapy were 5.8 and 11.4 months, respectively. The complete remission (CR) rate was higher in the low expression group than that of high expression group for BAALC (P = 0.011). The CR rate was not significantly different between low and high expression groups for FLT3, FHIT, MN1, or ERG, and between FLT3/ITD(+) and (-) groups. There were no significant difference in EFS or OS between patients with FLT3/ITD and those without. Patients with high expression of BAALC had a significantly shorter survival than those with low expression group; the median EFS was 10.3 mons (95% CI: 5.9-14.7 mons) vs. 0 mon, P = 0.044 and the median OS was 16.4 mons (95% CI: 8.3-25.5 mons) vs. 10.9 mons (95% CI: 6.5-15.3 mons), P = 0.031. Patients with high expression of MN1 also had a poor outcome compared with low expression group; the median EFS was 10.9 mons (95% CI: 0-28.3 mons) vs. 4.1 mons (95% CI: 0-11.7mons), P = 0.002 and the median OS was 29.7 mons (95% CI: 0-70.7mons) vs.11.0 mons (95% CI: 10.7-11.3 mons), P = 0.024. The EFS and OS were not significantly different between low and high expression groups for FLT3, BAALC, FHIT, and ERG. Conclusions:Our results showed that MLL-PTD was associated with overexpression of FLT3, BAALC, MN1, and ERG. The patients with lower expression level of BAALC had a higher CR rate and patients with overexpression of BAALC or MN1 had poor EFS and OS. FLT3/ITD had no prognostic impact.Supported by grants NSC97-2314-B-182 -011-MY3, NSC96-2314-B-195-006-MY3, and MMH-E-96009. Disclosures:No relevant conflicts of interest to declare.

Increased Expression of Macrophage Migration Inhibitory Factor (MIF) Receptor CD74 Is Associated with Inferior Outcome in Younger Patients (Pts) with Cytogenetically Normal Acute Myeloid Leukemia (CN-AML): a Cancer and Leukemia Group B (CALGB) Study.

Abstract 1616Poster Board I-642CD74 is a type II integral membrane protein receptor that binds its ligand MIF to induce phosphorylation of the extracellular signal-regulated kinase-1/2 (ERK-1/2) and drive cellular proliferation via nuclear factor-kappa B (NF-kB) activation. CD74 expression has been identified in human solid tumors, and its expression is associated with adverse prognosis in advanced pancreatic cancer. As CD74 is expressed and NF-kB constitutively activated in myeloblasts, we hypothesized that CD74 expression might also be associated with adverse outcome in AML. To investigate the prognostic impact of CD74 expression in the context of other predictive molecular markers in CN-AML, we assessed CD74 expression levels by Affymetrix HG-U133 Plus 2.0 microarray in 102 younger [<60 years (y)] adults with primary CN-AML, treated on the front-line CALGB 19808 trial with an induction regimen containing daunorubicin, cytarabine, etoposide and, in some cases, the inhibitor of multidrug resistance valspodar, and consolidation with autologous stem cell transplantation. Microarray data were analyzed using the Robust Multichip Average method, making use of a GeneAnnot chip definition file, which resulted in a single probe-set measurement for CD74. At diagnosis, CD74 expression, when assessed as a continuous variable, was significantly associated only with extramedullary disease involvement (P=.006) among clinical features, and with none of the molecular prognostic variables tested, including NPM1, WT1, CEBPA, FLT3 (FLT3-ITD and FLT3-TKD) mutations, MLL partial tandem duplication, or differential BAALC and ERG expression levels. Although CD74 expression levels were not associated with achievement of complete remission (CR; 83% vs 81%), higher levels of CD74 were associated with shorter disease-free survival [DFS; P=.046, hazard ratio (HR) 1.85, 95% confidence interval (CI) 1.12-3.08] and with shorter overall survival (OS; P=.02, HR 1.32, CI 1.04-1.67). In multivariable analyses, higher CD74 expression was independently associated with shorter DFS (P=.045, HR 1.98, CI 1.16-3.40), after adjusting for WT1 mutations (P<.001) and FLT3-TKD (P=.04), and shorter OS (P=.01, HR 1.58, CI 1.11-2.25) after adjusting for FLT3-TKD (P=.02), WT1 mutations (P=.007), BAALC expression levels (P=.02), white blood counts (P=.007), and extramedullary involvement (P=.04). As quartiles 2-4 had similar expression levels distinct from the lowest quartile, to display the impact of CD74 expression levels on clinical outcome only, pts were dichotomized into low (the lowest quartile) and high (the top three quartiles) CD74 expressers. The Kaplan-Meier curves for DFS and OS (Figures 1 and 2) are shown below. In conclusion, our study identifies elevated CD74 expression as associated with adverse prognosis in younger CN-AML pts. Since we previously reported that higher CD74 expression was favorably associated with achievement of CR in AML patients receiving chemotherapy plus bortezomib, an inhibitor of the proteasome and NF-kB (Attar et al., Clin Cancer Res, 2008;14:1446-54), it is possible that in future studies elevated CD74 levels can be used not only for prognostication, but also to stratify CN-AML pts to study of bortezomib-containing chemotherapy regimens. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Importance of MN1 Transcript Levels, and Biologic Insights From MN1-Associated Gene and MicroRNA Expression Signatures in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

