Transient Myeloproliferative Disorder Patients Research Articles

Hydrogel-Based 3D Culture Model for Down Syndrome Associated Transient Myeloproliferative Disorder Using Customized Induced Pluripotent Stem Cells

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent stem cells (iPSCs) provides an extraordinary tool for hematological disease modeling of rare disorders such as Down syndrome (DS) associated transient myeloproliferative disorder (TMD). TMD is a preleukemic condition observed in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. Hematopoiesis in the bone marrow (BM) is affected by cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs utilize either 2-dimensional (2D) monolayer of mouse stromal cells or animal tissue derived extracellular matrices. Generation of a 3-dimensional (3D) culture environment attempts to facilitate both cell-cell and cell-matrix interactions during iPSC differentiation. This study reports the development of a 3D culture system for hematopoietic differentiation of iPSCs to model TMD.iPSC colonies were encapsulated in 3D polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) (Fig. 1A) and cultured in maintenance medium (mTeSR1™, Stem Cell Technology) without feeder cells. There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig. 1B). The 3D encapsulation did not have an adverse effect on the viability of the iPSC colonies evaluated by in situ staining with viability dye (Fig. 1C). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG whereas colonies in 2D were more flattened (Fig. 1D). The pluripotency of the 3D encapsulated iPSCs was confirmed alkaline phosphatase staining (purple colonies) and by the presence of >96% population expressing pluripotency markers, Tra-1-60 and SSEA-4 (Fig. 1E).To test the efficiency of the 3D model system to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff Hematopoietic Kit. Following differentiation, immunophenotype analysis of single cells by flow cytometry revealed a 1.7-fold higher CD34+CD45+CD38-CD45RA- cell percentage in 3D hydrogels compared to 2D. Further delineation of sub-populations in HSPC compartment from 2D and 3D hydrogel revealed a 1.9-fold and 2.1-fold higher population of early HSPCs and multipotent progenitors (MPPs) in 3D compared to 2D respectively (Fig 1F, *P<0.05). In colony forming unit (CFU) assay, the 3D generated HSPCs gave rise to a 2.0-fold higher number of CFU-GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) colonies compared to 2D, with 2.0-fold decreased number of BFU-E (erythroid) colonies and a similar number of CFU-GM (granulocyte, macrophage) colonies (Fig. 1G). Thus, the low modulus synthetic matrix promoted hematopoietic differentiation producing higher percentage of early HSPCs as compared to the 2D culture system.We used this 3D system to model TMD by utilizing isogenic iPSCs with disomy 21 (D21), trisomy 21 (T21), and trisomy 21 bearing pathologic mutation in GATA1 (T21-G1). The megakaryoid population in the HSPCs generated by hematopoietic differentiation of 3D encapsulated iPSCs was characterized by the percentage of CD34+CD41+ population within the total CD41+ population, myeloid population as CD18+CD45+ and erythroid population as CD71+CD235+. T21 HSPCs showed increased erythroid and megakaryoid populations as compared to isogenic D21, consistent with the role of trisomy 21 in perturbing hematopoiesis. T21-G1 had elevated megakaryoid (93±6% vs 71±1%,) and myeloid (32±16% vs 8±4%) populations with reduced erythroid (27±12% vs 79±6%) population as compared to T21 HSPCs implicating GATA1s in altered hematopoiesis (Fig. 1H). T21-G1 HSPCs only produced CFU-GM colonies as compared to a high number of CFU-GEMM and BFU-E in T21 and D21 HSPCs (Fig. 1I). The expression of GATA1s in T21-G1 megakaryoid population was confirmed (Fig. 1J). The immunophenotype marker analysis of T21-G1 megakaryoid blasts showed expression of megakaryoid/erythroid antigens (CD41, CD61, CD42b, CD71) along with myeloid markers (CD11b, CD33, CD13) and increased expression of CD56 and CD117 consistent with TMD patients (Fig. 1K).In conclusion, our cost-effective tunable 3D hydrogel system promoted hematopoietic differentiation of iPSCs and generated TMD model mimicking the salient features of the disease. [Display omitted] DisclosuresBarwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Targeted Deep Sequencing of GATA1 more Sensitively Identifies Transient Myeloproliferative Disorder in Neonates with Down Syndrome

