Acute Megakaryoblastic Leukemia Research Articles

NUP98-KDM5A Fusion Gene Is Not Exclusive to Acute Megakaryoblastic Leukemia and Is an Independent Predictor of Poor Prognosis

NUP98 rearrangements are recurrent genetic events in pediatric acute myeloid leukemia (AML). We identified NPU98-NSD1 as a recurrent poor risk abnormality (Hollink et al, Blood 2011). Another recurrent fusion is NUP98-KDM5A, which was found in pediatric acute megakaryocytic leukemia (AMKL) and was associated with poor clinical outcome (de Rooij et al, Nature Genetics 2017). To date, it is unclear whether NUP98-KDM5A exclusively occurs in AMKL or also in other FAB-subtypes of childhood AML. The aim of this study was to determine the incidence of NUP98-KDM5A in large pediatric AML cohorts, and to define the biological and clinical characteristics of this subgroup.The study comprised a COG and a European cohort of patients, and included 997 patients from COG trials 03p1 and 0531 (Gamis et al, Blood 2013) and 354 European pediatric AML patients, previously described by Balgobind et al (Haematologica 2011), NUP98-KDM5A rearrangements were detected either by paired-end RNA sequencing or RT-PCR. Patients with FAB M3 AML were excluded from this study. For comparison of clinical characteristics between NUP98-KDM5A positive and negative patients, the χ-squared and the Fisher's exact test were used. Event-free survival (EFS) and overall survival (OS) were estimated using the Kaplan-Meier method; the Cox proportional-hazard model was used for multivariate analysis. For this analysis, age, white blood cell count (WBC), cytogenetic risk group, allogeneic hematopoietic stem cell transplant (HSCT) as time dependent variable and study group (COG vs European cohort) were used as co-variables.NUP98-KDM5A rearrangements were detected in 31 out of 1351 patients (2.3%). There was no significant difference in prevalence between the COG and European cohorts. 41% of the NUP98/KDM5A+ patients were male, compared to 53% of the NUP98-KDM5A- patients (p=0.24). The median age of NUP98-KDM5A patients was significantly lower as compared to patients without this fusion gene (5.8 vs 9.4 yrs; p=0.03). There was no difference in WBC between NUP98-KDM5A positive and negative patients (13.1*109/l vs.25.2*109/l, respectively, p=0.13). NUP98-KDM5A occurred in all FAB types, with FAB M7 being most frequent (33% vs 8% in NUP98-KDM5A - patients; p<0.001).The 5-year EFS of NUP98-KDM5A + patients was 36.8 ± 18.8%, compared to 48.1 ± 2.8% in the NUP98-KDM5A - patients (p=0.12). The 5-year OS of NUP98-KDM5A + patients was 41.1 ± 19.7%, a value significantly lower compared to the NUP98-KDM5A - patients (63.9 ± 2.8%; p=0.01), this finding suggesting that NUP98-KDM5A+ patients are difficult to rescue when they relapse. In multivariate analysis, NUP98-KDM5A was an independent risk factor for OS, but not for EFS, with hazard ratios of 1.76 (p=0.045) and 1.58 (p=0.083), respectively. Non-M7 (n=19) patients had lower survival rates with EFS of 26.4 ± 21.7% and OS of 30.0 ± 22.3% compared to M7 (n=9) patients (EFS 55.6 ± 33.1% and OS 64.8 ± 33.1%). These differences were not statistically significant.This study shows that NUP98-KDM5A is an aberration that occurs in all FAB types and not just in AMKL. NUP98-KDM5A is an independent risk factor for OS but not for EFS and identifies a poor prognostic subgroup. Although there was a big difference in survival between M7 and non-M7 NUP98-KDM5A rearranged patients, this was not statistically significant, probably because of the low number of patients in the two cohorts. Overall, these data suggest to screen for the presence of NUP98-KDM5A rearrangements all children with non-APL AML irrespectively of their AML FAB subtype, and to stratify these patients in the high risk group. DisclosuresNo relevant conflicts of interest to declare.

