Transient Myeloproliferative Disease Research Articles

A down syndrome patient diagnosed with transient myeloproliferative disease after presenting with cutaneous findings

A down syndrome patient diagnosed with transient myeloproliferative disease after presenting with cutaneous findings

A Concomitant Hemato-Oncologic Disorder of Patient with Down Syndrome

A patient with Down syndrome has hematologic and oncologic disorders of medical complication. Down syndrome shows that characteristic hematologic and oncologic abnormalities and developments of disorders are different at ages. Many hematologic disorders were related to Down syndrome. There are diseases of red blood cells, white blood cell disorders, platelet and bleeding disorders, aplastic anemia, and transient myeloproliferative disease. Acute myeloid leukemia, acute lymphoblastic leukemia, and rarely solid tumors (Neuroblastoma, Wilm’s tumor, Hodgkin’s lymphoma) are also associated with Down syndrome. We checked clinical manifestations of each disorder and we should make standard hematologic index of different age groups. In addition, the relations between chromosome 21 and hemato-oncologic disorders should be found. We need to investigate potential therapeutic interventions that can improve quality of life and life expectancy in patients with Down syndrome. Key words: Down syndrome; Lymphoblastic leukemia; Acute myeloid leukemia; Myeloproliferative disorders

Differences in Clinical Characteristics of Transient Myeloproliferative Disease and Leukemia in Down Syndrome: A Single University Hospital Study

Differences in Clinical Characteristics of Transient Myeloproliferative Disease and Leukemia in Down Syndrome: A Single University Hospital Study

Suggestion of MUC16 (c.39169C>T) Mutation As a Potential Candidate for Predisposition to Myeloid Malignancy Associated with Trisomy 21

While the majority of leukemia cases occur in the absence of any known predisposing factor, there are germline mutations that significantly increase the risk of developing hematopoietic malignancies in childhood. Germline testing for the predisposition to myeloid malignancies is becoming more common with the recognition of multiple familial syndromes. In USA, Clinical Laboratory Improvement Amendments (CLIA) approved testing exists for mutations in RUNX1, GATA2, CEBPA and the other genes of inherited bone marrow failure syndromes. Meanwhile, GATA1 mutation is almost associated with Down syndrome and in acute myeloid leukemia (AML) with acquired trisomy 21. We experienced a case of infant AML with trisomy 21 and coexisting mutation of MUC16.We experienced infant leukemia (FAB classification, AML M7, Acute megakaryoblasticleukemia) with trisomy 21 and coexisting MUC16, SCRIB, and CEBPA mutation in a 7-month-old boy. Peripheral blood (PB) was taken from the boy every day during hospitalization. Bone marrow (BM) examination was performed at initial diagnosis and after remission. All of PB and BM samples were collected with informed consent, and the study was reviewed and approved by the Institutional Review Board of Seoul National University College of Medicine. The selected five PB samples are demonstrated on Fig. 1. G-banding revealed trisomy 21 and fluorescent in situ hybridization (FISH) for chromosome enumeration 21 revealed 71% with trisomy 21 cells in his diagnosis. At first, we suspected transient abnormal myeloproliferative disease associated with mosaic Down syndrome and serially monitored the proportion of cells with trisomy 21 by FISH and the percentage of blast during hospitalization. Cells with trisomy 21 decreased as blast disappeared in PB during chemotherapy, suggesting trisomy 21 is acquired abnormality in leukemic cells.We performed target gene sequencing of 359 genes related to the hematologic neoplasm, bone marrow failure syndrome, and cancer susceptibility to selected five PB. In addition, to search for the inherited predisposition gene to AML, we also performed whole genome sequencing (WGS) with BM specimen at initial diagnosis and after achieving remission. These next generation sequencing (NGS) with the Illumina HiSeq2500 platform revealed MUC16 (c.39169C>T) is the only significant mutation that persisted throughout from initial diagnosis to post-remission status. Mutations which were seen at initial diagnosis but disappeared after achieving remission were SCRIB (c.2197G>A, p.Arg733Trp) and CEBPA (c.371G>A, p.Ala124Val).To investigate whether MUC16 (c.39169C>T) mutation is rare variant which can be detected in normal person, we screened MUC16 mutation in healthy control (n=365) and in patients with other hematologic diseases (adult myelodysplastic syndrome, n=155; adult aplastic anemia, n=57; adult myeloproliferative neoplasm, n=44; childhood myeloid neoplasm, n=26; inherited bone marrow failure syndrome, n=21; idiopathic eosinophilia, n=4; familiar hemophagocytic lymophohistiocytosis, n=10; congenital neutropenia, n=1) using allele-specific PCR. MUC16 (c.39169C>T) was found in none of them. Discovered site of MUC16 (c.39169C>T) was not reported in solid tumor as well, though the other sites of MUC16 were frequently reported. It is generally known that AML with trisomy 21 accompanies GATA1 mutation, but the infant AML in this study did not accompany GATA1 mutation, but rather, MUC16 mutation.In this patient, GATA1 mutation was not found throughout hospital course. Instead, here we suggest that the constitutional MUC16 (c.39169C>T) mutation coexerts with acquired trisomy 21 in the development of infant AML and that MUC16 (c.39169C>T) mutation is a potential candidate gene for predisposition to AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hematologic Malignancies in Children with Down Syndrome: Transient Myeloproliferative Disease and Acute Megakaryoblastic Leukemia