Down syndrome (DS) is caused by a constitutional trisomy 21 and up to 10% of neonates with DS develop a temporary accumulation of leukemic blasts in the peripheral blood, called transient myeloproliferative disorder (TMD). In most cases, TMD is a self-limiting disorder, however 20% of children develop serious symptoms and treatment with low dose cytarabine is then necessary. TMD is caused by a mutation in the GATA1 gene, occurring in cells with trisomy 21. Around 20% of children with TMD will develop a myeloid leukemia (ML) before the age of 4 years. It is a megakaryoblastic leukemia characterized by a GATA1 mutation identical to the mutation these children had in their TMD blast cells. The primary aim of this study was to define a population based frequency of TMD in DS neonates.In this nation-wide study we prospectively screened 368 peripheral blood (PB) samples from DS neonates (< 4 weeks old) in the Netherlands between January 1st 2008 and January 1st 2013 for TMD using morphology and immunophenotyping (IPT) (Dutch Trial Registry number NTR1667). TMD was defined as >5% myeloblasts in the PB. Patients with less than 5% of cells in the PB consistent with TMD using IPT were also included. PB samples of TMD positive patients were tested for GATA1 exon 2 mutations using Sanger sequencing. Patients with TMD were followed at least until the age of 4 years. TMD positive patients without a GATA1 exon 2 mutation using Sanger sequencing and TMD negative patients were retrospectively screened for GATA1 exon 2 and 3 mutations using targeted deep sequencing. Using this technique we could detect mutations with a frequency of 0.1%.In the prospective part of the study, TMD was diagnosed in 45 of the 368 cases screened (12.2%). In 41 of 45 neonates with TMD, GATA1 mutation analysis was performed. In 4 cases no GATA1 PCR was performed. In 28 cases a GATA1 exon 2 mutation was detected (28/41=68%), while in 13 cases no GATA1 mutation was detected.It was recently shown that there is a subgroup of DS neonates who do not have TMD by conventional criteria, but when more sensitively screened do harbour a GATA1 mutation (Roberts et al, Blood 2013). Therefore using targeted deep sequencing we retrospectively screened the 13 TMD cases without a GATA1 mutation by Sanger sequencing and 2 of the 4 cases that were not screened at TMD diagnosis. No GATA1 exon 3 mutations were detected. In 9 out of 15 patients a GATA1 mutation was detected using targeted deep sequencing. Both cases not prospectively Sanger sequenced were positive. 7 of 13 TMD samples tested negative using Sanger sequencing, using targeted deep sequencing were positive for a GATA1 mutation (median mutational frequency 6,8%). Overall, 43 TMD patient samples were screened using Sanger sequencing and/or targeted deep sequencing and 38 of these tested GATA1 positive (88%).In addition, we retrospectively screened 319 of the 323 peripheral blood samples of the TMD negative DS neonates for GATA1 using targeted deep sequencing. No GATA1 exon 3 mutations were detected. In 11 out of 319 TMD negative cases we detected a GATA1 exon 2 mutation with a median mutational frequency of 0,8%.14 children with DS born in the Netherlands between 1-1-2008 and 31-12-2013 were diagnosed with ML DS (follow-up until 31st of dec 2016). Of these 14 children, 7 were screened neonatally and in 6 cases TMD was diagnosed. Therefore 6 out of 45 TMD patients identified using conventional screening developed ML DS (13.3%). Out of the 11 ‘subclinical’ TMD cases only detected by targeted deep sequencing GATA1 screening, one patient was later diagnosed with ML DS (1/11=9.1%). The patient's neonatal blood sample harboured two different GATA1 exon 2 mutations (14 base pair (bp) insertion in 0,24% of cells and a 1 bp insertion in 2,6% of cells). In the ML DS cells of this patient the identical 1 bp exon 2 insertion in GATA1 was detected.In conclusion, conventional screening for TMD using immunophenotyping en morphology identified TMD in 12% of the screened DS neonates. An additional 3.4% can be detected using targeted deep sequencing. This is clinically relevant as these children are at risk of developing ML DS. Therefore, for future clinical studies we are developing targeted deep sequencing methods screening for GATA 1 in neonatal DS PB samples. DisclosuresKaspers:Janssen-Cilag: Research Funding.

Increased Mutagenesis during Fetal Hematopoietic Development in Down Syndrome

Children with Down syndrome are predisposed to leukemia during the first years of their life. 5-10% of newborns with Down syndrome are born with transient myeloproliferative disorder (TMD), which often spontaneously disappears. The majority of these patients achieves complete remission. However, in 20-30% of all TMD patients the disease progress in acute megakaryoblastic leukemia. In addition, they have a 20 fold higher risk of developing B-lymphocyte acute leukemia (B-ALL). Leukemic development in Down syndrome is initiated during fetal development. However, it is unclear why fetuses with a trisomy of chromosome 21 have an increased risk of developing leukemia. Previously, we have developed a method to study somatic mutations in single cells using clonal cultures. Here, we applied this method to human fetal hematopoietic stem and progenitor cells (HSPCs) from liver and bone marrow of Down syndrome human fetuses and control fetuses with two copies of chromosome 21 (D21). In addition, we characterized somatic mutation accumulation in not affected small intestine stem cells. Subsequently, we performed in depth mutational analyses to characterize active processes using mutational signatures in fetal stem cells, which potentially can drive leukemic development during early life. Recently, we have shown that that healthy adult HSPCs gradually accumulate somatic mutations in a linear fashion with an annual mutation rate of 14.2 base substitutions per year. Whereas the somatic mutation rate is significantly higher during fetal development. Subsequently, in Down syndrome fetuses the overall somatic mutation rate of fetal stem cells is significantly increased compared to D21 fetal stem cells (P-value: 0,024). We performed phylogenetic analysis to study relatedness of the cells and observed an higher somatic mutation rate in the first cell divisions. This elevated mutation rate can be explained by increased contribution of mutational process signature 1 and 5, which are already present in fetal stem cells. Therefore, Down syndrome fetal stem cells show enhanced activity or increased sensitivity to mutational processes that are normally active during development. The same mutational signatures are present in TMD blast cells, indicating that these processes can cause cancer driver mutations and subsequently contribute to leukemic development. Interestingly, some Down syndrome fetal stem cells showed very high mutation numbers that could partly be attributed to mutational signature 18, which likely reflect oxidative-stress induced mutagenesis. These findings, show increased mutagenesis in Down syndrome during fetal development in hematopoietic stem cells and small intestine stem cells. This increased mutagenesis can potentially explain why children with Down syndrome have an increased risk of developing leukemia in early life. Disclosures No relevant conflicts of interest to declare.

