Down Syndrome Acute Megakaryoblastic Leukemia Research Articles

Genomic Landscape of Pediatric Non-Down's Syndrome Acute Megakaryoblastic Leukemia in China

Acute megakaryoblastic leukemia (AMKL) accounts for 4% to 15% of newly diagnosed pediatric acute myeloid leukemia (AML), and different from Down's syndrome AMKL (DS-AMKL) and non-AMKL, clinical outcomes of non-DS-AMKL are characterized by an inferior prognosis. Although comprehensive genomic studies have revealed key genomic aberrations of non-DS-AMKL in Western cohort, there is still no reports about Chinese patients. Here we report a comprehensive transcriptomic study on 100 Chinese pediatric non-DS-AMKL cases through 2009 to 2022 in Shanghai Children's Medical Center. A total of 133 rearrangements were detected involving 78 patients, including previously reported recurrent fusion events which defining subgroups of AMKL, namely CBFA2T3-GLIS2 (n=20, 20%), NUP98-KDM5A (n=16,16%), rearrangements of HOX cluster genes (HOXr, n=11, 11%), RBM15-MKL1 (n=5, 5%) and MLLr (n=3, 3%). Two new recurrent gene translocations, XPO1-TRNC18 and STAG2 rearrangements (STAG2r) were detected in 5 (5%) and 11 (11%) cases, respectively. Further unsupervised clustering analysis was done with the most variant coding genes defined by standard deviation, the results showed that cases carrying STAG2r and XPO1-TNRC18 fusion exhibited distinguishable gene expression pattern from other subgroups and were clustered together respectively. It suggested these two rearrangements as potential driver genomic alterations in AMKL. By introducing XPO1-TNRC18 fusion and three STAG2 aberrations (STAG2 knock-out, STAG2-GNA12 and STAG2-OCRL) into 32D cell line, we proved these gene alterations promoted cell proliferation and colonies formation, suggesting potential function in tumor transformation. Based on these evidences, we defined XPO1-TNRC18 and STAG2r as two new subgroups of AMKL. We identified 222 nonsynonymous sequency mutations affecting 137 genes totally, including 202 single-nucleotide variants and 20 insertion/deletion, with a median of two mutations for per case (range 0-6). Among driver abnormalities, 7 aberrations occurred in more than 3% of cases in current cohort, including JAK2 (n=15, 15%), MPL (n=13, 13%), PTPN11 (n=7, 7%), NRAS (n=5, 5%), GATA1 (n=4, 4%), CTCF (n=4, 4%) and JAK3 (n=3, 3%). All the mutations could be clustered into 8 pathways, and the most frequently mutated pathways were JAK signaling (30%), transcription regulation (29%) and RAS signaling (14%). Mutation frequency varied across different subgroups. For example, JAK/STAT lesions were enriched in STAG2r subgroup (p<0.05). Distinct genomic aberration profile was revealed while comparing results of this non-DS-AMKL cohort to the mutation profile of non-AMKL cohort which we have published before. CBFA2T3-GLIS2, NUP98-KDM5A, STAG2r, XPO1-TNRC18 and RBM15-MKL1 subgroups were merely detected in AMKL, while driver aberrations like FLT3-ITD, CBFB-MYH1 and so on were not detected in AMKL. For driver mutations, JAK2 (15% vs 3.7%, p<0.0001), MPL (13% vs 0%, p<0.0001 ) and CTCF (4% vs 0%, p=0.007) were detected more frequently in non-DS-AML, while NRAS (5% vs 18.8%, p<0.001), KRAS (2% vs 10.6%, p=0.008), KIT (1% vs 21.6%, p<0.001), CSF3R (1% vs 6.1%, p=0.047), FLT3 (0% vs 22.5%, p<0.001), CEBPA (0% vs 9.8%, p<0.001), AXSL2 (0% vs 8.6%, p<0.001), GATA2 (0% vs 6.1%, p=0.007) and DHX15 (0% vs 4.5%, p=0.038) occurred less frequently in non-DS-AMKL cohort. Survival analysis revealed association between key aberrations and clinical outcomes. Carrying CBFA2T3-GLIS2 (p=0.018), NUP98-KDM5A (p<0.001) and FUS-ERG (p=0.018) was associated with unfavorable prognosis. Driver mutations in JAK2 (p=0.03) and RAS pathway (p=0.01) were associated with shorter overall survival. Among the two new subgroups, the new XPO1-TNRC18 subgroup demonstrated similar clinical outcomes to MLLr cases, with 5-year EFS and OS of 50%, and STAG2r was characterized by a favorable clinical outcome, with 5-year EFS and OS over 75%. As the first comprehensive genomic landscape of Chinese pediatric non-DS-AMKL, our results defined two new subgroups with distinct expression patterns and prognosis characteristics. We further revealed a distinct mutation profile between AMKL and non-AMKL and shed light on association between driver aberrations and clinical outcomes. These findings revealed non-DS-AMKL as a heterogenous disease, and suggested the importance of exploration of risk-stratification and targeted therapy in different mutations driven cases.

