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The homeobox (HOX) genes are a highly conserved family of transcription factors involved in embryonic patterning as well as adult hematopoiesis. Dysregulation of HOX genes, in particular upregulation of HOXA cluster genes, is a frequent event in Acute Myelogenous Leukemia (AML). Recently, we performed a detailed genomic analysis on pediatric non-Down Syndrome Acute Megakaryoblastic Leukemia (non-DS-AMKL) and identified novel fusions involving a HOX cluster gene in 14.9% of the cases. While most fusions were predicted to lead to an in-frame functional protein, several fusions included a non-coding HOX antisense gene (PLEK-HOXA11-AS, C8orf76-HOXA11-AS, HOXA10-AS-CD164) that were predicted to result in a loss of function of these regulatory transcripts. The functional consequence of these events, however, remain unknown. HOXA11-AS (human) and Hoxa11os (mouse) have been previously shown to have mutually exclusive expression with the Hoxa11 transcript throughout development. We therefore hypothesized that loss of function of non-coding HOX antisense genes as a result of these structural variations would cause upregulation of nearby coding HOXA genes that in turn promote leukemogenesis. To test this hypothesis, using CRISPR-Cas9 technology, we genome edited the human AMKL cell line CMK to carry the PLEK-HOXA11-AS translocation. qRT-PCR of HOXA11-AS and HOXA9-11 transcripts in this cell line recapitulated the pattern seen in patient specimens. Specifically, HOXA11-AS expression was significantly diminished while HOXA10 and HOXA11 transcripts were upregulated 1.8-2.5-fold when compared to parental CMK cells (p=0.0385 and p=0.006 respectively). To further investigate the loss of HOXA11-ASin vivo a CRISPR-Cas9 Hoxa11os knockout mouse model was established. qRT-PCR on bone marrow confirmed the loss of Hoxa11os transcripts in heterozygous (Hoxa11os1+/-) and homozygous (Hoxa11os-/-) mice of both genders (p= Combined these data demonstrate that loss of function alterations in Hoxa11os transcripts lead to upregulation of Hoxa11 and gender specific hematopoietic progenitor cell perturbations. Ongoing efforts include competitive transplant studies as well as RNA and ChIP sequencing to identify gender specific downstream targets of Hoxa11 in the hematopoietic compartment in order to understand the selective expansion of progenitor subsets and male specific self-renewal capacity of this protein. These data will contribute to our understanding on how HOXA11-AS translocations promote oncogenesis. Disclosures Zwaan: Daiichi Sankyo: Consultancy; Sanofi: Consultancy; Roche: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Incyte: Consultancy; Celgene: Consultancy, Research Funding; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Janssen: Consultancy. Gruber:Bristol-Myers Squibb: Consultancy.

Cornelia de Lange Syndrome (CdLS) is a rare autosomal dominant developmental disorder characterized by a distinctive facial dysmorphism and variable developmental anomalies including prenatal and postnatal growth delay, microcephaly, intellectual disability, upper limb anomalies and hirsutism. CdLS

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.

International audience; We report the outcome of 27 children with de novo acute megakaryoblastic leukemia (AMKL) (excluding Down syndrome) enrolled in the French multicenter prospective study ELAM02 (2005-2011). There was no difference in gender, initial leukocyte count, CNS involvement, and complete remission rate (88.9%), as compared to other acute myeloid leukemia (AML) subtypes. AMKL patients had a significantly poorer outcome (5-year overall survival 54% [CI 95% 33%-71%] than children with other AML subtypes (5-year overall survival 73% [CI 95% 68%-77%] p = 0.02). Gender, age, CNS leukemia, hyperleukocytosis, complete remission or cytogenetic subgroups were not significant prognostic factors of disease-free survival. AMKL (excluding Down syndrom) remains an AML subgroup with inferior outcome.

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].

Comprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age 50 chromosomes (n=44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): -7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1; 22)(p13; q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9; 11)(p22; q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk-7p abnormalities; poor risk-normal karyotypes, -7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, -13/13q-, and -15; and intermediate risk-others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.

Despite recent advances in the treatment of children with acute megakaryoblastic leukemia (AMKL) using intensified treatment protocols, clear prognostic indicators, and treatment recommendations for this acute myeloid leukemia (AML) subgroup are yet to be defined. Here, we report the outcome of 97 pediatric patients with de novo AMKL (excluding Down syndrome [DS]) enrolled in the prospective multicenter studies AML-BFM 98 and AML-BFM 04 (1998-2014). AMKL occurred in 7.4 % of pediatric AML cases, at younger age (median 1.44 years) and with lower white blood cell count (mean 16.5 × 109/L) as compared to other AML subgroups. With 60 ± 5 %, children with AMKL had a lower 5-year overall survival (5-year OS; vs. 68 ± 1 %, Plog rank = 0.038). Yet, we achieved an improved 5-year OS in AML-BFM 04 compared to AML-BFM 98 (70 ± 6 % vs. 45 ± 8 %, Plog rank = 0.041). Allogeneic hematopoietic stem cell transplantation in first remission did not provide a significant survival benefit (5-year OS 70 ± 11 % vs. 63 ± 6 %; PMantel-Byar = 0.85). Cytogenetic data were available for n = 78 patients. AMKL patients with gain of chromosome 21 had a superior 5-year OS (80 ± 9 %, Plog rank = 0.034), whereas translocation t(1;22)(p13;q13) was associated with an inferior 5-year event-free survival (38 ± 17 %, Plog rank = 0.04). However, multivariate analysis showed that treatment response (bone marrow morphology on day 15 and 28) was the only independent prognostic marker (RR = 4.39; 95 % CI, 1.97–9.78). Interestingly, GATA1-mutations were detected in six patients (11 %) without previously known trisomy 21. Thus, AMKL (excluding DS) remains an AML subgroup with inferior outcome. Nevertheless, with intensive therapy regimens, a steep increase in the survival rates was achieved. Electronic supplementary material The online version of this article (doi:10.1007/s00277-015-2383-2) contains supplementary material, which is available to authorized users.

