Childhood Acute Megakaryoblastic Leukemia Research Articles

Advances in molecular characterization of pediatric acute megakaryoblastic leukemia not associated with Down syndrome; impact on therapy development.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) in which leukemic blasts have megakaryocytic features. AMKL makes up 4%-15% of newly diagnosed pediatric AML, typically affecting young children (less than 2years old). AMKL associated with Down syndrome (DS) shows GATA1 mutations and has a favorable prognosis. In contrast, AMKL in children without DS is often associated with recurrent and mutually exclusive chimeric fusion genes and has an unfavorable prognosis. This review mainly summarizes the unique features of pediatric non-DS AMKL and highlights the development of novel therapies for high-risk patients. Due to the rarity of pediatric AMKL, large-scale multi-center studies are needed to progress molecular characterization of this disease. Better disease models are also required to test leukemogenic mechanisms and emerging therapies.

Transcriptomic and Network Signatures of Cryptic Fusion CBFA2T3-GLIS2 in Pediatric AML Reveals Clinical Considerations and Implications

IntroductionA cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion was initially reported in childhood acute megakaryoblastic leukemia (AMKL) and was shown to be associated with unfavorable outcomes. Although this fusion onco-protein is more prevalent in FAB M7 AML, it is reported in younger patients with all subtypes of AML. We present a comprehensive transcriptomic signature of an unselected cohort of children with CBFA2T3-GLIS2 fusion treated on COG AML trials.MethodsCBFA2T3-GLIS2 fusions were identified in 26 of 1042 (2.5%) patients enrolled in Children's Oncology Group (COG) studies AAML03P1 and AAML0531, with a prevalence of 8% in patients < 3 years of age. Whole transcriptome sequence data was available from 466 patients and 20 normal bone marrows (NBM) for expression profiling. Differentially expressed genes were identified using the Limma v.3.30.12 R package. Unsupervised hierarchal clustering was performed using Euclidean distance with the WPGMA linkage algorithm. Gene-set enrichment analysis was completed using the Broad Institute software v.2-2.2.4 with MSigDB C2 curated gene sets.ResultsOf 26 patients with CBFA2T3-GLIS2 fusions, 21 (81%) were < 5 years of age (Fig. 1A). KMT2A fusions co-occurred in only two patients, and none of the positive tumors carried core binding factor fusions. Few other variants accompanied CBFA2T3-GLIS2 fusions and overall, this subclass of AML had been categorized as cytogenetically normal (42% CN-AML, Fig. 1B). CBFA2T3-GLIS2 AML fared worse than the AML population as a whole; more than 75% of fusion-positive cases relapsed within two years (Fig. 1C, p = 0.001).Gene-set enrichment analysis between CBFA2T3-GLIS2 fusion and fusion-negative tumors revealed the top three positively enriched gene sets as 1) basal cell carcinoma (FDR = 0.003), 2) ECM receptor interactions (FDR = 0.006), and 3) hedgehog signaling pathways (FDR = 0.029). Consistent with previous reports, genes associated with WNT, hedgehog (HH), and BMP signaling were significantly up-regulated in CBFA2T3-GLIS2 tumors (Fig. 1D). CBFA2T3-GLIS2 fusions have been associated with induction of BMP signaling through downstream HH targets, BMP2 and BMP4 in pediatric AMKL. Moreover, concurrent dysregulation of WNT, BMP and HH signaling is a feature of CBFA2T3-GLIS2 AMKL (Gruber et al., 2015). Unsupervised hierarchical clustering of differentially expressed genes effectively clustered the fusion-positive samples, suggesting CBFA2T3-GLIS2 fusions drive a distinct transcriptional profile from other AML subtypes (Fig. 1E). CBFA2T3-GLIS2 tumors compared to NBM had upregulated expression of 4 cancer testes antigens (CTAs): CCNA1, PRAME, IGF2BP3, and KDM5B (fold-change > 2, adj. p < 0.001). CTAs are promising candidates for targeted T-cell therapies due their low expression in NBM and surrounding tissues, which could limit treatment related toxicity ( Ochsenreither et al., 2012) .We further performed unsupervised hierarchical clustering in 437 patients for whom flow cytometry data were available (Fig. 1F). Most fusion-positive tumors (7/10) clustered together by mean fluorescence intensity (MFI), indicating that CBFA2T3-GLIS2 tumors have highly distinctive immune profiles. Of particular interest, fusion-positive tumors expressed CD56 at high levels (median CD56 in CBFA2T3-GLIS2 : 1,505.0 MFI [range: 6.4 - 4,290.0] versus median CD56 in fusion-negative AML: 9.0 MFI [range: 2.4 - 3,360.0], p = 0.001). A novel immunophenotype in pediatric AML, termed RAM, has been associated with high CD56 expression and much lower event-free survival. A preponderance of CBFA2T3-GLIS2 AML displayed the RAM phenotype, with 70% of fusion-positive tumors identified as RAM compared to 0.23% (1/427) in the fusion-negative population (p < 0.001).ConclusionCBFA2T3-GLIS2 AML tends to have an aggressive disease pathology in much younger patients, who had been primarily classified as CN-AML. This suggests screening for CBFA2T3-GLIS2 in younger patients could be valuable as standard practice, though not yet recommended by the World Health Organization. Our associations with CBFA2T3-GLIS2 fusions provides a potential link between genomic and immunologic characteristics of a high-risk group of pediatric AMLs, which could be employed in the future for clinical screening through molecular or flow-cytometric methods. [Display omitted] DisclosuresBrodersen:Hematologics Inc: Employment. Loken:Hematologics Inc: Employment, Equity Ownership.