Identification of Candidate Progenitor Populations Which Causes Transient Abnormal Myelopoiesis

Transient abnormal myelopoiesis (TAM) is a temporal preleukemic status specific to neonates of Down syndrome (DS). TAM is characterized by leucocytosis and/or thrombocytopenia with the trisomy of chromosome 21 and somatic GATA1 mutation. Morphologically, most blasts found in TAM are myeloblasts, and some other types of blasts are similar to those typically found in DS-related acute megakaryoblastic leukemia (DS-AMKL). Although TAM patients usually experience spontaneous remission within 6 months, 20 to 30 % of develop DS-AMKL. The cell population that causes TAM phenotypes in vitro or in vivo mouse and human models has not yet been determined.Recently, the hematopoietic differentiation systems from human pluripotent stem cells (PSCs) have revealed the pathogenesis in various diseases including TAM. Previous TAM models have succeeded in recapitulating the phenotypes, such as arrest and/or skewness of differentiation from immature hematopoietic progenitor cells (HPCs). However, identification of the responsible cell population for TAM at a precise subpopulation level based on functional evaluations has not yet been accomplished. Therefore, we applied our 2D-hematopoietic differentiation system for identifying the responsible cell population through the progenitor assay. A comprehensive understanding of TAM pathogenesis will enable us to predict and eventually prevent the disease progression from TAM into life-threatening DS-AMKL.First, we differentiated artificial trisomy 21 embryonic stem cells (Ts21-ESCs) generated by transfer of human artificial chromosome (HAC) 21 in our 2D-differentiation system. Five subpopulations of HPCs were harvested at day6 and day9, and cells were subjected to expression analysis of hematopoiesis-related genes. Principal component analysis (PCA) successfully mapped each HPC subpopulation corresponding to the hematopoietic developmental pathways, proving that our system can be used for isolating functional HPC subpopulations.Next, we used genome edition to establish strictly controlled isogenic ES/induced pluripotent stem cell (iPSC) pairs derived from TAM, non-TAM/DS patients and the Ts21-ESCs. Then we differentiated ES/iPSCs into each hematopoietic lineage to identify the abnormal phenotypes in vitro . GATA1 -mutated clones (G1s-clones) showed obviously different phenotypes, compared to GATA1-WT clones (WT-clones). In particular, completely restricted erythroid lineage cells (CD42b-CD71+CD235a+) and increased myeloid lineage cells (CD34-CD235a-CD41-CD42b-CD43+CD45+) were observed in G1s-clones. Moreover, in megakaryocytic lineage, the frequency of immature (CD235a-CD41+CD42bdim) and mature (CD235a-CD41+CD42bbright) cells differed between G1s- and WT-clones. Notably, the proportion of immature megakaryoblasts increased in G1s-clones. These data indicated that we have succeeded in recapitulation the TAM phenotype with our in vitro differentiation system.Then, we performed progenitor assay based on in vitro TAM phenotypes. We differentiated 5 isolated HPCs subpopulation into erythrocyte, megakaryocyte and myeloid cells with a suspension culture and colony forming assay. As a result, we found strong correlations between the increased or decreased specific cell fractions and abnormal phenotypes. Correlation pattern is slightly different between clones.In conclusion, we have identified putative responsible cell populations that contribute to the pathological phenotype in TAM. These populations were located on a series of spectra in the differentiation stage. Ubiquitous expression of GATA1 during the differentiation process and wide heterogeneity of the differentiation systems in general may be the reason why precise identification of the single responsible population was not possible. However, our model of TAM using iPSCs enabled us to narrow down the candidate cell populations which have the potential to be a source of DS-AMKL. The identification of responsible progenitor cells is an important first step to predict the transformation from TAM into DS-AMKL and to prevent its progression. Moreover, the method used in this study, such as 2D-differentiation system and progenitor assay, is a promising way to identify the responsible cell population of various types of leukemia other than TAM. DisclosuresNo relevant conflicts of interest to declare.

Fusion gene detection by RNA-sequencing complements diagnostics of acute myeloid leukemia and identifies recurring NRIP1-MIR99AHG rearrangements.

Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, fluorescence in situ hybridization and reverse-transcriptase polymerase chain reaction. However, sequencing technologies are becoming more important in clinical routine as processing time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNAsequencing compared to standard diagnostic techniques, we analyzed 806 RNA-sequencing samples from patients with acute myeloid leukemia using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-sequencing detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-sequencing failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-sequencing. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-sequencing in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1- MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanisms of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-sequencing has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

Dual-Specificity, Tyrosine Phosphorylation-Regulated Kinases (DYRKs) and cdc2-Like Kinases (CLKs) in Human Disease, an Overview.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRK1A, 1B, 2-4) and cdc2-like kinases (CLK1-4) belong to the CMGC group of serine/threonine kinases. These protein kinases are involved in multiple cellular functions, including intracellular signaling, mRNA splicing, chromatin transcription, DNA damage repair, cell survival, cell cycle control, differentiation, homocysteine/methionine/folate regulation, body temperature regulation, endocytosis, neuronal development, synaptic plasticity, etc. Abnormal expression and/or activity of some of these kinases, DYRK1A in particular, is seen in many human nervous system diseases, such as cognitive deficits associated with Down syndrome, Alzheimer’s disease and related diseases, tauopathies, dementia, Pick’s disease, Parkinson’s disease and other neurodegenerative diseases, Phelan-McDermid syndrome, autism, and CDKL5 deficiency disorder. DYRKs and CLKs are also involved in diabetes, abnormal folate/methionine metabolism, osteoarthritis, several solid cancers (glioblastoma, breast, and pancreatic cancers) and leukemias (acute lymphoblastic leukemia, acute megakaryoblastic leukemia), viral infections (influenza, HIV-1, HCMV, HCV, CMV, HPV), as well as infections caused by unicellular parasites (Leishmania, Trypanosoma, Plasmodium). This variety of pathological implications calls for (1) a better understanding of the regulations and substrates of DYRKs and CLKs and (2) the development of potent and selective inhibitors of these kinases and their evaluation as therapeutic drugs. This article briefly reviews the current knowledge about DYRK/CLK kinases and their implications in human disease.

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.

Acute megakaryoblastic leukaemia with erythrophagocytosis

A 42-year-old man with Down syndrome presented with fever and facial ecchymosis. A full blood count showed: white blood cells 21.0 × 109/l, haemoglobin concentration 89 g/l and platelets 14 × 109/l. The blood film showed 30% small to medium sized blast cells with scant cytoplasm and anisopoikilocytosis with dacrocytes. A bone marrow aspiration was performed. The bone marrow was hypercellular, with the presence of 40% of heterogeneous blast cells. Most blast cells were similar to those in the peripheral blood, being small with round to slightly irregular nuclei and scant agranular cytoplasm, but medium to large blast cells with irregular nuclei and cytoplasmic blebs were also present. Moreover, a small proportion of blasts were very large, either multinucleated or with polylobulated nuclei with a moderate amount of cytoplasm with blebs and, sometimes, coarse myeloperoxidase (MPO)-negative granules (all images, ×100 objective). Dysplasia was present in 12% of erythroid cells. Abundant examples of erythrophagocytosis by leukaemic blast cells were present (top images). Multiple mitosis were observed with some mitotic figures indicating that the cells were hyperdiploid (bottom left image). The blasts were positive for cluster of differentiation (CD)117, CD33, CD34, CD41 and CD61, and negative for MPO, CD71 and CD14 by flow cytometry. There was a complex karyotype with monosomy of 7 and 16, a marker chromosome and the known germline trisomy of 21. GATA-binding protein 1 (GATA1) Sanger sequencing showed no mutations. A diagnosis of myeloid leukaemia associated with Down syndrome (acute megakaryoblastic leukaemia) was made. However, the age of the patient and the absence of a GATA1 mutation suggest that this case differs from the cases usually reported in the first 5 years of life. The patient received induction treatment with ‘3+7’ (idarubicin and cytarabine) and FLAG-IDA (fludarabine, cytarabine and idarubicin) regimens; however, the disease was refractory to treatment and the patient died at 4 months after diagnosis. Erythrophagocytosis by leukaemic blasts is a rare finding in acute myeloid leukaemia. It has been reported in leukaemias with monocytic or megakaryocytic morphology and the presence of t(8;16)(p11.21;13.3), inv(8)(p11.21q13.3) or tetraploidy.

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.