We encountered a case of neonatal acute megakaryoblastic leukemia not associated with Down syndrome (DS). Molecular cytogenetic analysis of leukemic blast cells indicated that increased blast cell status was caused by transient abnormal myelopoiesis with trisomy 21 and GATA1 mutation. Based on these molecular cytogenetic data, intensive chemotherapy was avoided, and the patient was successfully cured with low-dose cytarabine. Morphologically, leukemic blast cells of acute megakaryoblastic leukemia in a non-DS neonate are indistinguishable from a blast cell of transient abnormal myelopoiesis. The possibility of transient abnormal myelopoiesis should be carefully considered before intensive chemotherapy is adopted.

Mirror syndrome describes the association of fetal and placental hydrops with maternal edema. This case is the first reported case of mirror syndrome relative to fetal leukemia. We suggest that fetal leukemia can have a major impact on mirror syndrome, and provide a brief review of the literature related to this syndrome.

A subset of Down syndrome (DS) (trisomy 21) neonates is born with a unique erythromegakaryocytic myeloproliferative disorder that spontaneously resolves over the first few months of life (DS-transient abnormal myelopoiesis (DS-TAM); previously called DS-transient myeloproliferative disorder (DS-TMD) and DS-transient leukemia (DS-TL)). These infants are at high risk for developing subsequent acute megakaryoblastic leukemia (myeloid leukemia associated with Down syndrome (ML-DS); previously called DS-acute megakaryoblastic leukemia (DS-AMKL)). The molecular basis for DS-TAM/ML-DS remained mysterious for a long period of time. However, new genetic insights have been gained over the past 12 years that have begun to decipher the pathophysiology of this unusual disorder.

Key Clinical Message We describe a neonate with abdominal distension, massive hepatomegaly, and high serum neuron-specific enolase level suggestive of congenital neuroblastoma. The patient died of pulmonary hemorrhage after therapy. Autopsy revealed that the tumor cells in the liver indicated acute megakaryocytic leukemia with the RBM15-MKL1 fusion gene.

Background: Acute megakaryoblastic leukemia (AMKL) is a rare form of acute myeloid leukemia that encompasses three distinct subgroups, children with Down syndrome (DS-AMKL), children without DS (non-DS-AMKL) and adults (adult-AMKL). We hypothesize that the biological differences in the subtypes of AMKL may be reflected in distinct immunophenotypic (IP) features. Our aims were to employ mutiparameter flow cytometry to establish a comprehensive IP spectrum of AMKL in multiple demographic groups and to determine whether the subtypes of AMKL demonstrate distinct immunophenotypes. Methods: Thirty-seven AMKL cases were retrieved and subdivided into these three groups. Multiparameter flow cytometry was used to determine and compare the immunophenotypes. Available clinical, cytogenetic, and morphologic data were reviewed to confirm diagnoses. Results: While there is commonality in the expression pattern for a wide range of myelomonocytic markers in these three subtypes of AMKL, blasts in DS-AMKL were more likely to express CD7 and CD11b than those in non-DS-AMKL and adult-AMKL. Furthermore, blasts in DS-AMKL were more likely to express CD13, CD33 and CD36 than non-DS pediatric AMKL, and showed greater CD56 expression compared to adult AMKL. Conclusions: These results indicate that DS-AMKL is related to but immunophenotypically distinct from non-DS-AMKL and adult-AMKL. This distinct immunophenotypic pattern, co-expression of CD7 and CD11b, has practical implications for characterization of AMKL. © 2014 Clinical Cytometry Society.

Background Children with Down syndrome (DS) have an enhanced risk of developing acute leukemia, with the most common subtype being acute megakaryoblastic leukemia (AMKL). Myeloid leukemia in Down syndrome (ML-DS) is considered a disease with distinct clinical and biological features. There are few studies focusing on the clonal cytogenetic changes during evolution of ML-DS. Case presentation Here, we describe a complex karyotype involving a previously unreported set of chromosomal abnormalities acquired during progression of ML-DS in an infant boy: derivative der(1)t(1;15)(q24;q23), translocation t(4;5)(q26;q33) and derivative der(15)t(7;15)(p21;q23). Different molecular cytogenetic probes and probesets including whole chromosome painting (WCP) and locus specific probes, as well as, multicolor-FISH and multicolor chromosome banding (MCB) were performed in order to characterize the chromosomal abnormalities involved in this complex karyotype. The patient was treated according to the acute myeloid leukemia-Berlin-Frankfurt-Munich-2004 (AML-BFM 2004) treatment protocol for patients with Down syndrome; however, he experienced a poor clinical outcome. Conclusion The molecular cytogenetic studies performed, allowed the characterization of novel chromosomal abnormalities in ML-DS and possible candidate genes involved in the leukemogenic process. Our findings suggest that the complex karyotype described here was associated with the poor prognosis.