Detection and monitoring of minimal residual disease in acute megakaryoblastic leukemia in children

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), which is associated with poor prognosis for all patients except children with t(1;22) or Down syndrome. The frequency of complete remission in case of AMKL is comparable to the frequency of complete remission in other variants of AML, and the median survival is much lower. This determines the necessity to update criteria for assessment of the effect of treatment using flow cytometry definition of the level of minimal residual disease (MRD). Nowadays, there are no unified and standardized approaches for the measurement of MRD in case of myeloid leukemia, including AMKL, which prohibits adequate assessment of the therapy effect and in some cases determination of the indications for allogeneic hematopoietic stem cells transplantation. The article identifies diagnostic features and describes approaches for the measurement of the level of MRD in case of AMKL.&#x0D; Aim. The aim is to demonstrate the algorithms for diagnosing and measuring MRD in case of AML-M7 in children.&#x0D; Materials and methods. The article analyzes the clinical and immunological profile of 10 boys and 4 girls with the initial diagnosis of AMKL between the ages of 3 months 12 years old, 13 of them have received treatment in the FSBI N.N. Blokhin National Medical Research Center of Oncology and one in the GBUZ Morozovsky DGKB between 1995 and 2020, The measurement of MRD was carried out in 6 patients. The measurement of MRD was carried out using both morphocytochemical method and multiparameter flow cytometry with megakaryocyte markers (CD61, CD42, CD41) in combination with other myeloid markers (CD13, CD33), CD34, CD117 and aberrant markers (mainly CD7).&#x0D; Results. We showed that adequate measurement of the level of MRD had required detailed immunophenotyping during diagnosis to determine the aberration of megakaryoblasts. CD9 marker (100%), CD33 myeloid marker (69.2%), stem cell antigen CD34 (46.2%), CD13 (38.2%) in addition to megakaryocyte markers (100%) were most often expressed on blast cells in case of AMKL. The CD117 antigen was present on the blasts in 33.3% of cases. The expression of the T-cell-associated CD7 antigen (46.2%) was frequent. The measurement of MRD was carried out during the treatment (usually after an induction course) on the basis of the markers of megakaryocytic cell line (CD61, CD41, CD42a, CD42b), weak CD45 expression, as well as the immunophenotype characteristics during initial diagnosis. The level of MRD ranged from completely negative (0%; 0.006%) to evident (1.05%).&#x0D; Conclusion. The detection of residual tumor megakaryoblasts in case of AML-M7 using flow cytometry is a promising method to evaluate the effect of therapy. The adequate measurement of the level of MRD requires detailed immunophenotyping during the diagnosis to determine the aberration of megakaryoblasts.