PURPOSE To determine the prognostic importance of the meningioma 1 (MN1) gene expression levels in the context of other predictive molecular markers, and to derive MN1 associated gene- and microRNA-expression profiles in cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS MN1 expression was measured in 119 untreated primary CN-AML adults younger than 60 years by real-time reverse-transcriptase polymerase chain reaction. Patients were also tested for FLT3, NPM1, CEBPA, and WT1 mutations, MLL partial tandem duplications, and BAALC and ERG expression. Gene- and microRNA-expression profiles were attained by performing genome-wide microarray assays. Patients were intensively treated on two first-line Cancer and Leukemia Group B clinical trials. Results Higher MN1 expression associated with NPM1 wild-type (P < .001), increased BAALC expression (P = .004), and less extramedullary involvement (P = .01). In multivariable analyses, higher MN1 expression associated with a lower complete remission rate (P = .005) after adjustment for WBC; shorter disease-free survival (P = .01) after adjustment for WT1 mutations, FLT3 internal tandem duplications (FLT3-ITD), and high ERG expression; and shorter survival (P = .04) after adjustment for WT1 and NPM1 mutations, FLT3-ITD, and WBC. Gene- and microRNA-expression profiles suggested that high MN1 expressers share features with high BAALC expressers and patients with wild-type NPM1. Higher MN1 expression also appears to be associated with genes and microRNAs that are active in aberrant macrophage/monocytoid function and differentiation. CONCLUSION MN1 expression independently predicts outcome in CN-AML patients. The MN1 gene- and microRNA-expression signatures suggest biologic features that could be exploited as therapeutic targets.

Gene mutations and response to treatment with all-trans retinoic acid in elderly patients with acute myeloid leukemia. Results from the AMLSG Trial AML HD98B

In a previous randomized trial, AML HD98B, we showed that administration of all-trans retinoic acid in addition to intensive chemotherapy improved the outcome of older patients with acute myeloid leukemia. The objectives of this study were to evaluate the prognostic impact of gene mutations and to identify predictive genetic factors for the all-trans retinoic acid treatment effect. Data from mutation analyses of the NPM1, CEBPA, FLT3, and MLL genes were correlated with outcome in patients 61 years and older treated within the AML HD98B trial. The frequencies of mutations were: NPM1, 23%; CEBPA, 8.5% (analysis restricted to patients with a normal karyotype); FLT3 internal tandem duplications (ITD), 17%; FLT3 tyrosine kinase domain mutations, 5%; and MLL partial tandem duplications, 4.5%. The genotype mutant NPM1 was positively and adverse cytogenetics as well as higher white blood cell count negatively correlated with achievement of complete remission. In Cox regression analysis, a significant interaction between the genotype mutant NPM1 without FLT3-ITD and treatment with all-trans retinoic acid was identified, in that the beneficial effect of all-trans retinoic acid on relapse-free and overall survival was restricted to this subgroup of patients. Other significant factors for survival were age, adverse cytogenetics, and logarithm of white cell count. In elderly patients with acute myeloid leukemia, NPM1 mutations are associated with achievement of complete remission, and the genotype 'mutant NPM1 without FLT3-ITD' appears to be a predictive marker for response to all-trans retinoic acid given as an adjunct to intensive chemotherapy (ClinicalTrials.gov Identifier: NCT00151242).

