Cytogenetically Normal Acute Myeloid Leukemia Research Articles

Differential Clinical Impact Of Gene Mutations and Their Combinations In Primary Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

In CN-AML, mutations of specific genes have prognostic significance and are used for treatment guidance. However, these mutations are in general not mutually exclusive and CN-AML patients (pts) frequently harbor mutational combinations with unclear prognostic significance because of the concurrent presence of mutations with favorable (CEBPA, NPM1) and unfavorable (ASXL1, DNMT3A, IDH1, IDH2, FLT3-ITD, MLL-PTD, RUNX1, TET2, WT1) impact on outcome. Here, we report the frequency and clinical significance of isolated mutations and their combinations in 364 CN-AML pts. Younger [<60 years (y); n=163] and older (≥60 y; n=201) pts were intensively treated on frontline CALGB/Alliance protocols and, per protocol, did not undergo allogeneic stem cell transplantation in first complete remission (CR). Median follow-up for pts alive was 8.7 y. Pts were analyzed centrally for mutations in the ASXL1, CEBPA, DNMT3A, IDH1, IDH2, NPM1, RUNX1, TET2, and WT1 genes and the presence of FLT3-ITD, FLT3-TKD and MLL-PTD. We found that 99% of pts had ≥1 and 88% ≥2 of the 12 mutations. Specifically, 11% of pts had 1 gene mutated (n1), 40% had 2 (n2), 31% 3 (n3), and 18% harbored ≥4 mutated genes (n4). The distribution of the different n groups in younger and older pts was similar (Fig 1). The expected frequency of each mutated gene in every numerical (n) group was calculated as follows: [no. of pts with the mutated gene] x [total no. of mutations in the n group] / [total no. of mutations in all pts], and compared with the observed frequency. CEBPA, IDH2, NPM1, and RUNX1 mutations were overrepresented in the n1 and n2 groups (P<.001, P<.001, P=.002, P=.02, respectively) and thus likely represent “early” events in leukemogenesis. In contrast, DNMT3A, FLT3-ITD, and TET2 mutations were more frequent in the n3 and n4 groups (P=.008, P<.001, P=.02, respectively) suggesting that they are likely “late” events. The distribution among age groups and clinical impact of the “early” mutations (i.e., CEBPA, NPM1, IDH2) presenting as isolated mutations or in combination with other mutations were then investigated. Isolated RUNX1 mutations could not be evaluated due to small sample size. Younger pts were more likely to harbor an isolated “early” mutated gene than older pts (21% v 12%, P=.03). Specifically, isolated CEBPA and NPM1 mutations were more frequent in younger pts compared with the older (7% v 2%, P=.007; 7% v 3%, P=.07, respectively). In pts with isolated CEBPA mutations, these were always bi-allelic. Single “early” mutated genes varied in their impact on CR achievement (CEBPA: 100%, NPM1: 94%, IDH2: 67%), and overall survival (OS; 3-y OS: CEBPA: 87%, NPM1: 69%, IDH2: 22%). Acquisition of additional mutations adversely modified the prognostic significance of favorable “early” mutations in the CEBPA (Fig 2A) and NPM1 (Fig 2B) genes, but left unchanged the unfavorable effect of IDH2 mutations (Fig 2C). Two mutational combinations were exceptions to this: younger pts with both NPM1 and DNMT3A mutations (8 pts) had an excellent prognosis with 3-y OS of 100%; and older pts with concurrent NPM1 and IDH2 mutations (9 pts; all IDH2 mutations were in codon R140) had a high 3-y OS rate of 67%. We conclude that CEBPA and NPM1 mutations mainly define pts with favorable prognosis when they present as single markers, whereas mutated IDH2 predominantly associates with adverse outcome. The NPM1+DNMT3A and NPM1+IDH2 mutational patterns may define novel favorable molecular subtypes in younger and older CN-AML pts, respectively, but these results require corroboration in larger pt cohorts. [Display omitted] [Display omitted] Disclosures:Mrózek:AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 611 category Other. Eisfeld:AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 608 category Other.

ERG Overexpression Is Highly Associated With ERG Gene Amplifications In Patients With Myeloid Malignancies