Down sendromu (DS), 21. kromozomun tumu veya bir kisminin trizomisinden kaynaklanan ve hafiften agira kadar degisebilen mental yetersizlige yol acan bir kromozom anomalisidir. DS 1/700 canli dogum insidansi ile en sik gorulen insan anoploidisidir (1). DS’li olgularda hayat boyu losemi gelisme riski 10-20 kat daha yuksektir (2,3). Dunya Saglik Orgutu (DSO) tarafindan 2008’de yayinlanan hematopoetik ve lenfoid doku tumorleri siniflandirmasinda, DS ile iliskili 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. The Journal of Pediatric Research 2015;2(1):46-8

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus.

Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes.

Morphologic and GATA1 sequencing analysis of hematopoiesis in fetuses with trisomy 21.

Trisomy 21 alters fetal liver hematopoiesis and, in combination with somatic globin transcription factor 1 (GATA1) mutations, leads to development of transient myeloproliferative disease in newborns. However, little is known about the morphological hematopoietic changes caused by trisomy 21 in the fetus, and to date, the exact onset of GATA1 mutations remains uncertain. Therefore, we analyzed fetal liver hematopoiesis from second trimester pregnancies in trisomy 21 and screened for GATA1 mutations. We examined 57 formalin-fixed and paraffin-embedded fetal liver specimens (49 harboring trisomy 21 and 8 controls) by immunohistochemistry for CD34, CD61, factor VIII, and glycophorin A. GATA1 exon 2 was sequenced in fetal livers and corresponding nonhematologic tissue. Cell counts of megakaryocytes (P = .022), megakaryocytic precursors (P = .021), and erythroid precursors were higher in trisomy 21 cases. CD34-positive hematopoietic blasts showed no statistically significant differences. No mutation was detected by GATA1 exon 2 sequencing in fetal livers from 12 to 25 weeks of gestation. Our results suggest that GATA1 exon 2 mutations occur late in trisomy 21 fetal hematopoiesis. However, trisomy 21 alone provides a proliferative stimulus of fetal megakaryopoiesis and erythropoiesis. CD34-positive precursor cells are not increased in trisomy 21 fetal livers.

Blast cell deficiency of CD11a as a marker of acute megakaryoblastic leukemia and transient myeloproliferative disease in children with and without Down syndrome

The classification of acute myeloid leukemia (AML) FAB subtype M7 relies on immunophenotypic assessment. CD41 is expressed throughout all stages of maturation of megakaryocytes and has therefore been described as a specific blast cell marker in AML M7 as well as in transient myeloproliferative disease (TMD) of patients with Down syndrome (DS). However, technical difficulties underlie the need for new markers for these entities. We evaluated the expression of human lymphocyte function-associated antigen 1 (CD11a) in a large cohort of pediatric AML and TMD patients (n = 91) of the Austrian AML-BFM 98 and 2004 studies. We found a consistent deficiency of CD11a as assessed by mean fluorescence intensity in all patients with non-DS AML M7 (n = 8) and M6 (n = 1), all cases of classical DS-AML (n = 12) as well as TMD (n = 15) that was statistically significant in comparison to non-DS AML M0-M5 patients (n = 55; P < 0.001, sensitivity 100%). Only three of 55 Non-DS M0-5 patients were CD11a deficient (specificity 95%). Monocytic leukemias (M4/5) and normal monocytes typically showed a high CD11a expression, FAB types M1/2 and normal neutrophils an intermediate expression level, while all M3 leukemias were rather low in CD11a expression. We conclude, that deficiency of CD11a expression should be added to the diagnostic criteria of AML-M7, classical DS-AML and TMD.

