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Individuals with Down syndrome are at increased risk of myeloid leukemia in early childhood, which is associated with acquisition of GATA1 mutations that generate a short GATA1 isoform called GATA1s. Germline GATA1s-generating mutations result in congenital anemia in males. We report on 2 unrelated families that harbor germline GATA1s-generating mutations in which several members developed acute megakaryoblastic leukemia in early childhood. All evaluable leukemias had acquired trisomy 21 or tetrasomy 21. The leukemia characteristics overlapped with those of myeloid leukemia associated with Down syndrome, including age of onset at younger than 4 years, unique immunophenotype, complex karyotype, gene expression patterns, and drug sensitivity. These findings demonstrate that the combination of trisomy 21 and GATA1s-generating mutations results in a unique myeloid leukemia independent of whether the GATA1 mutation or trisomy 21 is the primary or secondary event and suggest that there is a unique functional cooperation between GATA1s and trisomy 21 in leukemogenesis. The family histories also indicate that germline GATA1s-generating mutations should be included among those associated with familial predisposition for myelodysplastic syndrome and leukemia.

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. Aim. The aim is to demonstrate the algorithms for diagnosing and measuring MRD in case of AML-M7 in children. 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). 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%). 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.

Background Acute 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 Methods Herein, 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. Results The 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 Conclusion AMKL 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.

Down syndrome (DS) is the most common chromosomopathy in humans, usually caused by constitutional trisomy 21. It associates with several developmental anomalies, as well as with an increased risk o...

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.

Young males have a unique but rare predilection to develop mediastinal nonseminomatous germ cell tumors (NSGCTs) and concomitant acute megakaryoblastic leukemia (AMKL). Common cytogenetic and molecular abnormalities such as isochromosome 12p and somatic Tumor Protein P53(TP53) and Phosphatase And Tensin Homolog (PTEN) mutations have been reported in the presumed mutual neoplastic clones of origin. We report the case of a 17-year-old male who presented with a mediastinal NSGCT with high-grade sarcomatous transformation and a diagnosis of AMKL approximately 4 months later. Next-generation sequencing revealed identical KRAS Proto-Oncogene, GTPase (KRAS) p.Ala146Thr, TP53 p.Leu257Pro, and PTEN p.Leu181Pro missense mutations at similar variant allele frequencies in both the NSGCT and AMKL samples. Cytogenetic and microarray analyses detected shared copy gains in all chromosomes except chromosomes 9, 13, and Y. Multiple additional clonal chromosomal alterations were detected in the AMKL sample when compared with the NSGCT. This case emphasizes the shared clonal origins of these malignancies and identifies KRAS and other copy number alterations as potential molecular drivers in a subset of these combined diseases.

Long noncoding ribonucleic acids (lncRNAs) are ribonucleic acid (RNA) molecules longer than 200 nucleotides without protein-coding capacity. Several studies have shown that lncRNAs play a pivotal role in the initiation, maintenance, and progression of acute myeloid leukemia (AML), which could make them a promising candidate in the diagnosis and treatment of leukemia. Acute Megakaryoblastic leukemia (AMKL) is a rare form of AML with a poor prognosis and low survival. It has been reported that lncRNA MIR100HG is involved several types of malignancies. In the present study, MIR100HG was downregulated in a human acute megakaryoblastic leukemia cell line (M-07e) using Antisense LNA GapmeRs. In order to assess the expression level of MIR100HG, cell viability, apoptosis, and necrosis (late apoptosis), quantitative reverse transcription polymerase chain reaction (qRT-PCR), Methyl-thiazol Tetrazolium assay, AnnexinV, and propidium iodide staining was performed at different time points after the transfection. In addition, the expression level of TGFβ was evaluated by qRT-PCR. Our results revealed that inhibition of MIR100HG might serve as a new method for inhibition of the proliferation of AMKL cells and therefore, could be a promising approach in medicine for targeted therapy in AMKL.

Acute megakaryocytic leukemia (AMKL) is a rare type of acute myeloid leukemia (AML), which is characterized by its effect on megakaryocytes in bone marrow. Despite standard doses of anthracycline plus cytarabine based regimen, AMKL is notorious for its poor prognosis. With the continuous development of targeted drugs, the choice of chemotherapy regimens for AML patients has been gradually enriched. However, as far as we known, there is little data with this regimen in AMKL with decitabine and Bcl-2 inhibitor combined with imatinib. Herein, we reported the first case of adult AMKL with BCR-ABL positive successfully treated with decitabine and venetoclax combined with imatinib.

