Acute Megakaryoblastic Leukemia Patients Research Articles

A Multicenter Clinical Study with Reduced Intensive Chemotherapy for Induction Remission of Pediatric Acute Megakaryoblastic Leukemia

Bcakground: Pediatric acute megakaryoblastic leukemia (AMKL) without Down syndrome (DS) is a genetically heterogenous myeloid malignancy and with dismal prognosis. Refractory and relapse remains a major challenge. Given to the young age distribution of AMKL and high frequency of abandonment in middle-income countries, we conducted a multicenter clinical trial (ChiCTR1800015875) in aims to lower the intensity of induction while without compromise the remission rate and survival. Methods: From June 2018 to December 2022, there were consecutively 43 cases with de novo non-DS AMKL, which accounted for 7.6% cases of acute myeloid leukemia (AML) during the same period, enrolled in this trial. All the patients received at least one cycle of low-dose induction with HAG regimen (homoharringtonine 1 mg/m 2, intravenous daily, days 1-7; cytarabine 10 mg/m 2, subcutaneously every 12 hours, 20 doses, and G-CSF 5µ/kg subcutaneous, daily, 10 doses) followed by 3 to 4 courses of intensive chemotherapy or HSCT as post-remission consolidation. Besides conventional molecule testing, RNA-sequencing and whole exon sequencing (WES) were performed as well to reveal underlying genetic landscape of this type of disease. Results: The median age of the 43 patients at diagnosis was 18.5 (range 4-114) months. The median while blood cell (WBC) counts and platelet at diagnosis were 17.2×10 9/L (range 1.8-105.7 ×10 9/L) and 36.5×10 9/L (range 4-472 ×10 9/L), respectively. Among them, 39.5% of cases exhibited hepatosplenomegaly. A diversity of genetic fusions and mutations were uncovered. The recurrent fusions in this cohort include CBFA2T3-GLIS2 (16.3%), MLLr (11.6%), NUP98-(9.3%), RBM15-MKL1 (7.0%), HOX fusions (7.0%) and PICALM-MLLT10 (4.7%). Other non-recurrent fusions accounted for 18.6% (Figure 1A). The common mutations detected in our pediatric AMKL were JAK2, NRAS, MPL, BCOR, CTCF, KRAS and PTPN11. Regarding treatment response, 20 of 43 cases (46.5%) attained complete remission (CR) or complete remission with incomplete blood cell recovery (CRi) after one cycle of induction. Among the 20 cases, 9 of them got minimal residual disease (MRD) negative by flow cytometry. Ten patients (23.2%) achieved partial remission (PR) while 13 patients (30.2%) showed no response (NR) to treatment. Thirty-one patients continued with another cycle of the same regimen and 21 of the 28 cases who were available for evaluation (75.0%) attained CR/CRi. Disease relapse was observed in 16/43 (37.2%) cases, with 7/43 (16.2%) patients relapsed after transplantation. The 3-year overall survival (OS) and event-free survival (EFS) were 49.6±8.2% and 29.5±7.1%, respectively (Figure 1B). In total, more than half of patients (22/43, 51.5%) underwent HSCT. The 3-year OS of transplant group and chemotherapy group were 57.9±11.7% versus 39.5±11.2%, P=0.036. As genetic characteristics often predict treatment response and prognosis, we wonder which subgroup could benefit from the reduced intensive induction. We further compared the features of patients who got CR/CRi after induction I with those achieved PR/NR. Interestingly, we found more patients with adverse fusions in PR/NR group, such as CBFA2T3-GLIS2 (26.0% vs. 5.0%), NUP98- (17.4% vs. 0%), MLLs (13.0% vs. 10.0%), and RBM15-MKL1 (8.7% vs. 5.0%); while other non-recurrent fusions were more common in CR/CRi group (4.3% vs. 35.0%). Two thirds of patients (67.4%) experienced refractory or relapse and almost half of them harbored adverse fusions or mutations such as CBFA2T3-GLIS2, MLLr, NUP98- fusions, NRAS and PTPN11. On the other hand, 14 patients without refractory or relapse had genetic features with more JAK2 and MPL mutations. No treatment-related mortality (TRM) occurred during induction. Only one patient with KRAS mutation died of infection after consolidation III. Conclusion: Insight into the genetic and molecular landscape improved our understanding of pediatric AMKL. Distinct molecular features can serve as a tool to tailor the therapy. Though treatment still lags behind and novel therapy is urgent for the adverse subgroups, our low-dose induction with HAG regimen could benefit for a subgroup with non-recurrent fusions, which showed efficiency while is tolerable for the young age group. Based on our results, we provide an induction option for these AMKL patients, especially for those in the middle-income countries.

A demographic study of acute megakaryoblastic leukemia using the national cancer database.

e19023 Background: Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia that, based on past studies, primarily affects children. Patients with Down syndrome (DS) have a higher risk of developing AMKL. While DS-associated AMKL has an overall survival rate of 80%, the prognosis of adult AMKL is much poorer (overall survival rate of < 40%). The prognostic heterogeneity of AMKL warrants an investigation of its diagnostic trends in order to gain epidemiological insights. In this study, demographic data of AMKL patients was obtained from the National Cancer Database (NCDB) and analyzed to determine factors that affect the outcome of AMKL patients. Methods: In this retrospective cohort study, data from the 2004-2020 NCDB was analyzed to identify cases of AMKL with ICD-O-3 code 9910. Demographic data attached to the identified cases assessed included age, sex, race, Hispanic status, insurance status, facility type, surgery status, palliative care status, and Charlson-Deyo score. Descriptive statistics were obtained, and the incidence trends of AMKL were interpreted using regression analysis. Results: A total of 765 patients diagnosed with AMKL were identified in the database between 2004 – 2020 with a decreasing incidence of patients diagnosed per year (64 cases in 2004 and 11 cases in 2020; R^2 = 0.86). The average age of diagnosis was 40.3 years (SD = 32.4, range = 0 – 90 years, median = 51 years). AMKL exhibits slight male prevalence (55.2% vs 44.8% for females). The majority of the patients were Non-Hispanic White (77.6%). Most individuals had Charlson-Deyo comorbidity scores of 0 (79.1%). The incidence rates were similar across household income quartiles. 72.8% of AMKL cases were diagnosed in urban areas with a population of > 250,000. Fewer cases were treated at community cancer programs as compared to either academic/research programs (45.1%) or integrated network cancer programs (20.6%). Private insurance (38.6%), Medicare (30.6%), and Medicaid (22.0%) were the most common insurance types. All patients received surgical intervention of the bone marrow and 3.2% of patients received palliative care. Conclusions: This NCDB-based demographic analysis on AMKL serves to improve the general understanding of AMKL. Surprisingly, the average and median ages of diagnosis are 40.3 and 51 years, respectively, which contradicts prior reports of AMKL being primarily a pediatric disease. Our analysis of the socioeconomic factors of AMKL patients showed that most AMKL patients were non-Hispanic Whites and diagnosed and treated in urban settings. In addition, the majority of AMKL patients had government insurance (54.6%; Medicare, Medicaid, and others). While this study provides initial insight into the role demographics play in the diagnosis and treatment of AMKL patients, further studies on how these factors contribute to their outcome would provide great value in understanding the social disparity in AMKL.