The Clonal Hematopoietic Spectrum of Down Syndrome and ML-DS

Introduction: Children with Down syndrome (DS) have a 150-fold higher risk of developing myeloid leukemia (ML-DS) (Wechsler J, Nat Gen 2002). Ten percent (10%) of DS children are also predisposed to developing a preleukemic self-limiting condition, transient myeloproliferative disorder (TMD). While the majority of these children achieve complete remission, 20-30% of TMD patients will eventually develop ML-DS (Gamis AS, Blood 2011). Unique to ML-DS and TMD is the association with mutations in the X-linked hematopoietic transcription factor, GATA1 (Alford KA, Blood 2011), which are pathognomonic, but perhaps not sufficient, for ML-DS. Along with GATA1 mutations, ML-DS has a distinct mutational landscape implicating the cohesin complex, epigenetic regulators, signal transducers and the RAS pathway (Yoshida K, Nat Gen 2013). While these findings have informed our understanding of ML-DS etiology, ~30% of healthy DS neonates - without overt TMD or ML-DS - also harbor GATA1 mutations (Roberts I, Blood 2013), suggesting that ML-DS arises from clonal outgrowth of these progenitors that acquire specific additional mutations. Because the spectrum of physiologic clonal hematopoiesis is unknown in DS, we sought to characterize clonal mutations in healthy DS children as compared to a cohort of ML-DS cases using our highly sensitive and specific error-corrected sequencing (ECS) methodology. A precise understanding of their altered hematopoietic development may inform risk stratification, therapeutic selection and outcomes.Methods: The Druley lab has developed a custom error-corrected sequencing (ECS) panel targeting 80 genes frequently mutated in both pediatric and adult myeloid malignancies (Young AL, Nat Comm 2016) that is validated to identify clonal mutations as rare as 0.0001 variant allele fraction (VAF), 100X below the error-rate of next-generation sequencing (Wong TN, Nature 2015; Young AL, Leukemia 2015). Using ECS, we surveyed 102 DS children (47 ML-DS enrolled from a current Phase III ML-DS study; 55 healthy DS enrolled at St. Louis Children's Hospital).Results: We identified 294 total clonal variants in 72/102 children (60.0%, healthy DS; 83.0%, ML-DS cases) at 0.0002-0.82 VAF. On average, we found 1.8 clonal variants per healthy DS control and 4.1 clonal variants per ML-DS case. Consistent with Yoshida et al., we find a spectrum of recurrent mutations in ML-DS cases: GATA1 (53.2%), EZH2 (25.5%), RAD21 (14.9%), and STAG2 (10.6%) etc. Surprisingly, we also identified frequent mutations in FAT1 (14.9%) and SETD2 (10.6%), genes previously unassociated with ML-DS. Moreover, we report a different set of recurrent mutations in healthy DS children: FAT1 (12.7%), BCOR (12.7%), TET2 (10.9%), SETD2 (7.3%), and TRIM24 (5.5%), and TP53 (5.5%) etc.Discussion: Earlier ML-DS studies could only detect more common mutations >0.02 VAF due to the error-rate of standard next-generation sequencing (NGS). Using ECS, we present the first characterization of the unique clonal hematopoietic spectrum in children with DS and ML-DS. These results reveal that clonal hematopoiesis is common amongst healthy DS children without hematologic conditions, and that children with ML-DS have a higher clonal mutation burden than those without. Notably, we identified two novel genes recurrently mutated in both ML-DS cases and healthy DS controls: FAT1 and SETD2. We suspect that these mutations were not identified in other ML-DS studies as the median VAFs of our detected FAT1 and SETD2 mutations were 0.001 and 0.0008, respectively. Mutations in FAT1 and SETD2 have been shown to lead to dysregulated Wnt signaling in numerous cancers (Morris LG, Nat Gen 2013; Yuan H, J Clin Invest 2017). We and others have previously shown that Wnt signaling is crucial in specifying a primitive or definitive hematopoietic program (Sturgeon CM, Nat Biotech 2014; Creamer JP, Blood 2017). We speculate that these mutant FAT1 and SETD2 clones, like mutant GATA1 clones, may inhibit the definitive hematopoietic potential in DS children. Lastly, we note a reduced incidence of GATA1 mutations in our healthy DS cohort and attribute this to an older average age (8.3 years), as compared to Roberts et al. studying neonates. Other groups have speculated that somatic mutations in GATA1 (with a DS background) may only occur during a restricted developmental window (<4 years) as TMD clones cycle into quiescence afterwards (Zhe L, Nat Gen 2005; Hasle H, Leukemia 2007). DisclosuresNo relevant conflicts of interest to declare.