Analysis of Clinical Characteristics and Prognosis in Children with Acute Megakaryoblastic Leukemia without Down Syndrome

To analyze the clinical characteristics and treatment effects of children with acute megakaryoblastic leukemia without down syndrome (non-DS-AMKL). The clinical data of 19 children with non-DS-AMKL treated in the Pediatric Hematology Ward in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from May 2008 to April 2018 were analyzed retrospectively. The clinical characteristics, laboratory test and treatment methods of the children were concluded. All patients were followed up to evaluate the effect of treatment. The 19 cases of children included nine male and ten female, the median age of onset was 2 years old. The clinical manifestations showed nonspecific. The median white blood cell of peripheral blood was 15.88×109/L, the median hemoglobin was 67 g/L and median platelet was 16×109/L. An increase of primitive and naive megakaryocytes was found in the bone marrow sample. The immunophenotypes of bone marrow detected by flow cytometry in 19 children were all positive expressed for CD41, CD61. Genetic tests showed that five cases carrying EVI1, including one complicating with MLL/AF10, one patient carrying HOX11, one carrying GATA1, IKZF1 and DDX11 mutations, one patient carrying missense mutation of NRAS, one with missense mutation of KRAS and two cases with high expression of WT1. Karyotype analysis were performed in 11 cases of children, including four with normal karyotype, four with complex karyotypes, one with trisomy 8, one with 7/13 trisomy 21 without Down syndrome manifestations (considered as abnormal somatic karyotype) and one Robertsonian translocation carrier involving chromosomes 14 and 21. Ten children received treatment, including three cases with allogeneic hematopoietic stem cell transplantation (allo-HSCT) after complete remission (CR), two cases with complete donor implantation, and one without implanted but hematopoietic recovered, these three children were followed up for 26, 15 and 12 months respectively and the minimal residual disease (MRD) were all less than 10-4. Another three cases achieving CR after chemotherapy but relapsed in 5, 10 and 12 months after the onset respectively, and all the three children were died eventually. One children died of pulmonary hemorrhage during chemotherapy and three children died from discontinuation of treatment after non remission. The remaining nine children died because of without chemotherapy treatment. Non-DS-AMKL was rare in children and difficult to be diagnosed. Determination of MICM classification as early as possible was helpful for diagnosis, and genetic testing played an important role for diagnosis and prognosis evaluation. Early hematopoietic stem cell transplantation in patients with CR after chemotherapy might be an effective way to cure AMKL.

EP066 - Trisomy 3 and CBFA2T3-GLIS2 fusion in a child with acute megakaryoblastic leukemia without Down syndrome

eP066 - Trisomy 3 and CBFA2T3-GLIS2 fusion in a child with acute megakaryoblastic leukemia without Down syndrome

Molecular Mechanisms of the Genetic Predisposition to Acute Megakaryoblastic Leukemia in Infants With Down Syndrome.

Individuals with Down syndrome are genetically predisposed to developing acute megakaryoblastic leukemia. This myeloid leukemia associated with Down syndrome (ML–DS) demonstrates a model of step-wise leukemogenesis with perturbed hematopoiesis already presenting in utero, facilitating the acquisition of additional driver mutations such as truncating GATA1 variants, which are pathognomonic to the disease. Consequently, the affected individuals suffer from a transient abnormal myelopoiesis (TAM)—a pre-leukemic state preceding the progression to ML–DS. In our review, we focus on the molecular mechanisms of the different steps of clonal evolution in Down syndrome leukemogenesis, and aim to provide a comprehensive view on the complex interplay between gene dosage imbalances, GATA1 mutations and somatic mutations affecting JAK-STAT signaling, the cohesin complex and epigenetic regulators.

Infant Acute Myeloid Leukemia: A Unique Clinical and Biological Entity.

Infant acute myeloid leukemia (AML) is a rare subgroup of AML of children <2 years of age. It is as frequent as infant acute lymphoblastic leukemia (ALL) but not clearly distinguished by study groups. However, infant AML demonstrates peculiar clinical and biological characteristics, and its prognosis differs from AML in older children. Acute megakaryoblastic leukemia (AMKL) is very frequent in this age group and has raised growing interest. Thus, AMKL is a dominant topic in this review. Recent genomic sequencing has contributed to our understanding of infant AML. These data demonstrated striking features of infant AML: fusion genes are able to induce AML transformation without additional cooperation, and unlike AML in older age groups there is a paucity of associated mutations. Mice modeling of these fusions showed the essential role of ontogeny in the infant leukemia phenotype compared to older children and adults. Understanding leukemogenesis may help in developing new targeted treatments to improve outcomes that are often very poor in this age group. A specific diagnostic and therapeutic approach for this age group should be investigated.