Revealing the intratumoral heterogeneity of non-DS acute megakaryoblastic leukemia in single-cell resolution.

Pediatric acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) characterized by abnormal megakaryoblasts, and it is divided into the AMKL patients with Down syndrome (DS-AMKL) and AMKL patients without DS (non-DS-AMKL). Pediatric non-DS-AMKL is a heterogeneous disease with extremely poor outcome. We performed single-cell RNA sequencing (scRNA-seq) of the bone marrow from two CBFA2T3-GLIS2 fusion-positive and one RBM15-MKL1 fusion-positive non-DS-AMKL children. Meanwhile, we downloaded the scRNA-seq data of normal megakaryocyte (MK) cells of the fetal liver and bone marrow from healthy donors as normal controls. We conducted cell clustering, cell-type identification, inferCNV analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and Monocle2 analysis to investigate the intratumoral heterogeneity of AMKL. Using canonical markers, we identified and characterized the abnormal blasts and other normal immune cells from three AMKL samples. We found intratumoral heterogeneity of AMKL in various cell-type proportions, malignant cells’ diverse copy number variations (CNVs), maturities, significant genes expressions, and enriched pathways. We also identified potential markers for pediatric AMKL, namely, RACK1, ELOB, TRIR, NOP53, SELENOH, and CD81. Our work offered insight into the heterogeneity of pediatric acute megakaryoblastic leukemia and established the single-cell transcriptomic landscape of AMKL for the first time.

Immunophenotypic Analysis of Acute Megakaryoblastic Leukemia: A EuroFlow Study.

Simple SummaryAcute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We show that such patients can be identified by flowcytometric immunophenotyping using the standardized EuroFlow panel. AMKL patients show a unique immunophenotypic profile, and among AMKL patients, various subgroups can be distinguished.Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML—not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL—other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile.

Epigenetically Enhanced MED12L in ETO2-GLIS2 Positive Pediatric Acute Megakaryoblastic Leukemia Is Associated with Resistance to the CDK8 Inhibitors

Introduction. ETO2-GLIS2 (aka CBFA2T3-GLIS2) is the most common (30%) alteration in pediatric de novo acute megakaryoblastic leukemia (AMKL). These patients have poor response to induction therapy, a high incidence of relapse (~90%), and dismal 5-year survival rates (<20%). Previous studies suggest that ETO2-GLIS2 induces leukemia through abnormal enhancer formation as a single oncogenic “hit”. Because ETO2-GLIS2 expression induces formation of leukemia-specific neo-superenhancer (SE) elements, we hypothesize that mediator (MED) proteins are involved in linking neo-SE elements to distal gene expression, and thus could be a therapeutic target. Here, we analyzed expression of MED-family genes in ETO2-GLIS2 positive AMKL patients in combination with enhancer-histone marks, and evaluated impact of MED-kinase inhibition with selective CDK8 inhibitors (CDKi) against cell viability.Methods. To address our hypothesis, we analyzed published RNA sequencing dataset (Smith et al. 2020) for human MED-genes expression from a pan-pediatric AML cohort (N=1476). The cohort consisted of subgroups expressing fusions of ETO2-GLIS2 (N=40), CBFB-MYH11 (N=174), DEK-NUP214 (N=49), KMT2A-ELL (N=50), KMT2A-MLLT10 (N=86), KMT2A-MLLT3 (N=114), KMT2A-MLLT4 (N=49), NUP98-NSD1 (N=107), RUNX1-RUNX1T1 (N=210) and no detectable fusions (N=526), compared to normal bone marrow (NBM) samples (N=71). Enrichment of histone marks overlapping MED-genes was analyzed from published chromatin immunoprecipitation (ChIP) sequencing in ETO2-GLIS2 positive M-07e AMKL line (Thirant et al. 2017). We tested efficacy of CDK8 inhibition with BI-1347 and CCT251545 against M-07e cells to determine their activity in the context of marked MED12L overexpression.Results. We examined expression of 29 MED-genes comprising the 4 major (head, middle, tail, and kinase) MED-modules. MED gene expression was variable across AML subtypes and NBM. However, MED genes were more commonly over-expressed in the ETO2-GLIS2 group, in particular MED 17, MED1, MED10, MED27, and MED12L (paralog of MED12) were upregulated in the subgroup. Most notably, we noted exceptional upregulation of MED12L (FC 4.9, log2), compared to NBM (Figure 1). Because MED12/12L plays an intrinsic biological role in establishing oncogenic enhancer-expression loops in hematopoietic stem or leukemic cells, we investigated the overlap of enhancer bound histones such as H3K27ac and H3K4me1 to MED12L in M-07e cells, compared to umbilical cord blood-derived normal megakaryoblasts (MK) (S004BT; Blueprint epigenome database). We found enrichment of H3K4me1 at MED12L transcription start site (TSS) and upstream promoter both in MK and M-07e cells. In addition, we observed a large region of H3K27ac enrichment spanning 89 Kb (16 kb upstream and 73 kb downstream) across MED12L TSS in M-07e cells, suggesting neo-enhancer activity at this locus. Considering the dependency of MED12 on CDK8 for MED-kinase activities (Klatt et al. 2020), we treated M-07e cells with CDK8i(s), to test our hypothesis if perturbation of epigenetically enhanced MED12L expression can impact leukemic growth. However, we observed a poor correlation between M-07e cell viability and IC 50 of BI-1347 (IC 50: 0.87 µM, R 2: 0.36) or CCT251545 (IC 50: 0.4 µM, R 2: 0.48). In contrast, ETO2-GLIS2 negative MV4-11 AML cells were susceptible to both BI-1347 (IC 50: 0.44 µM, R 2: 0.88) and CCT251545 (IC 50: 0.12 µM, R 2: 0.87). Given the inefficacy of CDK8i against M-07e, and cooperativity of bromodomain extra-terminal (BET)-BRD4 and MED12/12L in forming enhancer complexes, we tested possible inhibitory impact of BRD4 inhibitor JQ1 on MED12L expression and leukemic growth in target cells. We observed effective reduction in M-07e cell viability (IC 50:0.3 µM, R 2: 0.93) with concomitant reduction not only in BRD4 protein-expression, but diminished MED12L protein expression at IC 50 and higher doses of JQ1.Conclusion. Our findings revealed that MED12L is highly overexpressed and overlapped with strong neo-enhancer chromatin-marks in ETO2-GLIS2 positive cells, while maintaining resistance to CDK8i. Future studies will contribute to deeper insights into the preferential recruitment and role of MED12L in ETO2-GLIS2 bound enhancers and potential mechanisms of resistance to CDK8 inhibition in the disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The ETO2-GLIS2 Fusion Oncogene Alters Early Human Hematopoiesis in an Induced Pluripotent Stem Cells-Derived Model of Pediatric Acute Megakaryoblastic Leukemia