BackgroundA high ecotropic viral integration site 1 (EVI1) expression (EVI1high) is an independent prognostic factor in adult acute myeloid leukemia (AML). However, little is known of the prognostic value of EVI1high in pediatric AML. This study aimed to examine the biological and prognostic significance of EVI1high in uniformly treated pediatric patients with AML from a large cohort of seven centers in China.MethodsA diagnostic assay was developed to determine the relative EVI1 expression using a single real-time quantitative polymerase chain reaction in 421 newly diagnosed pediatric AML patients younger than 14 years from seven centers in southern China. All patients were treated with a uniform protocol, but only 383 patients were evaluated for their treatment response. The survival data were included in the subsequent analysis (n = 35 for EVI1high, n = 348 for EVI1low).ResultsEVI1high was found in 9.0% of all 421 pediatric patients with de novo AML. EVI1high was predominantly found in acute megakaryoblastic leukemia (FAB M7), MLL rearrangements, and unfavorable cytogenetic aberrance, whereas it was mutually exclusive with t (8; 21), inv (16)/t (16; 16), CEBPA, NPM1, or C-KIT mutations. In the univariate Cox regression analysis, EVI1high had a significantly adverse 5-year event-free survival (EFS) and overall survival (OS) [hazard ratio (HR) = 1.821 and 2.401, p = 0.036 and 0.005, respectively]. In the multivariate Cox regression analysis, EVI1high was an independent prognostic factor for the OS (HR = 2.447, p = 0.015) but not EFS (HR = 1.556, p = 0.174). Furthermore, EVI1high was an independent adverse predictor of the OS and EFS of patients with MLL rearrangements (univariate analysis: HR = 9.921 and 7.253, both p < 0.001; multivariate analysis: HR = 7.186 and 7.315, p = 0.005 and 0.001, respectively). Hematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) provided EVI1high patients with a tendential survival benefit when compared with chemotherapy as a consolidation (5-year EFS: 68.4% vs. 50.8%, p = 0.26; 5-year OS: 65.9% vs. 54.8%, p = 0.45).ConclusionIt could be concluded that EVI1high can be detected in approximately 10% of pediatric AML cases. It is predominantly present in unfavorable cytogenetic subtypes and predicts adverse outcomes. Whether pediatric patients with EVI1high AML can benefit from HSCT in CR1 needs to be researched further.

Acute megakaryocytic leukemia (AMKL) is a rare type of acute myeloid leukemia (AML), which is characterized by its effect on megakaryocytes in bone marrow. Despite standard doses of anthracycline plus cytarabine based regimen, AMKL is notorious for its poor prognosis. With the continuous development of targeted drugs, the choice of chemotherapy regimens for AML patients has been gradually enriched. However, as far as we known, there is little data with this regimen in AMKL with decitabine and Bcl-2 inhibitor combined with imatinib. Herein, we reported the first case of adult AMKL with BCR-ABL positive successfully treated with decitabine and venetoclax combined with imatinib.

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (

Myeloid leukemia associated with Down syndrome (ML-DS) is characterized by a predominance of acute megakaryoblastic leukemia, the presence of GATA1 mutations and a favorable outcome. Because DS children can also develop conventional acute myeloid leukemia with unfavorable outcome, detection of GATA1 mutations is important for diagnosis of ML-DS. However, myelofibrosis and the significant frequency of dry taps have hampered practical screening of GATA1 mutations using bone marrow (BM) samples. In response to those problems, 82 patients were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-D11 study. GATA1 mutations were analyzed by Sanger sequencing (SS) using genomic DNA (gDNA) from BM and cDNA from peripheral blood (PB) followed by targeted next-generation sequencing (NGS) using pooled diagnostic samples. BM and PB samples were obtained from 71 (87%) and 82 (100%) patients, respectively. GATA1 mutations were detected in 46 (56%) and 58 (71%) patients by SS using BM gDNA and PB cDNA, respectively. Collectively, GATA1 mutations were identified in 73/82 (89%) patients by SS. Targeted NGS detected GATA1 mutations in 74/82 (90%) patients. Finally, combining the results of SS with those of targeted NGS, GATA1 mutations were identified in 80/82 (98%) patients. These results indicate that SS using BM gDNA and PB cDNA is a rapid and useful method for screening for GATA1 mutations in ML-DS patients. Thus, a combination of SS and targeted NGS is a sensitive and useful method to evaluate the actual incidence and clinical significance of GATA1 mutations in ML-DS patients.

GLI-similar 1-3 (GLIS1-3), a subfamily of Krüppel-like zinc finger transcription factors, function as key regulators of several biological processes important to oncogenesis, including control of cell proliferation, differentiation, self-renewal, and epithelial-mesenchymal transition. This review provides a short overview of the critical roles genetic changes in GLIS1-3 play in the development of several malignancies. This includes intrachromosomal translocations involving GLIS2 and ETO2/CBFA2T3 in the development of pediatric non-Down's syndrome (DS), acute megakaryoblastic leukemia (AMKL), a malignancy with poor prognosis, and an association of interchromosomal translocations between GLIS3, GLIS1, and PAX8, and between GLIS3 and CLPTM1L with hyalinizing trabecular tumors (HTTs) and fibrolamellar hepatocellular carcinoma (FHCC), respectively. Targeting upstream signaling pathways that regulate GLIS signaling may offer new therapeutic strategies in the management of cancer.