Calcineurin inhibitor-free GVHD prophylaxis with sirolimus, mycophenolate mofetil and ATG in Allo-SCT for leukemia patients with high relapse risk: an observational cohort study

Certain leukemias have a high relapse risk even after allo-SCT, and GVHD prophylaxis with calcineurin inhibitors (CNIs) may interfere with a possible GVL effect. Therefore, we replaced CYA by sirolimus in patients with high relapse risk. In contrast to CNIs, sirolimus promotes the generation of regulatory T-cells and has potent antineoplastic activity. Sirolimus has been used in combination with CNI for GVHD prophylaxis in hematopoietic SCT. However, no CNI-free prophylactic regimen with sirolimus has been evaluated so far. Within the FLAMSA-RIC protocol, 15 patients received GVHD prophylaxis with sirolimus and mycophenolate mofetil (MMF). The underlying diagnoses were relapsed or refractory T-ALL (n=3), AML with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) or mixed-lineage leukemia-partial tandem duplication (MLL-PTD; n=10; 5 with refractory disease) and CML in refractory myeloid blast crisis (n=2). All evaluable patients (n=14) were engrafted. Grades II-IV acute GVHD occurred in 21% and chronic GVHD in 30% of patients. Non-relapse mortality rate was 14%. No thrombotic microangiopathy or sinusoidal obstruction syndrome was observed. Three patients with FLT3-ITD+ AML relapsed after a median of 112 days. At a median follow-up of 10 months after transplantation, 10 patients are alive and in complete remission. In conclusion, sirolimus-based GVHD prophylactic regimens deserve further investigation.

RUNX1 Mutations Play a Major Role in the Progression of MDS to S-AML Following MDS: A Genetic and Cytogenetic Analysis of Sequential Samples