De novo pediatric acute megakaryoblastic leukemia (AMKL) is a genetically heterogeneous subtype of acute myeloid leukemia (AML) expressing fusion oncogenes, including CBFA2T3 - GLIS2 (a.k.a. ETO2-GLIS2) present in 15-25% of patients and associated with a poor prognosis because of a high risk of relapse or refractoriness to chemotherapy. To characterize the consequences of ETO2-GLIS2 on early stages of human hematopoiesis, we developed a model of ETO2-GLIS2 expression in induced pluripotent stem cells (iPSC) derived from healthy human CD34+ cells. To this end, we introduced the human ETO2-GLIS2 cDNA tagged with the green fluorescent protein (GFP) under the control of the human CD43 promoter in iPSC through ZFN-mediated recombination at the AAVS1 safe-harbor locus. After puromycin selection, 23 independent clones were confirmed by PCR and sequencing to bear a correct homozygous integration and were amplified. Three independent iPSC clones were subsequently differentiated into hematopoietic progenitors and then into megakaryocytes using a 2D co-culture system on matrix adapted from Chou S et al. (PNAS 2012) and compared to control iPSC. We demonstrated that ETO2-GLIS2 is expressed in CD43+ hematopoietic cells both at the transcript and protein levels and localizes in the nucleus. In addition, ETO2-GLIS2-expressing cells showed an increased expression of several recently characterized direct targets of the fusion including ERG as compared to control cells. Hematopoietic differentiation of iPSC was then assessed at day 13, 15 and 18 using flow cytometry analyses for progenitors and megakaryocytic markers. As early as day 13, ETO2-GLIS2-expressing cells showed an increased number of CD43+ hematopoietic cells composed of a higher percentage of CD41+CD42+ megakaryocytic cells compared to controls, indicating that ETO2-GLIS2 enhances early human megakaryocyte development. Importantly, we observed strong differentiation alterations, including a lower expression of CD61 on ETO2-GLIS2 megakaryocytes compared to controls at all time points and a progressive accumulation of an abnormal CD41lowCD42+ population never detected in control conditions. This CD41lowCD42+ population was also observed in AMKL patients. Finally, preliminary clonogenic assays in methylcellulose indicate that ETO2-GLIS2 enhances serial replating activity for several weeks. These data support the idea that expression of ETO2-GLIS2 in human hematopoietic cells obtained from iPSC recapitulates several cellular and molecular features of human pediatric AMKL, including transcriptional activation of target genes, enhanced self-renewal of progenitors and aberrant differentiation of megakaryocytic cells. This model will facilitate further cellular and preclinical studies on this rare pediatric cancer. DisclosuresNo relevant conflicts of interest to declare.

Utility of CD36 as a novel addition to the immunophenotypic signature of RAM-phenotype acute myeloid leukemia and study of its clinicopathological characteristics.

In 2016, Children Oncology Group (COG) described a new high-risk subtype of acute myeloid leukemia (AML) with a distinct immunophenotypic-signature, RAM-phenotype (RAM-AML). Data on clinical and laboratory features of RAM-AML are still limited to COG report only. Herein, we report the clinicopathological characteristics and detailed immunophenotypic features of RAM-AML patients. In COG report, 38% of RAM-AML belonged to acute megakaryoblastic leukemia (AMKL)-subtype. Hence, we further compared the immunophenotypic features RAM-AML with non-RAM-AMKL diagnosed during the same study period. We included RAM-AML and non-RAM AMKL patients diagnosed between January 2017 and December 2019. We studied their morphological, cytochemical, immunophenotyping, cytogenetic, and molecular characteristics. Mean fluorescent intensity (MFI) and expression-pattern of immunophenotypic markers of RAM-AML were compared with non-RAM AMKLs patients. We identified 11 RAM-AML (1%) and 21 non-RAM AMKL (1.9%) patients in 1102 pediatric-AML patients. Seven of 11 (63.64%) patients belonged to FAB-M7-subtype. CD56, CD117, and CD33 demonstrated overexpression, whereas CD45 and CD38 showed under-expression in RAM-AML patients. CD36 was consistently negative in RAM-AML, whereas moderate-bright positive in non-RAM AMKLs patients (p < 0.0001). On principle component analysis, addition of CD36 enhanced the visual-separation between RAM-AML and non-RAM AMKL clusters. Cytogenetic and molecular studies did not show any recurrent abnormality; however, RNA-sequencing study revealed CBFA2T3-GLIS2-fusion in three of seven (42.8%) RAM-AML patients. We report the clinicopathological characteristics and the detailed immunophenotypic profile in the world's second series of RAM-AML patients. We further report a novel finding of CD36-negative expression as an additional parameter to the multidimensional immunophenotypic signature of this entity.

Clinical Characteristics and Prognosis of 27 Patients with Childhood Acute Megakaryoblastic Leukemia.

BackgroundThe aim of this study was to investigate the clinical features and prognostic factors of childhood acute megakaryoblastic leukemia (AMKL).Material/MethodsThe data of 27 cases of childhood AMKL admitted from November 2009 to July 2018 were retrospectively analyzed. The survival analysis and prognostic factors were analyzed by Kaplan-Meier method.ResultsThe median follow-up time was 26.4 months in 27 cases, and the complete response rate was 92.31% after 2 chemotherapy courses. Eight patients underwent bone marrow transplantation after 3–6 courses. Five patients died after transplantation, 4 of whom died due to recurrence after transplantation. Of the 27 patients, 10 developed recurrence (37.04%), and 8/10 had recurrence within 1 year. The 3-year overall survival rate and disease-free survival rates were (47±12)% and (36±14)%, respectively. Of the 27 AMKL cases, the 3 with Down syndrome (DS-AMKL) all survived after treatment, and the 3-year overall survival rate was 100%. However, of the other 24 AMKL patients without Down syndrome (non-DS-AMKL), 6 died and 6 abandoned treatment, and the 3-year overall survival rate was only 50%. Univariate analysis showed that 3-year overall survival rate was not correlated to gender, age, number of newly diagnosed white blood cells, karyotype, remission after 2 courses of treatment, and transplant after 3 courses of treatment of childhood AMKL cases. Nevertheless, recurrence and remission after 2 courses of treatment were significantly correlated with 3-year overall survival rate.ConclusionsChildren with non-DS-AMKL have a high degree of malignancy and are prone to early recurrence with a poor prognosis, whereas the prognosis of DS-AMKL is relatively good. Recurrence after treatment and remission after 2 courses of treatment are important factors influencing the prognosis of childhood AMKL. Recurrence after transplantation is the leading cause of death in transplantation patients.

