Down Syndrome Leukemia Research Articles

Transient Leukemia in Down Syndrome: Report of Two Cases with Review of Literature

Transient leukemia (TL) also referred to as transient abnormal myelopoiesis (TAM) or transient myeloproliferative disorder (TMD) is a unique syndrome that frequently occurs in newborns with Down syndrome (DS). It manifests in the first few days of life and shows leukocytosis with blast cells in the blood and bone marrow. This leukemia resolves spontaneously within first few months of life in the majority of cases. In this report we describe two newborns with a karyotype of 47,XY,+21, presented with marked leukocytosis and many blast cells in the peripheral blood. In both the cases, the blasts disappeared and the total leukocyte count reverted to normal without any specific treatment.

Retrospective analysis of risk factors for development of liver dysfunction in transient leukemia of Down syndrome

About 20% of patients with transient leukemia (TL), which is a disease noted in Down syndrome, are reported to develop hepatic fibrosis, which has a poor prognosis. The clinical factors related to the poor prognosis of TL were retrospectively analyzed in 25 patients, and criteria for starting chemotherapy were established. The initiation of chemotherapy was recommended when two or more of the following categories were fulfilled in the process of the disease: (1) a reduced hepatic functional reserve estimated by direct bilirubin, prothrombin time, and the presence of ascites, (2) an elevated level of hyaluronic acid (>500 U/mL), (3) respiratory failure or poor sucking associated with hepatosplenomegaly, and (4) demonstration of fibrosis by liver biopsy. When these criteria were applied to our cases, all patients who received chemotherapy remained alive. Our criteria are useful for selecting patients with TL at high risk of developing hepatic fibrosis and for starting chemotherapy.

ダウン症候群に合併した急性巨核球性白血病 (AMkL) の治療成績

ダウン症候群に合併した急性巨核球性白血病 (AMkL) の治療成績

Myeloid Leukemia in Down Syndrome

Although adults with Down syndrome (DS) show a decreased incidence of cancer compared to individuals without DS, children with DS are at an increased risk of leukemia. Nearly half of these childhood leukemias are classified as acute megakaryoblastic leukemia (AMKL), a relatively rare subtype of acute myeloid leukemia (AML). Here, we summarize the clinical features of myeloid leukemia in DS, review recent research on the mechanisms of leukemogenesis, including the roles of GATA1 mutations and trisomy 21, and discuss treatment strategies. Given that trisomy 21 is a relatively common event in hematologic malignancies, greater knowledge of how the genes on chromosome 21 contribute to DS-AMKL will increase our understanding of a broader class of patients with leukemia.

Down syndrome and GATA1 mutations in transient abnormal myeloproliferative disorder: mutation classes correlate with progression to myeloid leukemia

AbstractTwenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

Myeloid Leukemia of Down Syndrome: The Results of An International Retrospective Study

