Down syndrome (DS) children have a high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. At least 2 in utero genetic events are required, although not sufficient, for DS-AMKL: trisomy 21 (T21) and N-terminal-truncating GATA1 mutations. To investigate the role of T21 in DS-AMKL, we compared second trimester hemopoiesis in DS without GATA1 mutations to gestation-matched normal controls. In all DS fetal livers (FLs), but not marrows, megakaryocyteerythroid progenitor frequency was increased (55.9% ؎ 4% vs 17.1% ؎ 3%, CD34 ؉ CD38 ؉ cells; P < .001) with common myeloid progenitors (19.6% ؎ 2% vs 44.0% ؎ 7%) and granulocyte-monocyte (GM) progenitors (15.8% ؎ 4% vs 34.5% ؎ 9%) commensurately reduced. Clonogenicity of DS-FL versus normal FL CD34 ؉ cells was markedly increased (78% ؎ 7% vs 15% ؎ 3%) affecting megakaryocyte-erythroid (ϳ 7-fold higher) and GM and colony-forming unit-granulocyte, erythrocyte macrophage, megakaryocyte (CFU-GEMM) progenitors. Replating efficiency of CFU-GEMM was also markedly increased. These data indicate that T21 itself profoundly disturbs FL hemopoiesis and they provide a testable hypothesis to explain the increased susceptibility to IntroductionChildren with Down syndrome (DS) have a uniquely high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. [1][2][3] The leukemic cells acquire mutations in utero in the critical megakaryocyte transcription factor GATA1. [3][4][5][6][7][8][9][10] In many DS children, the first manifestation is neonatal transient myeloproliferative disorder (TMD), 1,2 which evolves to AMKL in 20% to 30% of infants. 3 TMD and AMKL represent 2 distinct steps in the pathogenesis of DS-AMKL. However, an additional necessary leukemogenic event (most probably the initiating event) is trisomy 21 (T21). T21 is essential for GATA1-associated TMD and AMKL 5,11,12 ; truncating GATA1 mutations in the absence of T21 are not leukemogenic. 13 GATA1 mutations also occur at high frequency in T21 (ϳ 5% of all DS neonates 14 ) and 25% of DS-associated AMKL patients have multiple, independent clones with GATA1 mutations. 6 These data suggest that GATA1 mutations and T21 specifically synergize to generate preleukemic TMD. The cellular and molecular mechanisms by which this occur are unclear.That a putative leukemia-initiating cell is present in T21 fetal liver (FL) is suggested both by the natural history of TMD (origin in utero, frequent liver involvement, and spontaneous resolution as FL hemopoiesis ceases 11,15 ) and by analysis of germline N-terminal mutant Gata1 phenotypes in mouse and humans. 16 To investigate the impact of T21, independent of GATA1 mutations, on human hemopoiesis, we studied myeloid progenitors from second trimester T21 FL and bone marrow. MethodsSecond-trimester FLs and marrow collected during elective surgical termination of pregnancy were processed immediately as previously described. 17 The study was approved by Hammersmith and Queen Charlotte's Hospitals Research Ethics Committee; written informed consent was obtain...

Children with Down syndrome (DS) have a uniquely high frequency of acute megakaryoblastic leukemia (AMKL)- ~500-fold increased compared to children without trisomy 21 (T21). At least two genetic events are required but are not sufficient for DS-AMKL: T21 and N-terminal truncating mutations in the key megakaryocytic transcription factor GATA1. This tight association of T21 with GATA1 mutations and the development of AMKL in a narrow temporal window (fetal life-5yrs) makes DS-AMKL a highly informative model of multi-hit leukemogenesis in which the first steps occur in utero. However, the individual contributions of T21 and mutant GATA1 in the leukemogenesis are unclear. To specifically investigate the role of T21 in DS-AMKL and why leukemia-initiation is confined to fetal (or early post-natal) life we have studied fetal hemopoiesis in DS during the second and third trimester in 16 fetuses (gestational age 15–37 weeks) where an antenatal diagnosis of DS with T21 was made by amniotic fluid fetal cell karyotyping. Samples of fetal blood (n=13), fetal liver (n=9) and fetal bone marrow (n=8) were screened for mutations in the GATA1 gene genomic DNA by DHPLC or direct sequencing (sensitivity of detecting a GATA1 mutation is 1–5% by DHPLC). No GATA1 mutations were detected. This allowed us to study the impact of T21 independent of GATA1 mutation on fetal hemopoiesis. DS fetuses showed marked qualitative and quantitative abnormalities in hemopoiesis. While the total number of CD34+ cells in DS and normal fetal liver were comparable, DS fetuses had a striking increase in bi-potential megakaryocyte-erythroid progenitors (MEP; CD34+CD38+FcgloCD45RA+− 74.4% vs 27.0% of fetal liver CD34+/CD38+ cells. Peripheral blood from all DS fetuses studied compared to normal fetal blood samples showed dysmegakaryopoiesis (abnormally shaped and/or giant platelets and MK fragments), dyserythropoiesis (macrocytes, poikilocytes, basophilic stippling), increased numbers of blast cells and also had an increased percentage of MEPs − 40.3% vs 26.9%. By contrast, there was no difference in the number of MEP nor erythroid or MK lineage morphology in DS fetal bone marrow compared to normal fetal bone marrow. CD34+ cells from DS fetal liver and fetal blood expressed both fl GATA1 and GATA1s mRNA indicating that dysmegakaryopoiesis and erythropoiesis were not due to lack of expression of fl GATA1. These data indicate, for the first time, that T21 by itself profoundly disturbs megakaryopoiesis and erythropoiesis and leads to an increased of frequency of MEP. This has important implications since it provides a testable hypothesis for the role of T21 in the initiating step of AMKL, namely that T21 expands a fetal liver-derived progenitor compartment which forms a substrate upon which GATA1 mutations then confer a further selective advantage.

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