High frequency of copy number alterations in myeloid leukaemia of Down syndrome

High frequency of copy number alterations in myeloid leukaemia of <scp>D</scp>own syndrome

A 15q24 microdeletion in transient myeloproliferative disease (TMD) and acute megakaryoblastic leukaemia (AMKL) implicates PML and SUMO3 in the leukaemogenesis of TMD/AMKL

Transient myeloproliferative disorder (TMD) of the newborn and acute megakaryoblastic leukaemia (AMKL) in children with Down syndrome (DS) represent paradigmatic models of leukaemogenesis. Chromosome 21 gene dosage effects and truncating mutations of the X-chromosomal transcription factor GATA1 synergize to trigger TMD and AMKL in most patients. Here, we report the occurrence of TMD, which spontaneously remitted and later progressed to AMKL in a patient without DS but with a distinct dysmorphic syndrome. Genetic analysis of the leukaemic clone revealed somatic trisomy 21 and a truncating GATA1 mutation. The analysis of the patient's normal blood cell DNA on a genomic single nucleotide polymorphism (SNP) array revealed a de novo germ line 2·58Mb 15q24 microdeletion including 41 known genes encompassing the tumour suppressor PML. Genomic context analysis of proteins encoded by genes that are included in the microdeletion, chromosome 21-encoded proteins and GATA1 suggests that the microdeletion may trigger leukaemogenesis by disturbing the balance of a hypothetical regulatory network of normal megakaryopoiesis involving PML, SUMO3 and GATA1. The 15q24 microdeletion may thus represent the first genetic hit to initiate leukaemogenesis and implicates PML and SUMO3 as novel components of the leukaemogenic network in TMD/AMKL.

Hematological disorders and leukemia in children with Down syndrome

Constitutional trisomy 21 inherent to Down syndrome (DS) is associated with several hematological disorders occurring at different ages. Neonates with DS may present with transient asymptomatic blood count abnormalities such as neutrophilia, thrombocytopenia and polycythemia. Within 1-2 months of life, 3-10% of DS infants develop transient myeloproliferative disease. Despite a spontaneous regression in most of the cases, TMD can be fatal or lead to the subsequent development of myeloid leukemia in 20% of DS children (DS ML). DS ML has clinical and biological features that define a unique entity with a high sensitivity to chemotherapy and a favorable outcome. Children with DS also have an increased risk of developing acute lymphoblastic leukemia (ALL) characterized by a more heterogeneous pattern of genetic findings and by a higher rate of treatment-related toxicities. These features highlight the role of trisomy 21 in leukemogenesis and confirm the need of specific and adapted therapeutic approach for DS children with leukemia.

Patient-Derived Induced Pluripotent Stem Cells Reveal Distinct Hematopoietic Defects Conferred by Trisomy 21 and Truncated GATA-1