BackgroundThe proto-oncogene ERG is located on chromosome 21q22 and overexpression of ERG was shown to have an adverse impact on outcome in cytogenetically normal AML (CN-AML). Acquired gain of chromosome 21 (+21) is a recurrent cytogenetic abnormality in AML, however, the pathogenetic impact remains elusive. Data indicate that +21 could be the underlying mechanism for altered expression of genes located in the respective regions, such as ERG, which might contribute to the pathogenesis in myeloid malignancies with +21. Aims1) To investigate a possible relationship between ERG copy numbers and variations in ERG mRNA expression levels in patients (pts) with myeloid malignancies. 2) Analysis of ERG expression in CN-AML to reveal a possible association between ERG overexpression and other relevant molecular markers and to ascertain the prognostic impact of ERGin context of these markers. MethodsERG mRNA expression and ERG copy numbers were analyzed using a hydrolysis probe based real-time PCR assay. 1) To address ERG expression in relation to +21 the following cohorts were analyzed: 62 AML pts with a complex karyotype including +21 (CK+21); 44 AML pts with a non-complex aberrant karyotype including +21 (AK+21); 19 pts with various myeloid malignancies (10 de novo AML, 3 MDS, 1 s-AML, 2 t-AML, 1 MDS/AML, 1 t-MDS, 1 MDS/MPN) all of them showing ERG amplification by interphase FISH and array CGH (ERG-Amp). In addition, 32 CK-AML and 330 CN-AML pts without +21 or ERGamplification were analyzed. Results were expressed as mean values±SEM. Expression levels/DNA copy numbers were compared by t-test. 2)ERG expression was assessed in a cohort of 330 patients (<65 years) with de novo CN-AML. Female/male ratio was 169/161. To distinguish low from high ERG expressers the median %ERG/ABL1 level was used. BAALC expression levels were analyzed accordingly. Expression levels were correlated with clinical outcome and with the presence of mutations (mut) in ASXL1 (n=330), CEBPA (n=330), DNMT3A (n=261), FLT3-TKD (n=330), IDH1R132 (n=328), IDH2R140 (n=328), IDH2R172 (n=328), NPM1 (n=330), NRAS (n=330), RUNX1 (n=329), TET2 (n=166), WT1 (n=329) and FLT3-ITD (n=330), and with MLL-PTD (n=330) and BAALCexpression (n=328). Results1) Analysis of pts with +21 or ERG-Amp revealed significantly higher expression levels of ERG in pts with +21 (AK+21 and CK+21 combined, 331 ± 28) and ERG-Amp (606 ± 127) as compared to pts with CN-AML (229 ± 10; p=0.001, p=0.008, respectively) or CK-AML (177 ± 36; p=0.001, p=0.004, respectively). Mean ERG expression was even higher in pts with ERG-Amp (606 ± 127) as compared to pts with +21 (331 ± 28, p=0.047). Quantification of ERG copy numbers on DNA level showed good correlation to ERG mRNA expression, with abundantly higher ERG DNA amount in +21 (3.25 ±0.14) and moreover in ERG-Amp (9.21 ±1.05) as compared to CN-AML and CK-AML (combined: 2.20 ± 0.05; p<0.001 and p<0.001, respectively). 2) In CN-AML, %ERG/ABL1 levels ranged from 0.078 to 1,016.027 (median: 188.904). High ERG expression was associated with lower age (mean: 48.7 vs. 52.4 years, p=0.002), higher white blood cell (WBC) count (mean: 64.8 vs. 44.4 x 109/L, p=0.019), FLT3-ITDmut/wt ratio≥0.5 (51/165, 30.9% vs. 24/165, 14.5%, p=0.001), IDH2R172mut (6/164, 3.7% vs. 0/164, 0.0%, p=0.030) and high BAALC expression (104/164, 63.4% vs. 59/164, 36.0%, p<0.001), as compared to low ERG expression. In contrast, NPM1mut (91/165, 55.2% vs. 118/165, 71.5%, p=0.003), IDH1R132mut (10/165, 6.1% vs. 29/163, 17.8%, p=0.001) and TET2mut (10/76, 13.2% vs. 22/90, 24.4%, p=0.077) were less frequent in high ERG expressers. Survival analysis revealed inferior overall survival (OS at 3 years: 52.2% vs. 68.7%, p=0.021) and event free survival (EFS at 3 years: 34.6% vs. 43.1%, p=0.052) for high ERG expressers as compared to low ERG expressers. In a multivariate analysis adjusted for age, WBC, BAALC expression, FLT3-ITDmut/wt ratio≥0.5, MLL-PTD and WT1mut, high ERG expression revealed a trend towards an adverse impact on OS (p=0.069), while no impact on EFS was observed. Conclusions1) Gain of chromosome 21 and especially amplification of chromosomal band 21q22 is a mechanism for ERG overexpression. This might indicate ERG as an important factor contributing to the pathogenesis and progression of myeloid malignancies with gain of chromosome 21. 2) In CN-AML, ERG overexpression is associated to several molecular markers and has a negative impact on OS and EFS. Disclosures:Weber:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Noel:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

IDH1 and IDH2 Mutations Are Frequent Genetic Alterations In Cytogenetically Normal Acute Myeloid Leukemia With Adverse Outcome

BackgroundThe isocitrate dehydrogenase (IDH1/IDH2) genes are recently identified to be frequently mutated in acute myeloid leukemia (AML) patients. The IDH1 is located in the cytosol and the IDH2 in the mitochondrion and are normally involved in citrate metabolism in the tricarboxylic acid cycle. The mutant IDH1 (R132) and IDH2 (R140 or R172) acquire neomorphic enzymatic activity by converting α-ketoglutarate (α-KG) to an accumulation of 2-hydroxyglutarate (2-HG) and receive epigenetic defects. TET2, a DNA demethylase and α-KG dependent enzyme and essential for demethylation, interrupt the function when the IDH1 or IDH2 genes are mutated and results in hypermethylation and impaired hematopoietic differentiation. AimTo investigate the frequency and outcome of the acquired IDH1/IDH2 mutations in cytogenetically normal AML (CN-AML) cohort. Materials and Methods207 de novo AML patients were genotyped for IDH1 and IDH2 mutations. The analysis was performed by a PCR reaction and the PCR product was purified and used for direct sequencing. Kaplan-Meier survival analysis with log rank test was used to evaluate the impact of different IDH1 and IDH2 genotypes on overall survival (OS) calculated as time from diagnosis until death, date of the latest follow-up, or date of bone marrow transplantation. ResultsIDH1 mutations in exon 4 codon132 were found in 16/207 (7.7%) of the patients and four different amino acid exchanges were detected (R132C, R132H, R132G, R132L). IDH2 mutations in exon 4 in codon 140 and codon 172 were found in 21/207 (10.1%) and 6/207 (2.9%) patients, respectively. Two different amino acid exchanges were identified in codon 140 (R140Q and R140G) and in codon 172 all exchanges were to the same amino acid (R172K). There was a significant impact on OS for patients carrying mutated IDH2 gene in codon 140 compared with patients carrying wildtype IDH2 gene (p=0.009), the mean OS was 3 vs. 21 months. This was seen in the intermediate risk patient group with CN-AML ((IDH2 mutated n=9, IDH2 wildtype n=67). Due to the low frequency with codon 172 mutations (n=6), no independent effect could be detected. Neither in the entire patient group nor subdivided in different risk groups, IDH1 mutations had any significance compared with the wildtype IDH1 patients. ConclusionsOverall, IDH1 and IDH2 mutations were identified in 20.8% in de novo AML in this cohort. IDH1 and IDH2 mutations were mutually exclusive. A significant impact in overall survival with adverse outcome was seen in patients with IDH2 mutations (R140) in the intermediate risk patient group with CN-AML. Previously, no other studies have reported any impact on overall survival for patients with IDH2 (R140) mutations. Other studies have reported that the accumulation of 2-HG is enough to promote leukemogenesis when IDH1 or IDH2 mutations arise. Further other studies have demonstrated that it is possible to target the 2-HG metabolite and get the AML blasts to differentiate. Thus, AML patients with IDH1 or IDH2 mutations may be a new patient group for risk stratification and new treatment options like upfront allogeneic stem cells transplantation. Disclosures:No relevant conflicts of interest to declare.