Neonatal and adult megakaryopoiesis

It has become increasingly clear that there are substantial biological differences between fetal/neonatal and adult megakaryopoiesis. Over the last 18 months, studies challenged the paradigm that neonatal megakaryocytes are immature and revealed a developmentally unique uncoupling of proliferation, polyploidization, and cytoplasmic maturation. Several studies also described substantial molecular differences between fetal/neonatal and adult megakaryocytes involving transcription factors, signaling pathways, cytokine receptors, and microRNAs. This review will summarize our current knowledge on the developmental differences between fetal/neonatal and adult megakaryocytes, and recent advances in the underlying molecular mechanisms, including differences in transcription factors, in the response to thrombopoietin (Tpo), and newly described developmentally regulated signaling pathways. We will also discuss the implications of these findings on the way megakaryocytes interact with the environment, the response of neonates to thrombocytopenia, and the pathogenesis of Down syndrome-transient myeloproliferative disorder (TMD) and Down syndrome-acute megakaryoblastic leukemia (DS-AMKL). A better characterization of the molecular differences between fetal/neonatal and adult megakaryocytes is critical to elucidating the pathogenesis of a group of disorders that selectively affect fetal/neonatal megakaryocyte progenitors, including the thrombocytopenia-absent radius (TAR) syndrome, Down syndrome-TMD or Down syndrome-AMKL, and the delayed platelet engraftment following cord blood transplantation.

Ikaros Is a GATA Switch Target Gene That Restrains Megakaryopoiesis In Part by Blocking Notch Signaling.

AbstractAbstract 1602The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures:No relevant conflicts of interest to declare.

Developmental stage-specific interplay of GATA1 and IGF signaling in fetal megakaryopoiesis and leukemogenesis

Oncogene-mediated transformation of hematopoietic cells has been studied extensively, but little is known about the molecular basis for restriction of oncogenes to certain target cells and differential cellular context-specific requirements for oncogenic transformation between infant and adult leukemias. Understanding cell type-specific interplay of signaling pathways and oncogenes is essential for developing targeted cancer therapies. Here, we address the vexing issue of how developmental restriction is achieved in Down syndrome acute megakaryoblastic leukemia (DS-AMKL), characterized by the triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21. We demonstrate overactivity of insulin-like growth factor (IGF) signaling in authentic human DS-AMKL and in a DS-AMKL mouse model generated through retroviral insertional mutagenesis. Fetal but not adult megakaryocytic progenitors are dependent on this pathway. GATA1 restricts IGF-mediated activation of the E2F transcription network to coordinate proliferation and differentiation. Failure of a direct GATA1-E2F interaction in mutated GATA1s converges with overactive IGF signaling to promote cellular transformation of DS fetal progenitors, revealing a complex, fetal stage-specific regulatory network. Our study underscores context-dependent requirements during oncogenesis, and explains resistance to transformation of ostensibly similar adult progenitors.

Developmental Stage-Specific Interplay Between GATA1 and IGF Signaling in Fetal Hematopoiesis and Leukemogenesis.