AbstractAbstract 2718Children with Down Syndrome (DS) have an increased risk of developing acute myeloid leukemia. Myeloid leukemia of Down Syndrome (ML-DS) has a better prognosis than sporadic pediatric AML, using reduced-intensity treatment protocols as the leukemic cells of these patients are relatively sensitive to chemotherapy. Moreover, ML-DS patients have an increased risk of side-effects, hence there is a delicate balance between anti-leukemic efficacy and treatment related mortality.So far, no prognostic factors in ML-DS have been identified that allow a risk-stratified approach in future ML-DS treatment protocols. The majority of the ML-DS cases are characterized by additional karyotypic changes in their leukemic cells besides the constitutional trisomy 21 (Forestier et al, Blood 2008), but the potential prognostic impact of these cytogenetic abnormalities is not known. We therefore conducted a large international retrospective study and collected clinical and cytogenetic data of 451 children with ML-DS from 13 collaborative study groups. Patients were eligible between 6 months and up to 5 years of age, and were diagnosed between Jan 1, 1995 and Jan 1, 2005. Patients who were not treated with curative intent were excluded. All karyotypes were centrally reviewed before assigning patients to cytogenetic groups.The Kaplan-Meier method was used to estimate the 5-years OS and 5-years EFS, and outcome estimates were compared using the log-rank test. Cumulative incidence functions of relapse (CIR) were compared by the Gray test. For multivariate analysis, the Cox proportional-hazard regression model was used.The median age of all ML-DS patients (n= 451) was 1.8 years (range 0.5 – 5.0) and the median white blood cell count (WBC) was 7.0 × 109/l (range 0.8 – 290). The overall 5-years EFS was 78%, the 5-years OS 79%, the 5-years CIR 8%, and treatment related mortality (TRM) was 11%. The main groups were the patients with a normal karyotype (NK) (n= 103; 29%), trisomy 8 (n= 63; 18%) and 'other’ patients (n= 187; 53%). Outcome estimates showed large differences across these different cytogenetic groups.There were no significant differences in age between the NK, trisomy 8, and the other patients. However, trisomy 8 patients did have a significantly lower median WBC (6.3 × 109/l; P25=4.0 – P75=10.3) compared to both other groups (7.2 × 109/l; P25=4.6 – P75=16.7); P= 0.03). The NK patients had a significantly worse 5-years OS, EFS, and CIR compared to the other patients and the trisomy 8 group (OS 69% vs. 83% vs. 90%; P= 0.002 and 0.006; EFS 68% vs. 81% vs. 88%; P= 0.006 and P= 0.005; and CIR 19% vs. 9% vs. 5%; overall p(Gray)= 0.002). TRM was highest in the NK patients (13%). In addition, patients with a loss of 7q/monosomy 7 (n=16) had a 5-years EFS of 69%, which is remarkably high compared to non-DS AML (39%) (Hasle et al, Blood 2007).Furthermore, younger patients, age < 3 years showed a significantly better 5-years EFS and CIR than patients aged ≥ 3 years (EFS 79% vs. 65%; P = 0.04; CIR 9% vs. 21%; P = 0.03). OS was not significantly different (80% vs. 69%; P = 0.10). Patients with a WBC < 20 × 109/l had a borderline significantly better 5-years EFS and CIR than patients with a WBC ≥ 20 × 109/l (EFS 80% vs. 70%; P = 0.05, CIR 9% vs. 16%; P = 0.05). OS was not significantly different. Multivariate analysis revealed NK patients (hazard ratio [HR] = 1.82, P = 0.027), WBC ≥ 20 × 109/l (HR = 1.77, P = 0.029) and age > 3 years (HR = 2.37, P = 0.005) as independent predictors for poor EFS. The trisomy 8 group was not independently associated with a favorable outcome, which may be explained by the fact that the WBC of these patients was significantly lower and hence that there is interference between these 2 characteristics.In conclusion, this study shows independent risk-groups within ML-DS that may benefit from stratified therapy. It has to be mentioned that patients were treated on different treatment protocols, which were sometimes adjusted for DS. As NK ML-DS patients have a higher CIR than TRM, therapy reduction is not an option for this group. To improve the prognosis of these patients, the biological background has to be investigated. As known in non-DS AML normal karyotype patients, mutations in WT1 and FLT3 are associated with poor outcome. FLT3 mutations are not found in ML-DS patients and as WT1 mutations have not yet been well studied in this group, this may be subject of further research. Disclosures:No relevant conflicts of interest to declare.

Good General Health In Survivors of Childhood AML Treated with Chemotherapy Only: A NOPHO-AML Study.

AbstractAbstract 1076 Background.More than 60% of children with acute myeloid leukemia (AML) become long-term survivors and most are cured with chemotherapy only. With this improvement, the late mortality and morbidity after the very intensive treatment deserve attention. Previous studies have reported late effects after childhood AML, but most side effects were probably caused by cranial irradiation or hematopoietic stem cell transplantation (HSCT). Limited data exist about the long-term mortality, morbidity, and social outcomes in AML survivors treated with chemotherapy only. The objectives of this study were to report the occurrence of late mortality and compare the self-reported health care use and health experience, lifestyle behavior, and social outcomes of AML survivors with those of their sibling controls. Methods.Children treated for AML according to the Nordic protocols NOPHO-AML-84, -88, and -93, and alive by 30 June 2007 were identified in the NOPHO-AML database. We excluded patients with myeloid leukemia of Down syndrome, Fanconi anemia, preceding myelodysplastic syndrome, therapy-related AML, patients receiving allogeneic or autologous HSCT, and patients who had experienced a relapse or had a secondary malignancy. A total of 102 (74%) of the 138 eligible AML survivors accepted the invitation for a physical and paraclinical examination and completed a questionnaire concerning health and social status. One sibling per patient was invited to complete a similar questionnaire which was accepted by 85 (91%) of 93 eligible siblings. Results.The median follow-up was 11 (range 4–25) years after diagnosis. Five-year AML survivors' overall survival rate was 94% at 20 years after diagnosis (95% confidence interval; 84–98%). Four children died more than five years after end of treatment; one from sequelae of initial cerebral bleeding, two from side effects of treatment including one with hearth failure, and one from secondary malignancy. AML survivors had no increased rate of hospitalization compared with sibling controls but more received prescription drugs, especially for asthma (23% vs. 9%, p=0.03). Self-reported health experience was excellent or very good in 77% and comparable with that of siblings (Table). The rate of current smokers was similar in the two groups (23% vs. 24%). Sixty-seven percent of the AML survivors >20 years of age and 73% of their siblings reported to be undertaking or having completed an education, defined as vocational training or academic education lasting at least three years (p=0.8). Among those >20 years of age, the frequency of marriage or cohabitation did not differ between AML survivors and siblings (39% vs. 37%). Conclusions.Children treated on NOPHO-AML protocols without HSCT and surviving at least five years after diagnosis had a very low mortality rate. Their self-reported health was good, and health care use was limited and comparable to that of sibling controls, but they were more often receiving prescription drugs for minor illnesses. Most AML survivors seemed to have coped well with their transition into adulthood, and they reported educational achievement, employment and marital status comparable to those of their sibling controls.TableSelf-reported health in AML survivors (age >10 years) and their siblings.Health statusAML survivorsSiblingsn=86%n=71%Excellent/very good66775577Good12141217Fair/poor8946 Disclosures:No relevant conflicts of interest to declare.