Acute megakaryoblastic leukemia is a rare subtype of pediatric acute myeloid leukemia (AML) with poor outcomes in patients with non-Down syndrome. The reported outcomes have been poor, and the prognostic factors have not been clearly determined.To evaluate the prognostic significance of various cytogenetic abnormalities and minimal residual disease status determined by flow cytometry after induction I, we retrospectively analyzed the data of 80 patients with non-Down syndrome with a diagnosis of M7 AML treated at Children's Cancer Hospital Egypt (CCHE-57357) from July 2007 through December 2016.Of the 80 patients, 15 died during induction I and were excluded from the survival analysis. The overall survival, event-free survival, and cumulative incidence of relapse at 2 years was 52.6% ± 12.7%, 45.2% ± 12.3%, and 31.8% ± 11.5% respectively. Of the 90 patients, 61 had cytogenetic abnormalities, including trisomy 19,13q, trisomy 8, complex karyotype, t(1;22), KMT2A rearrangements, and trisomy 21. None of these had an effect on the outcomes. In addition, 34 patients had minimal residual disease 0.1% after induction I, but the difference did not reach statistical significance. Patients with a delayed time to recovery (possibly due to myelofibrosis) had worse outcomes compared with those with early recovery (47% ± 19.2% vs. 63.2% ± 21.9%, respectively).Acute megakaryoblastic leukemia in patients with non-Down syndrome has a poor outcome with no clearly defined prognostic factors. However, future directions to risk stratify and tailor therapy should include assessment of the tumor biology according to the molecular pathways and study of the pathogenesis of myelofibrosis in this disease, which could affect the prognosis.

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.

Simple Summary Infant acute myeloid leukemia (AML) is a rare subgroup of AML of pediatric patients under two years of age. For a long time, they have been included in pediatric forms of AML. However, infant AML demonstrates unusual clinical and biological characteristics, and its prognosis differs from AML in older children. Most treatment protocols do not distinguish age subgroups in pediatric AML, when in reality these unusual forms of infancy might benefit from specific targeted therapeutics. Herein we summarize the various specificities of infant AML. Abstract Infant acute myeloid leukemia (AML) is a rare subgroup of AML of children

Background CBFA2T3-GLIS2 is a chimeric transcription factor in 18.4% of pediatric patients with non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL). It remains one of the poorest prognostic subtypes amongst pediatric acute myeloid leukemia patients with event free and overall survival probabilities of 8% and 14% respectively, despite intensive multi-agent chemotherapy and stem cell transplant treatment modalities.1 Adoptive cell therapies targeting the CBFA2T3-GLIS2 fusion may provide additional treatment options for AMKL patients. We hypothesize CBFA2T3-GLIS2 fusion junction specific neoantigen(s) are presented by surface HLA class I expression and elicit functional immune responses by CD8+ T cells. Methods To interrogate this hypothesis in silico, we used NetMHCcons to predict neoantigens spanning the fusion junction.2 To test our hypothesis in vivo, we established a small cohort (total N=12) of immunodeficient NSG-SGM3 mice engrafted with patient derived xenograft (PDX) cells from a CBFA2T3-GLIS2 positive AMKL patient for 20 days followed by the introduction of PBMCs from a healthy, unrelated donor with a 6/6 HLA class I match to the PDX cells for an additional 10, 15, or 20 days (figure 1).3Leukemia and T cell phenotype was characterized by flow cytometry. Levels of soluble analytes were measured in the serum by cytometric bead array. The TCR repertoire and transcriptional profile of the CD8+ T cells was determined through paired gene expression and 5’ single cell V(D)J sequencing using 10X Genomics. Results NetMHCcons identified two neoantigens spanning the fusion junction with intermediate binding affinities (IC50) of 304.37 and 384.09 nM. Flow cytometry analysis at Day 40 (figure 1) showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to Leukemia Only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at Day 40 in the PDX mice with PBMCs (9665.4 pg/ml) compared to the PBMC Only control (789.7 pg/ml), indicating a potential leukemia-directed cytokine lymphocyte response. TCR repertoire sequencing of CD8+ T cells from Day 40 revealed a decrease in alpha beta TCR diversity when exposed to PDX cells (104 TCRdiv) compared to the PBMC Only control (107 TCRdiv), consistent with a clonal expansion of neoantigen-specific TCRs4 (figure 2). Specificity of the TCRs for the putative neoantigens was assessed by cloning top expanded TCRs to measure tetramer binding and functional response.5 Conclusions Our novel murine model demonstrates a potential neoantigen-directed CD8+ T cell response against patient AMKL cells, highlighting the importance of adoptive cell therapy to improve patient outcomes. Acknowledgements We would like to thank Scott Perry in the Flow Cytometry and Cell Sorting Shared Resource (SJCRH) for his help with flow cytometry and the Animal Resource Center (SJCRH) staff for the care and housing of the murine model described here. This work is supported by the American Lebanese Syrian Associated Charities (ALSAC) (T.A.G., P.G.T.), R01 AI136514 (P.G.T.), and the Mark Foundation ASPIRE award (P.G.T.). Ethics Approval The vertebrate animal studies described here were approved by the Institutional Animal Care and Use Committee (IACUC); animal welfare assurance number A3325-01. The patient derived xenograft leukemia cells and healthy donor PBMCs used in the studies described here are exempt from requiring Institutional Review Board (IRB) approval because we receive no personal identifying information about the patient/donor from which the xenograft cells/PBMCs are derived. References de Rooij J, Branstetter C, Ma J, Li Y, et al. Pediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomes. Nat. Genet 2017; 49:451–456. Karosiene E, Lundegaard C, Lund O. NetMHCcons: a consensus method for the major histocompatibility complex class I predictions. Immunogenetics 2012; 64:269–281. Roth M, Harui A. Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model. J Immunother Cancer. 2015; 10:3-11. Dash P, Fiore-Gartland A, Wang G, et al. Quantifiable predictive features define epitope-specific T cell receptor repertoires. Nature 2017; 547:89–93. Zamora A, Crawford J, Allen E, Kuo X, et al. Pediatric patients with acute lymphoblastic leukemia generate abundant and functional neoantigen-specific CD8+ T cell responses. Sci Transl Med 2019; 11:eaat8549.