Low-dose cytarabine to prevent myeloid leukemia in children with Down syndrome: TMD Prevention 2007 study

AbstractApproximately 5% to 10% of children with Down syndrome (DS) are diagnosed with transient myeloproliferative disorder (TMD). Approximately 20% of these patients die within 6 months (early death), and another 20% to 30% progress to myeloid leukemia (ML-DS) within their first 4 years of life. The aim of the multicenter, nonrandomized, historically controlled TMD Prevention 2007 trial was to evaluate the impact of low-dose cytarabine treatment on survival and prevention of ML-DS in patients with TMD. Patients received cytarabine (1.5 mg/kg for 7 days) in case of TMD-related symptoms at diagnosis (high white blood cell count, ascites, liver dysfunction, hydrops fetalis) or detection of minimal residual disease (MRD) 8 weeks after diagnosis. The 5-year probability of event-free and overall survival of 102 enrolled TMD patients was 72 ± 5% and 91 ± 3%, respectively. In patients eligible for treatment because of symptoms (n = 43), we observed a significantly lower cumulative incidence (CI) of early death as compared with symptomatic patients in the historical control (n = 45) (12 ± 5% vs 33 ± 7%, PGray = .02). None of the asymptomatic patients in the current study suffered early death. However, the treatment of symptomatic or MRD-positive patients did not result in a significantly lower CI of ML-DS (25 ± 7% [treated] vs 14 ± 7% [untreated], PGray = .34 [per protocol analysis]; historical control: 22 ± 4%, PGray = .55). Thus, low-dose cytarabine treatment helped to reduce TMD-related mortality when compared with the historical control but was insufficient to prevent progression to ML-DS. This trial was registered at EudraCT as #2006-002962-20.

Long-term outcome for Down syndrome patients with hematopoietic disorders.

Although Down syndrome (DS) patients have a higher risk of developing transient myeloproliferative disorder (TMD) and acute leukemia, very little data is available on long-term outcome in Taiwanese patients. The current study was designed to determine the clinical characteristics and treatment outcome of DS patients with TMD or acute leukemia (AL). In 25 consecutive DS patients with TMD or AL enrolled from 1990 to 2012, clinical manifestations and treatment protocols were investigated and GATA1 (GATA binding protein 1) mutations were identified. Among 16 DS-acute myeloid leukemia (DS-AML) patients, clinical outcomes were compared between survivors and nonsurvivors. Most of our DS patients had TMD (32%), acute megakaryoblastic leukemia (24%), or acute erythromegakaryoblastic leukemia (16%). The median follow-up time was 22.5 months (1-230 months). The age was younger and the hemoglobin (Hb) level and platelet count were higher in TMD patients than in leukemia patients. Among DS-AML patients, the Hb level was higher in survivors than nonsurvivors (8.8±2.7g/dL vs. 5.8±2.4g/dL; p=0.044) and the age was older in relapsed patients than in nonrelapsed patients (43.8±18 months old vs. 21.6±8.6 months old; p=0.025). The 3-year overall survival (OS) rate was 44%, higher in patients receiving appropriate chemotherapy than in those receiving inadequate treatment (63.6% vs. 0%, p=0.001), and higher in those diagnosed with TMD or AL after 2008 than before 2008 (33.3% vs. 75%; p=0.119). Outcome in DS-AML patients is optimal if appropriate treatment is provided. With modification of the treatment strategy in 2008, OS increased in Taiwan.

Exploring the Pathogenesis of Down Syndrome-Related Myeloproliferative Disorders Using iPSCs