Abstract B03: Acute megakaryoblastic leukemia is associated with poor overall survival and enriched in JAK2 variants

Abstract In this study, we aimed to characterize the molecular mechanisms of two very rare subtypes of AML: acute megakaryoblastic leukemia (AMKL) and acute panmyelosis with myelofibrosis (APMF). Our goal is to describe the clinical-pathologic features of APMF and explore the genomic landscape AMKL to further understand these rare malignancies. We identified 35 patients diagnosed with AMKL (n=27) and APMF (n=8) at Mayo Clinic between 1995 and 2015 by retrospective chart review. For 10 of the AMKL patients, tissue was available for whole-exome sequencing and was analyzed in comparison to a published AML reference population. The median overall survival was shorter in patients with AMKL (3.9 months, 95% CI 1.7-7.4) than in patients with APMF (14.3 months, 95% CI 0.7-NE, p = 0.026,). For both AMKL and APMF, patients who underwent allogeneic hematopoietic stem cell transplantation had better overall survival (14.3 months, 95% CI 1.9-NE) compared to those not undergoing transplantation (4.6 months, 95% CI 1.5-7.4, p = 0.003). Patients with AMKL presented more frequently with splenomegaly, with higher peripheral blood blast counts, and with higher serum lactate dehydrogenase concentrations. 9 out of 10 AMKL patients with available tissue for sequencing demonstrated variants in 22 of 35 AML driver genes, with JAK2 V617F as the most common mutation. Although our small study numbers preclude firm conclusions, it does reflect the rarity of these AML subtypes. Furthermore, inferior survival in AMKL (compared to APMF) was reflected in its proliferative disease features such as splenomegaly, high blast counts, and high lactate dehydrogenase. Interestingly, the presence of JAK2 V617F variants in 40% of the interrogated samples was higher than expected. Although JAK2 is a known molecular driver in myeloproliferative neoplasms, it is actually very rare in de novo AML (~1%). Despite improved survival with allogeneic stem cell transplant, we can conclude that treatment outcomes for AMKL and APMF with standard-of-care therapy were sobering and that patients with these rare AML subtypes should undergo molecular evaluation to qualify them for clinical trials with novel targeted therapies. Citation Format: Marissa Li, Moritz Binder, Basma Basha, Ben Zhang, Alejandro Ferrer, Yuanhan Liu, Mrinal Patnaik, David Viswanatha, Naseema Gangat. Acute megakaryoblastic leukemia is associated with poor overall survival and enriched in JAK2 variants [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B03.

The Pediatric Acute Leukemia Fusion Oncogene ETO2‐GLIS2 Increases Self‐Renewal and Alters Differentiation in a Human Induced Pluripotent Stem Cells‐Derived Model

Supplemental Digital Content is available in the text.

3085 - Generation of a Novel Mouse Model Recapitulating Features of Human Acute Megakaryoblastic Leukemia

Non-Down syndrome (non-DS) acute megakaryoblastic leukemia (AMKL) represents approximately 10% of pediatric acute myeloid leukemia (AML) cases and is associated with poor prognosis (<40% 3-year survival). Transcriptome, whole-genome sequencing, FISH, and RT-PCR analyses of pediatric non-DS AMKL patients recently revealed the presence of cryptic oncogenic fusions. While the majority of pediatric non-DS AMKLs harbor only one fusion, none are recovered in adult AMKL. Despite intensive studies of these oncogenic proteins, the molecular mechanism(s) underlying the role of specific oncogenic fusions in tumor development remains poorly characterized. This is due, in part, to the lack of mouse models that mimic the development of human AMKL. Thus, the generation of new AMKL mouse models harboring specific oncogenic fusions is a prerequisite to study the in vivo transformation process underlying AMKL formation and to identify new therapeutic avenues. To generate AMKL mouse models, we reconstituted lethally irradiated mice with fetal liver cells transduced with oncogenic fusion proteins. AMKL was generated with a penetrance of 50–60% and affected mice displayed striking splenomegaly. Flow cytometric analysis of fusion positive cells revealed increased proportions of cells co-expressing CD41 and CD61, and a reduced proportion of cells expressing B cell, T cell, macrophage and erythroid lineage markers, indicating a skewing towards megakaryocytic differentiation. Moreover, we observed an enrichment of hematopoietic stem and progenitor cells, lineage committed progenitors, and immature megakaryocyte progenitors in the bone marrow and spleens of sick mice. Bioluminescent imaging allowed detection of leukemia cells in bone marrow and spleen. Finally, histology analysis of bone marrow, spleen and peripheral blood revealed the presence of cells with morphology consistent with megakaryoblasts, megakaryocytes and megathrombocytes. In summary, we developed and characterized a novel AMKL murine model with high penetrance that recapitulates many clinical features of human AMKL and provides a novel tool to conduct mechanistic and preclinical studies of this disease.