The work is devoted to the classification of tumor lesions of the hematopoietic organs - bone marrow and lymphoid system. These lesions are called hemopoietic neoplasia (hemoblastosis). Hemopoietic neoplasia is divided into leukecmia (leucosis) and lymphoma (hematosarcoma). Leukemia is a systemic blood disease of a tumor nature with primary bone marrow damage. Lymphomas are tumors of lymphoid cells with primary extra-bone marrow localization and localized tumor growth. Leukemias are divided into myeloid and lymphoid. Myeloids arise as a result of clonal proliferation of malignant progenitor cells of the myeloid series - CFU-GEMM, or its descendants. Lymphoid - as a result of proliferation of malignant progenitor cells of lymphoid series - CFU-L, or its descendants. Myeloid disorders include strictly myeloid (granulocytic), erythrocytic, monocytic, megakaryocytic leukemias, and myelodysplastic syndromes. Lymphoid leukemias are divided into T-cell, B-cell and plasma-cell. Leukemias are also divided into acute and chronic. Acute is called leukemia, in which the substrate of tumor growth form immature, blastic cells of I-IV classes of hematopoiesis, that lose their ability to maturation (differentiation). In chronic leukemia, this substrate consists of maturing and mature cells of V-VI classes of hematopoiesis, which have retained the ability to mature. The article presents a schematic classification of leukemias developed by the authors, which is based on modern conceptions about the pedigree of tumor cell clones. In 3 tables the FAB-classification of leukemias, adapted in 1991 by American Society of Veterinary Clinical Pathology (ASVCP) to the needs of veterinary medicine is stated. It is shown that today, along with morphological features, cytochemical (enzymatic) and immunological (genetic) markers are increasingly used for more detailed classification. The classification of hemopoietic neoplasias of certain species of domestic animals – cattle, sheeps, cats, dogs, horses, birds, etc. is also considered. Brief data on the prevalence of these diseases in them are given. References are given to the latest improvements and further development of the classifications of leukemias and lymphomas, in particular the revision of these WHO classifications in 2016.

Background Forced polyploidization is an effective strategy for acute megakaryoblastic leukemia (AMKL) therapy and factors controlling polyploidization are potential targets for drug development. Although bone morphology protein 2-inducible kinase (BMP2K) has been implied to be a potential target for fasudil, a potent polyploidy-inducing compound, the function of BMP2K in megakaryopoiesis and AMKL remains unknown. This study aimed to investigate the role of BMP2K as a novel regulator in megakaryocyte polyploidization and differentiation and its implication in AMKL therapy. Results BMP2K upregulation was observed in human megakaryopoiesis and leukemia cells whereas BMP2K was downregulated in AMKL cells forced to undergo terminal differentiation. Functionally, BMP2K suppressed MLN8237-induced megakaryocytic differentiation in AMKL cells and dampened megakaryocyte differentiation in primary mouse fetal liver cells. Furthermore, BMP2K overexpression conferred resistance to multiple chemotherapy compounds in AMKL cells. Mechanistically, cyclin-dependent kinase 2 (CDK2) interacted with BMP2K and partially mediated its function. In transient MLN8237 and nocodazole challenge cell model, BMP2K reduced cell percentage of G2/M phase but increased G1 phase, suggesting a role of BMP2K antagonizing polyploidization and promoting mitosis by regulating cell cycle in megakaryopoiesis. Conclusions BMP2K negatively regulates polyploidization and megakaryocyte differentiation by interacting CDK2 and promoting mitosis in megakaryopoiesis. BMP2K may serve as a potential target for improvement of AMKL therapy.

MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation of gene expression. In many cancers, up- or downregulation of different miRNAs is reported. In acute myeloid leukemia, upregulation of miR-92a-3p was reported in human in vitro studies. We performed blockage of miR-29a-3p in human acute megakaryoblastic leukemia cell line (M-07e) by using locked nucleic acid (LNA) and cell proliferation; apoptosis and necrosis were assessed. At different time points after LNA-anti-miR92a-3p transfection, miR-92a-3p quantitation was assessed by qRT-real-time PCR, MTT assay and annexin/propidium iodide staining were performed. The data were processed using the ANOVA test. At all three time points, the expression of miR-92a-3p was lower in the LNA-anti-miR group compared with the control groups. Cell viability between LNA-Anti-miR and the control group was statistically significant. Blockage of miR-92a-3p was associated with increment of the ratio of apoptotic cells in the LNA-anti-miR group was higher than the other group. The ratio of necrotic cells in the LNA-antimiR group was higher than the other groups. These assessments indicate that miR-92a-3p blockage can decrease the viability of M-07e cells, which is mainly due to induction of apoptosis and necrosis. Our findings could open up a path to a miRNA based therapeutic approach for treatment of acute megakaryoblastic leukemia.

Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML-DS). Defects in mesenchymal stromal cells (MSC) and cytokines specifically released by the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche (BMN). To model the BMN of pediatric patients with AMKL in mice, we first established MSCs from pediatric patients with AMKL (n = 5) and ML-DS (n = 9). Healthy donor control MSCs (n = 6) were generated from unaffected children and adolescents ≤18 years of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited decreased adipogenic differentiation potential and enrichment of proliferation-associated genes. Importantly, TGFB1 exposure in vitro promoted early profibrotic changes in all three MSC entities. To study BMF induction for longer periods of time, we created an in vivo humanized artificial BMN subcutaneously in immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, using a mixture of MSCs, human umbilical vein endothelial cell, and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this BMN after 8 weeks induced fibrosis grade I/II in a dose-dependent fashion over a time period of 4 weeks. Thus, our study developed a humanized mouse model that will be instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL blasts in future. Implications: TGFB1 supports fibrosis induction in a pediatric AMKL model generated with patient-derived MSCs. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/18/10/1603/F1.large.jpg.

Background The occurrence of a mediastinal germ cell tumor (GCT) and hematological malignancy in the same patient is very rare. Due to its rarity, there have been only two reports of the concurrent cases undergoing detailed genetic analysis with whole-exome sequencing (WES), and the possible clonal relationship between the both tumors remained not fully elucidated. Methods We performed whole-exome sequencing analysis of mediastinal GCT and acute myeloid leukemia (AML) samples obtained from one young Japanese male adult patient with concurrent both tumors, and investigated the possible clonal relationship between them. Results Sixteen somatic mutations were detected in the mediastinal GCT sample and 18 somatic mutations in the AML sample. Mutations in nine genes, including TP53 and PTEN both known as tumor suppressor genes, were shared in both tumors. Conclusions All in our case and in the previous two cases with concurrent mediastinal GCT and AML undergoing with whole-exome sequencing analysis, TP53 and PTEN mutations were commonly shared in both tumors. These data not only suggest that these tumors share a common founding clone, but also indicate that associated mediastinal GCT and AML harboring TP53 and PTEN mutations represent a unique biological entity.

The first event in origination of many childhood leukemias is a specific preleukemic fusion gene (PFG) that arises, often in utero, in hematopoietic stem/progenitor cells (HSPC) from misrepaired DNA double strand break (DSB). An immanently elevated level of DSB and impaired apoptosis may contribute to origination and persistence of PFG and donor cell-derived leukemia in recipients of allogeneic transplantation of umbilical cord blood (UCB). We investigated DSB, apoptosis and PFG in the backtracked UCB cells of leukemic patients. RNA from UCB of three patients with acute lymphoblastic leukemia, patient with acute megakaryoblastic leukemia and Down syndrome, and four healthy children was screened for common PFG by RT-qPCR. Presence of PFG was validated by sequencing. Endogenous γH2AX and 53BP1 DNA repair foci, cell populations, and apoptosis were analyzed in UCB CD34+/- cells with imaging and standard flow cytometry. We found MLL2-AF4 and BCR-ABL (p190) fusion genes in UCB of two out from four pediatric patients, apparently not detected at diagnosis, while UCB cells of TEL-AML1+ ALL patient were tested negative for this PFG and no PFG were detected in UCB cells of healthy children. No significant difference in DNA damage and apoptosis between UCB CD34+/- cells from healthy children and leukemic patients was observed, while Down syndrome trisomy increased DNA damage and resulted in distribution of cell populations resembling transient abnormal myelopoiesis. Our findings indicate increased genetic instability in UCB HSPC of leukemic patients and may be potentially used for diagnostics and exclusion of possibly affected UCB from transplantation.

The advent of large scale genomic sequencing technologies significantly improved the molecular classification of acute megakaryoblastic leukaemia (AMKL). AMKL represents a subset (∼10%) of high fatality pediatric acute myeloid leukemia (AML). Recurrent and mutually exclusive chimeric gene fusions associated with pediatric AMKL are found in 60%-70% of cases and include RBM15-MKL1, CBFA2T3-GLIS2, NUP98-KDM5A and MLL rearrangements. In addition, another 4% of AMKL harbor NUP98 rearrangements (NUP98r), with yet undetermined fusion partners. We report a novel NUP98-BPTF fusion in an infant presenting with primary refractory AMKL. In this NUP98r, the C-terminal chromatin recognition modules of BPTF, a core subunit of the NURF (nucleosome remodeling factor) ATP-dependent chromatin-remodeling complex, are fused to the N-terminal moiety of NUP98, creating an in frame NUP98-BPTF fusion, with structural homology to NUP98-KDM5A. The leukemic blasts expressed two NUP98-BPTF splicing variants, containing one or two tandemly spaced PHD chromatin reader domains. Our study also identified an unreported wild type BPTF splicing variant encoding for 2 PHD domains, detected both in normal cord blood CD34+ cells and in leukemic blasts, as with the fly BPTF homolog, Nurf301. Disease course was marked by rapid progression and primary chemoresistance, with ultimately significant tumor burden reduction following treatment with a clofarabine containing regimen. In sum, we report 2 novel NUP98-BPTF fusion isoforms that contribute to refine the NUP98r subgroup of pediatric AMKL. Multicenter clinical trials are critically required to determine the frequency of this fusion in AMKL patients and explore innovative treatment strategies for a disease still plagued with poor outcomes.