Down syndrome (DS) is a congenital syndrome due to the trisomy of chromosome 21. Transient myeloproliferative disorder (TMD) is its hematopoietic complication, affecting approximately 10% of DS-neonates. TMD is characterized by transient abnormal proliferation of blastic cells, and importantly, all TMD blasts bear mutations in GATA1 gene. Although TMD usually resolves spontaneously within 3 months after birth, twenty to thirty percent of TMD patients develop acute megakaryoblastic leukemia (AMKL) within several years afterward. This leukemogenic transition is considered as a good model for multi-step tumorigenesis. According to this putative multi-step model, the first hit should be additional chromosome 21, the second one is mutations on GATA1 gene which is requisite to the onset of TMD, and the “unknown” third hits are required for the progression into AMKL. However, it is still unclear that 1) how GATA1 mutation promotes TMD development, 2) what kinds of third hit are required for the onset of AMKL, and 3) why GATA1-mutated progenitors prevails during embryonic hematopoiesis only in trisomy 21 patients?In order to address these issues, a strictly controlled isogenic cell panels that can reproduce human emboryonic hematopoietic development is needed. Human induced pluripotent stem cells (iPSCs) derived from DS patients are a promising platform for this, but so far there is no report regarding GATA1-mutated TMD-associated iPSCs. Therefore, we set out to establish an iPSC panel that covers each genomic status of chromosome 21 and GATA1 gene. For this, we established both GATA1 mutant and wildtype clones from both trisomy 21 and disomy 21 clones. And we also established TMD patient derived iPSCs.First, we established isogenic iPSCs derived from EB virus immortalized B-lymphocytes of 2 mosaic DS patients. Frequency of trisomy 21 cells evaluated by FISH analysis and G-banding were 93% and 94% for each patient. We reprogrammed these cells by introducing 5 episomal vectors, pCE-hOCT3/4, pCE-hSK, pCE-hUL, pCE-mp53DD and pCXB-EBNA1, under feeder free condition. We genotyped each iPSC clones by digital-PCR analysis and found that the frequency of trisomy iPSC clones were comparable to that of trisomy cells in original EBV immortalized B-lymphocytes. There is no morphological difference between disomy and trisomy iPSC clones in both patients. We next introduced disease-associated GATA1 mutation into established isogenic trisomy and disomy iPS clones using transcription activator-like effector nuclease (TALEN) technology. We introduced a frameshift mutation in exon 2, which causes premature termination of the full-length transcript originated from 1st ATG and exclusively produces the shorter isoform of GATA1 (GATA1s) transcribed from 2nd ATG.Next, we obtained peripheral blood mononuclear cells (PBMCs) from a TMD patient in order to establish TMD-blast-derived iPSCs. Eighty-nine percent of nuclear cells in the PBMC fraction was CD117+CD45+ blastic cells, whereas only 5.6% were non-blast cells including CD3 positive T-lymphocytes, CD11b positive myeloid lineage cells and CD19 positive B-lymphocytes. The CD117+ cells showed TMD/AMKL blast-like appearance such as coarse choromatin pattern with nucleolus and bleb-like structures. We sorted out CD45+CD117+ blastic cells and CD45+CD117- non-blastic cells and successfully established iPSC clones from both populations.In conclusion, we successfully established a comprehensive panel of iPSC clones for evaluating the hematopoietic consequence associated with the GATA1 genotype and the ploidy of chromosome 21. We are currently evaluating hematopoietic differentiation potential of each clone and exploring the underlying pathophysiology of TMD/AMKL by using this platform. We believe that comprehensive understanding of TMD and AMKL pathogenesis provides a fruitful insight into our understanding of human leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Analysis of GATA1 mutations and leukemogenesis in newborns with Down syndrome

It has been reported that patients with Down syndrome (DS) frequently develop transient myeloproliferative disorder (TMD) and less commonly myeloid leukemia in DS (ML-DS). We examined the pathogenetic relationship of these conditions with somatic mutations of the GATA1 gene in children with both TMD and ML-DS. To determine the incidence of GATA1 mutations in a cohort of DS patients and the applicability of these mutations as a clonal marker to detect minimal residual disease, we screened 198 samples of 169 patients with DS for mutations in GATA1 exon 2 by direct sequencing. Novel mutations were detected in four of the 169 DS patients (2 with TMD and 2 with ML-DS). We examined spontaneous remission and response to therapy in TMD and ML-DS patients and concluded that these mutations can be used as stable markers in PCR analysis to monitor these events.

Hematopoiesis of Human Induced Pluripotent Stem Cells Derived From Patients with Down Syndrome

AbstractAbstract 881Trisomy 21, genetic hallmark of Down syndrome, is the most frequent human chromosomal abnormality. Infants and children with Down Syndrome (DS) are known to have some hematological disorders with an increased risk of developing leukemia. Ten to 20% of newborn with DS are diagnosed as neonatal preleukemic status, Transient Myeloproliferative Disorder (TMD), and approximately 30% of TMD patients are predisposed to acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in the N-terminal activation domain of the GATA1 gene, leading to expression of a shorter GATA1 isoform (GATA1s), have been reported in AMKL and TMD (Wechsler et al., 2002; Mundschau et al., 2003), but neither patients nor mice with germline mutations leading to expression of GATA1s developed AMKL and TMD in the absent of trisomy 21. These findings suggested that trisomy 21 itself directly contributes to the development of AMKL and TMD. However, the role of trisomy 21 in hematopoiesis, particularly in the human fetus remains poorly understood. To better understand the effects of trisomy 21 on hematopoiesis in embryonic stage and leukemogenesis, we employed human induced pluripotent stem cells (hiPSCs) derived from patients with DS (DS-hiPSCs). Six DS-hiPS and 5 hiPS cell lines (control) from healthy donors, which we used here, were all created from skin fibroblasts and reprogrammed by the defined 3 or 4 reprogramming factors (OCT3/4, KLF4, and SOX2, or c-MYC in addition to the 3 factors, respectively). We generated blood cells from DS-hiPSCs and controls with coculture system using murine aorta-gonad-mesonephros (AGM)-derived stromal cell line (Ma et al., 2009). The cells from hiPSCs were harvested at D11 or D12 of coculture and analyzed the presence of hematopoietic markers and the potentials of hematopoietic colony formation. In the experiments using hiPSCs reprogrammed by 3 factors, human CD34 expression in harvested cells from DS-hiPSCs or controls were detected 10.06 ± 4.35% and 3.04%, respectively. CD45 expression of CD34+ cells was small proportion in both DS-hiPSCs and controls. We next examined the hematopoietic colony formation. Both myeloid and erythroid colonies were detected. Number of colonies formed from DS-hiPSCs was 43.7±11.1 to 74.3±11.2 per an iPSC colony. It's approximately 2 to 3.5 folds numbers of control (p-value<0.05). Similar results were obtained in the experiments using hiPSCs reprogrammed by 4 factors. These results indicated that hiPSCs derived from patients with Down syndrome could differentiate into multiple hematopoietic cell lineages and the differentiation into hematopoietic lineage was promoted in DS patients. Further researches are under investigation to identify the responsible genes in trisomy 21 for acceleration of hematopoiesis with microarray analysis. Our study may contribute to understanding of the effects of trisomy 21 on hematopoiesis and effective use of patients derived hiPSCs in research and clinical application. Disclosures:No relevant conflicts of interest to declare.