Abstract 386Oncogene-mediated transformation of hematopoietic cells has been extensively studied, but little is known about the molecular basis for restriction of oncogenes to certain target cells and differential cellular context-specific requirements for oncogenic transformation between infant and adult leukemias. Understanding cell type-specific interplay between signaling pathways and oncogenes is essential for developing targeted cancer therapies.To study how developmental restriction is achieved in Down syndrome acute megakaryoblastic leukemia (DS-AMKL), characterized by a triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21, we generated a DS-AMKL mouse model (G1s-mAMKL) through retroviral insertional mutagenesis. Using cross-species integrated genomic analysis, we demonstrate overactivity of insulin-like growth factor (IGF) signaling in murine and authentic human DS-AMKL. ShRNA mediated IGF1R knockdown in DS-AMKL cell lines (CMK, CMY) led to a dramatic reduction of transduced cells, whereas no substantial effect was observed in “control” cell lines, including K562, CHRF288-11, and M-07. Pharmacological activation and repression of IGF1R signaling followed by in vitro culture, CFU assays and intracellular phosphoprotein staining showed that in both murine and primary human AMKL cells expressing GATA1s, the IGF/IGF1R/mTOR pathway is highly activated and is the major mitogenic pathway for growth. We observed a significant delay of leukemia onset upon IGF1R knockdown after transplantation of G1s-mAMKL cells into RAG2-/- recipients. Gene set enrichment analysis revealed that the mitogenic activity of IGF1R is exerted at the transcriptional level by activation of E2F target genes. Using a genetic approach we demonstrate that fetal, but not adult, hematopoietic progenitor cells are dependent on IGF1R signaling, showing a developmental difference in the activity of this pathway. Gata1 restricts IGF-mediated activation of the E2F transcription network to coordinate proliferation and differentiation. In contrast, mutated Gata1s is insufficient to control the IGF1R induced proliferation in fetal megakaryocytic progenitors (MPs), which were stimulated or retrovirally transduced with IGFR ligands (IGF1 and IGF2). The hyperproliferative phenotype of Gata1s fetal MPs could be rescued by shRNA mediated repression of global E2F transcription activity.Thus, by focusing on DS-AMKL, which is restricted to neonates and infants, we have discovered fetal stage-specific interplay between insulin-like growth factor (IGF) signaling and lineage-determining transcription factor, GATA1. Acquired mutation in GATA1 perturbs the regulatory function of GATA1 on IGF pathway. Together with overactive IGF signaling, mutant GATA1 leads to malignant transformation of DS fetal progenitors, underscoring context-dependent requirements during oncogenesis and explaining resistance to transformation of ostensibly similar adult progenitors. Disclosures:No relevant conflicts of interest to declare.

GATA-2 Controls Proliferation of Megakaryocyte Progenitors and Its Expression Contributes to Human DS-AMKL.

GATA2 is an essential transcription factor that regulates multiple aspects of hematopoiesis. Mutations in GATA2 are associated with blast crisis phase of chronic myelogenous leukemia (CML) while overexpression of GATA2 is a hallmark of Down syndrome acute megakaryoblastic leukemia (DS-AMKL), a malignancy that is defined by the combination of trisomic chromosome 21 and a GATA1 mutation. Here, we show thatGATA2 is essential for megakaryocyte development from both wild-type and GATA-1mutant hematopoietic progenitors. Furthermore, we reveal that GATA2 is indispensable for cell cycle progression and that its expression cooperates with activated JAK3 to promote TPO-independent expansion of megakaryocytes. Genome-wide microarray analysis revealed that GATA2 regulates a wide set of genes including cell cycle regulators and lineage-specific genes that are essential for terminal differentiation of megakaryocytes. Of note, a subset of genes within the GATA2 signature is contained within human DS-AMKL patient specimens. These observations implicate overexpression of GATA2 in the aberrant gene expression and proliferation of AMKL. Taken together, our data demonstrate thatGATA2 is a critical regulator of normal and malignant megakaryopoiesis.

Highly penetrant myeloproliferative disease in the Ts65Dn mouse model of Down syndrome