Abstract 911Infants with Down syndrome (DS, trisomy 21, T21) frequently exhibit hematological abnormalities including polycythemia and/or thrombocytopenia. About 10% of DS infants develop transient myeloproliferative disease (TMD), which usually self-resolves. However, approximately 30% of affected patients develop acute megakaryoblastic leukemia (AMKL) by age 4 years. Both TMD and AMKL are accompanied by somatic GATA1 gene mutations that give rise to GATA-1s (for “short”), an amino-truncated protein lacking amino acids 1–81. Thus, DS-associated AMKL requires at least three sequentially occurring genetic abnormalities in hematopoietic cells: germline T21, somatic GATA1 mutations in fetal progenitors, and postnatal mutations in additional, currently unidentified genes. To analyze this malignant progression step by step and better understand T21-associated hematopoietic abnormalities, we created induced pluripotent stem cells (iPSCs) from DS subjects (n=3), TMD blasts (n=5) and controls (n=3). All iPSC lines exhibited signature features of pluripotency and retained their relevant genotypes: T21, T21+GATA1s and normal euploid. We compared the blood-forming capacities of iPSC lines by generating embryoid bodies in defined medium containing hematopoietic cytokines. Stage-matched embryoid bodies of each genotype produced similar numbers CD41+/235+/43+ hematopoietic progenitors capable of erythroid, myeloid and megakaryocytic differentiation. However, in methylcellulose colony assays, progenitors from DS iPSCs contained 13.5-fold increased numbers of burst forming unit erythroid (BFU-E) progenitors compared to control iPSCs (p=.009) (Table). While the absolute numbers of colony forming unit-megakaryocytes (CFU-MK) were similar between DS and wild type iPSC-derived progenitors (p=0.21), the CFU-MK:CFU-myeloid ratio was increased in progenitors from DS iPSCs (p=0.014). Thus, DS iPSC-derived hematopoietic progenitors exhibit increased propensity for erythro-megakaryocytic differentiation, similar to what occurs in DS fetal liver (Chou et al; Tunstall-Pedoe et al, Blood v112, 2008). In contrast, CD41+/235+/43+ progenitors from all 5 DS TMD iPSC lines studied (T21/GATA1s) exhibited complete absence of erythroid developmental potential in liquid culture and methylcellulose colony assays (p<.001), despite robust production of myeloid and megakaryocytic cells. We confirmed this observation by comparing the hematopoietic potential of iPSCs generated from TMD blasts (T21/GATA1s) and normal blood cells (T21/GATA1wt) of the same DS patient (n = 2 different individuals). In each case, acquisition of the GATA1s mutation selectively blocked erythropoiesis and tended to increase megakaryopoiesis. Thus, the amino terminus of GATA-1, absent in GATA-1s, is required for primitive (yolk-sac type) erythropoiesis, the developmental stage that is recapitulated in our iPSC differentiation protocols. In agreement, loss of the GATA-1 amino terminus inhibits primitive erythropoiesis in mice (Li et al, Nature Genetics v37, 2005). Our findings illustrate distinct hematopoietic defects conferred by T21 and GATA-1s, and suggest how these might synergize in TMD and AMKL. More generally, our studies illustrate how analysis of patient-derived iPSCs can be used to analyze genetic blood disorders, particularly those that arise during fetal development.TableAverage number of colonies per 5000 CD41+/235+/43+ progenitors plated in methylcellulose assays for BFU-E and CFU-GM, and Megacult assay for CFU-MK. Standard deviation in parenthesesGenotype#patients#replicatesBFU-ECFU-GMCFU-MkEuploid31031 (+26)138 (+70)468 (+135)T2138417* (+201)52* (+32)574 (+211)T21+GATA1s5140** (+0)394** (+245)754 (+657)*p<0.05, euploid vs T21,**p<0.05 T21 vs T21+GATA1s. Disclosures:No relevant conflicts of interest to declare.

Serum thrombopoietin level and thrombocytopenia during the neonatal period in infants with Down's syndrome

The pathogenesis of thrombocytopenia during the neonatal period in Down's syndrome (DS) infants remains unclear. To elucidate kinetic changes of serum thrombopoietin (TPO) level and platelet count, and their correlation in DS neonates. Twelve DS infants (male/female: 7/5, term/late preterm: 10/2) born between 1997 and 2007 were included. Blood samples were serially collected during the neonatal period and serum TPO levels were determined in 44 sera using an enzyme-linked immunosorbent assay. Thrombocytopenia <150 x 10(9) per liter was observed in seven (58%) patients. In 12 DS patients, the median TPO value showed 2.86 fmol ml(-1) on day 0, rose to 4.64 fmol ml(-1) on day 2, and thereafter decreased to 4.30 fmol ml(-1) on day 5, 2.40 fmol ml(-1) on days 11-15, and 1.75 fmol ml(-1) on days 28-30. This kinetics parallels that in historical non-DS controls. In 35 pair sample analysis from 11 patients without transient myeloproliferative disease, TPO level inversely correlated with platelet count (r=-0.38, P=0.023). However, there was no significant difference in TPO concentrations between thrombocytopenic and non-thrombocytopenic DS individuals. This is the first study to describe the relationship between TPO level and platelet count in neonates with DS. Median TPO levels and their kinetic changes in DS neonates are comparable to those in non-DS controls. In contrast to earlier findings in several studies showing higher TPO concentrations in thrombocytopenic non-DS newborns than those in non-thrombocytopenic counterparts, the response of the TPO system to thrombocytopenia in DS during the neonatal period seems suboptimal.