CBFA2T3-GLIS2 is an oncogenic fusion gene that acts as the driver mutation in ~18% of pediatric patients with acute megakaryoblastic leukemia (AMKL) that do not have Down syndrome. CBFA2T3-GLIS2 driven leukemia carries the worst prognosis among all pediatric acute myeloid (AML) subtypes, with event free and overall survival probabilities of 8% and 14% respectively. Chemotherapy and stem cell transplantation fail to cure this disease, thus innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven't been realized in AML due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which use heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by MHC molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, we established a humanized patient derived xenograft (PDX) murine model by conditioning 15 immunodeficient NSG-SGM3 mice with 200 rads 24 hours prior to transplantation of 3.5 million CBFA2T3-GLIS2 positive PDX cells derived from an individual female patient. After 20 days of engraftment, 2.5 million HLA class I matched PBMCs from an unrelated female donor were introduced. Experimental mice (engrafted with both PDX cells and PBMCs), leukemia only control mice, and PBMC only control mice were sacrificed at days 30, 35, and 40. Flow cytometry analysis at day 40 showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to the leukemia only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at day 40 in the PDX mice with PBMCs compared to the PBMC only control (9665.4 pg/ml vs. 789.7 pg/ml, p Disclosures Gruber: Kura Oncology: Consultancy.

AML, frequently showing megakaryocytic differentiation, is known to arise in MGCT and has a dismal prognosis. Close inspection of MGCT is required to identify concurrent AML.

Pediatric leukemia presents specific clinical and genetic features from adult leukemia but the underpinning mechanisms of transformation are still unclear. Acute megakaryoblastic leukemia (AMKL) is the malignant accumulation of progenitors of the megakaryocyte lineage that normally produce blood platelets. AMKL is diagnosed de novo , in patients showing a poor prognosis, or in Down syndrome (DS) patients with a better prognosis. Recent data show that de novo AMKL is primarily associated with chromosomal alterations leading to the expression of fusions between transcriptional regulators. This review highlights the most recurrent genetic events found in de novo pediatric AMKL patients and, based on recent functional analyses, proposes a mechanism of leukemogenesis common to de novo and DS–AMKL.

Essential thrombocythemia (ET) is a Philadelphia chromosome-negative myeloproliferative neoplasm characterized by sustained thrombocytosis. Its transformation into acute leukemia is a rare event (5% at 20 years). Transformation can be spontaneous or related to therapy. Acute leukemias most often associated with transformation from ET are myeloblastic or myelomonoblastic leukemias. There are very few case reports of ET transforming into acute megakaryoblastic leukemia. Here we report a case of transformation to acute megakaryoblastic leukemia in an elderly female patient who was treated with hydroxyurea for ten years. After 10-years with stable disease she presented and rapidly deteriorated with acute megakaryoblastic leukemia with high JAK2V617F mutant allele burden and high p53 expression. This case provides another example of the poor outcome of acute megakaryoblastic transformation of essential thrombocythemia. Keywords: Essential thrombocythemia, Acute megakaryoblastic leukemia, Transformation

International audience; Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

Background Alpha-synuclein is a member of synuclein family of proteins with unidentified function localized in the cytoplasm, mitochondria of neurons, and presynaptic nerve endings. Although it is found in the Lewy bodies in synucleinopathies and in Alzheimer's disease, the protein could also be considered as a novel marker in diagnosis of diseases related to the hematopoietic system. Methods The current study evaluated alpha-synuclein expression in bone marrow sections obtained from 9 patients with acute myeloblastic leukemia (AML)-M6, 2 patients with AML-M7, and 56 patients with other forms of AML by immunohistochemical (IHC) analysis. Results Seven out of 9 cases with erythroleukemia (66.7%) and 1 of the 2 cases with M7 (50%) were positive. In contrast; the blasts in 2 out of 56 AML cases with non-M6/M7 (3.6%) showed positive staining. Accordingly, alpha-synuclein was positive in normal erythroid precursors and megakaryocytes (if existing) in these cases; while, it was negative in lymphoid and myeloid precursors. Conclusion Alpha-synuclein expression in non-neoplastic and neoplastic erythroid cells and megakaryocytes could be used as a complementary and useful marker for distinction between AML-M6/M7 and other types of AML.

Background Acute myeloid leukemia (AML) is a heterogeneous disease regarding morphology, immunophenotyping, genetic abnormalities, and clinical behavior. The overall survival rate of pediatric AML is 60% to 70%, and has not significantly improved over the past two decades. Children with Down syndrome (DS) are at risk of developing acute megakaryoblastic leukemia (AMKL), which can be preceded by a transient myeloproliferative disorder during the neonatal period. Intensification of current treatment protocols is not feasible due to already high treatment‐related morbidity and mortality. Instead, more targeted therapies with less severe side effects are highly needed. Procedure To identify potential novel therapeutic targets for myeloid disorders in children, including DS‐AMKL and non‐DS‐AML, we performed an unbiased compound screen of 80 small molecules targeting epigenetic regulators in three pediatric AML cell lines that are representative for different subtypes of pediatric AML. Three candidate compounds were validated and further evaluated in normal myeloid precursor cells during neutrophil differentiation and in (pre‐)leukemic pediatric patient cells. Results Candidate drugs LMK235, NSC3852, and bromosporine were effective in all tested pediatric AML cell lines with antiproliferative, proapoptotic, and differentiation effects. Out of these three compounds, the pan‐histone deacetylase inhibitor NSC3852 specifically induced growth arrest and apoptosis in pediatric AML cells, without disrupting normal neutrophil differentiation. Conclusion NSC3852 is a potential candidate drug for further preclinical testing in pediatric AML and DS‐AMKL.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2–GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2–GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2–GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. Significance: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state. See related commentary by Cruz Hernandez and Vyas, p. 1653. This article is highlighted in the In This Issue feature, p. 1631