The development of mature blood cell from hematopoietic stem cells is regulated by transcription factors that coordinate the expression of lineage-specific genes. GATA transcription factors are zinc finger DNA-binding proteins that play crucial roles in various biological processes, including hematopoiesis. Among GATA family proteins, GATA-1, GATA-2, and GATA-3 are essential for hematopoiesis. GATA-1 functions to promote development of erythrocytes, megakaryocytes, eosinophils, and mast cells. Mutations in GATA-1 are associated with acute megakaryoblastic leukemia (AMKL), congenital erythroid hypoplasia (Diamond-Blackfan anemia; DBA), and X-linked anemia and/or thrombocytopenia. Conversely, GATA-2 functions early in hematopoiesis and is required for maintenance and expansion of hematopoietic stem cells (HSCs) and/or multipotent progenitors. GATA-2 mutations are associated with immunodeficiency, lymphedema, myelodysplastic syndrome (MDS), and leukemia. Furthermore, decreased GATA-2 expression may contribute to the pathophysiology of aplastic anemia. GATA-3 has an important role in T cell development, and has been suggested to be involved in the pathophysiology of acute lymphoblastic leukemias. This review summarizes current knowledge on hematological disorders associated with GATA-1 and GATA-2 mutations.

Background/Aim: As the knowledgebase of acute myeloid leukemia (AML) has grown, classification systems have moved to incorporate these new findings. Methods: We assessed 32,941 patients with AML whose records are contained in the Surveillance, Epidemiology, and End Results (SEER) database. Results: Half of all patients diagnosed between 2001 and 2013 did not have a World Health Organization (WHO) classification. Acute promyelocytic leukemia and acute panmyelosis with myelofibrosis were associated with the longest leukemia-specific survival (110 and 115 months, respectively), and AML with minimal differentiation and acute megakaryoblastic leukemia with the shortest (30 and 28 months, respectively). For patients in the WHO groups AML not otherwise specified (AML-NOS) and AML with recurrent genetic abnormalities (AML-RGA), the risk of death was greater for older patients and less for married patients. Black patients with any type of AML-NOS also had a higher risk of death. Patients whose case of AML did not receive a WHO classification were older and this group had a higher risk of death when compared to patients with a WHO type of AML-NOS. Conclusion: Our findings highlight the divergent outcomes of patients with AML and the importance of using the WHO classification system and demographic factors to gauge their prognosis.

Patient: Female, 3-month-old Final Diagnosis: Acute megakaryoblastic leukemia Symptoms: Abdominal distension • fever Medication: — Clinical Procedure: — Specialty: Pediatrics and Neonatology Objective: Challenging differential diagnosis Background: The reciprocal translocation t(1;22)(p13;q13) involving the RBM15 and MKL1 genes is an uncommon abnormality that occurs in a subset of acute myeloid leukemia with megakaryocytic differentiation (AMKL). Variant translocations have been infrequently described in this subtype of leukemia. Case Report: We describe the case of a 3-month-old girl who presented with progressive abdominal distension, vomiting, and fever. Although there was no morphologic evidence of leukemia in the bone marrow, cytogenetic and metaphase fluorescence in situ hybridization analysis identified an insertion of p13p31 bands of chromosome 1 onto the long arm of chromosome 22, resulting in the karyotype: 46,XX,ins(22;1)(q13;p13p31). Subsequent liver biopsy demonstrated extensive involvement by AMKL. Conclusions: AMKL can present with fewer than 20% blasts in the peripheral blood or bone marrow, necessitating careful evaluation for extramedullary disease. In other situations, bone marrow fibrosis can result in difficult marrow aspirations and a falsely decreased blast count. This case report highlights the critical role of careful cytogenetic and FISH testing in the diagnosis of AMKL.

Objectives GATA binding factor 1 (GATA1) is a transcription factor essential for erythromegakaryocytic differentiation. Given its function in lineage specification, we sought to evaluate the immunohistochemical profile of GATA1 in normal marrow and acute leukemia and assess the use of GATA1 as a specific erythromegakaryocytic immunohistochemical marker. Methods Immunohistochemical studies for GATA1 expression were performed on bone marrow biopsy specimens to define its role in the evaluation of acute leukemia and other hematologic disorders. Results In normal marrows, intense nuclear reactivity is seen in immature erythroid precursors and megakaryocytes. Weak to moderate nuclear positivity is seen in eosinophils and mast cells. In marrows involved by acute leukemia, blasts of pure erythroleukemia and acute megakaryoblastic leukemia exhibit intense nuclear GATA1 positivity, while blasts of acute myeloid leukemia of other categories are negative. GATA1 is also absent in the blasts of acute lymphoblastic leukemia/lymphoma and in the neoplastic cells of metastatic carcinoma and plasma cell neoplasms. Conclusions Intense GATA1 nuclear expression is a sensitive and specific marker for cells of erythroid and megakaryocytic lineages and is an excellent marker for neoplastic cells of pure erythroleukemia and acute megakaryoblastic leukemia.

Background CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins. Methods We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile. Results As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions. Conclusions Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0396-0) contains supplementary material, which is available to authorized users.

Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathologic including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI, and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2 which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain of function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor (MEP) gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathologic drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts. While rare in adults, AMKL accounts for 4-15% of newly diagnosed childhood AML cases. AMKL in individuals without Down syndrome (non-DS-AMKL) is frequently associated with poor clinical outcomes. Previous efforts have identified chimeric oncogenes in a substantial number of non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements, and NUP98-KDM5A. However, the etiology of 30-40% of cases remains unknown. To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult). We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes. These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes. Overall, these data shed light on the etiology of AMKL and provide useful information for the tailoring of treatment.