Children with Down syndrome (DS) display macrocytosis, thrombocytosis, and a 500-fold increased risk of developing megakaryocytic leukemia; however, the specific effects of trisomy 21 on hematopoiesis remain poorly defined. To study this question, we analyzed blood cell development in the Ts65Dn mouse model of DS. Ts65Dn mice are trisomic for 104 orthologs of Hsa21 genes and are the most widely used mouse model for DS. We discovered that Ts65Dn mice display persistent macrocytosis and develop a myeloproliferative disease (MPD) characterized by profound thrombocytosis, megakaryocyte hyperplasia, dysplastic megakaryocyte morphology, and myelofibrosis. In addition, these animals bear distorted hematopoietic stem and myeloid progenitor cell compartments compared with euploid control littermates. Of the 104 trisomic genes in Ts65Dn mice, Aml1/Runx1 attracts considerable attention as a candidate oncogene in DS-acute megakaryoblastic leukemia (DS-AMKL). To determine whether trisomy for Aml1/Runx1 is essential for MPD, we restored disomy at the Aml1/Runx1 locus in the Ts65Dn strain. Surprisingly, trisomy for Aml1/Runx1 is not required for megakaryocyte hyperplasia and myelofibrosis, suggesting that trisomy for one or more of the remaining genes can promote this disease. Our studies demonstrate the potential of DS mouse models to improve our understanding of chromosome 21 gene dosage effects in human hematologic malignancies.

Leukemia-Initiating Cells in Acute Megakaryoblastic Leukemia and Transient Leukemia of Down Syndrome.

Background. Children with Down syndrome (DS) have a 500-fold greater risk of developing Acute Megakaryoblastic Leukemia (AMKL) than the general population. In addition, approximately one out of ten newborns with DS has circulating blasts in the blood, a condition termed Transient Leukemia (TL). Unlike AMKL, TL resolves spontaneously within three months but in about 20% of cases is followed by AMKL later in life. Both the blasts of TL and AMKL of DS (DS-AMKL) show megakaryocytic differentiation and both harbor somatic mutations of GATA1, resulting in the expression of the N-terminally truncated mutant protein GATA1s. We hypothesize that the difference between the reversible and irreversible phenotype of TL and AMKL in DS, respectively, is due to a functional difference of the leukemia-initiating cells in both conditions.Methods. To characterize the leukemia-initiating cells we established experimental models of AMKL and TL of DS by transplanting cryopreserved primary human cells into NOD/SCID mice. Cell doses ranging from 0.5 to 20x106 were injected intrafemorally into 8-week-old irradiated recipients, which had also been treated with anti-NK cell antibody (anti-CD122). Human hematopoietic growth factors (stem cell factor, interleukin-3 and thrombopoietin) were administered intraperitoneally during the first two weeks following transplantation. Phenotypic analysis using standard cytological, histological and flowcytometric methods was carried out approximately 8 weeks after transplantation.Results. Recipients transplanted with 2 (of a total of 7) AMKL samples showed engraftment with 32% (range 26–44%; n=3) and 73% (range 16–95%; n=8) human cells at the site of the original cell injection (right femur) and 15% (n=3) and 38% (n=8) at distant medullary sites. The engrafted human cells were trisomic for human chromosome 21, expressed the megakaryocytic marker CD61 and, compared with the transplanted primary AMKL cell population, harbored the concordant GATA1 mutation. Bone marrow biopsy revealed increased reticulin fibres 8 weeks after transplantation of AMKL cells. In our experiments, DS-AMKL-initiating cells were found to occur within a broad range of frequency (18x10−4 to 20x10−6) but were not defined by their expression of CD34 and/or CD38. In keeping with the self-renewal capacity of leukemia-initiating cells in human acute myeloid leukemia, DS-AMKL cells collected from the right femur (site of initial cell injection) and from distant bone marrow sites of primary recipients were able to engraft secondary recipients. In contrast, only one of five primary TL cell samples showed engraftment within the right femur (21%, range 3–81%; n=5), the site at which TL cells had been injected 8 weeks earlier. No TL cells or engrafted human cells were detected in any distal bone marrow site or extramedullary compartment such as the spleen.Conclusion. Our results indicate that the function of leukemia-initiating cells in DS-AMKL but not TL parallels those of non-DS human acute myeloid leukemia. Our model provides an experimental approach to distinguish the role of the cellular target vs. mutations cooperating with GATA1 mutations in the development of AMKL and TL in DS.