Clinical impact of genomic characterization of 15 patients with acute megakaryoblastic leukemia-related malignancies.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia but is approximately 500 times more likely to develop in children with Down syndrome (DS) through transformation of transient abnormal myelopoiesis (TAM). This study investigates the clinical significance of genomic heterogeneity of AMKL in children with and without DS and in children with TAM. Genomic evaluation of nine patients with DS-related TAM or AMKL, and six patients with non-DS AMKL, included conventional cytogenetics and a comprehensive next-generation sequencing panel for single-nucleotide variants/indels and copy-number variants in 118 genes and fusions involving 110 genes. Recurrent gene fusions were found in all patients with non-DS, including two individuals with complex genomes and either a NUP98–KDM5A or a KMT2A–MLLT6 fusion, and the remaining harbored a CBFA2T3–GLIS2 fusion, which arose from both typical and atypical cytogenetic mechanisms. These fusions guided treatment protocols and resulted in a change in diagnosis in two patients. The nine patients with DS had constitutional trisomy 21 and somatic GATA1 mutations, and those with DS-AMKL had two to four additional clinically significant somatic mutations. Comprehensive genomic characterization provides critical information for diagnosis, risk stratification, and treatment decisions for patients with AMKL. Continued genetic and clinical characterization of these rare cancers will aid in improving patient management.

Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21

AbstractPatients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s. We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL.

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.

Outcome and Genomic Characteristics of Non-Down Syndrome Acute Megakaryoblastic Leukemia in Children Treated with Low-Dose Chemotherapy for Induction Remission

Background: Acute megakaryocytic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) with a dismal outcome in children without Down syndrome. Refractory or relapsed disease accounts for treatment failure in most cases. Hematopoietic stem cell transplantation (HSCT) is recommended as post-remission therapy, although some patients have been cured with intensive chemotherapy alone. Genetic and genomic abnormalities are common in AMKL, but their prognostic significance is not known. In this study, we analyzed AMKL using high-throughput sequencing in addition to conventional morphology, immunophenotype, cytogenetic, and molecular (MICM) approaches. Methods: From February 2015 to April 2019, 19 cases of AMKL in non-Down syndrome patients were diagnosed according to conventional methods in our center. All patients, except one, were treated using a low-dose chemotherapy (LDC) regimen (cytarabine 10 mg/m2, subcutaneously every 12 h, 20 doses; mitoxantrone 5 mg/m2 days 1, 3 and 5, and G-CSF 5µ/kg subcutaneous, daily, 10 doses) for induction remission followed by four courses of intensive chemotherapy or hematopoietic stem cell transplantation (HSCT) as post-remission consolidation[1]. Patients were treated with consolidation chemotherapy if parents refused HSCT. High-throughput mutational sequencing of leukemia cells obtained at diagnosis was performed. The average sequencing depth was 500-1000× and analyses were performed using mGATK, SAMTools, SIFT, PolyPhen2, LRT, and MutatioinTaster methods. Results: The average age at diagnosis of AMKL of the nine boys and 10 girls was 19.7 months (7-53 months). Eighty-four percent of the cases were younger than 2 years of age. The 19 patients with AMKL represent 10% of cases diagnosed with AML during the study period. The median initial white blood cell (WBC) count was 13×109/L(2.38-126×109/L)and the average blasts at initial bone marrow (BM) was 54.5% (13-85%). In all cases of AMKL, more than 25% of cells were positive for CD41. Sixty-three percent of patients had recurrent fusion genes, including NUP98-KDM5A, HOXA11-AS/ANGPT1, CBFA2T3-GLIS2, KMT2A-MLLT3, RBM15-MKL1. Also, we identified PICALM-MLL10, TNIP1-CSF1R, NUP98-TAF3, and CBFA2T3-GLIS1 fusion genes which had not been previously reported in AMKL. EVI-1 expression was evaluated in all cases and was overexpressed in two cases. Forty-seven percent of patients showed at least single-gene mutations, including JAK2, MPL, KRAS, NRAS, TP53, NTRK3, MYO16, PDGFB, FLT3, and others. The response rate after remission induction I (IND-I) were 84.2% with 63.1% (12/19) attaining complete remission (CR) and 21.1% (4/19) partial remission (PR). After IND-II, the CR, PR, and no response (NR) rates was 68.4%, 10.5%, and 21.1%, respectively. Four patients (21.1%) were refractory to induction chemotherapy. Seven patients (36.8%) relapsed, three during consolidation chemotherapy courses, and four after transplantation (Table 1). Currently, 75% (6/8) patients who received chemotherapy only for consolidation were alive (median follow-up time 27 months). Eleven patients underwent allo-HSCT, and 8 patients are alive, including 6 cases living without evidence of disease. Four patients relapsed after allo-HSCT, and two of them have died. One additional patient died of transplantation related mortality. The 4-year OS, RFS, and EFS in this cohort were 62.4%±13.9%, 54.1±13.0% and 40.5±11.5%, respectively (Figure 1).Conclusion: Non-Down syndrome AMKL is genetically heterogeneous with recurrent and new fusion genes. Patients treated with LDC for induction followed by intensive chemotherapy or HSCT for consolidation have outcome comparable to those treated with intensive induction regimens. New treatment strategies are needed to improve the outcome of pediatric AMKL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Immunophenotypic characterization of acute megakaryoblastic leukaemia in children