Liver Fibrosis In Myeloid Leukemia of Down Syndrome

AbstractAbstract 874 Background.Fibrosis is a common complication of leukemia with a megakaryoblastic blast phenotype. Children with Down syndrome (DS) have an increased risk for two hematological disorders with a predominantly megakaryoblastic phenotype. First, approximately 10% of newborns with DS develop Transient Leukemia (TL; also termed Transient Myeloproliferative disorder, TMD), which is self-resolving in the majority but associated with life-threatening complications in approximately 15% of cases. Liver fibrosis and failure are predominant causes of fatal outcomes in these infants. Second, children with DS develop acute myeloid leukemia, predominantly with a megakaryoblastic phenotype, approximately 150-times more often than the general pediatric population during the first 4 years of life. Somatic mutations of GATA1 are specific for TL/TMD and myeloid leukemia of DS. We found significant liver fibrosis in NOD/SCID recipients of human DS myeloid cells. We are using this model to define the mechanisms that control the development of this potentially life-threatening complication. Methods.Recipients were engrafted with primary human DS myeloid leukemia cells and cells of the human DS myeloid leukemia cell line GRW (established at our institution). U937 human monocytic leukemia cells, which lack trisomy 21, were used in control experiments. Cells were injected into the right femur of 8-week-old irradiated NOD/SCID mice, which had also been injected with anti-NK (anti-CD122) antibody. Phenotypic analysis at 4 to 6 weeks after transplantation used standard cytological, histological and flowcytometric methods (CD45, CD61 and CD34 antibodies were obtained from B&D). Affymetrix human Gene ST1.0 expression arrays were used to compare GRW and U937 cells. Specific gene expression was quantified by TaqMan real time RT-PCR relative to b-actin (Applied Biosystems). Results.Organ fibrosis in recipients of DS myeloid leukemia cells. Primary cells from patients with DS myeloid leukemia engrafted murine recipients and caused moderate bone marrow fibrosis. Transplantation of GRW human DS myeloid cells resulted in significant infiltration of recipient liver and bone marrow (33±10% of mononuclear cells, n=6). The frequency of the leukemia-initiating cells was 1/87,448 by limiting dilution. Marked fibrosis of the liver developed within 6 weeks of transplantation. Fibrosis-associated gene expression in DS myeloid leukemia cells. DS myeloid leukemia cells (GRW) expressed significantly more PDGFD and FGF17 transcripts than non-DS myeloid leukemia control cells (U937). PDGFD transcripts were 4.3-fold more abundant in GRW cells than U937 cells (p=2.67 × 10-7; n=4). FGF17 expression levels were moderately increased (1.24, p=0.07). Quantitative real time RT-PCR analysis confirmed expression of PDGFD in DS myeloid leukemia cells (GRW) (5.55 × 10-4PDFGD/b-actin transcripts; n=4) and did not detect measurable expression in control (U937) cells. FGF17 transcripts were approximately 100-fold more abundant in DS myeloid leukemia cells (GRW) cells compared to U937 controls (4.85 × 10-4 and 4.91 × 10-6 for FGF17/b-actin transcripts, n=4). Analysis of fibrosis-associated gene expression in recipient (murine) liver cells in response to transplanted DS myeloid leukemia cells (GRW) and functional analysis of the role of PDGFD and FGF17 expression in DS myeloid leukemia cells is underway. Conclusion.Organ fibrosis is a significant cause of mortality of children with DS and megakaryoblastic disorders, particularly during the newborn period. Chemotherapy frequently is unable to achieve a timely reversal of this process. Better understanding of the mechanisms controlling the development of organ fibrosis is required to develop better anti-fibrotic intervention. The observed phenotype of marked liver fibrosis in murine recipients of the human DS myeloid leukemia cell line GRW provides an experimental tool to determine the components of the fibrogenic process both in infiltrating leukemia cells and recipient liver tissue. PDGFD and FGF17 expression by DS myeloid leukemia cells has been identified as possible inducer of organ fibrosis. The model can now be used to define tissue responses to infiltrating human DS myeloid leukemia cells in liver tissue of recipients and to determine the functional role of candidate genes in DS myeloid leukemia cells by altering expression levels prior to transplantation. Disclosures:No relevant conflicts of interest to declare.