Clinical features of 35 Down syndrome patients with transient abnormal myelopoiesis at a single institution

Transient abnormal myelopoiesis (TAM) is a unique clonal myeloproliferation characterized by immature megakaryoblasts that occurs in 5-10% of neonates with Down syndrome (DS). Although TAM regresses spontaneously in most patients, approximately 20% of TAM cases result in early death, and approximately 20% of survivors develop acute megakaryoblastic leukemia (AMKL). We retrospectively reviewed records of 35 DS patients with TAM to determine the correlation between clinical characteristics and blast percentage. Thirteen of the 35 patients were classified as low blast percentage TAM (LBP-TAM), defined as TAM with a peak peripheral blast percentage ≤ 10%. Although no patient with LBP-TAM experienced systemic edema, disseminated intravascular coagulation, or early death, eight patients had elevated direct bilirubin levels (> 2mg/dl) and one developed AMKL. All patients with LBP-TAM had serum markers of liver fibrosis that exceeded the normal limits, and two patients underwent liver biopsy to clarify the etiology of pathological jaundice. Taken together, our results suggest that patients with LBP-TAM may be at risk of liver fibrosis and liver failure, similarly to patients with classical TAM. Although these patients generally have a good prognosis, they should be carefully monitored for potential development of liver disease and leukemia.

Congenital Acute Megakaryoblastic Leukemia in a Neonate with Antenatal Hepatosplenomegaly

Various conditions can lead to antenatal hepatosplenomegaly. In addition to commonly known etiologies like hydrops fetalis or viral illnesses, hematologic disorders like congenital leukemia, although rare, should also be ruled out. We present a case of hepatosplenomegaly detected during antenatal screening in a neonate, in whom acute megakaryoblastic leukemia was diagnosed postnatally based on the immunophenotype.

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.

Distinctive Demography, Biology, and Outcome of Acute Myeloid Leukemia and Myelodysplastic Syndrome in Children With Down Syndrome: Children's Cancer Group Studies 2861 and 2891

AbstractIn recent pediatric trials of acute myeloid leukemia (AML), children with Down syndrome (DS) have had significantly more megakaryoblastic leukemia and have experienced better outcome than other children. To further characterize AML in DS, Children's Cancer Group Studies 2861 and 2891 prospectively studied demography, biology, and response in AML and myelodysplastic syndrome (MDS) of children with and without DS. These studies evaluated timing of induction therapy and compared postremission chemotherapy with marrow transplantation in 1,206 children. One-hundred eighteen (9.8%) had DS, a fourfold increase in 20 years. DS patients were younger, had lower white blood cell and platelet counts, more antecedent MDS, acute megakaryoblastic leukemia or undifferentiated AML, and an under-representation of chromosomal translocations (P < .001 for each variable). Four-year event-free survival in DS was 69% versus 35% in others (P < .001). Intensively timed induction conferred significantly higher mortality in DS patients; bone marrow transplantation offered no advantage. Conventional induction followed by chemotherapy achieved an 88%, 4-year, disease-free survival in DS patients versus 42% in others (P < .001). Megakaryoblastic leukemia was unfavorable in others but prognostically neutral in DS. AML in DS is demographically and biologically distinct from AML in other children. It is singularly responsive to conventional chemotherapy and may warrant even less therapy. The increasing proportion of DS patients with AML most likely reflects changes in attitudes about entering DS patients on AML trials and possibly increasing ability to distinguish megakaryoblastic leukemia from lymphoid leukemia.

SON inhibits megakaryocytic differentiation via repressing RUNX1 and the megakaryocytic gene expression program in acute megakaryoblastic leukemia.