Relapsed acute megakaryoblastic leukemia in Down syndrome (AMKL-DS) may be cured with chemotherapy alone, without stem cell transplantation (HSCT)

17543 Background: Children with recurrent AMKL -DS have an extremely poor prognosis without HSCT. Methods: We report a case of a relapsed AMKL-DS cured with intensive chemotherapy alone, without HSCT. Results: A 19 month old boy with Down syndrome, who had transient leukemia as a newborn, developed AMKL-DS (GATA1 positive) and was treated with four cycles of CI-TAD: continuous infusion of cytarabine and daunorubicin, and oral thioguanine for four days, and intrathecal cytarabine; followed by two cycles of high dose cytarabine plus L-Asparaginase; and three weekly doses of intrathecal cytarabine. Although he achieved remission at the completion of first cycle of the induction chemotherapy, he relapsed within 6 weeks after the completion of therapy. He then received the following chemotherapy over the next six months: Cycle 1 and 2. High dose Ara-C ( 33. 3 mg/kg q 12 hours x 8 doses) on days 0 to 3 plus mitoxantrone 0. 4 mg/kg/day x 4 on days 3 to 6 Cycle 3. High dose Ara-C 33. 3 mg/kg q 12 hours x 8 doses Cycle 4 and 5 (FLAG): Fludarabine 0. 8 mg/kg/day x 4 plus Ara-C 67 mg/kg/d x 4 and G-CSF 10 mcg/kg/day x 4 all on days 0 to 3. He achieved complete remission after the 1st cycle of chemotherapy, and underwent further cycles of chemotherapy while waiting for HSCT from an unrelated donor. But after the 5th cycle of chemotherapy, he developed a pulmonary lesion of fungal etiology for which he received three months of therapy with liposomal amphotericin. Because of a persistent lung lesion on imaging, he was considered ineligible for HSCT, and no further therapy was given. His bone marrow aspirate after the 3rd cycle of therapy was negative for GATA1. He has been disease-free for 59 months after the completion of his last cycle of chemotherapy, and he is currently in good health without any evidence of pulmonary or cardiac dysfunction. Conclusions: Intensive chemotherapy alone, without HSCT, may be curative for relapsed AMKL- DS. No significant financial relationships to disclose.

CD36 (thrombospondin receptor) expression in childhood acute megakaryoblastic leukemia: In vitro drug sensitivity and outcome

The outcome for children with acute megakaryoblastic leukemia (AMKL) remains poor, except for cases associated with Down syndrome (DS). This study compared immunophenotypic and drug sensitivity patterns of childhood AMKL cases with or without DS. All DS-AMKL cases showed high expression of CD36 and were very sensitive to cytarabine and daunorubicin in vitro. In children without DS, high expression of CD36 on AMKL blasts identified a sub-group with immunophenotypic and drug sensitivity patterns similar to that of DS AMKL. Among non-DS AMKL patients, CD36-high cases had a superior outcome compared with CD36-negative cases. CD36 expression in acute myeloid leukemia cases other than AMKL was not associated with increased in vitro drug sensitivity. CD36 expression in AMKL may be an indicator of megakaryoblast maturation and chemotherapy sensitivity.