High PRMT1 Expression Promotes the Progression of Acute Megakaryocytic Leukemia Via Controlling Metabolic Pathways

Metabolic reprogramming is needed not only to accommodate but also to drive leukemia progression. Yet very little is known on genetic factors other than IDH1 mutations, which can drive leukemogenesis via metabolic reprogramming. Here, we will present data to suggest that protein arginine methyltransferases 1 (PRMT1) is a driver for acute megakaryocytic leukemia via reprogramming metabolism. PRMT1 is highly expressed in megakaryocyte-erythrocyte progenitor cells and downregulated during the terminal differentiation of megakaryocytes. Constitutively high expression of PRMT1 in acute megakaryoblastic leukemia (AMKL) blocks megakaryocyte differentiation. PRMT1 upregulates RBM15 protein level via methylation-dependent ubiquitylation pathway (Zheng et al. Elife, 2015). In this presentation, we discovered that metabolic stress such as hypoxia downregulates PRMT1 protein level. Thus, metabolic stress is the upstream signal for the PRMT1-RBM15 pathway. We have identified that RBM15 specifically binds to 3'UTR of mRNAs of genes involved in metabolic pathways. Using RNA-immunoprecipitation with anti-RBM15 antibody and real-time PCR assays, we validated that RBM15 binds to mRNAs of genes involved in fatty acid oxidation and glycolysis. We transduced PRMT1 into an RBM15-MKL1 expressing cell line 6133. Overexpression of PRMT1 renders 6133 cells to grow in a cytokine-independent manner with increased mitochondria biogenesis, which in turn produces higher concentration of ATP in our metabolomic analysis. Based on the analysis of metabolomics data and RBM15-target genes, we conclude that PRMT1 promotes the usage of glucose as bioenergy via oxidative phosphorylation and inhibits fatty acid oxidation. Given that acetyl-coA is higher in PRMT1 expressing 6133 cells, we asked whether histone acetylation is upregulated in PRMT1 overexpressed 6133 cells. Indeed, we found higher histone acetylation level in PRMT1 highly expressed cells. We also found that propionylated histone is reduced, which is consistent with reduced fatty acid oxidation. Propionyl-CoA molecules are produced from fatty acids with odd carbon numbers. Thus PRMT1-mediated metabolic reprogramming changes epigenetic programming during leukemia progression. Intriguing, we also found PRMT1 overexpression enhances histone H3S10 phosphorylation via methylation-dependent ubiquitylation of DUSP4. DUSP4 promotes polyploidy during megakaryocyte differentiation. Thus PRMT1 caused profound epigenetic changes to promote leukemogenesis. In this vein, we established mouse AMKL models by bone marrow transplantation of 6133 cells as well as human AMKL patient samples respectively. Using this mouse model, we tested PRMT1 inhibitors, acetyltransferase inhibitors as well as other metabolic inhibitors. Treating cells with PRMT1 inhibitors as well as metabolic inhibitors promote MK differentiation of AMKL leukemia cells. Metabolomics analysis of cells recovered from mouse models will be discussed in the presentation. In summary, our data demonstrated that PRMT1 is a major sensor for metabolic stress and that PRMT1 in turn reprograms metabolic pathways to bring epigenetic changes in leukemogenesis. Therefore, targeting PRMT1 and downstream PRMT1-regulated metabolic pathways will offer new avenues in treating acute megakaryocytic leukemia and other hematological malignancies with defective megakaryocyte differentiation. DisclosuresNo relevant conflicts of interest to declare.

Analysis on the laboratory examination characteristics in 22 patients with acute megakaryoblastic leukemia

目的分析急性巨核细胞白血病（AMKL）患者实验室检查特点。方法采集22例AMKL患者骨髓，分离有核细胞，一部分用于制备普通透射电镜标本，观察AMKL患者巨核细胞的形态结构；另一部分用于制备血小板过氧化物酶（PPO）细胞组化电镜标本，分析AMKL患者巨核细胞PPO阳性反应。同时结合分析患者骨髓细胞形态学、细胞化学和染色体核型等检查结果。结果22例患者中，17例患者骨髓巨核细胞以Ⅰ期巨核细胞为主，核圆，胞质含少量微管、膜性囊腔及微小致密颗粒，无分界膜和表面连接管道系统等特征性结构，致密颗粒和α颗粒少；5例患者骨髓巨核细胞大小不等，除Ⅰ期巨核细胞外，同时含有大量Ⅱ、Ⅲ期巨核细胞。22例患者巨核细胞PPO阳性率0~80%。22例患者骨髓涂片中均可见原始和幼稚巨核细胞，CD41免疫组化染色呈阳性反应。复杂染色体核型异常多见。结论大部分AMKL患者骨髓巨核细胞以Ⅰ期巨核细胞为主，少数患者除Ⅰ期巨核细胞外还伴有大量Ⅱ、Ⅲ期巨核细胞；PPO阳性反应差别明显；巨核细胞CD41免疫组化染色具有特异性；染色体核型复杂。

Prognostic Relevance of Recurrent Genetic Aberrations in Pediatric Acute Megakaryoblastic Leukemia

Genetic abnormalities and early treatment response are the main prognostic factors in pediatric acute myeloid leukemia (AML). Non-Down Syndrome (non-DS) acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML, with a poor prognosis. These cases present with diverse cytogenetic aberrations, which might be important for risk-group stratification. A well-known genetic aberration in non-DS-AMKL is t(1;22)(p13;q13), resulting in expression of the RBM15/MKL1(OTT/MAL) fusion transcript. In recent years, targeted and genome-wide sequencing has identified novel, recurrent molecular abnormalities in pediatric AMKL. New fusions identified are inv(16)(p13.3q24.3) resulting in CBFA2T3/GLIS2 (Gruber et al, Cell, 2012; Masetti et al, Blood, 2013) and t(11;12)(p15;p13) resulting in NUP98/KDM5A (de Rooij et al, Leukemia, 2013) as specific aberrations in pediatric non-DS AMKL. Also KMT2A(=MLL)-rearrangements are recurrent in non-DS AMKL. The prognostic relevance of these novel abnormalities is not determined in a large cohort, since this disease is rare.To assess frequencies, clinical characteristics and outcome parameters of recurrent cytogenetic aberrations of pediatric non-DS AMKL, databases of the BFM-SG, DCOG, AIEOP, and COG were combined. In this study, we analyzed 151 newly diagnosed pediatric non-DS AMKL cases diagnosed between 1998 and 2014 of whom a sample was available. All patients included were screened for NUP98/KDM5A, CBFA2T3/GLIS2 and RBM15/MKL1 with reverse transcriptase(RT-) PCR,and for KMT2A-rearrangements using split signal FISH and RT-PCR. To assess outcome, probability of event-free survival (pEFS) and probability of overall survival (pOS) were estimated by the Kaplan-Meier method, and groups were compared with the log-rank test; the cumulative incidence of non-response or relapse (pCIR) was analysed by the Kalbfleisch and Prentice method, and groups were compared with the Gray's test. A Cox regression analysis was done for EFS and relapse incidence with as co-variables cytogenetic subgroup, age, sex, WBC and stem cell transplantation (time dependent variable).Patients were treated with different protocols; however, all protocols consisted of intensive chemotherapy using an anthracycline and cytarabine backbone for both induction and consolidation; 26% of patients received stem cell transplantation (SCT) in first remission. Median age was 1.6 years (range 0.1-17.1 years), with 62% of the patients younger than 2 years at diagnosis; 45% of patients were males, and median white blood cell count was 13.7x109/L (range 1.1-378.5x109/L). Translocation NUP98/KDM5A was identified in 9%; the CBFA2T3/GLIS2 translocation in 16%; RBM15/MKL1 in 12% and KMT2A-rearrangements in 9%, and hence in 54% none of these abnormalities were detected. All these aberrations were mutually exclusive. Comparing patients with NUP98/KDM5A, CBFA2T3/GLIS, RBM15/MKL1, or KMT2A-rearrangements with other pediatric AMKL patients, no significant differences in sex, age, and white blood cell count were found. Outcome between the included collaborative groups did not differ significantly. The 5-year pOS of the entire pediatric non-DS AMKL cohort was 56±4%, 5-year pEFS was 51±4%. NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearranged positive patients showed a poor outcome (pOS 36±13%, 38±10%, and 38±13% respectively), compared to RBM15/MKL1 positive cases and other pediatric non-DS AMKL (70±11% and 65±5% respectively, p=0.04, Figure 1). Harboring NUP98/KDM5A, CBFA2T3/GLIS2 or a KMT2A-rearrangement resulted in an increased risk of experiencing an event (EFS; HR1.65, 95%CI;1.03-2.66, p=0.039) or relapse (RFS; HR2.14, 95%CI;1.12-4.11, p=0.022). SCT was not an independent factor for event or relapse free survival (HR1.20, 95%CI;0.65-2.20, p=0.565 and HR 1.13, 95%CI;0.57-2.24, p=0.717, respectively), nor did sex, age at diagnosis or WBC.Our results indicate that non-DS AMKL is a heterogeneous group within pediatric AML. Although the overall survival is poor for non-DS AMKL in general with a 5-yr pOS of ~55%, the poor outcome is specifically determined by cases with NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearrangements. Other variants, including those with RBM15/MKL1, are associated with a better outcome. These data show that international collaboration allows the identification of prognostic subgroups, which may lead to new and more refined risk-group stratification. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MN1-Fli1 oncofusion transforms murine hematopoietic progenitor cells into acute megakaryoblastic leukemia cells.