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia, in which the bone marrow produce increased numbers of immature abnormal megakaryoblasts. AMKL is rare both in children and adults, but is the most frequent subtype of acute myeloid leukemia (AML) in children with Down syndrome. Morphological diagnosis of this disease could be complicated, thus flow cytometry plays a crucial role in the diagnostics of AMKL. The aim of the present study was to investigate the immunophenotypic characteristics of AMKL in children. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. The study group included 103 patients with AMKL. Antigen expression profile was assessed by multicolor flow cytometry. We identified three groups of patients according to different levels of CD45 expression, and in majority of patients (74%) high level of CD45 expression was detected. Significant immunophenotypic differences between these groups were found. In 56% of patients trisomy of 21 chromosome was detected. Among these patients, 86% belonged to group of high CD45 expression. Moreover, children with trisomy 21 represented the majority in the group with high level of CD45 expression (64%). Also, there were found several significant differences between patients with and without trisomy 21 within the group of high CD45 expression. This study demonstrated the wide immunophenotypic heterogeneity of AMKL. In general, the revealed diversity obviously reflects the biological heterogeneity of this AML subtype.

ACUTE MEGAKARYOBLASTIC LEUKEMIA

Acute Megakaryoblastic Leukemia (AMKL) is a subtype of acute myeloid leukemia triggered by megakaryocytes. Acute megakaryoblastic leukemia is divided into three groups, AMKL in children with Down syndrome (DS-AMKL), AMKL in children who do not have Down Syndrome (non-DS-AMKL), and AMKL in non-DS adults (AMKL adults).The basis of the diagnosis of AMKL or AML-M7, according to FAB, is the presence of megakaryocyte line cells as many as 30% or more of all cells. Meanwhile, the diagnosis of AMKL, according to the 2016 WHO guidelines, is acute leukemia with blasts, about &gt; 20%, &gt; 50% of which is megakaryocyte line cells. Megakaryocyte cells can be more clearly seen with electron microscopes that react positively to platelet peroxidase or use marker antibodies to CD41/gpIIb, CD42b/gpIb, CD61/gpIIIa, von Willebrand factors, and linker for T cell activation.Based on the results of this research, there are differences in cytogenetics between the three types of AMKL according to their different pathophysiology. The World Health Organization (WHO) argued that AMKL was categorized into not otherwise specific (NOS) AML criteria. These criteria exclude AML with myelodysplasia (AMLMRC), AML associated with therapy, and AML with recurrent genetic abnormalities, such as AML with t (1; 22) (p13.3; q13.1), inv (3) (q21.3q26.2), or t (3; 3) (q21.3; q26.2). DS-AMKL is also classified into myeloid leukemia associated with DS. In conclusion, AMKL in adults is not only considered as a rare subtype of AMKL, only 1% of AML cases in population-based clinical experiments and data but also has a poor prognosis.

The changing scenario of non-Down syndrome acute megakaryoblastic leukemia in children.

Pediatric non-Down-syndrome acute megakaryoblastic leukemia (non-DS-AMKL) is a heterogeneous subtype of leukemia that has historically been associated with poor prognosis. Until the advent of large-scale genomic sequencing, the management of patients with non-DS-AMKL was very difficult due to the absence of reliable biological prognostic markers. The sequencing of large cohort of pediatric non-DS-AMKL samples led to the discovery of novel genetic aberrations, including high-frequency fusions, such as CBFA2T3-GLIS2 and NUP98-KDM5 A, as well as less frequent aberrations, such as HOX rearrangements. These new insights into the genetic landscape of pediatric non-DS-AMKL has allowed refining the risk-group stratification, leading to important changes in the prognostic scenario of these patients. This review summarizes the most important molecular pathogenic mechanisms of pediatric non-DS-AMKL. A critical discussion on how novel genetic abnormalities have refined the risk profile assessment and changed the management of these patients in clinical practice is also provided.