Análisis de GATA1 en los trastornos mieloproliferativos asociados a la trisomía 21

Introduction Neonatal transient myeloproliferative disorder and acute megakaryoblastic leukaemia of Down syndrome are considered different manifestations of the same disease. In most cases, transient myeloproliferative disorders require no treatment, while acute megakaryoblastic leukaemia of Down's syndrome is characterised by an increased sensitivity to chemotherapy and its treatment should be adapted with a reduction in dose intensity. Both entities share specific mutations at exón 2 of the transcription factor GATA1. Patients and methods We analysed biological features and GATA1 mutations in 4 patients with transient abnormal myelopoiesis (2) and acute megakaryoblastic leukaemia (2) including one phenotypically normal trisomy 21 mosaicism. We found abnormal GATA1 mutated clones in each case, and a specific point mutation at exón 2 was detected in three cases. Given the heterogeneous phenotype of megakaryoblastic blasts and the low percentage of blasts at presentation, the recognition of GATA1 mutations was helpful for diagnosis. In addition, molecular remission was established in 2 patients after subsequent normal mutational GATA1 analysis. Conclusions We conclude that GATA1 mutational study is a useful tool for the diagnosis and management of trisomy 21 associated myeloproliferative disorders.

High Frequency of Copy Number Variations in Myeloid Leukemia of Down Syndrome.

Abstract 3242Poster Board III-179Pediatric acute myeloid leukemia (AML) is a heterogenous disease, with current therapy resulting in cure rates of approximately 60%. The prognosis depends on cytogenetics and early response to therapy. Recently, Radtke et al. (PNAS 2009) reported that (by using single-nucleotide-polymorphism microarrays and candidate resequencing) de novo AML is characterized by a low frequency of copy number variations (CNVs), with a mean of 2.38 somatic CNVs per case. This is in contrast to pediatric acute lymphoblastic leukemia (ALL), with an average of 6 per case (Mullighan et al, Leukemia 2009). The only exception in AML was acute megakaryocytic leukaemia (AMKL, n=9 cases), which is characterized by a high number of CNVs (average of 9.33). Of these 9 AMKL cases, only one patient had a GATA1-mutation, typical of Myeloid Leukemia in Down syndrome (ML-DS). Most ML-DS cases are classified as AMKL, and can be preceded by a pre-leukemic clone in newborns (transient leukaemia- TL), which in approximately 80% resolves spontaneously. Treatment outcome for non Down syndrome AMKL is poor, whereas Down AMKL has a good prognosis.We performed array-based comparative genomic hybridization (Array-CGH) to detect copy number variations (CNV) in Down syndrome related leukemias, with the aim to study whether progression from TL to ML-DS is associated with clonal evolution. We also compared the data obtained with array-CGH from DS patients with two other subgroups of non-DS pediatric AML, i.e. MLL-rearranged AML, and cytogenetically normal AML (CN-AML). This may provide insight in differences in genomic stability between these subgroups. For this analysis, 66 MLL-rearranged AML, 41 CN-AML, 7 TL and 14 ML-DS samples taken at initial diagnosis were available. TL and ML-DS samples were unpaired. Genomic aberrations were scored as a minimum of three (44K arrays, Agilent) and as a minimum of five (105K arrays, Agilent) adjacent probes deviating beyond the threshold of ∼0.5. Results are given in table 1 and 2 below.Copy number variations in different subgroups of pediatric AML in percentages of the total number of samplesSubgroupAmplificationDeletionTotal CNVMLL-rearranged AML n = 6638%55%71%CN-AML n =417%34%37%TL n = 729%57%57%ML-DS n =1464%93%100%Copy number variations in different subgroups of pediatric AML, expressed per caseSubgroupAmplificationDeletionTotal CNVMLL-rearranged AML n = 660.670.641.36CN-AML n =410.070.460.54TL n = 70.432.002.43ML-DS n =141.862.934.79ML-DS patients had a significantly higher frequency of samples with CNVs compared to TL-patients (100% vs. 57%; p=0.026). Most amplifications were (sub)chromosomal (e.g. extra copies of chromosome 8, 11 and 21), whereas the deletions were mostly focal. Except for the (sub)chromosomal amplifications, the other aberrations were mostly found in single cases. Overall, deletions were more common than amplifications. When we analyzed the number of CNVs per case, ML-DS patients had a trend to have a higher number of CNVs per patient (p=0.06) than TL-patients. When the TL and ML-DS patients were taken together and compared to the other non-DS AML-subgroups taken together, there was a significantly higher number of patients with copy variations (86% vs. 58%; p=0.013) in the Down leukemia group. Also, the number of CNV per case was significantly higher in the Down syndrome patients compared to the non-DS AML subgroups (p=< 0.001).In conclusion, we observed significantly less patients with CNVs and less CNVs per patient in MLL- rearranged and CN-AML compared to Down syndrome related leukemia. This suggests that the Down syndrome related leukemias are more genetically unstable when compared with their non-DS controls. Our data are consistent with the findings in AMKL in non-Down syndrome patients as reported by Radtke et al, and may suggest that genomic instability is a general phenomenon of AMKL. This is noteworthy because ML-DS and non-DS AMKL differ in various other aspects. The mechanisms behind the genetically more instable character of the AMKL subgroup is still unclear, and requires further study. Within the Down syndrome patients, there was a statistically significant increase in patients with CNVs in ML-DS compared to TL, and a trend for more CNVs per patient, suggestive of clonal evolution. DisclosuresNo relevant conflicts of interest to declare.