Chromosome copy number and leukemia–Lessons from Down's syndrome

Cancer is a genetic disease of the somatic cell. Virtually all cancers, including leukemias, harbor multiple acquired genetic abnormalities. These abnormalities are commonly manifested by growth chromosomal abnormalities. These abnormalities can be either structural or numerical. Aneuploidy, change in the normal chromosomal copy number, is one of the frequent abnormalities in cancer. For example high hyperdiploid acute lymphoblastic leukemia (ALL) is the most common leukemia observed in young children. The leukemic cells contain extra copies of multiple chromosomes. The chromosomes involved are non-random. Chromosomes 21, X, and 6 are almost always involved, as are chromosomes 4,10,17 and occasionally 18. This specific leukemia syndrome suggest a causative role for the chromosomal numerical aberrations. Yet it the mechanisms by which acquired trisomies contribute to leukemia is unknown. Children with Down Syndrome (DS) have a 20-fold risk and about 600 risk for acute metakaryocytic leukemia. This strong association between germline trisomy 21 and leukemia strongly suggest that trisomy 21 is leukemogenic. Elucidation of the pathogenesis of the leukemias of DS is likely to reveal the mechanisms by which aneuploidy contribute to leukemia and cancer. Approximately 10% of children with DS are born with a megakaryocytosis syndrome commonly called ‘‘transient myeloproliferative disorder’’ (TMD) or ‘‘transient abnormal myelopoiesis’’ (TAM) or ‘‘transient leukemia’’. As suggested by the different names the disorder is usually transient and resolves spontaneously within up to several months. The term ‘‘transient leukemia’’ is occasionally used because the blasts in this transient disorder are mostly of clonal megakaryoblastic origin. The biological mechanism of the spontaneous resolution is unclear. About 1 /2% of DS patient will develop, however, full blown malignant acute megakaryoblastic leukemia (AMKL) during their first four years of life that will not regress without chemotherapy. In fact, the risk of AMKL is about 600 times higher in children with DS. The factor(s) underlying the transformation from ‘‘benign’’ TMD into ‘‘malignant’’ AMKL are largely unknown. Thus the megakaryocytic malignancies of DS provide a ‘‘natural genetic model’’ of multistep lineage specific leukemogenesis. Both the congenital disorder and the full blown AMKL are characterized by differentiation arrest of the megakaryocytic lineage. The marrow and the liver of infants with TMD contain a large number of dysplastic micromegakaryocytes. Identical cells are observed in the early stages of the AMKL of DS. Both disorders are also characterized by thrombocytopenia, indicative of poor platelets formation by the dysplastic megakaryocytes. The peculiar association between DS and childhood megakaryoblastic disorders has led to intensive search for gene or genes on chromosome 21 than may cause the differentiation arrest and initiate the leukemia. A surprising twitch in this story came with the discovery that a gene on chromosome X , GATA1, was mutated in the megakaryoblasts from all the patients with DS and either TMD or AMKL. The mutations were also found in fetal liver of aborted DS fetuses. The mutations are acquired as they are not found in remission samples, and are specific to the megakaryoblastic disorders associated with trisomy 21. No GATA1 mutations were found in other AMKLs, in sporadic acute myeloid leukemia (AML) or in the acute lymphoblastic leukemia (ALL) associated with DS. Thus a clear model for multistep

Natural history ofGATA1mutations in Down syndrome

AbstractAcquired mutations in megakaryocyte transcription factor GATA1 have recently been reported in Down syndrome (DS), transient myeloproliferative disorder (TMD), and acute megakaryoblastic leukemia (AMKL). To provide novel insight into GATA1 mutations in DS, genomic DNA was assayed from 12 AMKL and 4 TMD cases (including neonatal, prediagnosis samples in 4 of 16), neonatal blood spots from 21 DS children without clinically evident TMD or AMKL, and 62 non-DS cord blood samples, using techniques not previously employed with such samples. GATA1 mutations were present in all TMD and AMKL cases and at birth in 3 of 4 children without known clinical TMD, who later developed AMKL. They were present at birth in 2 of 21 DS neonates, who have not yet, but could still, develop AMKL (now 26 and 31 months). GATA1 mutations were not detected in 62 non-DS cord blood samples. In 4 AMKL patients multiple independent GATA1 mutations were observed. These data show GATA1 mutations occur in utero in most DS TMD and AMKL, that they may occur without clinical signs of disease, and that multiple separate GATA1 mutant clones can occur in an individual. The findings have implications for pathogenesis of DS TMD and AMKL and highlight parallels between DS AMKL and other childhood leukemias.

Interphase cytogenetic analysis of clonality in peripheral blood cells from a patient with Down syndrome and acute megakaryoblastic leukemia

A combination of fluorescence-activated cell sorting and interphase fluorescence in situ hybridization (FISH) techniques was used to detect a clonal chromosomal marker in blasts, granulocytes, and T and B lymphocytes of the peripheral blood from a patient with Down syndrome and acute megakaryoblastic leukemia (AMKL) associated with trisomy 8 as a karyotypic abnormality. Immunophenotypic studies with flow cytometry showed two populations of leukemic blasts distinguished by their expression of the CD34 antigen. Interphase FISH studies revealed clonal trisomy 8 FISH signals in almost all blast cells, regardless of CD34 expression, as well as in a small subpopulation of granulocytes. Normal chromosome 8 signal patterns were detected in T and B cells and in a great majority of granulocytes. The present study provides evidence for the clonal involvement of leukemic blasts in AMKL of Down syndrome, indicating that a trisomy 8 abnormality may be a primary event in leukemogenesis. The transformation occurs in progenitor cells with limited myeloid differentiation and without involvement of lymphoid lineage cells.