Prognostic significance of chimeric fusion gene analysis in pediatric acute megakaryoblastic leukemia

Acute megakaryoblastic leukemia in children without Down syndrome (non-DS AMKL) is considered to be a poor prognostic subtype in acute myeloid leukemia. Recently, some chimeric fusion genes were found in pediatric non-DS AMKL; therefore, we attempted to detect chimeric fusion genes RBM15-MKL1, CBFA2T3-GLIS2, and NUP98-KDM5A from 10 pediatric non-DS AMKL diagnostic samples using polymerase chain reaction and Sanger sequencing methods. Two samples were positive for RBM15-MKL1, four had CBFA2T3-GLIS2, and only one case had NUP98-KDM5A. Both RBM15-MKL1-positive patients showed long-term remission after chemotherapy. The eight RBM15-MKL1-negative patients received hematopoietic stem cell transplantation (HSCT). In four CBFA2T3-GLIS2-positive patients, three had HSCT without complete remission and two of themdied. Additional treatment stratification depending on chimeric fusion genes and development of new therapeutic drugs are required for non-DS AMKL.

Global Chromatin Occupancy and Epigenetic Signature Analysis Reveal New Insights into the Function of GATA1 N-Terminus in Erythropoiesis

Mutations in GATA1 are seen in rare cases of dyserythropoietic anemia and in a subset of patients with Diamond Blackfan Anemia (DBA). Of note the truncation mutations in DBA, known as GATA1s, closely resemble those that are more commonly associated with acute megakaryoblastic leukemia in children with Down syndrome (DS). Studies with a mouse model of the Gata1s mutation revealed that replacement of the full-length protein by the shortened isoform led to a marked yet transient enhancement in megakaryopoiesis, similar in some respects to transient myeloproliferative disorder in DS. Furthermore, these mutant mice displayed impaired embryonic erythropoiesis but ostensibly no defects in adult hematopoiesis. In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in the Gata1s mouse strain. We observed a striking impairment in erythropoiesis in fetuses at E10.5 though E12.5, but saw improvement as the animals progressed through E14.5 and beyond. Defects included impaired terminal maturation and reduced numbers of erythroid progenitors, likely at the expense of expanded megakaryopoiesis. RNA-sequencing revealed that both erythroid genes and megakaryocytic genes were altered by the Gata1s mutation. Epiproteomic histone modification analysis further revealed there was an accumulation of H3K27 methylation in the R3 (CD71hiTer119hi) erythroid progenitor population, which suggests that GATA1 has a link to the epigenetic machinery that is altered in Gata1s mutant cells. Despite a global increase in H3K27me3, critical Gata2 regulatory elements in Gata1s mutant erythroid progenitors were marked by substantially less H3K27me3 than in wild-type littermates. Given that overexpression of GATA2 has been reported to impair erythropoiesis, we investigated whether reducing the GATA2 levels would ameliorate the phenotype. Indeed, we observed that haploinsufficiency for Gata2 rescued the erythroid defects of Gata1s fetuses. Next, to comprehensively study the effect of absence of the GATA1 N-terminus genome-wide, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with H3K27me3, GATA1 or GATA1s antibodies on wild-type versus Gata1s expressing fetal erythroid cells. Our data indicated that there is a substantial reduction in H3K27me3 along regulatory elements of the Runx1 gene at the late stage (R3) of fetal erythropoiesis in Gata1s mice. Along with an increase in Runx1 expression we observed strong downregulation of Klf1, a repressive target of RUNX1. Thus, failure of GATA1s to facilitate trimethylation of Runx1 and Gata2 regulatory elements appears to cause the defects in erythroid cell and megakaryocyte development. In parallel, we performed an in-depth analysis of the phenotype of adult Gata1s mice and discovered that they have reduced red cell counts, lower hemoglobin and hematocrit, increased extramedullary hematopoiesis and impaired stress erythropoiesis compared to control littermates. Although there were significantly more megakaryocyte erythrocyte progenitors (MEPs, Lin-c-Kit+Sca-1-CD34-FcgR-) in Gata1s mouse bone marrow, there were fewer pre-colony-forming unit erythroid cells (preCFU-E, Lin-c-Kit+Sca-1-CD41-FcgR-CD150hiCD105hi), likely at the expense of expanded megakaryocyte progenitors (MkP, Lin-c-Kit+Sca-1-CD41+CD150hi).Gata1s mice also developed an MDS-like disease with age. Together, our integrated genomic analysis of transcriptome, GATA1/GATA1s chromatin binding profile and chromatin signature reveal that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in gene transcriptional regulation, lineage determination and red blood cell maturation. DisclosuresCrispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Heterogeneous cytogenetic subgroups and outcomes in childhood acute megakaryoblastic leukemia: a retrospective international study

AbstractComprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age ≤18 years) with non–Down syndrome de novo AMKL diagnosed from 1989 to 2009. Patients with AMKL (median age 1.53 years) comprised 7.8% of pediatric AML. Five-year event-free (EFS) and overall survival (OS) were 43.7% ± 2.7% and 49.0% ± 2.7%, respectively. Patients diagnosed in 2000 to 2009 were treated with higher cytarabine doses and had better EFS (P = .037) and OS (P = .003) than those diagnosed in 1989 to 1999. Transplantation in first remission did not improve survival. Cytogenetic data were available for 372 (75.9%) patients: hypodiploid (n = 18, 4.8%), normal karyotype (n = 49, 13.2%), pseudodiploid (n = 119, 32.0%), 47 to 50 chromosomes (n = 142, 38.2%), and >50 chromosomes (n = 44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): –7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1;22)(p13;q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9;11)(p22;q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk—7p abnormalities; poor risk—normal karyotypes, –7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, –13/13q-, and –15; and intermediate risk—others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.

Split End Family RNA Binding Proteins: Novel Tumor Suppressors Coupling Transcriptional Regulation with RNA Processing

Split End (SPEN) family proteins have three members: SPEN, RBM15, and RBM15B. SPEN family proteins contain three conserved RNA recognition motifs on the N-terminal region and an SPOC domain on the C-terminal region. RBM15 is fused to MKL1 in chromosome translocation t (1;22), which causes childhood acute megakaryoblastic leukemia (AMKL). Haploinsufficiency of RBM15 in AMKL indicates that RBM15 is a tumor suppressor. Both SPEN and RBM15 are mutated in a variety of cancer types, implying that they are tumor suppressors. SPEN and RBM15are required for the development of multiple organs including hematopoiesis partly via regulating the NOTCH signaling pathway, as well as the WNT signaling pathway in species ranging from Drosophila to mammals. Besides transcriptional regulation, RBM15 regulates RNA export and RNA splicing. In this review, we summarized data in the literature on how the members in SPEN family regulate gene expression at transcription and RNA processing steps. The crosstalk between epigenetic regulation and RNA metabolism is increasingly appreciated in understanding tumorigenesis. Studying the SPEN family of RNA binding proteins will create new perspectives for cancer therapy.

MiR-125b and miR-99a encoded on chromosome 21 co-regulate vincristine resistance in childhood acute megakaryoblastic leukemia

Vincristine (VCR) resistance remains a major challenge for the successful treatment of leukemias. MicroRNAs (miRNAs) have a role in proliferation, differentiation and apoptosis, and are associated with poor prognosis phenotypes. Co-expression of miR-125b and miR-99a contribute to VCR resistance to CMK cells due to actively transcribed miR99a/miR-125b locus on chromosome 21q21.1. Here, we show that miR-99a downregulates the targets FKBP51 and IGF1R in CMK cell lines which, in turn, increase p-Akt (Ser473) levels. Our results suggest that these miRNAs can possibly be used in the development of therapies for the treatment of leukemia.

Acute Megakaryoblastic Leukemia in Children: Diagnosis and Management Challenges in Resource-Poor Countries

Acute Megakaryoblastic Leukemia (AMKL) is a rare subtype of acute myeloid leukemia classified as AML M7 by the French-American-British (FAB) Leukemia Cooperative Study Group. We reviewed the records of children aged less than 18 years with AMKL during the period from January 2003 to December 2010. The diagnosis of AMKL was established on the basis of the FAB criteria and confirmed by immunophenotyping. Eight cases were assigned for this period. The incidence rate of AMKL was 5.5 % of AML. The median age at the time of diagnosis was 1 year. The karyotype presented translocation (1, 22) in two cases. One girl had Down syndrome (DS). Treatment included two inductions based on daunorubicin and cytosine arabinoside (7+3) followed by two consolidations with high dose of cytosine arabinoside (16 g/m 2 ). Complete remission (CR) was achieved in 3 patients (43%). One patient underwent allogeneic stem cell transplant (SCT) and is still enjoying continuous CR 5 years after treatment. Other patients died from failure or relapse. In this small series from a single institution, reported incidence of AMKL is underestimated probably due to the difficulties of diagnosis. The prognosis was poor, particularly because of unavailability of intensified chemotherapy treatment and allogeneic SCT.