A high incidence of acute megakaryoblastic leukemia (AMKL) in Down syndrome patients implies that chromosome 21 genes have a pivotal role in AMKL development, but the functional contribution of individual genes remains elusive. Here, we report that SON, a chromosome 21-encoded DNA- and RNA-binding protein, inhibits megakaryocytic differentiation by suppressing RUNX1 and the megakaryocytic gene expression program. As megakaryocytic progenitors differentiate, SON expression is drastically reduced, with mature megakaryocytes having the lowest levels. In contrast, AMKL cells express an aberrantly high level of SON, and knockdown of SON induced the onset of megakaryocytic differentiation in AMKL cell lines. Genome-wide transcriptome analyses revealed that SON knockdown turns on the expression of pro-megakaryocytic genes while reducing erythroid gene expression. Mechanistically, SON represses RUNX1 expression by directly binding to the proximal promoter and two enhancer regions, the known +23 kb enhancer and the novel +139 kb enhancer, at the RUNX1 locus to suppress H3K4 methylation. In addition, SON represses the expression of the AP-1 complex subunits JUN, JUNB and FOSB which are required for late megakaryocytic gene expression. Our findings define SON as a negative regulator of RUNX1 and megakaryocytic differentiation, implicating SON overexpression in impaired differentiation during AMKL development.

Outcome and Prognostic Features in Pediatric Acute Megakaryoblastic Leukemia Without Down Syndrome: A Retrospective Study in China

BackgroundAcute megakaryoblastic leukemia (AMKL) is a biologically heterogeneous subtype of acute myeloid leukemia that originates from megakaryocytes. Patients with AMKL with non-Down syndrome (DS) had a poorer prognosis. However, clear prognostic indicators and treatment recommendations for this subgroup remain controversial. Patients and MethodsHerein, we performed a retrospective study on 40 patients (age ≤ 18 years) with non-Down syndrome AMKL at our institution. We assessed the effect of different prognostic factors, such as their cytogenetic abnormalities, early treatment response, and the role of hematopoietic stem cell transplantation (HSCT) as post-remission treatment on the outcomes. ResultsThe complete remission (CR) rate of the patients was 57.9% and 81.1%, respectively, at the end of induction therapy 1 and 2. The overall survival (OS) and event-free survival rates at 2 years were 41% ± 13% and 41% ± 10%, respectively. An analysis of the cytogenetic features showed that patients with +21 or hyperdiploid (> 50 chromosomes) had significantly better OS than those in other cytogenetic subgroups (Plog-rank = .048 and Plog-rank = .040, respectively). Besides cytogenetics, an excellent early treatment response (CR and minimal residual disease < 1% after induction therapy 1) also provided a significant survival benefit in univariate analysis in our study. However, multivariate analysis indicated that allogeneic HSCT was the only independent prognostic marker (relative risk, 11.192; 95% confidence interval, 2.045-61.241; P = .005 for OS and relative risk, 5.400; 95% confidence interval, 1.635-17.832; P = .006 for event-free survival, respectively). ConclusionAMKL in patients with non-Down syndrome has a poor outcome. With poor OS but CR rates comparable with other acute myeloid leukemia subtypes, allogenic HSCT may be a better option for post-remission therapy than conventional chemotherapy, especially for those having a poor response to induction therapy.

A Human Model of Down Syndrome Associated Leukemia Reveals Different Cell of Origins for Initiation and Progression