The genetic and cytogenetic events underlying the development of myelodysplastic syndrome (MDS) as well as the mechanisms leading to progression of MDS into acute myeloid leukemia (AML) are largely unknown. Activating mutations in receptor tyrosine kinases like FLT3 or members of the RAS family have been implicated in this process. Recently, mutations of RUNX1 (AML1) have been detected in MDS as well as in AML. As the development of AML is considered as a multistep process, in which mutations of genes with proliferative potential (class I) cooperate with gene mutations in hematopoietic transcription factors (class II), we hypothesized that similar mechanisms might be responsible for the progression of MDS into s-AML. Therefore, we investigated a cohort of 21 MDS patients (pts) (14 male, 7 female; median age at diagnosis of MDS=67.1/range 49.1–76.5), with WHO categories: MDS RARS (n=1), MDS RCMD-RS (n=1), MDS RAEB-1 (n=8), MDS RAEB-2 (n=3), MDS/AML (n=1), CMML-1 (n=1), MDS/MPS overlap (n=1), MDS not further classifiable (n=4) and one patient with reactive condition in morphology only, but del(5q) in chromosome banding analysis. In all cases specimens at the time of progression into s-AML following MDS were available and all specimens were characterized for RUNX1 mutations (entire coding region), FLT3-ITD, MLL partial tandem duplications (MLL-PTD), NPM1-exon 12 mutations and NRAS (codons12/13 and 61) mutations. Furthermore, in all cases chromosome banding analyses (CBA) was performed. At diagnosis of MDS, a normal karyotype (NK) was present in 11/21 cases, whereas cytogenetic aberrations were detected in the remaining 10 cases (del(5q) (n=2), del(20q) (n=1), +8 (n=2), complex (n=2), −Y (n=1), t(1;3)(p36;q21) (n=1) and t(1;14) (p34;q32), −7 (n=1)). The following molecular aberrations were detected at diagnosis of MDS: RUNX1 (n=5), MLL-PTD (n=1), NRAS (n=1), NPM1 (n=1), but never FLT3-ITD. Only one pt had two of these molecular aberrations in combination at the MDS stage (RUNX1/NPM1). After progression to s-AML, 3 of the 11 pts with NK at the MDS stage had developed cytogenetic aberrations (+8 (n=1), +11 (n=1), del(5q), del(7q) (n=1)). The other 10 already at the MDS stage cytogenetically aberrant cases remained unchanged. Thus at the s-AML stage there were 8 pts with NK and 13 with cytogenetic aberrations. At this stage the cohort was again characterized for molecular aberrations and following gains were detected: RUNX1 (n=3), MLL-PTD (n=2), NRAS (n=1), NPM1 (n=1) and FLT3-LM (n=2). The median time between diagnosis of MDS and s-AML was 207 days (d) (range 27–960 d). However, no significant difference could be detected for pts with RUNX1 mutations at the MDS stage (n=5, mean=210 d) vs. the remaining cohort (n=16, mean=266 d) (p=0.536, t-test) and pts with NK at the MDS stage (n=11, mean=200 d) vs. pts with cytogenetic aberrations (n=10, mean=324 d) (p=0.190). The pattern of acquisition of the different aberrations was complex and therefore the cohort was divided in three different subgroups based on the cytogenetic profile:cytogenetically stable, NK: this group included pts without molecular aberration at all stages (n=5), pts with RUNX1 mutation at the MDS stage (n=2), where one acquired secondary a MLL-PTD and the other a FLT3-ITD and one pt, who acquired a RUNX1 mutation at progression.cytogenetically stable, AK: 3 pts without molecular aberrations, 4 pts with molecular aberrations (RUNX1, RUNX1/NPM1, MLL-PTD, NRAS), where the pt with RUNX1/NPM1 acquired FLT3-ITD at progression and 3 pts, who acquired molecular aberrations at progression (RUNX1, NPM1, NRAS).cytogenetically unstable: 2 pts without aberrations at MDS, who progressed with an AK with or without RUNX1 mutation and one pt with a RUNX1 mutation, who progressed with an additional AK plus MLL-PTD.In conclusion, RUNX1 mutations, when present at the MDS stage, seem to play a role in the progression to s-AML following MDS. Cooperating molecular events for progression to s-AML are the acquisition of FLT3-ITD (n=2) and MLL-PTD (n=2). FLT3-ITD was never detected at the MDS stage in our cohort and seems to be a strong indicator for the progression to s-AML, whereas MLL-PTD can be already detected at earlier stages. Finally, the combination of RUNX1 mutation with FLT3-ITD or MLL-PTD was always associated with progression.

Importance of FLT3 Mutations and Other Genetic Abnormalities in Normal Karyotype AML.

Cytogenetic findings at diagnosis are currently considered the most important prognostic factor in AML. By far, the most common cytogenetic finding, found in approximately 40–50% of patients, is a normal karyotype (defined as detection of only normal chromosomes in 20 or more marrow cells). Recent molecular analyses of leukemic blasts in cytogenetically normal AML (CN-AML) have revealed a striking heterogeneity with regard to the presence of acquired gene mutations and changes in gene expression. Multiple submicroscopic genetic alterations with prognostic significance have been discovered, including mutations of the FLT3 gene, both internal tandem duplications (ITDs) and point mutations (TKDs), partial tandem duplication (PTD) of the MLL gene, mutations in the NPM1, CEBPA, and WT1 genes, and high expression of the BAALC, ERG, and MN1 genes. Most of these abnormalities, except for mutations in NPM1 and CEBPA, have been associated with an adverse prognosis. CN-AML patients with FLT3 ITD, NPM1 wild type, or both have worse outcome in the range of 30% long-term survival, unless they also harbor CEBPA mutations. A high expression of the FLT3 ITD allele compared to the FLT3 wild type allele confers an especially poor prognosis. In contrast, CN-AML patients without a FLT3 ITD and with NPM1 mutations have a particularly good outcome in the range of 60% long-term survival, unless they present with high ERG expression. The prognostic significance of FLT3 TKD mutations is controversial, and, recently, the outcome of patients with the MLL PTD has improved when treated with induction therapy, including etoposide and post induction therapy with autologous transplantation. As a result, the 2008 World Health Organization classification of AML recommends evaluation of FLT3, NPM1, and CEBPA mutations in CN-AML. Application of gene expression profiling has also identified signatures that appear to separate CN-AML patients into prognostic subgroups, and, more recently, a microRNA expression signature was found to be associated with outcome in molecular high-risk CN-AML patients (i.e., those with FLT3 ITD, NPM1 wild type, or both). These and similar future findings are likely to have a major impact on the clinical management of CN-AML not only in prognostication, but also in selection of appropriate treatment, since many of the identified genetic alterations already constitute (e.g., FLT3 mutations for tyrosine kinase inhibitors) or will potentially become targets for specific therapeutic interventions.