ダウン症児は巨核芽球性白血病（DS－AMKL）のリスクが高い．DS－AMKL ではほぼ全例でGATA1 遺伝子変異があり，N末端領域を欠くGATA1s だけが発現する．非ダウン症例 （non－DS－AMKL）にはこの変異は稀である．最近我々は，19例中1例のnon－DS－AMKL にインフレーム欠失を見出した．患児は出生時より白血球増多を認め，生後1 ヶ月時にAMKL と診断された．芽球の核型は46,XY であった．変異確認の際，第3 エクソンのスプライシングを起こす別の欠失を認め，両変異は同アレルに座位していた．この変異体（#135）の転写活性化能は60%に減弱していた．完全長GATA1 発現レトロウィルスの導入でDS－AMKL 細胞株KPAM1 の増殖が著しく抑制されたが，GATA1s や#135では緩やかな抑制だった．これらの結果は#135変異体がnon－DS－AMKL 発症に関与していることを示している, 弘前医学. 65(2-4), 2014, p.227-237

GATA1 mutations are found almost exclusively in children with myeloid proliferations related to Down syndrome (DS). Here, we report two phenotypically and cytogenetically normal children with acute megakaryoblastic leukemia (AMKL) whose blasts had both acquired trisomy 21 and GATA1 mutation. Patient 1 was diagnosed with transient abnormal myelopoiesis in the neonatal period. Following spontaneous improvement of the disease, leukemic blasts increased 7 months later. He received less intensive chemotherapy, and he is now 6 years old in complete remission. Patient 2 was diagnosed with AMKL at the age of 18 months. Although he received intensive chemotherapy and a cord blood transplantation, he died without gaining remission. In both cases, trisomy 21 and GATA1 mutation were detected only in leukemic blasts, but not in germline samples. Based on a literature review, we identified reports describing 14 non-DS AMKL with GATA1 mutation and acquired trisomy 21. Of those, 12 cases were diagnosed during the neonatal period, whereas the remaining 2 cases were diagnosed at the age of 22 and 31 months, respectively.These cases suggest that GATA1 mutation may cooperate with the additional chromosome 21 in developing myeloid proliferations even in non-DS patients.

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

Therapy-related AML (t-AML) is one of the newly expanded disease entities in the 2008 WHO classification, accounting for 10-20% of all cases of AML, and its incidence is increasing worldwide because of improved survival rates following treatment for other primary cancers [1, 2]. Acute megakaryoblastic leukemia (AMKL) (M7) is the least common of the t-AML French-American-British (FAB) subtypes, and only two such cases have been reported to date [3, 4], neither of which was in Korea. AMKL accounts for about 7-10% of childhood AML cases (frequently associated with Down syndrome), but only about 1% of adult AML cases [5]. Here, we describe a rare case of therapy-related acute megakaryoblastic leukemia (t-AMKL) with chromosome 5 and 7 abnormalities that presented ten years after chemoradiotherapy in an elderly lung cancer patient.

After a peak during the first 2 years of life, the incidence of acute myelogenous leukemia (AML) is low (five per million 5- to 9-year-olds per year in the United States) until after 9 years of age, when it slowly increases during adolescence and adulthood (to nine per million per year among 15- to 19-year-olds in the United States). Biological features including the prevalence of some genetic abnormalities appear to differ between pediatric and young adult AML. Treatment results in AML have improved during the last 30 years for all age groups; however, survival decreases with advancing age even when genetic risk factors are considered. In contrast to data about children and older adults, data on biological features and outcome are scarce in the adolescent and young adult (AYA) age group. This is partly due to the low number of patients of this age group participating in clinical trials. Differences in outcome for AYAs participating in pediatric trials compared to adult trials seem to be significant when different protocols are used, but minor with similar or identical protocols. As the needs of AYAs are different from those of young children and those of older adults, it is recommended to treat these patients in special units whenever possible.

Acute megakaryoblastic leukemia (AMKL) is a rare malignancy affecting megakaryocytes, platelet-producing cells that reside in the bone marrow. Children with Down syndrome (DS) are particularly prone to developing the disease and have a different age of onset, distinct genetic mutations, and better prognosis as compared with individuals without DS who develop the disease. Here, we discuss the contributions of chromosome 21 genes and other genetic mutations to AMKL, the clinical features of the disease, and the differing features of DS- and non-DS-AMKL. Further studies elucidating the role of chromosome 21 genes in this disease may aid our understanding of how they function in other types of leukemia, in which they are frequently mutated or differentially expressed. Although researchers have made many insights into understanding AMKL, much more remains to be learned about its underlying molecular mechanisms.

Non-down syndrome pediatric acute megakaryoblastic leukemia (AMKL, AML M7; FAB classification) is an aggressive disease, and classical protocols used for the treatment of AML in children with AMKL have proven unsuccessful. This is largely due to current poor understanding of the molecular mechanisms by which oncogenics fusions – which are frequently occurring in pediatric but not adult AMKL - drive leukemogenesis and sustain cellular self-renewal. We recently developed a mouse model of AKML as a tool to identify and characterize novel therapeutic targets. AMKL was generated with a penetrance of 50-70% and affected mice displayed striking splenomegaly. Our model recapitulates many features of the human disease which is characterized by an accumulation of megakaryocytic blasts in the peripheral blood and bone marrow as well as infiltration into extramedullary tissues such as the spleen and liver. Blasts also express one or more megakaryocyte markers (CD61, CD41, CD42, factor VIII), and the morphology of bone marrow and spleen cells is consistent with megakaryoblasts and megakaryocytes. In addition, increased proportions of stem and progenitor cells (c-Kit+/Sca1+), progenitors (c-Kit+/Sca1-) and immature megakaryoblasts (c-Kit+/CD41+) were detected in the spleen and bone marrow of leukemic mice. Finally, genome-wide RNA sequencing of leukemic blasts revealed a megakaryocytic signature (Itga2b, Itgb3, Gp1ba, Vwf), while gene set enrichment analysis uncovered, among other pathways, increased Notch and Wnt/s-catenin signalling with an altered metabolic gene expression profile. To conclude, by taking advantage of our mouse model, we are currently investigating signalling pathways that are altered in leukemic megakaryoblasts to ultimately uncover unique therapeutic vulnerabilities specific to pediatric non-DS AMKL.

Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.

Whole-exome sequencing identifies a novel somatic mutation in MMP8 associated with a t(1;22)-acute megakaryoblastic leukemia

Acute Megkaryoblastic Leukemia (AMKL) is a rare entity accounting for 1-2% of all Acute myeloblastic Leukemia (AML) (1). It is known as a distinct entity since a very long time but still difficult to diagnose correctly due to lack of distinct clinical features and morphological criteria. Here we present the clinical, morphological, cytochemical features of five cases of AMKL. Certain morphological features such as presence of abnormal platelet count, giant platelets and cytoplasmic blebbing in blasts were found to be important pointers towards the diagnosis.

Acute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development.

Acute megakaryoblastic leukemia (AMKL) accounts for 10% of childhood acute myeloid leukemia. Although AMKL patients with Down syndrome (DS-AMKL) have an excellent survival rate, patients with non-DS-AMKL experience poor outcomes. Despite therapeutic advances, including use of intensified treatment protocols, there is no consensus on the optimal treatment strategy for non-DS-AMKL. Some study groups recommend hematopoietic cell transplantation (HCT) for patients with non-DS-AMKL in the first complete remission (CR1); however, it is unclear whether HCT in CR1 improves outcomes of these patients. To assess outcomes and risk factors of HCT for patients with non-DS-AMKL, we retrospectively analyzed 203 patients with non-DS-AMKL (male, 95; female, 108) who underwent first HCT between 1986 and 2015 using data from the Japan Society for Hematopoietic Cell Transplantation. The median age at the time of HCT was 2 years (range, 0-16). Out of 203 patients, 12 had translocation t(1;22)(p13;q13), 18 had monosomy 7, 21 had complex karyotype, and 109 had other chromosomal abnormalities. Bone marrow transplantation (BMT) was performed for 114, cord blood transplantation (CBT) for 74, and peripheral blood stem cell transplantation (PBSCT) for 15 patients. Seventy-eight patients underwent HCT from related (HLA-matched, 43; HLA-mismatched, 35), 108 from unrelated (HLA-matched, 56; HLA-mismatched, 52), and 17 from autologous donors. Ninety-two patients underwent HCT in CR1, 21 in the second CR (CR2), and 90 in non-CR. Myeloablative conditioning (MAC) regimen was defined as the use of total body irradiation (TBI) ≥8 Gy, administration of busulfan >8 mg/kg or melphalan >140 mg/m2. All other regimens were included in reduced intensity conditioning (RIC) regimen. MAC regimen was used for 192 and RIC regimen for 11 patients. Median duration of follow-up after HCT was 713 days (range, 3-10008). Out of 203 patients, 183 (90%) exhibited engraftment. Sixteen out of 20 patients who did not exhibit engraftment underwent HCT in non-CR. In the entire cohort, 5-year overall survival (OS) and event free survival (EFS) rates were 43% and 38%, respectively. Seventy-two patients died of disease relapse and 42 died of transplantation-related complications. Five-year OS/EFS rates were not significantly different between BMT (48%/43%), CBT (35%/31%), and PBSCT (31%/27%) (p = 0.221/p = 0.087). In allogeneic HCT, 5-year OS/EFS rates were significantly lower in patients who underwent HCT from HLA-mismatched donors (27%/22%) than those from HLA-matched donors (52%/47%) (p < 0.001/p < 0.001). Five-year cumulative incidence (CI) of relapse after HLA-mismatched HCT (64%) was significantly higher than that after HLA-matched HCT (45%) (p = 0.006). Five-year OS/EFS rates were not significantly different between patients with translocation t(1;22)(p13;q13) (50%/42%), those with monosomy 7 (65%/59%), and those with complex karyotype (61%/52%) (p = 0.094/p = 0.111). Five-year OS/EFS rates of patients who underwent HCT in CR1 (72%/64%) were significantly higher than those of patients who underwent in CR2 (23%/13%) and non-CR (16%/16%) (p < 0.001/p < 0.001). Five-year CI of relapse was significantly lower in CR1 (28%) than in CR2 (73%) and non-CR (73%) (p < 0.001). Five-year non-relapse mortality was significantly higher in non-CR (27%) than in CR1 (15%) and CR2 (14%) (p = 0.022). Among the 92 patients who underwent HCT in CR1, 3-year CI of relapse in patients who received RIC (67%) was significantly higher than that in patients who received MAC regimen (25%) (p = 0.021). Five-year OS/EFS rates associated with HCT from HLA-mismatched (70%/63%) and HLA-matched donors (77%/59%) were not significantly different (p = 0.609/p = 0.761). In the multivariate analysis for 5-year OS, HCT in CR2 and non-CR was found to be a significant risk factor (hazard ratio, 5.16; 95% CI, 3.36-7.92; p < 0.001). In conclusion, multivariate analysis confirmed that HCT in CR1 was a good prognostic factor for 5-year OS. Risk stratification in patients with non-DS-AMKL should be established to determine the indication for HCT in CR1. Disclosures No relevant conflicts of interest to declare.