Congenital leukemia is rare disease with an incidence of one to five cases per million births. Transient abnormal myelopoiesis (TAM), also called transient myeloproliferative disorder, is a pre-leukemia disorder that may occur in Down syndrome (DS) or non-DS infants. TAM may enter spontaneous remission; however, continual monitoring is required, as this disorder has been observed to develop into acute megakaryoblastic leukemia in 16-30% of cases. In the literature, 16 cases of TAM in non-DS infants have been reported. The case presented in the current study is, to the best of our knowledge, the first case of an Italian non-DS newborn presenting with clinical manifestations of acute leukemia at five days after birth, exhibiting a normal karyotype, trisomy 21 only in blast cells, and spontaneous remission. Chromosomal analyses on peripheral blood cells, bone marrow cells and dermal fibroblasts were conducted using a G-banding technique, and fluorescence in situ hybridization (FISH) was used to identify the critical regions of DS. Amplification of GATA binding protein 1 (GATA1) exon 2 genomic DNA was performed using polymerase chain reaction. Cytogenetic analysis of 50 peripheral blood cells and dermal fibroblasts from the patient revealed a normal karyotype: 46, XX. Conversely, cytogenetic analysis of the patient's bone marrow revealed an abnormal karyotype 47, XX+21. In order to investigate this result, FISH was performed, which identified the presence of three signals in 70% of the cells and two signals in 30% of bone marrow cells. GATA1 sequencing revealed the substitution of a single base (c.150delG) in exon 2. Seven months after the initial analysis, FISH and cytogenetic analyses of the stimulated/unstimulated peripheral blood cells and bone marrow cells were performed, revealing that each exhibited diploid signals, as observed in a normal karyotype.

Acute erythroid leukemia (FAB-M6) and acute megakaryoblastic leukemia (FAB-M7) exhibit closely related properties in cells regarding morphology and the gene expression profile. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the mainstay of the treatment for both subtypes of leukemia due to their refractoriness to chemotherapy and high rates of relapse, it remains unclear whether allo-HSCT is curative in such cases due to their scarcity. We retrospectively examined the impact of allo-HSCT in 382 patients with M6 and 108 patients with M7 using nationwide HSCT data and found the overall survival (OS) and relapse rates of the M6 patients to be significantly better than those of the M7 patients after adjusting for confounding factors and statistically comparable with those of the patients with M0/M1/M2/M4/M5 disease. Consequently, the factors of age, gender, performance status, karyotype, disease status at HSCT and development of graft-vs.-host disease predicted the OS for the M6 patients, while the performance status and disease status at HSCT were predictive of the OS for the M7 patients. These findings substantiate the importance of distinguishing between M6 and M7 in the HSCT setting and suggest that unknown mechanisms influence the HSCT outcomes of these closely related subtypes of leukemia. © 2016 Elsevier Ltd., Embargo Period 12 months

Ataxia telangiectasia mutated (ATM) is a tumor suppressor gene, and its somatic inactivation plays a role in the pathogenesis of lymphoid malignancies. However, the role of ATM in patients with myeloid malignancies is still unknown. We herein report a case of acute megakaryoblastic leukemia (AMKL) with ATM gene deletion. An 84-year-old Japanese woman presenting with a pale face and pancytopenia was admitted to our institution and diagnosed to have AMKL with ATM gene deletion. She was treated with intravenous azacitidine. The azacitidine treatment was effective for approximately 1 year. Somatic inactivation of the ATM gene may therefore be involved in the pathogenesis of AMKL.

MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation of gene expression. In many cancers, up- or downregulation of different miRNAs is reported. In acute myeloid leukemia, upregulation of miR-92a-3p was reported in human in vitro studies. We performed blockage of miR-29a-3p in human acute megakaryoblastic leukemia cell line (M-07e) by using locked nucleic acid (LNA) and cell proliferation; apoptosis and necrosis were assessed. At different time points after LNA-anti-miR92a-3p transfection, miR-92a-3p quantitation was assessed by qRT-real-time PCR, MTT assay and annexin/propidium iodide staining were performed. The data were processed using the ANOVA test. At all three time points, the expression of miR-92a-3p was lower in the LNA-anti-miR group compared with the control groups. Cell viability between LNA-Anti-miR and the control group was statistically significant. Blockage of miR-92a-3p was associated with increment of the ratio of apoptotic cells in the LNA-anti-miR group was higher than the other group. The ratio of necrotic cells in the LNA-antimiR group was higher than the other groups. These assessments indicate that miR-92a-3p blockage can decrease the viability of M-07e cells, which is mainly due to induction of apoptosis and necrosis. Our findings could open up a path to a miRNA based therapeutic approach for treatment of acute megakaryoblastic leukemia.