Mutations in GATA1 in both transient myeloproliferative disorder and acute megakaryoblastic leukemia of Down syndrome

Mutations in transcription factors often contribute to human leukemias by providing a block to normal differentiation. To determine whether mutations in the hematopoietic transcription factor GATA1 are associated with leukemia, we assayed for alterations in the GATA1 gene in bone marrow samples from patients with various subtypes of acute leukemia. Here we summarize our findings that GATA1 is mutated in the leukemic blasts of patients with Down syndrome acute megakaryoblastic leukemia (DS-AMKL). We did not find mutations in GATA1 in leukemic cells of DS patients with other types of acute leukemia, or in other patients with AMKL who did not have DS. Furthermore, we did not detect GATA1 mutations in DNAs from over 75 other patients with acute leukemia or from 21 healthy individuals. Since the GATA1 mutations were restricted to DS-AMKL, we also investigated whether GATA1 was altered in the “preleukemia” of DS, transient myeloproliferative disorder (TMD). TMD is a common myeloid disorder that affects 10% of DS newborns and evolves to AMKL in nearly 30% patients. We detected GATA1 mutations in TMD blasts from every infant examined. Together, these results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD and DS-AMKL, and that mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis. We hypothesize that disruption of normal GATA-1 function is an essential step in the initiation of megakaryoblastic leukemia in DS.

GATA1 mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome

AbstractChildren with constitutional trisomy 21 (Down syndrome) have an approximately 500-fold increased risk of developing acute megakaryoblastic leukemia (AMKL), a form of acute myeloid leukemia. Unique to newborn infants with Down syndrome is a transient leukemia (TL), also referred to as transient myeloproliferative syndrome, that undergoes spontaneous remission in the majority of cases but in approximately 20% is followed by AMKL later in life. Recently mutations of the gene encoding the hematopoietic transcription factor GATA1 were shown to be specific for AMKL of Down syndrome. Here, we demonstrate that GATA1 mutations are present in blasts of TL and show the identical GATA1 mutation in sequential samples collected from a patient during TL and subsequent AMKL. These findings suggest a model of malignant transformation in Down syndrome AMKL in which GATA1 mutations are an early event and AMKL arises from latent TL clones following initial apparent remission.

Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome.

Children with Down syndrome have a 10-20-fold elevated risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL). While a subset of pediatric AMKLs is associated with the 1;22 translocation and expression of a mutant fusion protein, the genetic alterations that promote Down syndrome-related AMKL (DS-AMKL) have remained elusive. Here we show that leukemic cells from every individual with DS-AMKL that we examined contain mutations in GATA1, encoding the essential hematopoietic transcription factor GATA1 (GATA binding protein 1 or globin transcription factor 1). Each mutation results in the introduction of a premature stop codon in the gene sequence that encodes the amino-terminal activation domain. These mutations prevent synthesis of full-length GATA1, but not synthesis of a shorter variant that is initiated downstream. We show that the shorter GATA1 protein, which lacks the N-terminal activation domain, binds DNA and interacts with its essential cofactor Friend of GATA1 (FOG1; encoded by ZFPM1) to the same extent as does full-length GATA1, but has a reduced transactivation potential. Although some reports suggest that the activation domain is dispensable in cell-culture models of hematopoiesis, one study has shown that it is required for normal development in vivo. Together, these findings indicate that loss of wildtype GATA1 constitutes one step in the pathogenesis of AMKL in Down syndrome.

Leukemic blasts with markers of four cell lineages in Down's syndrome ("megakaryoblastic leukemia").

Among 16 patients with Down's syndrome (DS) and acute leukemia admitted to our department during a ten year period, 6 were diagnosed as acute megakaryoblastic leukemia (AMkL). The diagnosis was based on clinical and hematologic criteria, confirmed in three patients with the use of monoclonal antibodies (MoAb) specific for megakaryocytic antigens. In these three, and in a fourth patient, the leukemic blasts were positive for other myeloid, lymphoid and erythroid markers in MoAb testing. We suggest that AMkL in DS is a mixed lineage leukemia with blasts presenting a variety of cell surface antigens, indicating origin from an early progenitor cell with the capability of megakaryocytic differentiation. Of the 6 patients with AMkL, 4 treated with standard AML protocols are in complete continuing remission (CCR) with observation periods from 57+ to 148+ months.