The Mll partial tandem duplication: differential, tissue-specific activity in the presence or absence of the wild-type allele

AbstractThe partial tandem duplication of MLL (MLL-PTD) is found in 5% to 10% of patients with acute myeloid leukemia (AML) and normal cytogenetics. Its expression in leukemic blasts is coincident with a silenced wild-type (WT) MLL allele. We therefore generated mice expressing the Mll-PTD in the absence of Mll-WT. These MllPTD/− mice die at birth unlike the normal life expectancy of MllPTD/WT, MllWT/−, and MllWT/WT mice. Using MllWT/WT fetal liver cells (FLC) as baseline, we compared MllPTD/− with MllPTD/WT FLC and found both had increased HoxA gene expression and granulocyte-macrophage colony-forming progenitor cells (CFU-GM); in contrast, only MllPTD/WT FLC had increased pluripotent hemopoietic progenitors (CFU-GEMM). The similarities between MllPTD/WT and MllPTD/− mice suggest that the Mll-PTD mutation can up-regulate target genes in a dominant, gain-of-function fashion. The differences between these 2 genotypes suggest that in select tissues the Mll-PTD requires cooperation with the Mll-WT in the genesis of the observed abnormality.

DNA hypermethylation and epigenetic silencing of the tumor suppressor gene, SLC5A8, in acute myeloid leukemia with the MLL partial tandem duplication

AbstractPosttranslationally modified histones and DNA hypermethylation frequently interplay to deregulate gene expression in cancer. We report that acute myeloid leukemia (AML) with an aberrant histone methyltransferase, the mixed lineage leukemia partial tandem duplication (MLL-PTD), exhibits increased global DNA methylation versus AML with MLL-wildtype (MLL-WT; P = .02). Among the differentially methylated genes, the SLC5A8 tumor suppressor gene (TSG) was more frequently hypermethylated (P = .003). In MLL-PTD+ cell lines having SLC5A8 promoter hypermethylation, incubation with decitabine activated SLC5A8 expression. Ectopic SLC5A8 expression enhanced histones H3 and H4 acetylation in response to the histone deacetylase inhibitor, valproate, consistent with the encoded protein—SMCT1—short-chain fatty acid transport function. In addition, enhanced cell death was observed in SMCT1-expressing MLL-PTD+ AML cells treated with valproate. Within the majority of MLL-PTD AML is a mechanism in which DNA hypermethylation silences a TSG that, together with MLL-PTD, can contribute further to aberrant chromatin remodeling and altered gene expression.

Wilms’ Tumor 1 Gene Mutations Independently Predict Poor Outcome in Adults With Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