Stage Selective Requirement For The N-Terminus Of GATA1 During Erythropoiesis

The X-linked blood transcription factor GATA1 is required for the survival and maturation of erythroid cells. Loss of Gata1 causes profound anemia and related mid-gestation lethality in mouse models and human GATA1 mutations are associated with congenital dyserythropoietic anemia, congenital thrombocytopenia, porphyria, Diamond-Blackfan anemia (DBA) and acute megakaryoblastic leukemia in children with Down syndrome (DS-AMKL). In DBA, DS-AMKL, and a subset of congenital anemia, GATA1 mutations cause the exclusive production of a shorter GATA1 isoform, termed GATA1s. The GATA1s isoform retains both zinc fingers and binds DNA and cofactors accordingly, but lacks the N-terminal 83 residues of full-length GATA1. Intriguingly, exclusive production of GATA1s promotes AMKL in the context of Down syndrome (trisomy 21), while disomic individuals harboring the same type of mutations suffer from anemia and severe red cell defects. A mutation causing exclusive production of Gata1s in mice does not appear to affect adult hematopoiesis, but has been shown to cause a marked expansion of megakaryocytes in the fetal liver. The effects of the replacement of Gata1 with Gata1s during fetal liver erythropoiesis, however, remain uncharacterized.To investigate the effects of exclusive Gata1s production during fetal hematopoiesis, we performed comprehensive phenotypic and mechanistic studies using Gata1s knock-in embryos. We found significant changes in myelo-erythroid differentiation beyond the known expansion of megakaryocytes. Flow cytometric analysis revealed altered erythroid differentiation at embryonic days 12.5 and 14.5, evidenced by decreased Ter119 and increased CD71 expression, characteristic of delayed erythroid maturation. Changes in expression of myelo-erythroid progenitor commitment markers were also discovered. Specifically, we observed marked decreases in pre-CFU-E and CFU-E committed progenitors and an increase in pre-GM and pre-MegE populations. Our findings are consistent with a bias towards megakaryopoiesis at the expense of erythroid commitment caused by the expression of Gata1s in place of full-length Gata1. A shift in differentiation was also observed in the embryonic granulocyte/ macrophage lineage, with an increased generation of macrophages with fewer developing granulocytes. Mechanistically, we found that expression of erythroid -specific Gata1 target genes such as Alas2, Slc4a1 and Klf1 are markedly reduced in the Gata1 knock-in erythroid cells, indicating that Gata1s is a less effective transcriptional activator than full-length Gata1. In particular, given that Klf1 functions to promote erythroid specification downstream of the megakaryocyte-erythrocyte progenitor, our results suggest that Gata1s may promote megakaryocyte differentiation at the expense of erythroid differentiation, in part, by failing to activate Klf1.Taken together, our studies demonstrate a stage-specific requirement for full-length Gata1 during embryonic erythropoiesis. Furthermore, erythroid defects associated with exclusive production of Gata1s in humans may result from an incomplete activation of the erythroid transcriptional program. Disclosures:Crispino:Sanofi: Research Funding.

The Chromosome 21 Kinase DYRK1A Controls Cell Cycle Exit and Survival During Lymphoid Development and Is a Novel Therapeutic Target In Acute Lymphoblastic Leukemia