Functional analysis of JAK3 mutations in transient myeloproliferative disorder and acute megakaryoblastic leukaemia accompanying Down syndrome

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS-AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS-AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient-derived JAK3 mutation (JAK3(I87T)), as well as two novel JAK3 mutations (JAK3(Q501H) and JAK3(R657Q)) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof-of-principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS-AMKL patients carrying activating JAK3 mutations.

Activating JAK3 Mutations in Transient Myeloproliferative Disorder and Acute Megakaryoblastic Leukemia Accompanying Down Syndrome.

Children with Down syndrome (DS) have an approximately 20-fold higher incidence of leukemia than the general population. The majority of leukemia cases associated with DS are acute megakaryoblastic leukemia (AMKL). Although GATA1 mutations have been found in almost all cases of transient myeloproliferative disorder (TMD) “a preleukemia” that may be present in as many as 10% of newborn infants with DS and AMKL accompanying DS (DS-AMKL), GATA1 mutation alone may not be sufficient for development of leukemia. Following identification of acquired activating JAK3 mutations in DS-AMKL, JAK3 mutations have been reported also in TMD patients. However, the frequency and functional consequence of JAK3 mutations in TMD remain unknown. To further understand how JAK3 mutations are involved in the development and/or progression of leukemia in DS, we screened TMD patients and two DS-AMKL cell lines for JAK3 mutation, and examined the functional consequences of these JAK3 mutations. In one out of the two DS-AMKL cell lines, MGS, we identified novel JAK3 mutations (JAK3Q501Hmutation in the SH2 domain and JAK3R657Qmutation in the psuedokinase domain in the same allele). JAK3Q501H and JAK3R657Q each constitutively phosphorylates STAT5 and transformes Ba/F3 cells to factor-independent growth, whereas the double mutant (JAK3Q501Hand JAK3R657Q) has more potent transforming activity than each mutant. Biochemichal analysis in Ba/F3 cells revealed that the degrees of phosphorylation of STAT5 in the cells transduced with each JAK3 mutant were correlated with its transforming activity. Although we previously identified a JAK3I87Tmutation in one of two TMD patients, no JAK3 mutations were detected in another 9 TMD patients. Together with the previous results, we found JAK3 mutations in each of 11 TMD and 11 DS-AMKL patients. Although the number of the patients analyzed was small, these results indicate that there are no significant differences in the frequency of JAK3 mutations between TMD and DS-AMKL. In this study, we showed for the first time that the TMD patient-derived JAK3 mutation was also an activating one. JAK3I87T transformed Ba/F3 cells to factor-independent growth. Treatment with JAK3 inhibitors (WHI-P131 and WHI-P154) resulted in a significant decrease in the growth and viability of Ba/F3 cells expressing each activating JAK3 mutant. These results suggest that the JAK3 activating mutation is an early event during the development of AMKL in DS. Furthermore these results provide a proof-of-principle that JAK3 inhibitor should have therapeutic effects on the AMKL and TMD patients carrying the activating JAK3 mutations.

JAK3, JAK2 and GATA1 Mutations in Childhood Acute Megakaryoblastic Leukemia with and without Down Syndrome.