Is Prevention of Myeloid Leukemia Possible?.

Abstract 481Childhood leukemia frequently originates prenatally. Only a small percentage (<1%) of children with recurrent leukemia associated aberrations detected at birth suffer leukemia later on. In addition, no option to treat the preleukemic clone is availabel. Therefore, neither general screening at birth is useful nor preemptive treatment is possible.The high incidence (5 to 10%) of the transient leukemia (TL) in newborns with trisomy 21 and the high risk to develop a myeloid leukemia of Down Syndrome (ML-DS) within the first 4 years of life supported the hypothesis that the elimination of the preleukemic GATA1 positive clone might prevent leukemia. Prerequisites are the high sensitivity of TL-blasts to cytarabine, the recurrence of the same GATA1-mutated clone and the feasibility to monitor the preleukemic clone.Since 4/2007 69 children with TL were enrolled the study “Prevention of Myeloid Leukemia in Children with Down Syndrome and Transient Leukemia” (EudraCT 2006-002962-20) ; Germany n=50, The Netherlands n=16, Slovakia n=1, Czech Republic n=2).Inclusion criteria were met by 52 children (study patients), 17 children were observed only (protocol patients). Table 1 summarizes the patients' characteristics. The TL and ML-DS specific mutations of the transcriptions factor GATA1 have been detected in 60 children (87%), failure of detection were caused by low percentage of blasts (<2%) combined with late diagnosis (≥20 days after birth). The median follow-up within the study group was 1 year (0.2 to 2.3 years). [Display omitted] Totally, 58 % of the children showed clinical symptoms associated to the TL, severe complications have been reported in 22 children (table 2). According to the study guidelines 20 out of these 22 children were treated with low dose cytarabine (1.5mg/kg body weight 1 week). [Display omitted] Enrollment to the study including reference diagnostics and consulting, and a consequent treatment seems to improve the prognosis of this particular group. Compared to the historical group of children with similar characteristics (Klusmann et al. Blood 111(6):2991-8, 2008), the overall survival (2 years) significantly increased from 55±7% to 84±8%, p=0.03.MRD diagnostics by qRT-PCR and/or immunophenotyping was performed in 53 children (77%). Reasons for failure were early deaths (n=9; cardiac defects n=1, prematurity/MOV n=7, liver fibrosis n=1), refusal of monitoring by the parents (n=3), lack of material (n=4). If the MRD-level at week 8 and/or 10 exceeded 10-3 (qRT-PCR) or 10-2 (immunophenotyping), respectively, intervention with low-cytarabine was recommended. Currently, 39 children were already analyzed at week 12 (1st endpoint).In 7 children (13%) treatment recommendation according to high MRD levels were given. With exception of transient myelosuppression (CTC Grade II) no severe side effects occurred. All children but two became MRD negative at week 12. To date one child with persistent detectable MRD levels suffered ML-DS (1 year after TL).In summary, participating in the study and treatment of children with TL causing severe clinical symptoms seems to improve the prognosis.Although the recruitment into the study is faster than expected and the results to date are promising, the follow-up is much too short to draw definitive conclusion. Disclosures:No relevant conflicts of interest to declare.