The Clinical and Prognostic Influence Of Mutations In The Cohesin Complex In Acute Myeloid Leukemia

BackgroundThe cohesin complex presents a ring structure that regulates chromosome segregation during meiosis and mitosis and is thus essential for cell division. Genes that belong to the cohesin complex in somatic vertebrate cells are SMC1A, SMC3, RAD21 (SCC1), STAG2 (SA-2) and STAG1 (SA-1). Recently, mutations in the cohesin complex have been reported to occur in AML. The aim of this study was to evaluate the clinical and prognostic implications of cohesin mutations in 389 uniformly treated younger AML patients. Patients and MethodsDiagnostic bone marrow or peripheral blood samples were analyzed from 389 younger adult patients with de novo (n=348) or secondary AML (n=41) with French-American-British (FAB) classification M0-M2 or M4-M7, who were entered into the multicenter treatment trials AML SHG 0199 (n=276) or AML SHG 0295 (n=113). Patient samples were assessed for frequently occurring mutations including FLT3-ITD, NPM1, DNMT3A, IDH1, IDH2. In the subgroup of cytogenetically normal AML (CN-AML), additional mutation analyses were performed for CEBPA, MLL-PTD, WT1 and WT1 SNP rs16754, NRAS, and expression levels of BAALC, ERG, EVI1, MN1, MLL and WT1. Sequencing of all coding exons of the cohesin genes was performed with the SOLiDTM system (Life Technologie, Darmstadt, Germany). Sequences were analyzed twice separately using the DNAnexus software and a separate pipeline of bioinformatics software. Identified mutations were validated by Sanger sequencing. Results27 patients (7%) were identified to carry a mutation in one of the cohesin genes. 10 patients harboured a mutation in STAG1. The second most frequently mutated gene in the cohesin complex was SMC3 with 7 patients showing this mutation. 5 patients harboured a mutation in RAD21, 3 patients in STAG2 and 2 patients in SMC1A. All mutations were mutually exclusive. The majority of mutations were missense mutations, apart from 2 frameshift mutations and 3 nonsense mutations. All evaluable patients lost their cohesin mutation during remission and with the exception of one patient regained their mutation during relapse.Patients with mutations in the cohesin genes were older (47 vs 44 years, P=.04), had a higher white blood cell count (WBC) (P=.046) and a higher peripheral blast count (P=.048). We observed a strong correlation between cohesin gene and NPM1 mutations (P=.007). BAALC expression was lower in patients with a mutation compared to patients without a mutation in the cohesin genes (P=.026).In order to get a better understanding of when mutations in the cohesin complex occur during clonal evolution we evaluated the allelic burden of mutations in the cohesin complex. Because of the strong association between NPM1 and cohesin mutations, the allelic ratio of mutated and wildtype NPM1 was compared to the allelic ratio of mutated and wildtype cohesin genes. Interestingly, we found a similar mutation burden between NPM1 and genes of the cohesin complex (Pearson correlation coefficient R=0.67), suggesting that cohesin gene mutations occurred in the same clone as NPM1 mutations.When considering all mutations in the cohesin complex as one group, overall survival (OS), relapse free survival (RFS) and complete remission rate (CR) were not influenced by the presence of cohesin mutations (OS: HR 1.23; 95%CI 0.77-1.99; P=.4; RFS: HR 0.92; 95%CI 0.51-1.65; P=.77; CR: mutated 77% vs wildtype 76%, P=.83). We next evaluated the prognostic influence of each gene in the cohesin complex separately in all patients. STAG1, SMC3 and RAD21 mutations had no influence on OS and RFS. A trend for a reduced OS was observed for STAG2 mutated patients (OS: HR 3.02; 95%CI 0.97-9.46; P=.057), although the analysis is limited by the small number of mutated patients. In the subgroup of patients with cytogenetically normal AML we did not identify a difference in OS, RFS or CR rates for patients with (n=18) or without mutations (283) in the cohesin complex (OS: HR 1.00; 95%CI 0.52-1.92; P=.99; RFS: HR 0.64; 95%CI 0.30-1.39; P=.26; CR rates: mutated 80% vs wildtype 73%, P=.64). ConclusionMutations in the cohesin genes represent novel, recurrent mutations in AML. We found a strong association between these mutations and mutations in NPM1. Cohesin complex mutations as a group had no prognostic impact in all and in cytogenetically normal AML patients. Disclosures:Schlenk:Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Chugai: Research Funding; Ambit: Honoraria.