Long-term outcome of acute megakaryoblastic leukemia (AMKL) patients without Down's syndrome remains poor. Founding mutations and chimeric oncogenes characterize various AMKL subtypes. However, for around one third of all cases the underlying mechanisms of AMKL leukemogenesis are still largely unknown. Recently, an in-frame fusion of meningeoma 1–friend leukemia virus integration 1 (MN1–Fli1) gene was detected in a child with AMKL. We intended to investigate the potential role of this oncofusion in leukemogenesis of acute myeloid leukemia. Strikingly, expression of MN1–Fli1 in murine hematopoietic progenitor cells was sufficient to induce leukemic transformation generating immature myeloid cells with cytomorphology and expression of surface markers typical for AMKL. Systematic structure function analyses revealed FLS and 3′ETS domains of Fli1 as decisive domains for the AMKL phenotype. Our data highlight an important role of MN1–Fli1 in AMKL leukemogenesis and provide a basis for research assessing the value of this oncofusion as a future diagnostic marker and/or therapeutic target in AMKL patients.

Novel AMKL Chimeric Transcription Factors Are Potent Inducers of Leukemia with Unique Mechanisms of Leukemogenesis

Acute megakaryoblastic leukemia (AMKL) accounts for ~10% of childhood AML. AMKL patients without Down syndrome have a poor outcome with a 3 year survival of less than 40%. To gain insight into the biology of this disease, we previously performed transcriptome sequencing on diagnostic blasts from a discovery cohort of 14 pediatric cases and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult samples. This analysis identified novel fusion transcripts restricted to pediatric AMKL including CBFA2T3-GLIS2,GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9.To confirm their role in oncogenesis and gain insight into the mechanism whereby these fusions promote disease, we introduced each of them into murine hematopoietic cells and assessed their effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a control retrovirus failed to form colonies after the second replating. By contrast, expression of each of the fusion genes resulted in a marked increase in self-renewal capacity, with colony formation persisting through 10 replatings. Immunophenotypic analysis revealed evidence of megakaryocytic differentiation in CBFA2T3-GLIS2 and MN1-FLI1 cohorts, whereas NIPBL-HOXB9 and GATA2-HOXA9 cells carried markers consistent with myeloid progenitors. Transplantation of fusion gene modified bone marrow cells into syngeneic recipients induced overt leukemia in all cohorts with the exception of CBFA2T3-GLIS2, suggesting an essential requirement for cooperative mutation(s) in cases expressing this chimeric gene. To assess self-renewal activity of the leukemia generated in our murine models, we conducted secondary transplants for all cohorts. In all cases, the leukemia was transplantable with a shorter latency than in the primary transplant setting.To characterize the tumors at the molecular level, 5 samples from each of the 3 fusions underwent array comparative genomic hybridization, transcriptome, and whole exome sequencing. Samples demonstrated a small number of cooperating mutations with 1.5 copy number alterations (range 0-6) and 6.4 single nucleotide variations (range 2-13) per case. Overall, cases carried an average of 7.9 mutations (range 2-14). Despite the low number of lesions, recurrently mutated genes were identified. These include activating mutations in Flt3, Kras, and cMet, as well as loss of function mutations in the tumor suppressors Phactr4, Wt1, and Tet2. A comparison between fusion subtypes did not reveal any statistically significant differences, although there was a trend towards a greater number of mutations in the GATA2-HOXA9 cohort. Transcriptome sequencing of cohorts, along with normal hematopoietic progenitor subsets, confirmed unique gene expression patterns between each of the fusions. Consistent with immunophenotyping, MN1-FLI1 demonstrated enrichment of the MEP signature while NIPBL-HOXB9 and GATA2-HOXA9 were enriched for CMP and monocyte precursor signatures respectively. ChIP-seq analysis of each of the fusions is underway to definitively identify the genomic targets whose expression is directly altered by their binding.A common characteristic between all fusions is the presence of protein interaction domains contributed by the N term partner, and DNA binding domains contributed by the C term partner. To determine if these fusions have a novel gain of function distinct from their independent counterparts, we introduced each partner gene into murine bone marrow cells for transplantation experiments. As previously described, introduction of MN1 into hematopoietic cells led to a highly penetrant leukemia. In contrast, HOXA9, HOXB9, and FLI1 all had >75% disease free survival with few myeloid leukemias resulting from their over expression, while GATA2 failed to induce any disease at all. NIPBL’s size precluded transplant assays. Therefore, to evaluate its contribution we introduced a point mutation previously shown to disrupt binding of NIPBL to the cohesion component MAU2. This alteration abrogated the ability of the fusion to induce leukemia in our transplant model, demonstrating the importance of this interaction in the pathogenesis of disease. In conclusion, our data confirms a pathogenic role for GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9 in AMKL. Further studies delineating the cooperating mutations required for CBFA2T3-GLIS2 are indicated. DisclosuresNo relevant conflicts of interest to declare.