Introduction: Leukemia is the most common cancer in children and sequencing data suggest that the first genetic alterations often occur in utero. Children with Down syndrome (Trisomy 21, T21) have a 150-fold increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient myeloproliferative disorder (pre-leukemia) occurs, which is characterized by a clonal proliferation of immature megakaryoblasts carrying somatic mutations in the GATA1 transcription factor (GATA1s) and resolves spontaneously in most cases. In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemic clone by acquisition of additional mutations, such as in the cohesin subunit STAG2. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1s and STAG2. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the interplay between each genetic event and the cellular origin of transformation. Methods: Human long-term hematopoietic stem cells (LT-HSCs) were sorted from normal karyotype and T21 fetal livers (N-FL and T21-FL) and subsequently CRISPR/Cas9 edited to try to establish a humanized model of Down Syndrome associated pre-leukemia and AMKL. To model the initiation of the pre-leukemic state, GATA1s mutations were introduced, while additional STAG2- mutations were overlaid to model the progression to fully transformed AMKL. CRISPR/Cas9-edited control, GATA1s, STAG2- and GATA1s/STAG2- LT-HSCs were functionally interrogated in near-clonal xenograft assays, along with transcriptional and epigenetic profiling. Results: T21 status in combination with GATA1s had a profound synergistic effect on megakaryocytic lineage output in vivo compared to normal karyotype with GATA1s. Moreover, a high percentage of blasts were found in xenografts of GATA1s edited T21-FL LT-HSCs (&amp;gt;30%) but not in xenografts of GATA1s edited N-FL LT-HSCs. Conversely, GATA1s/STAG2- edited LT-HSCs generated grafts with &amp;gt;50% of blasts, regardless of T21 status. The immunophenotype of these blasts recapitulated those observed in patients diagnosed with Down Syndrome pre-leukemia and AMKL (CD117+CD34+CD41+CD71+CD33+CD4+CD7+). Thus, T21 is required for pre-leukemia development, but seems dispensable for AMKL as both N- and T21-FL LT-HSCs underwent leukemic transformation upon GATA1s/STAG2-. Serial xenotransplantation assays from primary engrafted mice were carried out to assess self-renewal properties of GATA1s-induced pre-leukemia and GATA1s/STAG2- induced AMKL. Only GATA1s/STAG2- edited N- and T21-FL grafts were able to propagate the leukemic phenotype with a high stem cell frequency, which was endowed by the additional STAG2- knock-out. ATACseq and RNAseq profiling of blast populations revealed an enrichment of GATA-binding sites with concomitant up-regulation of genes implicated in translation. To assess the role of progenitor cells in pre-leukemic initiation and leukemic progression, we CRISPR/Cas9 edited short-term HSCs, common myeloid progenitors and myelo-erythroid progenitors with GATA1s and/or STAG2- and subjected them to xenotransplantation. Strikingly, all progenitor subsets with combined GATA1s/STAG2- editing were able to drive leukemic transformation, while single GATA1s editing in the same subsets did not initiate pre-leukemia. This data strongly suggests that the initial GATA1s mutation must occur in T21 LT-HSCs, but subsequent STAG2 mutations can occur further downstream in progenitors. Lastly, to gain insight into how chromosome 21 predisposes towards pre-leukemia, three chromosome 21 miRNAs (miR-99a, -125b-2 and -155) were identified to be up-regulated in T21-FL LT-HSCs compared to N-FL LT-HSCs. Over-expression of these miRNAs in N-FL LT-HSCs induced a T21-like state with increased myeloid and megakaryocytic skewing. Dramatically, CRISPR/Cas9-edited knock-out of these miRNAs in GATA1s edited T21-FL LT-HSCs resulted in a block of pre-leukemia initiation. Conclusion: Our findings demonstrate that T21 is required for pre-leukemia initiation, which is mediated by over-expression of chromosome 21 miRNAs in LT-HSCs. Further, this data demonstrates different cell of origins between pre-leukemia initiation and AMKL progression. Ongoing studies focus on preventing the progression of pre-leukemia to AMKL by pharmacological targeting. Figure Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Modeling Acute Megakaryoblastic Leukemia of Down Syndrome Using Induced Pluripotent Stem Cells