Heterogeneity Of Patients With Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) and Nucleophosmin-1 (NPM1) Gene Mutation: An Acute Leukemia French Association (ALFA) Study

▪ BackgroundCN-AML with NPM1 mutation accounts for 25% of AML cases and is individualized as a provisional entity in the WHO classification. In this large AML subset, additional gene mutations could influence patient outcome, like it has been shown for bad-prognosis FLT3 internal tandem duplication (FLT3-ITD). We thus reviewed the characteristics and outcome of 409 patients with de novo NPM1-mutated CN-AML treated in sequential ALFA studies. MethodsPatients were treated between 1990 and 2012 (median follow-up, 31 months) according to ALFA-9000 (#16), 9801 (#91), 9802 (#89), 9803 (#19) and 0701 (#84) published protocols or in the ongoing 0702 study (#110). In addition to NPM1, the following mutations were centrally assessed using standard-PCR methods and Sanger sequencing: FLT3-ITD, mutations of FLT3 tyrosine kinase domain (FLT3-TKD, i.e. FLT3D835/I836), IDH1R132, IDH2R140, IDH2R172, TET2 (exons 3-11), and DNMT3A (exons 8-9, 11-23). Primary endpoint was cumulative incidence of relapse (CIR) after censoring the 73 patients who received allogeneic stem cell transplantation (SCT) in first CR at SCT time. Analyses were performed with multiple imputations of missing values, followed by multivariable logistic regression or Cox model. ResultsMedian age was 52 years (17-79). Median white blood cell count (WBC) was 23.5 x 109/L (0.8-377). Gene mutations are indicated in Table 1. Multivariable analyses showed that: 1) FLT3-ITD was associated with higher median WBC (60 versus 18 x 109/L, P<0.001) and less frequent FLT3-TKD, IDH1R132 and IDH2R140 mutations (P=0.003, 0.037 and <0.001, respectively); 2) IDH2R140 mutations were associated with higher median WBC (42 versus 24 x 109/L, P<0.001) and less frequent IDH1R132 and DNMT3AR882 mutations (P<0.001 and 0.001, respectively); 3) TET2 mutations were associated with higher median WBC (37 versus 22 x 109/L, P=0.003) and less frequent FLT3-ITD, FLT3-TKD, and IDH1R132 mutations (P=0.007, 0.028 and 0.022, respectively); and 4) non-R882 DNMT3A mutations, but not R882 mutations, were markedly associated with advanced age (22% in patients ≥ 50 years versus 9% in younger patients; P<0.001). Overall, 372 patients (91%) reached complete remission (CR). In multivariable models, advanced age and higher WBC negatively influenced CR achievement (P=0.044 and 0.037, respectively), but none of the additional mutations. In CR patients, 3-year CIR was 51% (45-58). In a first multivariable model not considering TET2 mutational status (analyzed in 232 patients only), advanced age was associated with a higher specific hazard of relapse (SHR) (P<0.001). At 3 years, CIR was 63% in patients ≥ 50 years versus 35% in younger patients. The other factors associated with a higher CIR were FLT3-ITD (SHR, 2.04 [1.35-3.09]; P=0.001) and non-R882 DNMT3A mutations (SHR, 1.94 [1.10-3.42]; P=0.023), while FLT3-TKD was associated with a lower CIR (SHR, 0.38 [0.16-0.94]; P=0.036). Conversely, WBC, IDH1/2 mutations and R882 DNMT3A mutations did not influence CIR. Advanced age, FLT3-ITD and non-R882 DNMT3A mutations were also identified as independent risk factors in a second multivariable model performed in the subset of patients tested for TET2 mutations. Here, TET2 mutations had no impact on CIR. Overall 3-year CIR estimates were 41%, 61%, 68% and 94% in the FLT3-ITD-/non-R882 DNMT3A-, FLT3-ITD+/non-R882 DNMT3A-, FLT3-ITD-/non-R882 DNMT3A+, and FLT3-ITD+/non-R882 DNMT3A+ subsets, respectively.Table 1Gene mutations.PatientsIncidencePositive/Tested (N)OverallFLT3-ITD+no FLT3-ITDNPM1409/409---FLT3, all186/40746%--FLT3-ITD150/40737%--FLT3-TKD36/4049%3%12%IDH1/2, all101/37227%18%32%IDH1R13252/37414%11%15%IDH2R14049/37113%7%17%IDH2R1720/3710%0%0%DNMT3A, all148/31048%47%48%DNMT3AR882100/31032%30%34%DNMT3A other48/31016%17%15%TET233/23214%11%18% ConclusionsThis study confirms the heterogeneity of patients with NPM1-mutated CN-AML, independently of the known poor prognosis of FLT3-ITD. Higher WBC was associated with FLT3-ITD, IDH2R140 and TET2 mutations, but did not represent per se an independent risk factor for relapse. Higher incidence of unfavorable non-R882 DNMT3A mutations in older patients may explain, at least in part, why older patients do worse than younger patients in this peculiar AML subset. A potential favorable prognostic role of FLT3-TKD mutations in these patients needs to be confirmed. Disclosures:No relevant conflicts of interest to declare.

BAALC Acts As a Scaffold-Like Protein In The ERK Signaling Pathway and Promotes Leukemogenesis By Facilitating MEK–mediated ERK Activation