The cryptic fusion CBFA2T3-GLIS2 was initially described in infants with acute megakaryoblastic leukemia (AMKL) and is associated with adverse outcome. This fusion was subsequently shown to be present in other AML subsets with similar adverse associations. In order to define genes and pathways that are dysregulated and define therapeutic targets in these high-risk patients, diagnostic specimens from 1,049 children and young adults with AML underwent whole transcriptome sequencing (mRNA and miRNA Seq). Initial interrogation of the mRNA transcriptome identified 25 patients (2.4%) with the cryptic CBFA2T3-GLIS2 fusion. Patients with CBFA2T3-GLIS2 were significantly younger (median age 1.51 vs 10.2 years, p < 0.001), with 92% of fusion-positive patients < 3 years old, and an overall incidence of 9% in all patients < 3 years (23/255, p < 0.001). Half of all CBFA2T3-GLIS2 cases were classified as AMKL (13/25, 52%). Patients with CBFA2T3-GLIS2 lacked other risk features and the overwhelming majority (88%, p < 0.001) were classified as standard risk by conventional cytogenetic and molecular classifications. However, fusion-positive patients had an overall survival of 10% compared to 67.3% for the fusion-negative cohort (hazard ratio (HR) = 3.31, p < 0.001, Fig. 1A), with similar adverse event-free survival (HR = 2.48, p < 0.001). Genomic interrogation of fusion-positive patients by whole genome or targeted exome capture sequencing showed a lack of recurrent somatic mutations or other structural events in this patient population (Bolouri et al. 2017). Transcriptional profiling contrasting CBFA2T3-GLIS2 positive (N = 25) and negative cohorts (N = 1,024) identified several differentially enriched pathways, including TGFB/BMP (FDR < 0.001), WNT (FDR < 0.001), and Hedgehog signaling (HH, FDR = 0.002). Fusion-positive cases highly expressed GLIS2, the HH pathway genes, HHIP, PTCH1, and GLI1, as well as BMP2 (Fig. 1B). In addition, CASP1 and TRAIL-R2, which are involved in apoptosis, as well as the tumor suppressor GLIPR1 were significantly down-regulated. Integration of the miRNA with mRNA transcriptome demonstrated that expression of both CASP1 and TRAIL-R2 were significantly anti-correlated with and both are putative targets of the overexpressed miR-181b-5p; while downregulation of GLIPR1 was associated with an increase in miR-130a-3p, as well as being a predicted target (Fig. 1C). Gene-set enrichment analysis revealed highly up-regulated cell-adhesion and cell-surface markers, including extracellular matrix binding (FDR < 0.001), cell-adhesion molecule binding (FDR < 0.001), and integrin binding genes (FDR < 0.001). Investigating the most highly differentially expressed genes (90th percentile log2 fold-change (LFC), FDR < 0.001) with demonstrated localization to the plasma membrane and extracellular matrix, showed that CBFA2T3-GLIS2 exhibits a unique expression profile with a distinct set of dysregulated genes with plasma membrane localization (Fig. 1D). One of the most highly expressed cell-surface genes included NCAM1 (aka CD56; LFC = 6.97, FDR < 0.001). Given the extreme level of CD56 transcript expression in fusion-positive cases, we studied CD56 protein level using multidimensional flow cytometry (Hematologics, Seattle, WA). Surface CD56 in fusion-positive cases was massively over-expressed, with a median mean fluorescent intensity (MFI) of 1,960.0 versus 9.0 MFI for those without the fusion (p < 0.001, Fig. 1E). We evaluated CD56 as a potential therapeutic target using an anti-CD56 antibody-drug conjugate (m906-PBD-ADC) developed by NCI. Leukemic blasts or control lymphocytes from a patient with relapsed fusion-positive AML were incubated with varying doses of m906 and cytotoxicity was assessed after 72 hours (Notable Labs, Foster City, CA). The CD56-ADC exhibited a dose-dependent and CD56-specific toxicity on leukemic blasts (LIN-CD33+CD14-CD38-, p < 0.001, Fig. 1F), suggesting that CD56 might prove to be a therapeutic target in this high-risk cohort of patients. This study provides comprehensive transcriptome profiling of CBFA2T3-GLIS2 that defines a distinct and highly lethal subset of AML predominantly seen in infants. We also identify pathways and networks that further provide potential therapeutic options. Most importantly, CD56 may present the most immediately actionable target in this cohort of high risk patients. Figure 1. Figure 1. Disclosures Gekas: Notable Labs: Employment. Kolb:Roche- Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Eidenschink Brodersen:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

Acute megakaryoblastic leukemia (AMKL) constitutes ∼5%–15% of cases of non–Down syndrome AML in children, and in the majority of cases, chimeric oncogenes resulting from recurrent gene rearrangements are identified. Based on these rearrangements, several molecular subsets have been characterized providing important prognostic information. One such subset includes a group of patients with translocations involving the KMT2A gene, which has been associated with various fusion partners in patients with AMKL. Here we report the molecular findings of a 2-yr-old girl with AMKL and t(11;17)(q23;25) found to have a KMT2A–SEPT9 fusion identified through targeted RNA sequencing. A KMT2A–SEPT9 fusion in this subset of patients has not previously been reported.