Acute myeloid leukemia (AML) in children with Down syndrome (DS) exhibits unique characteristics, which includes the predominance of megakaryoblastic leukemia (AMKL). In about 10% of DS infants, transient myeloproliferative disorder (TMD) develops and then spontaneously resolves in most cases. However, 20% of TMD cases develop AMKL within 3 years, suggesting that additional genetic alterations might occur during the progression from TMD to AMKL. GATA1 mutations contribute to both the pathogenesis of TMD and AMKL, suggesting that the acquisition of additional genetic alterations might be necessary for the progression from TMD to AMKL. Recently, activating mutations of the JAK3 gene have been found in each of 16 non-DS and 3 DS-AMKL patients. These results suggest that the activating JAK3 mutations are involved in the pathogenesis of AMKL both with and without DS. In this study, we simultaneously analyzed JAK3, JAK2V617F which plays a critical role in the pathogenesis of myeloproliferative disorders and GATA1 mutations in non-DS children with AMKL and in DS children with AMKL or TMD. The study population included 11 non-DS children with AMKL and 20 AMKL and 6 TMD children with DS. After obtaining informed consent from the parents of the children for the purpose of sample banking and molecular analyses, bone marrow (BM) or peripheral blood (PB) samples were obtained from all of the patients with AMKL and TMD at the time of diagnosis. High-molecular weight DNA was extracted from the samples using standard methods. For the screening of the JAK3 mutations, we amplified genomic DNA that corresponded to all 23 exons of the JAK3 gene by using polymerase chain reaction (PCR) that employed 22 primer pairs. Amplified products were subjected to the denaturing HPLC (DHPLC) analysis using a WAVE Maker System (Transgenomic Inc., San Jose, CA). The DHPLC gradients and temperatures were determined using WAVE Maker System software. When heterozygous profiles were identified after a visual inspection of the chromatograms, amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced on a DNA sequencer (310; Applied Biosystems, Foster City, CA) using a BigDye terminator cycle sequencing kit (Applied Biosystems). For screening of GATA1 mutations and JAK2V617F mutations, we amplified the genomic DNA that corresponded to exon 2 of GATA1 and exon12 of JAK2 by using PCR that employed 1 primer pair respectively. Amplified products were cloned into the pGEM-T Easy vector and sequenced on a DNA sequencer using a BigDye terminator cycle sequencing kit. Mutations of JAK3 were found in 2 of the DS-AMKL patients and in 1 DS-TMD patient. A573V and A593T substitutions in the JH2 pseudokinase domain along with Q501H substitutions in the JH3 domain were observed in the DS-AMKL patients and an I87T substitution in the JH7 domain of the receptor-binding domain was observed in DS-TMD. In one of the DS-AMKL patients, A573V and A593T substitutions were found in the same allele. GATA1 mutations were observed in 19 of the 20 DS-AMKL patients, in all of the DS-TMD patients and in 1 non-DS-AMKL patient. JAK2V617F mutation was only found in 1 DS-AMKL patient who had also JAK3 mutation (Q501H). The fact that the JAK3 mutations were found in both the AMKL and TMD patients indicates that this is an early event that occurs during the development of AMKL in DS patients and that other genetic changes may contribute to the development of AMKL.

Distinct Clones are Associated with the Development of Transient Myeloproliferative Disorder and Acute Megakaryocytic Leukemia in a Patient with Down Syndrome

Children with Down syndrome (DS) have an approximately 20-fold higher incidence of leukemia than unaffected children, and most leukemia cases with DS present as acute megakaryocytic leukemia (AMKL). At least 10% of neonates with DS develop transient myeloproliferative disorder (TMD), and 20% to 30% of patients with TMD develop AMKL. Mutations in the GATA1 gene are identified not only in AMKL patients but also in TMD patients; however, sequential analysis of GATA1 is not often performed in the same patients. We describe a child with DS who developed TMD followed by AMKL and have identified different mutations in the GATA1 gene during the course of TMD and AMKL. Distinct clones were associated with the development of TMD and AMKL in this patient.

Down myeloid disorders: A paradigm for childhood preleukaemia and leukaemia and insights into normal megakaryopoiesis

Newborns and children with Down Syndrome are predisposed to a range of blood disorders, which include acute lymphoblastic leukaemia and acute megakaryocytic leukaemia (AMKL). Over the last four years there has been considerable progress in our understanding of DS AMKL. Like other childhood leukaemias DS AMKL is initiated in utero and can present in the neonatal period as a clinically overt preleukaemic condition, transient myeloproliferative disorder (TMD). In addition to trisomy 21, fetal haemopoietic progenitors acquire N-terminal truncating mutations in the key megakaryocyte-erythroid transcription factor GATA1. These are the minimum required events for TMD to develop. In ∼ 30% of TMD patients, additional as yet unidentified (epi)genetic mutations are required for progression to AMKL. Thus, DS TMD and AMKL provide a unique model of childhood leukaemia where the preleukaemic and leukaemic phases are ascertainable and separable allowing distinct steps in leukaemogenesis to be studied individually. These findings also have implications for the clinical management of DS TMD and AMKL specifically and also of childhood leukaemia more generally.

Mutagenesis of GATA1 is an initiating event in Down syndrome leukemogenesis

AbstractAs many as 10% of infants with Down syndrome (DS) present with transient myeloproliferative disorder (TMD) at or shortly after birth. TMD is characterized by an abundance of blasts within the peripheral blood and liver, and notably undergoes spontaneous remission in the majority of cases. TMD may be a precursor to acute megakaryoblastic leukemia (AMKL), with an estimated 30% of TMD patients developing AMKL within 3 years. We recently reported that mutations in the transcription factor GATA1 are associated with DS-AMKL. To determine whether the acquisition of GATA1 mutations is a late event restricted to acute leukemia, we analyzed GATA1 in DNA from TMD patients. Here we report that GATA1 is mutated in the TMD blasts from every infant examined. These results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD, and mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis.