Insights into the manifestations, outcomes, and mechanisms of leukemogenesis in Down syndrome

AbstractChildren with Down syndrome (DS) show a spectrum of clinical anomalies, including cognitive impairment, cardiac malformations, and craniofacial dysmorphy. Moreover, hematologists have also noted that these children commonly show macrocytosis, abnormal platelet counts, and an increased incidence of transient myeloproliferative disease (TMD), acute megakaryocytic leukemia (AMKL), and acute lymphoid leukemia (ALL). In this review, we summarize the clinical manifestations and characteristics of these leukemias, provide an update on therapeutic strategies and patient outcomes, and discuss the most recent advances in DS-leukemia research. With the increased knowledge of the way in which trisomy 21 affects hematopoiesis and the specific genetic mutations that are found in DS-associated leukemias, we are well on our way toward designing improved strategies for treating both myeloid and lymphoid malignancies in this high-risk population.

Malignancy in Children with Trisomy 21

Patients with Down syndrome (DS) display a unique spectrum of malignancies, with a 10- to 20-fold higher risk of acute leukemias, and a markedly lower incidence of solid tumors. This review discusses the current understanding of the basis for this distinctive pattern of cancer incidence and the clinical and biologic features of the malignant disorders most frequent in DS individuals: transient myeloproliferative disease, acute megakaryoblastic leukemia, and acute lymphoblastic leukemia. We also review distinctive pharmacogenetic issues, highlighting the differential chemosensitivity and toxicity profiles of DS patients compared with the general population, and epidemiologic studies of protective and adverse environmental risk factors for the development of leukemia.

OTT-MAL Activates the Notch Signaling Transcription Factor RBPJ and Cooperates with Mutant MPL to Induce Acute Megakaryoblastic Leukemia

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous subtype of acute myeloid leukemia (AML) and is more frequent in children than in adults. The molecular basis of AMKL is poorly understood in adults, whereas two major molecular subtypes are recognized in pediatric AMKL. The first group occurs in infants and is associated with the t(1;22)(p13;q13) chromosomal translocation resulting in expression of the OTT-MAL fusion protein (a.k.a. RBM15-MKL1). The second group is represented by Down syndrome (DS) patients with transient myeloproliferative disease and AMKL, who present with acquired GATA-1 mutations. We generated a murine knock-in model of OTT-MAL expression and observed that expression of OTT-MAL results in abnormal megakaryopoiesis during embryonic and adult development, but rarely causes AMKL. We hypothesized that transformation to AMKL associated with OTT-MAL requires cooperating mutations and used a candidate gene approach with previously reported activating alleles of signaling molecules. Our data indicate that OTT-MAL cooperates only with a limited subset of activated signaling molecules, including MPLW515L, to generate a fully penetrant and rapidly fatal disease closely resembling human AMKL. OTT has homology with SHARP, which inhibits Notch signaling through direct interaction with RBPJ. In light of our recent observation that canonical Notch signaling specifies megakaryocyte fate of hematopoietic stem cells (Mercher et al, Cell Stem Cell, in press), we investigated whether OTT-MAL could interfere with the Notch pathway. We observed that OTT-MAL interacts with RBPJ and aberrantly increases its transcriptional activity in vitro and in vivo. The N-terminal portion of OTT is required for interaction of OTT-MAL with RBPJ and the transactivation domain of MAL is necessary for activation of RBPJ-mediated transcription, suggesting that OTT-MAL aberrantly recruits transcriptional co-activators to regulatory elements of genes normally inhibited by OTT/RBPJ. As a confirmation of clinical relevance, we have observed that several Notch pathway genes are specifically upregulated in human AMKL patient cells associated with the t(1;22)(p13;q13) as compared to DS-AMKL. Together our data suggest that aberrant activation of RBPJ by OTT-MAL results in abnormal commitment of hematopoietic progenitors to the megakaryocytic lineage, and is the basis for the specific association between t(1;22)(p13;q13) and AMKL. These results provide important biological insights into a novel mechanism of leukemogenesis of the megakaryocyte lineage, and indicate that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage. Furthermore, these findings suggest that specific targeting of these pathways could be of therapeutic value for human AMKL.