Overexpression of GATA1 Confers Chemotherapy Resistance in Pediatric Acute Megakaryocytic Leukemia.

Abstract 2039Poster Board II-16Acute megakaryocytic leukemia (AMkL; M7) is a biologically heterogeneous form of AML, representing ∼10% of pediatric and 1-2% of adult AML cases. AMkL is the most common AML subtype of children with Down syndrome (DS). DS children with AMkL have an excellent prognosis with EFS rates of 80-100% when treated with ara-C/anthracycline-based protocols, in contrast to the <30% EFS rates of non-DS children with AMkL. This also contrasts to the ∼50% EFS rates of non-DS children with AML overall, indicating that AMkL is an extremely poor risk group amongst non-DS children with AML despite the use of intensive chemotherapy-based protocols. These clinical data make a compelling argument that new therapies are essential to improve the treatment outcome of this aggressive disease. Acquired somatic mutations of the transcription factor gene, GATA1 (localized to Xp11.23), have been detected uniformly in nearly all DS AMkL cases, but not in non-DS AML and non-AMkL DS leukemia cases. The net effect of GATA1 mutations is an introduction of early stop codons and synthesis of a shorter GATA1 protein (designated GATA1s) that has altered transactivation activity, potentially contributing to the uncontrolled proliferation of immature megakaryocytes. It is conceivable that the altered GATA1 function between DS and non-DS AMkL may account for differential expression of GATA1 target genes in these two groups of patients. On the other hand, overexpression of GATA1 in megakaryoblasts from non-DS children with AMkL compared to myeloblasts from non-DS children with other subtypes of AML may contribute to differences in chemotherapy sensitivity via regulation of GATA1 target genes. We previously reported that GATA1 mutations in DS AMkL are associated with decreased expression of cytidine deaminase (encodes an enzyme which can convert ara-C to ara-U, the inactive form of the drug), thus contributing to the enhanced ara-C sensitivity of DS AMkL blasts. Further, when GATA1 was ectopically expressed in a DS AMkL cell line, CMK, it caused significantly increased resistance to ara-C. In the present study, we confirmed overexpression of GATA1 in non-DS AMkL blasts compared to non-DS AML blasts by real-time RT-PCR quantitation of GATA1 transcripts in our cohort of patient samples. shRNA knockdown of GATA1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivities to ara-C and daunorubicin, the two main drugs used for AML treatment, and significantly increased basal level apoptosis. This was accompanied by significantly decreased Bcl-xL transcript and protein levels in the GATA1 shRNA knockdown clones compared to a shRNA negative control. Binding of GATA1 to the two GATA elements in Bcl-x promoter and transactivation of Bcl-x promoter activity by GATA1 was demonstrated by ChIP assays and luciferase reporter assays, respectively, in Meg-01 cells. In our cohort of non-DS AMkL and AML patient samples, significant overexpression of Bcl-xL in non-DS AMkL compared to non-DS AML cases and a significant correlation between Bcl-xL and GATA1 transcripts were detected. Besides Bcl-xL, additional GATA1 targets (e.g. TNF) related to apoptosis were also identified by gene expression and ChIP-on-ChIP microarray analyses. Interestingly, our microarray data also suggest that GATA1 may have an impact on PI3-kinase/Akt pathway through modulating directly or indirectly a group of genes within the pathway. Western blotting revealed increased phosphorylation of Akt in the GATA1 knockdown clones compared to the negative control cells. Previous studies reported that histone deacetylase inhibitors (HDACIs) treatment causes hyperacetylation and subsequent degradation of GATA1, suggesting that these agents may be effective in targeting GATA1 in AMkL. Treatment of Meg-01 cells with an HDACI, valproic acid (VPA), resulted in decreased protein levels for GATA1 and Bcl-xL and increased phosphorylation of Akt. Co-treatment of Meg-01 cells with VPA and ara-C resulted in synergistic induction of apoptosis and activation of caspase-3. This drug synergy was amplified when a non-toxic dose of the PI3-kinase inhibitor LY294002 was added. Our results demonstrate that GATA1 causes resistance to chemotherapy in non-DS AMkL by promoting AMkL blast survival through regulating its target genes. Treatment of AMkL may be improved by integrating HDACI and PI3-kinase or Akt inhibitors into the chemotherapy of this disease. Disclosures:No relevant conflicts of interest to declare.