High expression of platelet-derived growth factor and transforming growth factor-beta 1 in blast cells from patients with Down Syndrome suffering from transient myeloproliferative disorder and organ fibrosis.

To determine whether platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1) are involved in organ fibrosis in patients with transient myeloproliferative disorder (TMD) in Down syndrome, the expression of PDGF and TGF-beta 1 mRNA in blast cells of TMD was investigated using real-time quantitative reverse transcription polymerase chain reaction. Blasts and liver tissue from TMD patients with hepatic fibrosis showed a significantly elevated expression of PDGF gene. The expression of TGF-beta 1 gene was higher in TMD and acute megakaryoblastic leukaemia than in the control group. These results suggest that PDGF in combination with TGF-beta 1 plays a role in organ fibrosis of TMD.

Thrombopoietin level is inversely related to blast count, not platelet number, in Down syndrome neonates with transient myeloproliferative disorder

Transient myeloproliferative disorder (TMD) in neonates with Down syndrome is characterized by increased megakaryoblastic cells in the peripheral blood. Despite their spontaneous regression in weeks, prognosis is not always favorable because of fatal hepatic fibrosis. In this study, blood thrombopoietin (TPO) levels were measured by ELISA in six TMD patients and the expression of c-Mpl, a ligand for TPO, was examined on the blast cells from four patients by flow cytometer. At the onset, TPO level was undetectable in one patient and significantly lower in five patients than six neonatal controls (mean 0.52 fmol/ml, range 0.30–0.93 vs. 3.70, 1.38–8.33, P < 0.001), although platelet counts were similar (mean 321 × 109/l, range 42–1,040 vs. 253 × 109/l, 124–381). Two patients died of hepatic failure. TPO levels were measured in five patients after regression of the blast cells. With regression of blast cells, TPO levels were remarkably increased in four survived patients. In one patient with hepatic failure, TPO level was poorly elevated and relatively lower compared to the others. TPO levels were inversely correlated with blast numbers (r = −0.85, P < 0.001), but not with platelet counts (r = 0.426). Blast cells from four patients were all positive for c-Mpl. Our findings suggest that megakaryocyte mass is a major regulator of TPO levels and hepatic failure may affect the TPO level because liver is a major source of TPO production. Am. J. Hematol. 58:267–272, 1998. © 1998 Wiley-Liss, Inc.

Thrombopoietin level is inversely related to blast count, not platelet number, in Down syndrome neonates with transient myeloproliferative disorder.

Transient myeloproliferative disorder (TMD) in neonates with Down syndrome is characterized by increased megakaryoblastic cells in the peripheral blood. Despite their spontaneous regression in weeks, prognosis is not always favorable because of fatal hepatic fibrosis. In this study, blood thrombopoietin (TPO) levels were measured by ELISA in six TMD patients and the expression of c-Mpl, a ligand for TPO, was examined on the blast cells from four patients by flow cytometer. At the onset, TPO level was undetectable in one patient and significantly lower in five patients than six neonatal controls (mean 0.52 fmol/ml, range 0.30-0.93 vs. 3.70, 1.38-8.33, P < 0.001), although platelet counts were similar (mean 321 x 10(9)/l, range 42-1,040 vs. 253 x 10(9)/l, 124-381). Two patients died of hepatic failure. TPO levels were measured in five patients after regression of the blast cells. With regression of blast cells, TPO levels were remarkably increased in four survived patients. In one patient with hepatic failure, TPO level was poorly elevated and relatively lower compared to the others. TPO levels were inversely correlated with blast numbers (r = -0.85, P < 0.001), but not with platelet counts (r = 0.426). Blast cells from four patients were all positive for c-Mpl. Our findings suggest that megakaryocyte mass is a major regulator of TPO levels and hepatic failure may affect the TPO level because liver is a major source of TPO production.

Ultrastructural and ultracytochemical differences between transient myeloproliferative disorder and megakaryoblastic leukaemia in Down's syndrome

Ultrastructural and ultracytochemical studies were performed on blast cells from 12 Down's syndrome neonates with transient myeloproliferative disorder (TMD) and 13 Down's syndrome patients with megakaryoblastic leukaemia (MKL), in order to clarify the cytological characteristics of these cells. Average platelet peroxidase-positivity in blast cells of TMD patients was similar to that found in cases of MKL. Blast cells from subjects with TMD contained a number of different granules, namely, alpha granules, those that were myeloperoxidase (MPO)-positive, electron-lucent or basophil-like, and those containing membrane components or ferritin particles. On the other hand, granules found in the blast cells of MKL patients with Down's syndrome included the electron-lucent variety, those with membrane components and a few that were basophil-like, but not alpha and MPO-positive granules nor those containing ferritin particles. A demarcation membrane system was observed in blasts from the TMD group, but not in the MKL group. These findings suggest that blast cells in TMD patients differentiate to megakaryocytes, neutrophils, basphils and erythroblasts, while those in cases of MKL show limited differentiation to immature megakaryocytes, erythroblasts and, sometime, basophils. Such results correspond well with those of culture studies, in which TMD blasts were found to be precursors of various types of blood cells.