To analyze the prognostic impact of Wilms' tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS We studied 196 adults younger than 60 years with newly diagnosed primary CN-AML, who were treated similarly on Cancer and Leukemia Group B (CALGB) protocols 9621 and 19808, for WT1 mutations in exons 7 and 9. The patients also were assessed for the presence of FLT3 internal tandem duplications (FLT3-ITD), FLT3 tyrosine kinase domain mutations (FLT3-TKD), MLL partial tandem duplications (MLL-PTD), NPM1 and CEBPA mutations, and for the expression levels of ERG and BAALC. Twenty-one patients (10.7%) harbored WT1 mutations. Complete remission rates were not significantly different between patients with WT1 mutations and those with unmutated WT1 (P = .36; 76% v 84%). Patients with WT1 mutations had worse disease-free survival (DFS; P < .001; 3-year rates, 13% v 50%) and overall survival (OS; P < .001; 3-year rates, 10% v 56%) than patients with unmutated WT1. In multivariable analyses, WT1 mutations independently predicted worse DFS (P = .009; hazard ratio [HR] = 2.7) when controlling for CEBPA mutational status, ERG expression level, and FLT3-ITD/NPM1 molecular-risk group (ie, FLT3-ITD(negative)/NPM1(mutated) as low risk v FLT3-ITD(positive) and/or NPM1(wild-type) as high risk). WT1 mutations also independently predicted worse OS (P < .001; HR = 3.2) when controlling for CEBPA mutational status, FLT3-ITD/NPM1 molecular-risk group, and white blood cell count. We report the first evidence that WT1 mutations independently predict extremely poor outcome in intensively treated, younger patients with CN-AML. Future trials should include testing for WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML.

Low Meningioma 1 (MN1) gene expression to predict outcome in cytogenetically normal acute myeloid leukemia (CN-AML): A Cancer and Leukemia Group B (CALGB) study

7011 Background: Expression of the MN1 gene, which encodes a transcription co-activator implicated in myeloid leukemogenesis, has been shown to predict outcome in CN-AML patients (pts) in 1 study (Heuser et al. Blood 2006;108:3898). Here we present the first validation of the prognostic impact of MN1 expression in CN-AML in the context of not only mutated (mut) FLT3 and NPM1 and MLL-partial tandem duplication but also other established prognostic markers (i.e., CEBPAmut, WT1mut, ERG and BAALC expression levels). Methods: MN1 expression was measured by quantitative real-time RT-PCR in pretreatment bone marrow samples from 119 de novo CN-AML adult pts <60 years with material available for MN1 expression analyses who were treated on CALGB protocols 9621 and 19808. MN1 copy numbers were measured and normalized to ABL (internal control) copy numbers. Pts were dichotomized at the MN1 median expression and analyzed for outcome. Results: At diagnosis, low MN1 expression was associated with NPM1mut (P<0.0001) and the molecular low risk group (FLT3-internal tandem duplication (ITD)negative and NPM1mut; P<0.001). Complete remission (CR) rate was higher in low MN1 pts (93% v 73%); low MN1 pts were 6 times more likely to achieve a CR than high MN1 pts (P=0.005), once adjusting for hemoglobin (P=0.01) (Table). With a median follow-up of 4.6 years, low MN1 pts also had longer disease-free survival (DFS) (P<0.005; 3-year DFS: 60% v 32%) and overall survival (OS) (P<0.001; 3-year OS: 68% v 40%) than high MN1 pts. In multivariable analyses, MN1 expression independently predicted for DFS (P=0.01) and OS (P=0.03). For low MN1 pts, the risk for an event was reduced by approximately 50% compared with high MN1 pts (Table). Conclusions: MN1 expression predicts outcome independent of other established molecular markers in CN-AML pts. Particularly, low MN1 expression defines a group of CN-AML pts with favorable clinical outcome. Multivariable analyses for achievement of CR, DFS and OS Variables in Final Models CR DFS OS OR * P HR ** P HR ** P MN1 expression, low v high 6.4 0.005 0.46 0.01 0.54 0.03 WT1, mutated v wild-type – – 5.6 <0.0001 7.0 <0.0001 FLT3-ITD/NPM1 status, high v low risk – – – – 3.0 0.005 WBC, each 50 unit increase – – 2.1 0.002 1.7 <0.0001 Hemoglobin 1.7 0.01 – – – – * OR: odds ratio; ** HR: hazard ratio. Variables considered in the models were those significant at α=0.20 in univariable analyses as follows: for CR, MN1 expression, FLT3-ITD/NPM1 status (molecular high risk is FLT3-ITDpositive and/or NPM1wt; molecular low risk is FLT3-ITDnegative and NPM1mut), WBC, hemoglobin, age, race; for DFS, MN1 expression, FLT3-ITD/NPM1 status, ERG expression, WT1 and CEBPA mutations, WBC, race; for OS, MN1 expression, FLT3-ITD/NPM1 status, ERG and BAALC expression, WT1 mutations, WBC, age, race, hemoglobin, % blood blasts, extramedullary involvement. No significant financial relationships to disclose.