Introduction Development of Acute megakaryoblastic leukemia in Down syndrome children (DS-AMKL) is a multi-step process. Acquired GATA1s mutation during fetal hematopoiesis is responsible of a transient myeloproliferative disorder (TMD) characterized by an accumulation of megakaryoblasts. Although most of TMD regress around birth, some TMD can progress from the initial GATA1s clone to AMKL through the acquisition of additional mutations, including in (i) the cohesin complex (i.e: SMC3), (ii) the JAK/STAT signaling pathway, such as MPL and (iii) the polycomb repressive complex 2 (EZH2). How these mutations cooperate to deregulate megakaryocyte (MK) differentiation and to induce a full-blown AMKL, along with the precise role of trisomy 21 (T21) during this transformation process remain unclear. Because modeling of DS-AMKL is particularly difficult in mice, we performed a step-wise introduction of GATA1s, a gain of function mutation of MPL (MPLW515K) and a heterozygous loss of function mutation in a cohesin (SMC3), separately or in combination, in T21 and isogenic disomic 21 (Dis21) human induced Pluripotent Stem Cells (iPSCs). Methods Trisomy 21 iPSCs were kindly provided by M. Weiss (Memphis, TN). CRISPR/Cas 9 genome editing of GATA1 or SMC3 allowed the generation of GATA1s T21, SMC3+/- T21 and GATA1s SMC3+/- T21 iPSC clones. CRISPR/Cas9-mediated knock-in of MPLW515K was performed in T21 GATA1s iPSCs. The subsequent T21 GATA1sMPLW515K/W515Kclones were selected as well as a revertant Dis21 GATA1sMPLW515K/W515Kclone. Finally, SMC3 insertion/deletion were obtained in isogenic T21 and Dis21 GATA1s MPLW515K/W515K SMC3+/-iPSCs clones. Hematopoietic differentiation was induced in 2D cultures in presence of a matrix and a cocktail of cytokines followed by a MK differentiation with SCF and TPO. MK differentiation was studied by clonogenic assays, flow cytometry, confocal microscopy and ultrastructural studies. Gene expression analyses were performed by RNA-seq on highly purified MK from all genotypes. Results GATA1s alone blocked MK maturation characterized by a persistent CD34 expression, an accumulation of abnormal large granules, a defect in the development of demarcation membranes (DMS), and a marked decrease in proplatelet formation. The typical GATA1s MK were large megakaryoblasts with numerous large granules and rare DMS. However, GATA1s alone had no effect on the clonogenic activity in CFU-MK assays and MK numbers. The introduction of the MPLW515K mutation did not modify this phenotype either in Dis21 or T21 GATA1s MK, but induced a complete TPO independence. SMC3+/- alone enhanced the MK maturation allowing the generation of a higher number of proplatelets-generating MK. Importantly, the combination of GATA1s and SMC3+/- mutations had a marked cooperative effect that worsened the MK maturation defect, led to the generation of abnormal megakaryoblasts with only a pre-DMS and resulted in enhanced proliferation and ploidization both in Dis21 and T21 iPSCs. Interestingly, the proliferation was markedly higher in T21 clones compared to Dis21 counterparts. RNA-seq and GSEA analyses showed that T21 GATA1s SMC3+/- mutant MK exhibited transcriptional signatures consistent with a dramatic decrease in the expression of maturation genes, including GATA1 target genes, while DNA replication gene markers were increased compared with GATA1s alone. T21 GATA1s MPLW515K/W515K SMC3+/- MK were enriched for AMKL signatures as compared to isogenic Dis21 GATA1sMPLW515K/W515K SMC3+/- MK. Ongoing ATAC-seq analyses will define the consequence of the different mutations on chromatin accessibility. Conclusion Using iPSC modeling, we analyzed in a human cell-context the consequences of the different combination of mutations associated with DS-AMKL that would be difficult to model using human primary cells. Our data demonstrate that GATA1s expression cooperates with SMC3+/- to enhance proliferation of megakaryoblasts from T21 iPSCs and isogenic Dis21 iPSCs hence reproducing the abnormalities observed in DS-AMKL. T21 is not directly involved in the MK differentiation defects but rather give a proliferative advantage supporting its role in leukemia development. Disclosures No relevant conflicts of interest to declare.

Cases of transient abnormal myelopoiesis

Transient abnormal myelopoiesis (TAM) is a unique hematological syndrome specific for neonates with Down syndrome. Clinical and hematological manifestations of ТАМ are similar manifestations of acute leukemia, but they may resolve spontaneously by few weeks/months after birth. Summation trisomy 21 and GATA1 mutation in blast clone is a required element for development TAM. Presentation of this syndrome occurs in the first days of life; clinical manifestations may be absent (“silent” TAM) or even lead to death of fetus and neonate. The main interest in the study of this issue is the fact that after spontaneous regression there in 20% of cases at the age of 3–4 years developing acute megakaryoblastic leukaemia (AMKL). The basic transformation factors TAM to AMKL are unknown. In this article we represent 6 cases of TAM identified in Dmitry Rogachev National Research Center for Pediatric Hematology, Oncology, and Immunology from 2012 to 2019. Parents of these patients gave their agreement to use personal data in research and publications.