GATA1s Mutant Protein Contributes to “Down” Syndrome Megakaryoblastic Leukemia by Derepression of E2F Targets.

Somatic mutations in the hematopoietic transcription factor GATA1, leading to the production of a GATA1 variant (GATA1s) lacking its N-terminal domain, are a critical event in the development of Down syndrome (DS) associated transient myeloproliferative disease (TMD) and myeloid leukemia (ML-DS). Mice carrying Gata1s germline mutation exhibit a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors, hyperproliferating both in vitro and in vivo. However, either Gata1s mice alone or in conjunction with two DS mouse models (Ts65dn, Ts1Cje) fail to recapitulate human TMD or leukemia. Using a retroviral insertional mutagenesis screening in Gata1s fetal progenitors, we generated murine acute megakaryoblastic leukemia (AMKL) closely resembling human ML-DS. We demonstrate that retroviral integrations at the Evi1 and Plag1 loci cooperated with Gata1s in leukemogenesis. Through an integrated genomic approach, we show that restoration of full length Gata1 expression in these murine Gata1s AMKL blasts leads to downregulation of gene sets containing E2F target genes. In addition, most of these same gene sets are also enriched in Gata1s primary fetal liver-derived megakaryocytes compared to wild type megakaryocytes, in flow sorted human ML-DS leukemic blasts compared to sorted non-DS M7 leukemic blasts, and in ML-DS CMK cell line compared to other leukemic cell lines. Using a genetic approach, we could demonstrate that the hyperproliferative phenotype of Gata1s FL megakaryocytic progenitor cells can be rescued by broadly repressing the function of E2F factors. Additionally, the CMK cell line ceased to proliferate upon E2F inactivation. Thus, we discovered a new mechanism of GATA1 mediated growth regulation by controlling E2F function. GATA1s mutant protein contributes to leukemogenesis through insufficient repression of E2F targets, perturbing the coordination of proliferation and terminal differentiation. Our study reveals a previously unknown function of the N-terminal domain of GATA1 and adds a significant step towards our understanding of the role of GATA1 mutants in leukemogenesis.

Acute megakaryoblastic leukemia in Down syndrome

Children with Down syndrome (DS) have a 10- to 20-fold increased risk of developing acute leukemia. An estimated 10% of newborns with DS develop Transient Myeloproliferative Disease (TMD) or Transient Leukemia (TL), a clonal accumulation of megakaryoblasts that resolves spontaneously within months. Acute megakaryoblastic leukemia (AMKL) develops in approximately 20% of cases of TMD/TL by 4 years of age. Both the blasts of AMKL and TMD/TL in DS harbor somatic mutations of GATA1, an essential transcriptional regulator of megakaryocytic differentiation. The distinct phenotypes of megakaryoblastic leukemia in DS are a unique biological model of the incremental process of leukemic transformation.

Absence of JAK2 V617F Activating Mutations in Children with Acute Megakaryoblastic Leukemia with and without Down Syndrome.

