Acute Megakaryoblastic Leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts. While rare in adults, AMKL accounts for 4–15% of newly diagnosed childhood AML1–3. AMKL in patients without Down syndrome (non-DS-AMKL) is frequently associated with poor outcomes. Previous efforts have identified chimeric oncogenes in a significant number of cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements and NUP98-KDM5A4–6. The etiology of 30–40% of cases, however, remains unknown. To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult). We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes. These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes. Overall, these data shed light on the etiology of AMKL and provide useful information for treatment tailoring.

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Systematic assessment of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients has been hampered by lack of a reliable, uniform MRD marker applicable to all patients. We evaluated next-generation sequencing (NGS) for MRD assessment in AML patients (n = 80 samples). The ability of NGS technologies to generate thousands of clonal sequences makes it possible to determine the allelic ratio of sequence variants. Using NGS, we were able to determine the allelic ratio of different FLT3-internal tandem duplication (ITD) clones within one patient sample, in addition to resolution of FLT3-ITD insertion site, length, and sequence in a single analysis. Furthermore, NGS allowed us to study emergence of clonal dominance. Parallel assessment of MRD by NGS and quantitative real-time polymerase chain reaction in NPM1 mutated patients was concordant in 95% of analyzed samples (n = 38). The frequency of mutated alleles was linearly quantified by NGS. As NGS sensitivity is scalable depending on sequence coverage, it reflects a highly flexible and reliable tool to assess MRD in leukemia patients.

IntroductionThe t(6;9)(p22;q34), frequently reported with a breakpoint in 6p23 but now known to involve the DEK gene mapping to 6p22.3, is a rare translocation, estimated to occur in 1-2% of cases of childhood acute myeloid leukemia (AML). 1 The translocation was first identified in 1976, and the first pediatric patient was described in 1982.2,3 The World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia from 2008 listed the t(6;9)(p22;q34) as a distinct entity. 4 However, our current knowledge of t(6;9)(p22;q34) in AML is drawn from relatively small series of patients, predominantly adults, associating t(6;9) with young age at onset and a poor outcome. 1,[5][6][7] Typically, the t(6;9) presents as de novo AML, morphologically associated with FrenchAmerican-British (FAB) type M2, bone marrow basophilia, Auer rods, and dysplasia. 1,5,7,8 The translocation is primarily the sole cytogenetic abnormality (80%); among the 20% of ©2014 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2013 The online version of this article has a Supplementary Appendix. Manuscript received on September 24, 2013. Manuscript accepted on January 13, 2014 Acute myeloid leukemia with t(6;9)(p22;q34) is listed as a distinct entity in the 2008 World Health Organization classification, but little is known about the clinical implications of t(6;9)-positive myeloid leukemia in children. This international multicenter study presents the clinical and genetic characteristics of 62 pediatric patients with t(6;9)/DEK-NUP214-rearranged myeloid leukemia; 54 diagnosed as having acute myeloid leukemia, representing <1% of all childhood acute myeloid leukemia, and eight as having myelodysplastic syndrome. The t(6;9)/DEK-NUP214 was associated with relatively late onset (median age 10.4 years), male predominance (sex ratio 1.7), French-AmericanBritish M2 classification (54%), myelodysplasia (100%), and FLT3-ITD (42%). Outcome was substantially better than previously reported with a 5-year event-free survival of 32%, 5-year overall survival of 53%, and a 5-year cumulative incidence of relapse of 57%. Hematopoietic stem cell transplantation in first complete remission improved the 5-year event-free survival compared with chemotherapy alone (68% versus 18%; P<0.01) but not the overall survival (68% versus 54%; P=0.48). The presence of FLT3-ITD had a non-significant negative effect on 5-year overall survival compared with non-mutated cases (22% versus 62%; P=0.13). Gene expression profiling showed a unique signature characterized by significantly higher expression of EYA3, SESN1, PRDM2/RIZ, and HIST2H4 genes. In conclusion, t(6;9)/DEK-NUP214 represents a unique subtype of acute myeloid leukemia with a high risk of relapse, high frequency of FLT3-ITD, and a specific gene expression signature. t(6;9)(p22;q34)/DEK-NUP214-rearranged pediatric myeloid leukemia: an international study of 62 patients

Mutations in the NADP þ -dependent isocitrate dehydrogenase genes 1 and 2 (IDH1 and IDH2) have recently been found in adult acute myeloid leukemia (AML) patients with a prevalence rising up to 33%. To investigate the frequency of IDH1/2 mutations in pediatric AML, we characterized the mutational hotspot (exon 4) of these genes in diagnostic samples from 460 pediatric AML patients. Our analysis identified somatic IDH1/2 mutations in 4% of cases (IDH1 R132 n ¼ 8; IDH2 R140 n ¼ 10) and the minor allele of single-nucleotide polymorphism (SNP) rs11554137 in 47 children (10.2%). IDH mutations were associated with an intermediate age (P ¼ 0.008), FAB M1/M2 (P ¼ 0.013) and nucleophosmin1 mutations (P ¼ 0.001). In univariate analysis, IDH mutated compared with IDH wildtype patients showed a significantly improved overall survival (OS; P ¼ 0.032) but not event-free survival (EFS; P ¼ 0.14). However, multivariate analysis did not show independent prognostic significance. Children with at least one minor allele of IDH1 SNP rs11554137 had similar EFS (P ¼ 0.27) and OS (P ¼ 0.62) compared with major allele patients. Gene expression profiles of 12 IDH mutated were compared with 201 IDH wildtype patients to identify differentially expressed genes and pathways. Although only a small number of discriminating genes were identified, analysis revealed a deregulated tryptophan metabolism, and a significant downregulation of KYNU expression in IDH mutated cases.