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.

Chromosomal translocations of the Nucleoporin 98 gene (NUP98) are associated with multiple hematopoietic malignancies. In particular, NUP98 rearrangements can be found in up to 6.6% of de novo cases of pediatric AML. Specific subpopulations of pediatric AML appear to be even more enriched for NUP98 fusions, with over 16% of cytogenetically normal pediatric AML patients and 15% of non-Down syndrome acute megakaryoblastic leukemia (AMKL) patients harboring these fusions. Clinically, the NUP98-KDM5A fusion is associated with erythroid and megakaryocytic leukemias while the NUP98-NSD1 fusion is associated with myelomonocytic leukemias. When NUP98 fusions are present in AML, they confer a poor prognosis. To better characterize the role of these fusions in human hematopoiesis, we used cord blood CD34+ (cb-CD34+) hematopoietic stem and progenitor cells (HSPCs) as an in vitro model system. We transduced cb-CD34+ HSPCs with NUP98-NSD1, NUP98-KDM5A, or empty vector control and studied the effects on cell proliferation and differentiation in liquid culture media containing cytokines (IL-6, FLT3-Ligand, SCF, TPO, and SR1 (an aryl hydrocarbon receptor antagonist)). NUP98-NSD1 and NUP98-KDM5A fusions increase the proliferation rate in liquid culture by three-fold and 19-fold, respectively (p Citation Format: Ryan Hiltenbrand, Jonathan Miller, Jeffery Klco. Cord blood CD34+ HSPCs: An in vitro model system for characterizing NUP98 fusions [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B34.

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.

Several conventions have been established in order to define and characterize Mixed Phenotype Acute Leukemia (MPAL). However, megakaryocytic markers have not been included in the definition of MPAL neither in the European Group for the Immunological Characterization of Leukemias (EGIL) proposal nor in any of the WHO Classification of Tumors issues. We report four pediatric acute leukemia (AL) cases (prevalence: 0.18%) with megakaryoblasts co-expressing the T-specific antigen CD3 (cytoplasmic), together with a very homogeneous antigen profile of immature cells and other lymphoid traits. In one case, the presence of epsilon CD3 mRNA was confirmed as well on sorted CD34+ blasts. All four cases were infants, and two of them disclosed trisomy 21 in the blast population (not constitutional) without being children with Down Syndrome. They were homogeneously treated with AML schemes, achieving all four CR. However, 3 patients relapsed early. Only one patient is alive and remain disease-free, with a long follow-up. Even though cyCD3 was the only T cell marker expressed, its specificity entails the consideration of these cases as a new subtype of MPAL Megakaryoblastic/T, keeping this in mind when designing diagnostic panels. Detection and report of these cases are necessary so as to further characterize them in order to define the most appropriate treatment.

The RUNX1-EVI1 gene generated by the t(3;21) translocation encodes a chimeric transcription factor and is a causative gene in the development of de novo acute megakaryoblastic leukemia and leukemic transformation of hematopoietic stem cell tumors. Heterozygous RUNX1-EVI1 knock-in mice die in utero due to hemorrhage in the central nervous system and spinal cord and complete abolishment of definitive hematopoiesis in the fetal liver. On the other hand, the chimeric knock-in mouse develops acute megakaryoblastic leukemia. We created another mouse model of RUNX1-EVI1 using transplantation of retrovirus-infected bone marrow cells. Some mice transplanted with RUNX1-EVI1-expressing bone marrow cells developed acute megakaryoblastic leukemia within eight months, and the other non-leukemic mice showed thrombocytosis at around a year. In the non-leukemic mice, dysplastic megakaryocytes proliferated in the bone marrow and frequently infiltrated into the spleen, which was not associated with marrow fibrosis. In the leukemic mice, their tumor cells were positive for c-kit and CD41, and negative for TER119. Although they were negative for platelet peroxidase in the electron microscopic analysis, they had multiple centrioles in the cytoplasm, which are characteristic of megakaryocytes that undergo endomitosis. The leukemic cells were serially transplantable, and gene-expression analyses using quantitative RT-PCR arrays revealed that they showed significantly elevated expression of stem cell, primitive hematopoietic cell and endothelial cell-related genes compared with normal bone marrow cells. All these data suggested that RUNX1-EVI1 caused dysplastic hematopoiesis or leukemia of the megakaryocytic lineage and endowed gene expression profiles distinctive of immature hematopoietic cells.

Individuals with Down syndrome (DS) have a markedly increased risk of developing unique myeloid proliferations such as transient abnormal myelopoiesis (TAM) and myeloid leukemia associated with Down syndrome (ML-DS) [1, 2]. These proliferations occur in the first 3 years of life and are a result of several transforming genetic events that arise during the fetal and newborn period. The initial event, an additional chromosome 21, leads to increased megakaryocytic proliferation in the fetal liver. Subsequent mutation of GATA-binding protein 1 (GATA1) results in the development of TAM. Further acquisition of additional mutations of epigenetic regulators and common signaling pathways such as JAK family kinases, MPL, and multiple RAS pathway genes leads to the transformation to MS-DS [3].