Activating mutations at codon 617 of the Janus-2 tyrosine kinase (JAK2 V617F) have recently been described in hematological malignancies. In adult acute myeloid leukemia (AML), the reported frequencies vary, and JAK2 V617F mutations have mainly been detected in secondary AML following a myeloproliferative disorder. In adult de novo AML, the mutation was less frequent, and detected in 2/11 (18%) acute megakaryoblastic leukemia (FAB M7) samples (Jelinek et al., Blood 2005), and occasionally in other FAB-types.This prompted us to analyze a cohort of pediatric AML FAB M7 samples for this particular mutation. In children, at least 3 different subsets of AML M7 can be identified: infants with AML M7 characterized by t(1;22)(p13;q13), older children with random cytogenetic aberrations, and myeloid leukemia of Down syndrome (DS ML). DS ML is often preceded by transient myeloproliferative disease (TMD), hence we also screened TMD samples to detect whether JAK2 V617F mutations would be involved in clonal evolution from TMD to DS ML. To exclude germ-line mutations in DS, we tested normal mononuclear bone marrow cells (NBMC) from children with DS. These NBMC were obtained from a sternal aspirate from children undergoing cardiac surgery, after informed consent was obtained. Genomic DNA was harvested from leukemic cells, and JAK2 exon 12, including the intron-flanking regions, was amplified and sequenced to screen for the JAK2 V617F mutation. As a positive control for the JAK2 V617F mutation, we used HEL 92.1.7 cells (an erythroleukemic cell line). In a dilution experiment we could still detect the mutation, using direct sequencing, if 10% HEL/JAK2 mutated cells were mixed with 90% wild-type control cells. We tested 49 samples, comprising of 9 NBMC, 11 TMD, 14 DS-ML M7, 11 non-DS AML M7 and 4 relapsed non-DS AML M7 samples (including 2 initial diagnosis-relapse pairs). The median age of the TMD cohort was 3 days, for DS-ML children 1.9 years (range 0.9–3.8 yrs), and for non-DS AML 1.5 years (range 1.2–13.7 yrs). The median white blood cell count for TMD was 25.8x109/l, for DS-ML 13.8x109/l, and for non-DS AML 12.4x109/l. Cytogenetic data were available in 5/11 non-DS AML cases only, which showed no cases with a t(1;22). No JAK2 V617F mutations were detected in any of the clinical samples. We conclude that the role of JAK2 V617F mutations in pediatric DS and non-DS acute megakaryoblastic leukemia is limited at best. However, we were not able to screen the subgroup of non-DS AML cases with t(1;22).

CREB Transgenic Mice Develop Myeloproliferative Disease/Myelodysplastic Syndrome after a Prolonged Latency.

The cyclic AMP Response Element Binding Protein, CREB, is a basic leucine zipper transcription factor that induces genes that regulate cell proliferation and survival. CREB is a downstream target of GM-CSF signaling pathways in myeloid cells, resulting in transactivation of critical target genes. We previously demonstrated that both CREB protein and mRNA levels are increased in the bone marrow cells of patients with AML at diagnosis. In contrast, CREB is expressed at low levels in normal bone marrow cells. To examine the expression of CREB in specific bone marrow and leukemia stem cell populations, we performed quantitative real-time PCR. AML blast cells were sorted and were shown to have increased CREB mRNA expression in CD34+CD33+ and CD34-CD33+ population compared to normal bone marrow progenitor cells. To understand the role of CREB in normal hematopoiesis and leukemogenesis, we created transgenic mice in which the myeloid specific hMRP8 promoter controls CREB expression. Within months, these mice developed increased monocytes and neutrophils in the peripheral blood. The increased cells in the peripheral blood and bone marrow were Gr1+Mac1+. We did not observe increases in other cell lineages using FACS analysis and the markers, B220, CD3, and Ter119. Bone marrow cells from CREB transgenic mice displayed increased colony size, greater numbers of colonies, and immortalization in colony replating assays with methylcellulose containing IL-3, IL-6, and Stem Cell Factor, compared to age-matched littermate controls. Bone marrow from CREB transgenic mice grew in the absence of cytokines, demonstrating factor-independent growth. Increased numbers of CFU-M was observed with CREB transgenic mouse bone marrow in colony assays with methylcellulose containing M-CSF. Although the mice did not develop acute leukemia, 7 out of 8 CREB transgenic mice compared to 0 out of 13 control mice developed enlarged spleens and myeloproliferative disease (MPD) after 12 months of age. Histology of the spleens showed destruction of the normal architecture with aberrant myeloid cells, suggestive of a myeloproliferative disease/myelodysplastic syndrome. Both the indolent course and phenotype of our mice were similar to Chronic Myelomonocytic Leukemia (CMML) observed in humans. Interestingly, myeloid cells from patients with CML in chronic phase also express higher levels of CREB. Studies are underway to characterize CREB expression in peripheral blood or bone marrow cells from patients with MPD/MDS, including CML, CMML, and transient myeloproliferative disease of Down Syndrome. We are also analyzing the spleens from CREB transgenic mice that develop MPD/MDS. Our results demonstrate that overexpression of CREB is sufficient to induce myeloid cell transformation to a preleukemic state in vivo. Therefore, the CREB transgenic mouse provides a useful model to test novel therapies to treat MPD/MDS.