Significance of BAALC (brain and acute leukemia, cytoplasmic) as a poor prognostic factor for patients with cytogenetically normal acute myeloid leukemia (CN-AML) and acute lymphoblastic leukemia (ALL) has now been widely established, however, its molecular mechanisms are still mostly unknown. Available evidence suggests that BAALC is a cytoplasmic protein. Based on this finding, we carried out phosphoprotein array of cytoplasmic signal transduction-related proteins and sought to find out the deregulated signaling underlying BAALC-mediated leukemogenesis. Using BAALC-overexpressed HEK293T cells, we found enhanced phosphorylation of Extracellular signal-regulated kinase (ERK), and its downstream target p90RSK. Besides, Jurkat cells, a human ALL cell line, showed sustained phosphorylation of ERK when BAALC is overexpressed. These results suggest that BAALC acts as a scaffold-like molecule for the cytoplasmic mitogen-activated protein kinase kinase (MEK), upstream ERK activator and activates ERK signaling pathway. Immunoprecipitation assay revealed that BAALC binds to MEK in HEK293T cells. Co-localization of BAALC and MEK in the cytoplasm of HEK293T was also detected by immunofluorescence experiment. The proportion of the phosphorylated form in the MEK-bound ERK was increased compared to its control, indicating that BAALC binds to MEK and directly up-regulates its kinase activity to phosphorylate ERK. BAALC accelerated cell growth and increased the phosphorylation of ERK when overexpressed in Jurkat or HEL cells. Those cells showed accelerated cell cycling with no change of apoptotic status. On the other hand, BAALC knockdown decreased cellular proliferation as well as phosphorylation of ERK in the human leukemia cell line expressing a high level of BAALC, such as Kasumi-1 cells. Decelerated cell cycling was also observed in the BAALC-knocked down cells. Notably, the proportion of the phosphorylated ERK bound to MEK was decreased when BAALC was knocked down, which is consistent with BAALC-overexpressed experiments. Interestingly, in AML cell lines without active Ras mutations, the expression of BAALC was positively correlated to its sensitivity to MEK inhibitor (U0126). Moreover, growth advantage of BAALC overexpression in leukemic cell lines was canceled by the treatment with U0126, implying that BAALC-induced growth advantage depends on MEK-ERK signaling activity. Analysis of deletion mutants of BAALC revealed that binding ability of BAALC to MEK resides in its N-terminal region, which is highly conserved among mammals. This N-terminal region of BAALC was necessary for phosphorylation of ERK and the mutant lacking this region failed to promote proliferation of the leukemic cell lines. Taken together, BAALC acts as a scaffold-like protein that binds to MEK in the cytoplasm and directly up-regulates MEK-ERK signal transduction pathway. The fact that overexpression of BAALC significantly promotes proliferation of leukemic cells is consistent with the clinical observation that BAALC-high leukemia has a poor prognosis. The novel function of BAALC as a scaffold-like protein that activates MEK-ERK signal transduction pathway may account for the underlying mechanism for the generation of aggressive form of leukemia and would be a promising therapeutic target of BAALC-high leukemia. Disclosures:Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

Leukemia-Associated Mutations Of DNMT3A Inhibit Differentiation Of Hematopoietic Stem Cells and Promote Leukemic Transformation Through Abberant Recruitment Of Bmi1

Whole genome sequencing has revealed DNMT3A mutation is present in over 20% of cytogenetically normal acute myeloid leukemia (CN-AML) and R882 is the most frequent and recurrent mutated site. Cumulating clinical data have emphasized the importance of the mutation as a poor prognostic factor of AML. Since the functional role of DNMT3A mutation in leukemogenesis remains largely unknown, we aimed to elucidate the impact of DNMT3A mutation on the development and maintenance of AML. To investigate the effect of exogenous expression of DNMT3A R882 mutant (Mut) in hematopoiesis, we transplanted 5-FU primed mouse bone marrow cells transduced with empty vector (EV), DNMT3A wild type (WT), or DNMT3A Mut to lethally irradiated mice. Recipients transplanted with DNMT3A Mut-transduced cells exhibited hematopoietic stem cell (CD150+CD48-Lin-Sca1+c-Kit+) accumulation and enhanced repopulating capacity compared with EV and DNMT3A WT recipients. To identify the downstream target genes of DNMT3A Mut that evoked hematopoietic stem cell accumulation, we sorted vector-transduced LSK cells from transplanted mice and conducted quantitative PCR (Q-PCR) of various hematopoiesis-related genes. Q-PCR revealed that Hoxb cluster expression was up-regulated and differentiation-associated genes, such as PU.1 and C/ebpa, were down-regulated in DNMT3A Mut-transduced LSK cells. Targeted bisulfite sequencing showed hypomethylation of the Hoxb2 promoter-associated CpG island in DNMT3A Mut-transduced cells compared with EV-transduced cells, which suggests dominant-negative effect of DNMT3A R882 mutation. DNMT3A Mut caused no change in methylation status of PU.1 promoter-associated CpG island, indicating that DNA methylation-independent mechanism underlies PU.1 downregulation. Given that DNMT3A interacts with several histone modifiers to regulate target gene transcription, we performed co-immunoprecipitation to investigate whether these interactions are altered by DNMT3A mutation. We found that DNMT3A Mut has the emhanced capacity to interact with polycomb repressive complex 1 (PRC1), which is thought to be a potential mechanism of the DNMT3A Mut-induced differentiation defect. Co-immunoprecipitation experiments showed that DNMT3A R882H and R882C mutant exhibited augmented interaction with BMI1 and MEL18, respectively. In addition, RING1B, an essential component of PRC1, co-localized with DNMT3A Mut more frequently than WT, irrespective of the type of amino acid substitution. Furthermore, heterozygosity of Bmi1 restored the PU.1 mRNA to the normal level and canceled the effect of stem cell accumulation in mice transplanted with DNMT3A Mut bone marrow cells. Chromatin immunoprecipitation in AML cell lines showed that BMI1 and RING1B were more efficiently recruited to the upstream regulatory element of PU.1 upon expression of DNMT3A Mut than WT, while the amount of DNMT3A recruited were comparable between DNMT3A WT and Mut. In the murine transplantation model, we found that exogenous PU.1 expression impaired repopulating capacity in both EV and R882H-transduced cells to the similar level. Exogenous expression of DNMT3A WT inhibited proliferation and induced terminal myeloid differentiation, whereas DNMT3A Mut-transduced cells remained immature in AML cell lines. DNMT3A Mut-transduced cells were resistant to ATRA-induced differentiation compared to EV-transduced cells. Furthermore, R882 mutation promoted blastic transformation of murine c-Kit+ bone marrow cells in vitro in combination with HOXA9 which is highly expressed in clinical cases harboring DNMT3A mutation. Morphological and surface marker analysis revealed these cells were F4/80+ monocytic blasts, consistent with clinical observation that DNMT3A mutation is found frequently in FAB M4/M5 leukemia. These results indicate a distinct role for DNMT3A Mut as well as a potential collaboration between DNMT3A Mut and HOXA9 in malignant transformation of hematopoietic cells. Interestingly, Bmi1 heterozygosity impaired this monoblastic transformation of R882H and HOXA9 co-transduced progenitors. Taken together, our results highlight the functional role of DNMT3A mutation in differentiation block of hematopoietic stem cells and in promoting leukemic transformation via aberrant recruitment of Bmi1 and other PRC1 components. Disclosures: Kurokawa: Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding.