Dear Editor We report an unusual case of myeloperoxidase (MPO)-positive acute megakaryoblastic leukemia (AMKL). The patient was a 63-yr-old woman referred to our hospital with anemia and dyspnea. General physical examination revealed no lymphadenopathy or hepatosplenomegaly. An initial complete blood count indicated white blood cell counts of 26.78×109/L, including 47% blasts, hemoglobin of 7.2 g/dL, and platelets of 519×109/L. A peripheral blood smear showed blasts with prominent nucleoli and blue cytoplasm. Some giant platelets were also observed. A bone marrow (BM) biopsy showed hypercellularity with no fibrosis, packed with blasts, and revealed an increase in megakaryocyte number. BM aspirates showed that blasts accounted for 70% of all nucleated elements. Most blasts had medium-to-large, finely chromatinated nuclei with distinct nucleoli. Some showed multiple clear cytoplasmic projections (Fig. 1A, B) or Auer rods. No dysplastic features of hematologic precursors were observed. Cytochemical stain showed some blasts were MPO-positive (Fig. 1C) but negative for periodic acid-Schiff (PAS) stain and non-specific esterase (NSE, α-naphthyl butyrate). Fig. 1 (A, B) Bone marrow aspirates showing megakaryoblasts with cytoplasmic blebs (red arrows) (Wright-Giemsa stain, ×1,000). (C) Bone marrow megakaryoblasts positive for myeloperoxidase stain (×1,000). Immunophenotyping showed a distinct population in the CD45 vs. SSC plot that was positive for CD7, CD11c, CD13, CD33, CD41a, CD117, cytoplasmic MPO, and HLA-DR and negative for CD2, CD3, CD5, CD10, CD14, CD19, CD20, CD22, CD34, CD56, CD64, and cytoplasmic CD79a. About 57.0% of blasts were CD41a-positive, and 57.7% were cytoplasmic MPO-positive. A blast population co-expressing CD41a and cytoplasmic-MPO was also observed (Fig. 2). Conventional karyotyping revealed a 46,XX karyotype. Molecular studies did not reveal any genetic abnormalities. As the patient refused treatment, only supportive care was provided, and the patient died from pneumonia four months later. Fig. 2 Flow cytometry of the bone marrow sample. (A) CD45/SSC dot plot with the blast population highlighted. (B) FSC/SSC plot of the sample. Blasts are positive for (C) CD41a (57.0%) and (D) cytoplasmic myeloperoxidase (MPO) (57.7%). (E) Back-gating of CD41a. ... Diagnosis of AMKL relies on multiple criteria including morphology, immunophenotyping, cytochemical stain, and ultrastructural studies [1]. The standard for diagnosis is demonstration of platelet glycoprotein (GP)-CD41 (GP IIb/IIIa) and/or CD61 (GP IIIa) by immunophenotyping. Cytoplasmic expression is more specific owing to possible contamination of platelets [2]. In cytochemical stain, megakaryoblasts are not reactive with MPO or Sudan Black B. Cells of the megakaryocytic lineage are usually positive in PAS stain owing to glycogen granules in the cytoplasm, and they are typically present in the periphery on stain, with prominent cytoplasmic blebs [3]. Reactivity with α-naphthyl acetate, but not with α-naphthyl butyrate, a different substrate for NSE, is characteristic of megakaryoblasts [4]. An ultrastructural platelet peroxidase reaction by cytochemistry, while difficult to perform, is also diagnostic for megakaryoblasts [1]. Megakaryoblasts also show acid phosphatase reactivity localizing to the Golgi [4]. There are no distinct chromosomal abnormalities, but inv(3)(q21q26.2) and t(3:3)(q21q26.2) are associated with megakaryocytic/megakaryoblastic differentiation [2]. Although MPO is an exclusive marker for leukemia of the megakaryoblastic lineage, few MPO-positive AMKL cases have been reported [5,6,7]. Park et al. [5] reported one Korean case in 1996, where blasts showed morphology typical of megakaryoblasts, and CD61 expression was confirmed by flow cytometry. The blasts also were weakly positive for MPO in cytochemical stain, and some Auer rods were observed. The sample was PAS-negative, but positive foci in the Golgi were observed with NSE stain (α-naphthyl acetate). Tallman et al. [6] reviewed 20 patients previously diagnosed as having AMKL using morphologic evidence and found two MPO-positive patients. One patient expressed factor VIII, a megakaryocytic lineage marker, and karyotyping revealed t(3:3)(q21;q26). The other patient had a normal karyotype and no evidence of the megakaryocytic lineage. In another study, AMKL was diagnosed by using another platelet marker, CD31, with MPO [7]. CD31, also known as PECAM-1 (platelet endothelial cell adhesion molecule 1), is a 130-kDa transmembrane glycoprotein on the surface of platelets, monocytes, macrophages, and neutrophils [8]. Immunolocalization of CD31 is limited to megakaryocytes in normal BM or in cases of myelofibrosis [9]. A failed BM aspiration prevented analysis by flow cytometry. The sample was negative for factor VIII on immunohistochemistry. However, the authors diagnosed the case as AMKL on the basis of typical blast morphology and positive immunohistochemical reactivity for CD31, CD43, and MPO in the BM biopsy [7]. In this case, blasts showed typical megakaryoblastic morphology with some Auer rods. Unlike most cases, the blasts co-expressed CD41a and cytoplasmic-MPO, were MPO-positive on cytochemical stain, and PAS-negative. According to the 2008 WHO classification, megakaryocytic lineage markers are not included in the diagnosis of mixed phenotype acute leukemia [10]. On the basis of previous reports, we conclude that this was an MPO-positive AMKL. AMKL is rare, and its diagnosis is not clearly defined compared with other types of AML. Morphologic evidence is still important, and comprehensive analyses are required when diagnosing acute leukemia, especially with a megakaryocytic lineage.