The dual specificity tyrosine-regulated kinase 1A (DYRK1A) phosphorylates a growing number of proteins to influence various aspects of cell biology, including survival, apoptosis, proliferation, and neuronal development and function. Encoded on human chromosome 21, the DYRK1A locus resides in the Down syndrome critical region (DSCR) and has been implicated in the pathogenesis of neurocognitive deficits that result from trisomy 21. We recently demonstrated a prominent tumor-promoting role for DYRK1A in acute megakaryoblastic leukemia in children with DS (DS-AMKL). This discovery prompted us to ask whether DYRK1A is important for normal hematopoiesis, and if additional types of leukemia result from enhanced DYRK1A activity.Apart from our study in DS-AMKL, the involvement of DYRK1A in normal and malignant hematopoiesis has not been described. Dyrk1a germline knockout mice die mid-gestation; therefore, we generated conditional knockout (CKO) mice to allow inducible deletion of the kinase in hematopoietic cells with Mx1-Cre. In the bone marrow, CKO mice exhibit significant reductions of the hematopoietic stem cell-enriched LSK and common lymphoid progenitor populations (5-fold and >20-fold reductions vs. controls, respectively). In B cell lineage, CKO mice have 2-fold reductions of small pre-B and immature B cells, and a 5-fold reduction of mature recirculating B cells. In the thymus, CKO mice show dramatic loss of CD4+/CD8+double positive cells (DP, >30-fold fewer at 4 weeks). Furthermore, DYRK1A-deficient cells fail to reconstitute lymphoid cells in competitive bone marrow transplantation experiments and give rise to virtually no pre-B cell colonies in ex vivo assays. In contrast to these lymphoid defects, myeloid development is largely normal, with slightly increased numbers of erythroid precursors, granulocytes, and monocytes.To understand the mechanisms by which DYRK1A controls lymphopoiesis, we performed microwestern blot arrays. This approach allowed us to simultaneously screen >200 antibodies to identify differentially expressed proteins between CKO and control lymphoid cells. We found increased levels of cell cycle regulatory proteins, such as CDK1, suggesting that DYRK1A modulates cell cycle status during lymphoid development. Indeed, cell cycle analysis revealed that there were 20-30% fewer quiescent (G0) cells among CKO small pre-B cells and DP thymocytes, two developmental stages characterized by exit from the cell cycle following a proliferative burst. Among its targets, DYRK1A is known to contribute to DREAM (DP, Rb-like, E2F, and MuvB) complex assembly via phosphorylation of LIN52. The DREAM complex, not previously described in hematopoiesis, promotes quiescence by repressing the transcription of numerous cell cycle-associated genes during G0. RNA sequencing analysis in flow-purified G0 cells confirmed that DREAM complex targets are markedly de-repressed in the affected CKO populations, highlighting the importance of DYRK1A/DREAM complex interaction during lymphoid cell cycle exit.Both B- and T-lineage ALL express high levels of DYRK1A relative to other tumor types. Having observed that DYRK1A is essential for lymphoid cells, we next asked whether targeting DYRK1A with small molecule inhibitors would be an effective therapy for ALL. We found that EHT 1610, a highly selective DYRK1 inhibitor (Leblond B., Casagrande A.-S., Désiré L., Foucourt A., Besson T., DYRK1 inhibitors and uses thereof, WO 2013026806, 2013.02.28), dose-dependently induced apoptosis in B- and T-ALL cell lines and primary human pediatric ALL samples. Thus far, all cell lines and all primary samples tested were exquisitely sensitive to EHT 1610. Moreover, EHT 1610 induced apoptosis of primary ALL cells that were resistant to cytarabine, suggesting that DYRK1A inhibitors may be used in combination with standard ALL therapies for refractory or relapsed cases. Together, our data establish novel essential roles for DYRK1A in both normal and malignant lymphoid development and provide rationale for the design of DYRK1A-targeted ALL therapies. Disclosures:Leblond:Diaxonhit Therapeutics: Employment. Casagrande:Diaxonhit Therapeutics: Employment. Désiré:Diaxonhit Therapeutics: Employment. Crispino:Sanofi: Research Funding.

Prognostic features in acute megakaryoblastic leukemia in children without Down syndrome: a report from the AML02 multicenter trial and the Children’s Oncology Group Study POG 9421

Prognostic features in acute megakaryoblastic leukemia in children without Down syndrome: a report from the AML02 multicenter trial and the Children’s Oncology Group Study POG 9421

Acute megakaryoblastic leukemia and severe pulmonary fibrosis in a child with down syndrome: Successful treatment with ultra low-dose cytarabine using GATA1 mutation to monitor minimal residual disease

Acute megakaryoblastic leukemia and severe pulmonary fibrosis in a child with down syndrome: Successful treatment with ultra low-dose cytarabine using GATA1 mutation to monitor minimal residual disease