Oncogenic Wilms Tumor 1 (WT1) Mutation Augments Hematopoietic Progenitor Cell Clonogenicity and Promotes Expansion Of The Long-Term Hematopoietic Stem Cell (LT-HSC) Compartment: Implications For WT1-Mediated Leukemogenesis

BackgroundThe WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/ResultsWe obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. ConclusionOncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. [Display omitted] ▪ Disclosures:No relevant conflicts of interest to declare.

DHH-RHEBL1fusion transcript: a novel recurrent feature in the new landscape of pediatricCBFA2T3-GLIS2-positive acute myeloid leukemia

Childhood Acute Myeloid Leukemia (AML) is a clinically and genetically heterogeneous malignant disease. Despite improvements in outcome over the past decades, the current survival rate still is approximately 60-70%. Cytogenetic, recurrent genetic abnormalities and early response to induction treatment are the main factors predicting clinical outcome. While the majority of children carry recurrent chromosomal translocations, 20% of patients do not show any recognizable cytogenetic alteration and are defined to have cytogenetically normal AML (CN-AML). This subset of patients is characterized by a significant heterogeneity in clinical outcome, which is influenced by factors only recently started to be identified. In this respect, genome-wide analyses have been used with the aim of defining the full array of genetic lesions in CN-AML. Recently, through whole-transcriptome massively parallel sequencing of seven cases of pediatric CN-AML, we identified a novel recurrent CBFA2T3-GLIS2 fusion, predicting poorer outcome. However, since the expression of CBFA2T3-GLIS2 fusion in mice is not sufficient for leukemogenesis, we speculated that further unknown abnormalities could contribute to both cancer transformation and response to treatment. Thus, we analyzed, by whole-transcriptome sequencing, 4 CBFA2T3-GLIS2-positive patients, as well as 4 CN-AML patients. We identified a new fusion transcript in the CBFA2T3-GLIS2-positive patients, involving Desert Hedgehog (DHH), a member of Hedgehog family, and Ras Homologue Enrich in Brain Like 1 (RHEBL1), a gene coding for a small GTPase of the Ras family. Through the screening of a validation cohort of 55 additional pediatric AML patients, we globally detected DHH-RHEBL1 fusion in 8 out of 20 (40%) CBFA2T3-GLIS2-rearranged patients. Gene expression analysis performed on RNA-seq data revealed that DHH-RHEBL1-positive patients exhibited a specific signature. These 8 patients had an 8-year overall survival worse than that of the remaining 12 CBFA2T3-GLIS2-rearranged patients not harboring DHH-RHEBL1 fusion (25% vs 55%, respectively, P=0.1). Taken together, these findings are unprecedented and indicate that the DHH-RHEBL1 fusion transcript is a novel recurrent feature in the changing landscape of CBFA2T3-GLIS2-positive childhood AML. Moreover, it could be instrumental in the identification of a subgroup of CBFA2T3-GLIS2-positive patients with a very poor outcome.

Meningioma 1 (MN1) expression: Refined risk stratification in acute myeloid leukemia with normal cytogenetics (CN-AML)

Prognostic stratification of cytogenetic normal acute myeloid leukemia (CN-AML) is an area of active research. The aim of this study was to determine the prognostic importance of the meningioma 1 (MN1) gene expression levels in CN-AML. One hundred patients with CN-AML were diagnosed; MN1 expressions were analyzed using quantitative real-time polymerase chain reaction. High expressions were detected in 48 (48%) patients (expression range: 2.35–31.99, mean: 13.9 ± 8.49) in comparison with 52 (52%) patients with low expression (expression range: 0.02–2.3, mean: 0.68 ± 0.77). The course of the disease in patients with high MN1 expression was unfavorable. Patients with high MN1 expression was associated with significant low complete remission rate (62.5 vs. 8.4%, high vs. low MN1, P = 0.001) and high mortality rate (75% vs. 46.1, P = 0.03). AML patients with high MN1 expression tended to be refractory (37.5 vs. 19.2%, P = 0.00) and relapse risk (54.1 vs. 23%, P = 0.02). Multivariable analysis confirmed high MN1 expression as an independent risk factor for disease-free survival and overall survival. In conclusion, MN1 overexpression independently predicts bad clinical outcome in CN-AML patients.

WT1 mutation in pediatric patients with acute myeloid leukemia: a report from the Japanese Childhood AML Cooperative Study Group

Mutations in Wilms tumor 1 (WT1) have been reported in 10-22 % of patients with cytogenetically normal acute myeloid leukemia (CN-AML), but the prognostic implications of these abnormalities have not been clarified in either adults or children. One hundred and fifty-seven pediatric AML patients were analyzed for WT1 mutations around hotspots at exons 7 and 9; however, amplification of the WT1 gene by the reverse transcriptase-polymerase chain reaction was not completed in four cases (2.5 %). Of the 153 evaluable patients, 10 patients (6.5 %) had a mutation in WT1. The incidence of WT1 mutations was significantly higher in CN-AML than in others (15.2 vs. 4.5 %, respectively, P = 0.03). Of the 10 WT1-mutated cases, eight (80 %) had mutations in other genes, including FLT3-ITD in two cases, FLT3-D835 mutation in two, KIT mutation in three, MLL-PTD in three, NRAS mutation in one, and KRAS mutation in two (in some cases, more than one additional gene was mutated). The incidences of KIT and FLT3-D835 mutations were significantly higher in patients with than in those without WT1 mutation. No significant differences were observed in the 3-year overall survival and disease-free survival; however, the presence of WT1 mutation was related to a poor prognosis in patients with CN-AML, excluding those with FLT3-ITD and those younger than 3 years.