A Comparison of Acute Lymphoblastic Leukemia in Down Syndrome and Non— Down Syndrome Children: The Role of Trisomy 21

Down syndrome (DS), which occurs once in every 800 births, is associated with a trisomy on locus 21. Among the many aberrations caused by DS, including shortened stature and distorted facies, are several blood dyscrasias, including childhood leukemias-namely, acute myeloid leukemia (AML) and acute lymphoblastic, or lymphocytic, leukemia (ALL). One focus of the diagnosis of ALL is to distinguish it from AML.The benefits of immunophenotyping extend to treatment as well. ALL is associated with an inherited trisomy 21 in DS children (ALL-DS) and with acquired trisomies, +21, 8, and 13, in non-DS children (ALL-NDS). The differences in treatment, outcome, and prognosis between ALL-DS and ALL-NDS can be attributed to the interaction of their respective trisomies with several genetic mutations, including one on the GATA1 growth factor transcription gene. Other mutations are the gene fusion at TEL/AML1, and a new mutation found, which labels the Janus Kinase gene or JAK2 as on oncogenic precursor, which when associated with the B-cell precursor gene or BCP is highly leukomogenic. The treatments for the 2 groups have been based on quality of risk, with ALL-DS children having the highest risk along with the poorest prognosis, but alterations in medication regimens have brought treatment outcomes to near equality. It is worthwhile to study the trisomy 21 because in the future it may provide an understanding of all blood dyscrasias.

Breastfeeding and early infection in the aetiology of childhood leukaemia in Down syndrome

Background:For a child to develop acute leukaemia (AL), environmental exposure may not be sufficient: interaction with a susceptibility factor to the disease, such as Down syndrome (DS), may also be necessary. We assessed whether breastfeeding and early infection were associated with the risk of developing AL in children with DS.Methods:Children with DS in Mexico City, and either with or without AL, were the cases (N=57) and controls (N=218), respectively. Population was divided in children with AL and with acute lymphoblastic leukaemia (ALL) and also in children ⩽6 and >6 years old.Results:Breastfeeding and early infections showed moderate (but not significant) association for AL, whereas hospitalisation by infection during the first year of life increased the risk: odds ratios (confidence interval 95%) were 0.84 (0.43–1.61), 1.70 (0.82–3.52); and 3.57 (1.59–8.05), respectively. A similar result was obtained when only ALL was analysed.Conclusion:We found that breastfeeding was a protective factor for developing AL and ALL, and during the first year of life, infections requiring hospitalisation were related to a risk for developing the disease in those children with DS >6 years of age. These data do not support the Greaves's hypothesis of early infection being protective for developing ALL.

Sustained Remission Achieved With ATRA and Chemotherapy in Second Relapse of Acute Promyelocytic Leukemia in Down Syndrome

Children with Down syndrome (DS) are at an increased risk of developing acute leukemia. Acute myeloid leukemia predominates among DS children below 4 years of age but acute promyelocytic leukemia (APL) has rarely been reported in DS. Acute myeloid leukemia in DS is extremely sensitive to treatment but the optimum treatment of de novo or relapsed APL in DS is not known. We describe a child with DS and APL, who despite having a multiply relapsing course, achieved a third remission with ATRA and chemotherapy, which is sustained with maintenance therapy. A brief review of literature is also presented.

Cross-talk of GATA-1 and P-TEFb in megakaryocyte differentiation

AbstractThe transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclin-dependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy.

GATA1 Mutations in Transient Leukemia and Myeloid Leukemia in “Down” Syndrome.