Dear Editor Down syndrome (DS) patients exhibit a 50-fold increase in acute leukemia incidence during the first five years of life, compared with those without DS. Trisomy 21, with or without other cytogenetic abnormalities, is reported in ~3% of AML and MDS cases; however, tetrasomy 21 and pentasomy 21 are unusual [1, 2]. Isodicentric chromosome 21 is a rare but recurrent cytogenetic abnormality in acute leukemia patients [3]; however, pentasomy 21q with two isochromosome 21s is very rare and has not yet been reported in pediatric DS patients with acute megakaryoblastic leukemia (AMKL). Here, we report a first case of 19-month-old girl diagnosed as having DS, who was later diagnosed as having AMKL with pentasomy 21. The patient was admitted at 14 months of age, because bicytopenia was detected during a routine visit. Complete blood count (CBC) revealed the followings: Hb 8.6 g/dL; white blood cell (WBC) count, 4.6×109/L; and platelet count, 27×109/L. Peripheral blood smear showed anisopoikilocytosis with some elliptocytes and dacrocytes. DS was diagnosed at birth, and the patient's kayotype was 46,XX,+21c,der(21;21)(q10;q10)c. Although an attempted bone marrow aspiration resulted in a "dry tap," a needle biopsy specimen showed moderate fibrosis and megakaryocytic hyperplasia (4.9/high power field) with dysmegakaryocytic features. She was diagnosed as having transient myeloproliferative disorder, received one unit of packed red blood cells, and was subsequently discharged. Five months later, a follow-up CBC revealed leukoerythroblastic reaction, and a physical examination revealed hepatosplenomegaly. Bone marrow study revealed persistent megakaryocytic hyperplasia and grade 3 fibrosis (Fig. 1). Cytogenetic analysis revealed a 49,XX,+11,+19,+21c,der(21;21)(q10;q10)c,+der (21;21)[19]/46,XX,+21c,der(21;21)c[9] karyotype, per the International System for Human Cytogenetic Nomenclature (ISCN) 2013. FISH confirmed pentasomy 21 (Fig. 2). JAK2 mutation and BCR/ABL fusion were not detected. She received conservative therapy, and one month later, the disease progressed such that the peripheral blood presented 15% blasts. The patient was transferred to another hospital where she was diagnosed as having DS-associated AML. The blast count was 30% on the bone marrow aspirate, and the immunophenotype of the neoplastic cells was characteristic of megakaryoblasts: CD13+, CD33+, CD34+, CD64w+, CD117+, CD41w+, CD7+, HLA-DR+, MPO-, and nTdT-. GATA1 mutation was not detected; however, an additional trisomy 11-associated MLL gain was detected by FISH. Calreticulin gene mutation test was not performed. The patient underwent cytarabine plus daunorubicin induction chemotherapy but failed to respond to the treatment and expired six months later. Fig. 1 (A) Bone marrow needle biopsy specimen, showing grade 3 fibrosis (H&E stain, ×100). (B) Reticulin staining showing grade 3 fibrosis (Reticulin stain, ×200). Fig. 2 Patient with Down syndrome exhibiting primary myelofibrosis with pentasomy 21. (A) Karyogram (Karyotype: 49,XX,+11,+19, +21c,der(21;21)(q10;q10)c, +der(21;21)[19]/46,XX,+21c,der(21;21)(q10;q10)c[9]. Arrows indicate abnormal chromosomes of 11, 19, and ... We identified pentasomy 21 with two isochromosome 21s in DS-associated transient abnormal myeloproliferative disorder, which was later transformed into AMKL. Transient myeloproliferative disorder occurs in ~10% of newborns with DS, and non-transient AML develops one to three years later in ~20-30% of such cases [4]. Fifty percent of cases of DS-associated acute leukemia are usually AMKL. In -70% of AMKL cases preceded by a pre-leukemic phase, which is characterized by thrombocytopenia and dysplastic changes, and often accompanied by marrow fibrosis, the disease may last several months or even years [5]. The present patient showed a similar disease course, transforming eventually into AMKL a few months later. Trisomy 19 that was observed in this patient might be due to some unknown role played by the leukemic transformation, as the most common abnormality in AMKL [6], and in the AKT2, cyclin E, and MLL2 genes in the 19q12 region, which have been implicated in solid tumors and are under investigation in hematologic malignancies [7]. Genes on chromosome 21 play an important role in leukemogenesis [8]. One gene --RUNX1(AML1) at 21q22-- plays a crucial role in hematopoiesis during embryonic development, and is frequently involved in chromosomal rearrangements observed in various myeloid malignancies [9]. Isochromosome 21q, formed as idic(21)(p10;p10), could result in an increased gene copy number on the entire long arm of chromosome 21 [10]. Furthermore, the duplication of idic(21)(p10;p10) gave rise to pentasomy 21q and to amplification of oncogenes at 21q, such as RUNX, ETS, and ERG, which may be involved in the development of leukemia. Moreover, the MLL gene gain due to trisomy 11, might be associated with the poor prognosis in this particular patient. To date, 16 cases of pentasomy 21 with two isochromosomes have been reported in hematologic malignancies, but the clinical features common to these cases are unknown [3]. This case shows us that hematologic surveillance of peripheral blood is needed in DS newborns, and close follow-up as well as optimal molecular and cytogenetic studies should be performed in cases with abnormal peripheral blood findings. Clarification of the mechanism underlying myelofibrosis and leukemogenesis in pentasomy 21q with two isochromosomes, in myeloid leukemia associated with DS, requires further studies.

WOS: 000219054200011, Down Syndrome (DS) is an important genetic disease resulting from partial or total trisomy of chromosome 21 and characterized by dysmorphic facial features, intellectual disabilities and multiple congenital anomalies. Children with DS are at increased risk of developing leukemia. Specifically, 3-10% of newborns with DS are diagnosed with transient myeloproliferative disease, and children with DS are 500 times more likely to develop acute megakaryoblastic leukemia (AMKL), and 20 times more likely to develop acute lymphoblastic leukemia (ALL) than children without DS. In this study, we report two children with DS presented with transient myeloproliferative disease.