Prognostic relevance of Wilms tumor 1 (WT1) gene Exon 7 mutations in-patient with cytogenetically normal acute myeloid leukemia.

This study aimed to assess the prognostic influences of Wilms tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) among Egyptian patients. Exon 7 of WT1 was screened for mutations in samples from 82 CN-AML patients out of 203 newly diagnosed AML patients, using a high-resolution capillary electrophoresis. Seven out of 82 AML patients (8.3%) harbored WT1 mutations. There was no significant difference between the mutant WT1 and wild type AML patients as regard age, sex, French-American-British subtypes and the prevalence of success of induction remission therapy (P<0.5). AML patients with mutant WT1 had shorter overall survival as compared to those patients with wild WT1 (HR=1.38; 95% CI 4.79-6.86; P=0.004). In conclusion, CN-AML patients with WT1 gene mutation have poor clinical outcome. We recommend testing the WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML.

DNMT3A mutation is a poor prognosis biomarker in AML: Results of a meta-analysis of 4500 AML patients

Somatic DNA methyl transferase 3A (DNMT3A) mutations have been recognized recently as recurrent molecular aberrations in acute myeloid leukemia (AML). The precise role of these mutations in leukemogenesis remains elusive but a number of studies have already been conducted to study their potential prognostic value in AML patients with variable results. We performed a meta-analysis on published data from over 4500 AML patients to provide robust evidence supporting DNMT3A mutation testing in clinical setting for AML patients. Our meta-analysis showed that DNMT3A mutations were associated with M4 and M5 AML subtypes. Those mutations conferred significantly worse prognosis with both shorter OS (p=0.0004) and shorter RFS (p=0.002). Notably, DNMT3A mutations appeared to be an independent adverse prognostic factor also in younger patients with normal cytogenetics AML (OS (p=0.01) and RFS (p=0.0005)) and also in the subgroup of patients with high risk genotypes defined according to the criteria of the European Leukemia Net (ELN) (OS (p=0.002)). Therefore, DNMT3A mutational status can improve the risk stratification of AML patients in the setting of integrated mutational profiling.

A model for prediction of FLT3‐ITD and NPM1 (without FLT3‐ITD) positivity in patients with newly diagnosed acute myeloid leukaemia

Acute myeloid leukaemia (AML) varies greatly in response to therapy. Although cytogenetics is the most important predictor of response, it is least informative in the 40 - 50% of adults with normal cytogenetics (Grimwade et al, 1998). However, an increasing number of molecular markers with prognostic significance, particularly in cytogenetically normal AML (CN-AML), have been identified. The two most common are internal tandem duplications (ITD) in the FLT3 gene (FLT3-ITD) and mutations in the nucleophosmin gene (NPM1) with frequencies of approximately 50% and 30%, respectively, in CN-AML. While FLT3-ITD is associated with short relapse-free and overall survival, the prognosis of CN-AML with NPM1 mutations without FLT3-ITD (NPM+/FLT3-ITD-) is similar to that of “favourable” cytogenetics AML (Kottaridis et al, 2001;Schnittger et al, 2005; Thiede et al 2006).

Entinostat Prevents Leukemia Maintenance in a Collaborating Oncogene-Dependent Model of Cytogenetically Normal Acute Myeloid Leukemia

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis, and therapeutic intervention based on improved patient stratification. Relevant preclinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML), and a conditional transplantation mouse model was developed that demonstrated oncogene dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity map analysis identified Entinostat as a drug with the potential to alter the leukemic condition toward the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation, and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML.

Implication of higher BAALC expression in combination with other gene mutations in adult cytogenetically normal acute myeloid leukemia

Data for 125 patients with cytogenetically normal acute myeloid leukemia (CN-AML) regarding BAALC and combinatorial molecular markers at diagnosis were evaluated. Fewer patients with higher BAALC expression at diagnosis achieved a complete remission (CR) (49.2 vs. 75.8%, p = 0.002) after the first cycle of chemotherapy, and there were more primary refractory cases (37.3 vs. 18.2%, p = 0.017). In a combinatorial analysis, FLT3-ITD-positive patients with higher BAALC showed more refractoriness and the worst overall survival (OS) (p < 0.001) and disease-free survival (DFS) (p < 0.001) in CN-AML. When NPM1-mutated CN-AML was combined with either FLT3-ITD mutation or higher BAALC expression, both OS (p = 0.043) and DFS (p = 0.008) were worse; when combined with both, it showed the worst OS (p < 0.001) and DFS (p = 0.004). Higher BAALC expression and FLT3-ITD mutation, both individually and in combination, were associated with worse survival outcomes in CN-AML, and this was also applicable in NPM1-mutated CN-AML, known as a favorable-risk group.