Introduction: Newborns with trisomy 21 have a 5 to 10% risk to develop transient leukemia (TL). More than 20% of these infants progress to myeloid leukemia of Down syndrome (ML-DS) within the first 4 years of life. Mutations of the hematopoietic transcription factor GATA1 have been identified in almost all patients with TL and ML-DS. Here we report the biological and follow up data of a large cohort of children with proven GATA1 mutation reported to date either as TL (n=43) or ML-DS (n=28).Results:GATA1 mutations (point mutations, insertion, deletion, duplication; including 45 mutations not yet published) were identified in 42/43 TL (98%) and in 23/28 ML-DS (83%) patients.nage madiangastation weekWBC/μlblasts %outcometransient leukemia433 days (0 to 57)37 (31 to 40)33450 (1000 to 321000)45 (7 to 91)death n = 3, 7% ML-DS n = 9, 22%ML-DS281-3 yrs (0.8 to 3)38 (37 to 38)4900 (1000 to 160000)PB1 7(1-87) BM1 24 (4-78)death n = 2, 7% relapse n=01PB: peripheral blood; BM: bone marrowIn 9 patients multiple mutations were noted in the same clone as confirmed by subcloning. In one patient with TL two different GATA1 mutations were detected in two independent clones. When this patient progressed to ML-DS only the minor clone was present. The majority of the mutations was localized in exon 2 (n=59). Only a few mutations could be found in intron 1 and 2 (n=5) or in exon 3 (n=1). As a result, these mutations led to the introduction of a premature stop codon within exon 2 (n=40), frameshift (n=14), altered splicing (n=7), or lack of an initiation codon (n=4). Interestingly, children with TL and splicing mutations were significantly older at diagnosis than patients with other mutations (day 38 vs. day 3 p <0.05). No differences between mutational types were evident regarding gestational age, white blood cell count, platelet count, hemoglobin levels, or risk of death or ML-DS.In children with a myeloproliferative disease (MPD; n=7) or acute megakaryoblastic leukemia (AMKL; n=1) without stigmata of Down syndrome, GATA1 mutations could be detected. All of them were diagnosed as trisomy 21 mosaic. In this group the frequency of frameshift and altered splice mutations (5/7 vs. 9/36) was significantly higher compared to those with premature stop codons (2/7 vs. 27/36); pFishers exact =0.03).In 20 children (TL n=13, ML-DS n=7) the GATA1 mutant clone has been prospectively monitored by quantitative PCR using patient specific TaqMan probes. Seventeen TL patients showed decreasing minimal residual disease (MRD) levels and became negative (<10−4) during follow-up, whereas three children, who later developed ML-DS, remained positive at all time points. After two treatment elements all ML-DS patients had undetectable levels of GATA1s. After a median follow up of 1.5 years (0.9 to 2 years), no child suffered relapse however, the follow-up is much too short to draw definitive conclusions.Conclusion: In conclusion, we confirmed the high frequency of GATA1 mutations in children with TL or ML-DS. The occurrence of splicing mutations correlated with the age at diagnosis underlining the biologic relevance of the kind of mutation. We demonstrated the feasibility of a leukemia specific monitoring of MRD. As those children with sustaining detectable levels of GATA1s progressed to leukemia, these results might have therapeutic consequences for TL and later for ML-DS. In addition it may serve as a proof of principle for the feasibility of MRD monitoring in other AML-associated mutations. The identification of GATA1s positive MPD and AMKL in children without obvious stigmata of Down syndrome, all confirmed as trisomy 21 mosaic, implicate the necessity of GATA1s diagnostics in all newborn and infants with megakaryoblastic leukemia.

Epidemiology, classification and prognosis of adults and children with myelodysplastic syndromes

Myelodysplastic syndromes (MDS) belong to the most frequent bone marrow diseases with a crude incidence of about 4 in 100,000 per year. The diagnosis of MDS still is mainly based on morphologic findings in blood and marrow. The new WHO classification system takes into account the medullary and peripheral blast count as well as the degree of dysplasia in the different cell lines. To correctly identify MDS types, cytogenetic evaluation is of importance, as the WHO classification introduced the entity MDS with del(5q), which is characterized by special morphologic and hematologic features. The separation of MDS from acute leukemias has been redefined using a cutoff value of 20% peripheral and/or medullary blasts. The International Prognostic Scoring System still is the gold standard in prognostication, but new items like transfusion need will be used more and more and have been incorporated into the WHO adapted Prognostic Scoring System. In childhood, MDS is uncommon, accounting for less than 5% of all hematopoietic neoplasms in patients less than 14 years of age. To accommodate for the characteristics of pediatric MDS, a simple classification scheme based on morphological features and conforming with the WHO suggestions was proposed. The dysplastic prodrome of acute myeloid leukemia in Down syndrome is classified within myeloid leukemia in Down syndrome and excluded from the population-based studies of MDS.

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.

Tetrasomy 21 as a sole acquired abnormality without GATA1 gene mutation in pediatric acute megakaryoblastic leukemia: A case report and review of the literature

We report a case of pediatric acute megakaryocytic leukemia (AMKL) showing 48,XX,+21,+21 as a sole acquired cytogenetic abnormality without the mutation of GATA1 gene. A physical examination showed a phenotypically normal female. Bone marrow findings showed diffuse infiltration of leukemic blasts having scanty cytoplasm with budding blebs and prominent nucleoli, which were negative for myeloperoxide (MPO) stain, Sudan black B stain and periodic acid–Schiff stain. Immunophenotyping of leukemic cells revealed positive expression of CD34, CD13, CD33, CD117, CD41, CD61, CD7 and negative expression of TdT, anti-MPO, CD64, CD56, CD2, CD3, CD5, CD10, CD19, CD20 and CD22. A fluorescence in situ hybridization analysis showed four distinct AML1 signals in 284 of 300 interphase nuclei. The entire six exons of the GATA1 gene (7757 bp) were directly sequenced. We could not find any mutations, including known polymorphisms, which are known to be involved in transient myeloproliferative disorder and acute megakaryocytic leukemia of Down syndrome. After achieving complete remission, the tetrasomy 21 disappeared.