Pediatric Acute Megakaryoblastic Leukemia without Down Syndrome: A Retrospective Study by the International Berlin-Frankfurt-Munster Study Group (I-BFMSG)

Acute megakaryoblastic leukemia (AMKL) comprises up to 10% of childhood acute myeloid leukemia (AML) cases. However, no large-scale studies have comprehensively evaluated the clinical characteristics and outcomes of patients with AMKL. We performed a large-scale international retrospective study of pediatric patients (diagnosed at age ≤18 years) with de novoAMKL without Down syndrome treated from 1989 to 2009.The study included 490 patients with AMKL, which comprises 7.9% of the pediatric AML patients treated by 19 members of the I-BFMSG. At diagnosis, the median age of patients was 1.5 years (range, 0.0–16.5 years), median white blood cell count was 12.0×109/L (range, 0.6–188.0×109/L), 23 (4.7%) patients showed central nervous system involvement, and both sexes were equally represented. Complete remission (CR) was achieved in 417 (85.1%) patients, and 5-year event-free (EFS) and overall survival (OS) rates were 43.7%±2.7% and 49.0%±2.7%, respectively. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed in 206 (42.0%) patients. The 5-year disease-free survival and OS rates for patients who received allo-HSCT in first CR (n=112, 56.3%±5.2%, and 57.7%±5.2%, respectively) and for those who did not receive transplantation in first CR (n=298, 50.2%±3.6% and 55.2%±3.5%) were not significantly different (P=0.12 and P=0.57).Complete cytogenetic data were available for 372 (75.9%) patients: diploid (n=49, 13.2%), hypodiploid (n=18, 4.8%), pseudodiploid (n=119, 32.0%), 47–50 chromosomes (n=142, 38.2%), and >50 chromosomes (n=44, 11.8%). Chromosome gain occurred in 195 (52.4%) patients: +21 was the most common (n=106, 28.5%), followed by +19 (n=93, 25.0%), and +8 (n=77, 20.7%). Losses occurred in 65 cases (17.5%), in the decreasing order of –7 (n=13, 3.5%), –9 (n=9, 2.4%), –13, and –15 (n=7 each, 1.9%). Structural chromosomal aberrations were observed in 278 (74.7%) patients, most commonly t(1;22) (n=51, 13.7%) and 11q23 rearrangements (n=38, 10.2%) with t(9;11) in 21 patients. Other abnormalities included 7p (n=43, 11.6%) and 13q (n=31, 8.3%; 16 with deletions) breakpoints. Patients with t(1;22) were significantly younger at diagnosis (P<0.001; median 0.6 years, range 0.0–6.3 years) and more often female (P=0.05; 64.7%) while those with –7 were significantly older (P=0.006; median 4.2 years, range 0.4–15.0 years) than those with other cytogenetic subgroups.Multiple regression analysis for EFS and OS with clinical and cytogenetic features showed that treatment period (1989–1999 vs. 2000–2009), normal cytogenetics, –7, t(9;11), 13q-, and –15 were associated with significantly worse outcomes than those for other subgroups, whereas abnormalities in 7p were associated with better outcomes (Table). Patients with –13 and 9p abnormalities other than t(9;11) had a poorer EFS and OS, respectively. Patients with +21 or t(1;22) did not show a significant difference in survival rates.This international study on the largest cohort of AMKL patients analyzed to date shows heterogeneity in cytogenetic findings and identifies some subgroups with a particularly dismal outcome. The advent of newer methods to evaluate genetic lesions can help identify therapeutic targets for improving the outcome in this subgroup of patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NUP98/JARID1A is a novel recurrent abnormality in pediatric acute megakaryoblastic leukemia with a distinct HOX gene expression pattern

Cytogenetic abnormalities and early response to treatment are the main prognostic factors in acute myeloid leukemia (AML). Recently, NUP98/NSD1 (t(5; 11)(q35; p15)), a cytogenetically cryptic fusion, was described as recurrent event in AML, characterized by dismal prognosis and HOXA/B gene overexpression. Using split-signal fluorescence in situ hybridization, other NUP98-rearranged pediatric AML cases were identified, including several acute megakaryoblastic leukemia (AMKL) cases with a cytogenetically cryptic fusion of NUP98 to JARID1A (t(11;15)(p15;q35)). In this study we screened 105 pediatric AMKL cases to analyze the frequency of NUP98/JARID1A and other recurrent genetic abnormalities. NUP98/JARID1A was identified in 11/105 patients (10.5%). Other abnormalities consisted of RBM15/MKL1 (n=16), CBFA2T3/GLIS2 (n=13) and MLL-rearrangements (n=13). Comparing NUP98/JARID1A-positive patients with other pediatric AMKL patients, no significant differences in sex, age and white blood cell count were found. NUP98/JARID1A was not an independent prognostic factor for 5-year overall (probability of overall survival (pOS)) or event-free survival (probability of event-free survival (pEFS)), although the 5-year pOS for the entire AMKL cohort was poor (42 ± 6%). Cases with RBM15/MLK1 fared significantly better in terms of pOS and pEFS, although this was not independent from other risk factors in multivariate analysis. NUP98/JARID1A cases were characterized by HOXA/B gene overexpression, which is a potential druggable pathway. In conclusion, NUP98/JARID1A is a novel recurrent genetic abnormality in pediatric AMKL.

NUP98/JARID1A Is a New Recurrent Genetic Abnormality in Pediatric Acute Megakaryoblastic Leukemia with a Distinct HOX-Gene Expression Pattern