Mutations of GATA1, FLT3, MLL-partial tandem duplication, NRAS, and RUNX1 genes are not found in a 7-year-old Down syndrome patient with acute myeloid leukemia (FAB-M2) having a good prognosis

The prognosis of leukemia developed in Down syndrome (DS) patients has improved markedly. Most DS leukemia occurs before 3 years of age and is classified as acute megakaryocytic leukemia (AMKL). Mutations in the GATA1 gene have been found in almost all DS patients with AMKL. In contrast, it has been shown that occurrence of DS acute myeloid leukemia (DS-AML) after 3 years of age may indicate a higher risk for a poor prognosis, but its frequency is very low. Age is one of the significant prognostic indicators in DS-AML. The prognostic factor of gene alterations has not been reported in older DS-AML patients. We here describe the case of a 7-year-old DS boy with AML-M2, who had no history of transient abnormal myelopoiesis or any clinical poor prognostic factors, such as high white blood cell counts or extramedullary infiltration. We molecularly analyzed the GATA1, FLT3, MLL-partial tandem duplication, NRAS, and RUNX1 (previously AML1) genes and did not detect any alterations. The patient has lived for more than 5 years after treatment on the AML99-Down protocol in Japan. This suggests that a patient lacking these genes alterations might belong to a subgroup of older DS-AML patients with good prognosis. Accumulation of more data on older pediatric DS-AML patients is needed.

Differences in Cyto- and Molecular Genetic Abnormalities between Children

Young children (defined as <2 years old) with acute myeloid leukemia (AML) do not differ in outcome when compared with older children with AML. Previously, distinct cytogenetic aberrations specific for AML in young children have been reported, such as t(7;12), and t(1;22), which is found exclusively in FAB M7. Moreover, young children with AML are characterized by a high frequency of 11q23-rearrangements. However, so far, no information is available on differences in the molecular genetic background of these two age groups. We therefore retrospectively investigated the distribution of different cytogenetic and molecular aberrations in a large cohort (n=435) of pediatric AML cases, of which 75 (17%) were young children. The predominant cytogenetic aberration in infant AML consisted of 11q23-rearrangements, which occurred in 44% of young children versus 17% in older children (p=<0.005), without differences in the distribution of 11q23-translocation partners. We also found significant differences in other cytogenetic subgroups of AML between young and older children, i.e. normal karyotype, 5% vs. 18%, respectively (p=0.008) and complex karyotype, 12% vs. 5% (p=0.03). t(7;12) (n=3) and t(8;16) (n=3) were only detected in young children, in contrast to t(15;17) (n=16) and t(8;21) (n=44), which were only seen in older children. Patients were also screened for molecular abnormalities, including the mutational hotspots of c-KIT (n=229), FLT3 (n=230), N-RAS (n=187), K-RAS (n=187), PTPN11 (n=216), MLL-partial tandem duplications (MLL-PTD) (n=240) and NPM1 (n=291). In the overall cohort, a significantly different age distribution was found for NPM1 mutations (0% young vs. 9% in older children; p=0.05) and FLT3-ITD (0% vs. 21%, respectively; p=0.005). Mutations in the other genes showed no clear correlation with age. Several non-random associations between molecular and cytogenetic abnormalities were detected. 89% of c-KIT mutations were associated with core-binding factor AML in children ≥2 years old. In young children, 2/4 c-KIT-mutated cases were associated with an MLL-rearrangement. NPM1 and FLT3-ITD mutations in older children were significantly correlated with normal karyotype AML (57% of NPM1 mutations, and 75% of FLT3/ITD; p=<0.005). In young children, 71% of RAS mutations were associated with an 11q23-rearrangement vs. 28% in older children (p=0.08). In older children however, 41% of the RAS mutations were associated with a normal karyotype. These data suggest that young children with AML are characterized by differences in the type and frequency of cytogenetic and molecular genetic abnormalities when compared with older children with AML, possibly reflecting differences in underlying biology between these age-groups. These differences may become clinically relevant in the era of molecularly targeted therapy.