Establishment of xenotransplantation model of human CN-AML with FLT3-ITD mut /NPM1 − in NOD/SCID mice

Patients with FLT3-ITD (mut) /NPM1 (-) cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD (mut) /NPM1 (-) CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells. The FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD (mut) /NPM1 (-) CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

MicroRNA Dysregulation in Acute Myeloid Leukemia

In the article that accompanies this editorial, Marcucci et al present data on the clinical significance of the overexpression of miR-155 in patients with cytogenetically normal acute myeloid leukemia (CN-AML). miR-155 is a microRNA that is dysregulated in aggressive lymphomas, in the aggressive form of CLL, and in some solid malignancies such as lung, breast, and colorectal cancer. miR-155 has been shown to inhibit expression of tumor suppressors and genes involved in DNA repair. Interestingly, dysregulation of miR-155 in B cells of mice causes acute lymphoblastic leukemia and high-grade lymphomas. The authors carried out mRNA and microRNA profiling by using microarrays and measured the expression of the transcripts of the miR-155 gene by nCounter technology (NanoString, Seattle, WA), a sensitive amplification independent method. They investigated CN-AML patients, including 153 younger ( 60 years) and 210 older ( 60 years) patients, with untreated primary CN-AML who received intensive first-line therapy on CALGB trials and had diagnostic bone marrow or blood specimens available for molecular analyses. The results of their careful analysis show that miR-155 expression levels constitute an independent prognostic factor in patients with CN-AML. While higher miR-155 expression levels had a negative impact on the outcome of younger and older patients, higher miR-155 expression was associated with lower complete response (CR) rate, and shorter disease-free survival (DFS) and overall survival (OS) in younger patients. Higher miR-155 expression level was associated only with lower CR and shorter OS in older patients. Such findings could be related to biologic differences and differences in the intensity of consolidation therapy. At diagnosis, high miR-155 levels occurred more frequently in patients with AML who were FLT3-ITD positive, RUNX1-mutated, WT1 mutated, and had high ERG and BAALC expressors, and less frequently in CEBPA-mutated, IDH2-mutated and FLT3-tyrosine kinase–positive patients with AML. Thus, higher miR-155 levels associate with unfavorable prognosticators and predict worse CR and survival in both young and old patients. In multivariable analysis, patients with CN-AML with higher levels of miR-155 were approximately 50% less likely to achieve CR. Higher miR-155 expressors had also a 60% increased risk of death and were independent of FLT3-ITD. An international panel of experts on CN-AML on behalf of the European LeukemiaNet recommended a modified classification of patients with CN-AML. When the authors analyzed the prognostic significance of miR-155 in patients classified in the ENL favorable genetic group, younger patients with high miR-155 expression had significantly lower CR rates and shorter event-free survival and OS than patients with low expression, respectively. These correlations were not found with any other microRNA. This is the first clinical study, to our knowledge, where expression of miR-155 was evaluated by using the nCounter technology to understand how different expression levels of miR-155 may affect the aggressiveness of the disease, outcome, and response to treatment of patients with CN-AML. The results indicate that high levels of expression of miR-155 positively correlate with antiapoptotic activity and NF B activation. Although dysregulation of miR-155 in B cells and myeloid cells has been shown to be associated to aggressive malignancy and myeloproliferative disorders respectively in mouse models, the present study tries to assess the role of the dysregulation of this microRNA in patients with CN-AML. Their results support the hypothesis that miR-155 expression contributes to different degrees of aggressiveness in leukemia by affecting proliferation and survival of the leukemia cells. A conclusion of this study, as clearly indicated by the authors, is that if we want to have an effect in the treatment of this group of AML patients, we should try to revert miR-155 overexpression, for example, by using anti-miR-155 oligonucleotides or by inhibiting the pathways that regulate the levels of miR-155 expression. In addition, given that miR-155 is relatively easy to measure at diagnosis, we should consider the levels of miR-155 as a biomarker for risk stratification and guidance for treatment.

CBFA2T3-GLIS2 fusion transcript is a novel common feature in pediatric, cytogenetically normal AML, not restricted to FAB M7 subtype

AbstractPediatric cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous subgroup of myeloid clonal disorders that do not harbor known mutations. To investigate the mutation spectrum of pediatric CN-AML, we performed whole-transcriptome massively parallel sequencing on blasts from 7 CN-AML pediatric patients. In 3 patients we identified a recurrent cryptic inversion of chromosome 16, encoding a CBFA2T3-GLIS2 fusion transcript. In a validation cohort of 230 pediatric CN-AML samples we identified 17 new cases. Among a total of 20 patients with CBFA2T3-GLIS2 fusion transcript out of 237 investigated (8.4%), 10 patients (50%) did not belong to the French–American–British (FAB) M7 subgroup. The 5-year event-free survival for these 20 children was worse than that for the other CN-AML patients (27.4% vs 59.6%; P = .01). These data suggest that the presence of CBFA2T3-GLIS2 fusion transcript is a novel common feature of pediatric CN-AML, not restricted to the FAB M7 subtype, predicting poorer outcome.

Identification of emerging FLT3 ITD‐positive clones during clinical remission and kinetics of disease relapse in acute myeloid leukaemia with mutated nucleophosmin

FLT3 internal tandem duplication (ITD) mutations are frequently detected at diagnosis in cytogenetically normal acute myeloid leukaemia (CN-AML) and predict unfavourable outcome. FLT3 ITD is an unstable aberration and may be lost or acquired at relapse. Recent whole genome sequencing studies have suggested that FLT3 ITD(+)ve AML relapse may evolve from small subclones undetectable at diagnosis by routine polymerase chain reaction (PCR). We developed a patient-specific real-time quantitative-PCR (RQ-PCR) to implement FLT3 ITD detection in six AML patients whose blasts carried wild-type FLT3 at diagnosis and who relapsed with FLT3 ITD by routine PCR. Patient-specific forward primers were designed after cloning and sequencing the FLT3 ITD in each case. The assay allowed retrospective detection of FLT3 ITD in diagnostic samples of 4/6 cases and to establish the kinetics of clonal evolution preceding relapse. After conventional chemotherapy, all patients had early relapse despite having been classified as NPM1(+)ve/FLT3 ITD(-)ve at presentation, with shorter remissions being observed in four patients re-classified as FLT3 ITD(+)ve by the new assay. Notably, FLT3 ITD clone became detectable by conventional PCR in three patients tested during remission after initial treatment. Our data underscore the need of identifying low FLT3 ITD levels, which are probably associated with relapse in otherwise good prognosis CN-AML.