AbstractAbstract ▪537▪This icon denotes a clinically relevant abstract Introduction:Cure rates in pediatric AML are currently in the 60–70% range despite treatment with intensive chemotherapy. To improve prognosis new treatment targets need to be identified, hence there is a need to better understand the underlying biology. It is hypothesized that AML results from at least two types of mutations which non-randomly collaborate in leukemogenesis. The type-I aberrations confer a proliferative advantage, type-II mutations lead to impairment of hematopoietic differentiation (Kelly et al, 2002). We recently described NUP98/NSD1 as recurrent event in cytogenetically normal AML (Hollink et al, 2011). Patients with NUP98/NSD1 had dismal outcome, and a stem-cell phenotype characterized by overexpression of homeobox (HOX) A and –B genes. Using split-signal FISH on 122 pediatric AML cases without driving oncogenic mutation, 26 NUP98- rearranged cases were identified, including 1 patient with acute megakaryoblastic leukemia (AMKL). We previously reported a patient with fusion of JARID1A, located on chromosome 12p13, to NUP98, located on chromosome 11p15, in a non-Down Syndrome (DS) AMKL case (Van Zutven et al, 2006). Therefore, a large series of non-DS AMKL patients was screened for NUP98/JARID1A and for other abnormalities, including the novel CBFA2T3/GLIS2 translocation (Gruber et al, ASH2011; #757). Methods:Samples from 105 pediatric non-DS AMKL cases, diagnosed between 1998 and 2011, were obtained from the DCOG, the AML-BFM SG, the Saint-Louis Hospital in Paris, and the COG. AMKL is more common in DS patients, therefore we also screened a series of DS AMKL (n=16). Centrally reviewed clinical and cell-biological data were provided by these study groups. Translocation of NUP98/JARID1A, MLL-rearrangements, RBM15/MKL1, and CBFA2T3/GLIS2 were identified using RT-PCR, as well as molecular characterization including hospots for the following mutations: FLT3, KIT, RAS, PTPN11, NPM1, WT1, and CEBPA. HOXA and –B expression levels were analyzed using gene expression profiling (Affymetrix) in 274 pediatric AML patients (Balgobind et al, 2011) including 9 AMKL patients, and validated with quantitative real-time PCR (n=37). Results:NUP98/JARID1A translocations were identified in 11 patients (11%). Four other patients had a NUP98- aberration with unknown translocation partner based on split signal FISH. We identified 16/105 patients with RBM15/MKL1, 13/105 with CBFA2T3/GLIS2 translocation, and 13/96 harbouring an MLL-rearrangement. Hence, specific non-random abnormalities could be defined in 61% of pediatric AMKL cases. Only 3/45 cases harboured a type-I mutation, all localized in the RAS gene. Comparing NUP98/JARID1A positive cases with negative cases in pediatric AMKL, no significant differences in patient characteristics including sex, age, and white blood cell count (WBC) were found. Considering prognosis, 5-year pEFS (22±14% vs. 36±6%, p=0.50) did not differ significantly from all other AMKL patients, nor did the cumulative incidence of relapse (56±19% vs. 54±7% p=0.9). CBFA2T3/GLIS2 translocated patients also did not differ from other AMKL patients (pEFS 19±16% vs. 36±6%, p=0.63). However, 5-year pEFS for RBM15/MKL1 translocated patients was significantly better (73±13% vs. 28±6%, p=0.043), but not in multivariate analysis adjusted for age and WBC. Gene expression analysis showed significantly higher HOXA5/A9/A10 and HOXB2/B3/B4/B5/B6 expression in NUP98/JARID1A compared to other pediatric AML cases. We did not identify any NUP98/JARID1A cases in the 16 DS AMKL patients. Discussion and conclusion:NUP98/JARID1A is a recurrent cryptic translocation in approximately 11% of pediatric AMKL cases. In 61% of all AMKL cases a type-II mutation could now be identified. Similar to NUP98-NSD1 a stem-cell phenotype was detected with persistent HOXAB-gene expression. Although NUP98/JARID1A did not influence prognosis, outcome in pediatric AMKL is unsatisfactory. NUP98 is known to recruit CREBBP/p300 resulting in histone acetylation, and transcriptional activation of HOX genes (Wang et al, 2007), suggesting that histone acetyltransferase inhibitors may be active. Moreover, JARID1A is unable to demethylate H3K4me2/3, which also results in sustained up regulation of HOX genes. This may provide options for targeted therapy. Disclosures:No relevant conflicts of interest to declare.

High Frequency of GATA1 Mutations in Childhood Non-Down Syndrome Acute Megakaryoblastic Leukemia

AbstractAbstract ▪888▪This icon denotes a clinically relevant abstract Introduction:Pediatric acute megakaryoblastic leukemia (AMKL) occurred in 6.6% (84/1271) of the children enrolled to the AML-BFM98 and 2004 studies.Despite a similar phenotype in morphology and immunophenotype, AMKL shows a heterogenous cytogenetic distribution (normal karyotype 23%, complex karyotype 21%, t(1;22) 9%; MLL-rearrangement 8%; monosomy 7 5%, trisomy 8 5%; other aberrations 29%).Mutations of the hematopoietic transcription factor GATA1 have been identified in almost all children suffering myeloid leukemia of Down syndrome (ML-DS). In addition, GATA1 mutations (GATA1mut) could be identified in children with trisomy 21 mosaic.Here, AMKL without evidence of Down syndrome or Down syndrome mosaic were analyzed for mutations in exon 1, 2 or 3 of the transcription factor GATA1. Patients:Seventy-one children from the AML-BFM Study group (n=51; 2000–2011), the Netherlands (n=10), France (n=3) and Scandinavia (n=7) were included.Within the AML-BFM Group the 51 analyzed patients showed similar characteristics compared to the total cohort of 84 children with AMKL of the AML-BFM 98 and 2004 studies.AMKL was confirmed according to the WHO classification by genetics (t(1;22)); morphology and immunophenotyping. Table 1a) summarizes the patientxs characteristics and b) the cytogenetic results. Methods:For GATA1 mutation screening genomic DNA was amplified by PCR reaction for exon 1, 2, and 3. PCR amplicons were analyzed by direct sequencing or following denaturing high-performance liquid chromatography (WAVE). Results:Seven different GATA1 mutations were detected in 8 children (11.1%; table 2).In all GATA1mut leukemia, a trisomy 21 within the leukemic blasts could be detected.Seven out of these 8 children and all other 64 AMKL patients have been treated with intensive chemotherapy regimens according the study group protocols. The results are given in table 2. All achieved continuous complete remission (CCR; 0.4 to 4.2 years). [Display omitted] In one newborn with typical morphology and immunophenotype a GATA1mut associated transient leukemia was supposed. The child achieved CCR (follow-up 6 years).In total, allogeneic stem cell transplantation in 1st CR was performed in 6 children with AMKL (GATA1mut leukemia n=1). [Display omitted] Conclusions:GATA1 mutations occurred in 11% of children with AMKL without any symptoms or evidence of trisomy 21 or trisomy 21 mosaic. GATA1 mutations are associated with a trisomy 21 within the leukemic blasts. Although non-response occurred, prognosis was significant better compared to other AMKL. Therefore, analysis of GATA1 mutation in infant AMKL is strongly recommended. Whether treatment reduction similar to ML-DS Down syndrome is feasible needs to be confirmed. Disclosures:No relevant conflicts of interest to declare.