Down Syndrome Leukemia Research Articles

Clinical and hematological profile of acute megakaryoblastic leukemia: a 2year study.

Acute megakaryoblastic leukemia is a rare subtype of acute myeloid leukemia with a characteristic morphologic and immunophenotypic profile. It has to be distinguished from other subtypes of acute myeloid leukemia as well as acute myeloid leukemia with t (1; 22) (p13;q13) and acute megakaryoblastic leukemia in Down Syndrome because of its poor prognosis. We studied ten cases diagnosed over a period of 2years (from July 2011 to June 2013). All the ten cases were in the pediatric age group ranging from 4months to 2years. On morphology, pointers to the diagnosis were clustering of blasts, presence of cytoplasmic blebs and platelet budding. An additional interesting morphological feature observed in our study was nuclear blebs which were seen in nine cases. Diagnosis was confirmed in all cases by positive immunostaining for CD61. Two of the cases had an extremely rare clinical presentation as granulocytic sarcoma. Although rare, acute megakaryoblastic leukemia should be kept in mind especially in leukemia in infants.

Clinical and genetic features of pediatric acute lymphoblastic leukemia in Down syndrome in the Nordic countries.

BackgroundChildren with Down syndrome (DS) have an increased risk for acute lymphoblastic leukemia (ALL). Although previous studies have shown that DS-ALL differs clinically and genetically from non-DS-ALL, much remains to be elucidated as regards genetic and prognostic factors in DS-ALL.MethodsTo address clinical and genetic differences between DS-ALL and non-DS-ALL and to identify prognostic factors in DS-ALL, we ascertained and reviewed all 128 pediatric DS-ALL diagnosed in the Nordic countries between 1981 and 2010. Their clinical and genetic features were compared with those of the 4,647 B-cell precursor (BCP) ALL cases diagnosed during the same time period.ResultsAll 128 DS-ALL were BCP ALL, comprising 2.7% of all such cases. The 5-year event-free survival (EFS) and overall survival (OS) were significantly (P = 0.026 and P = 0.003, respectively) worse for DS-ALL patients with white blood cell counts ≥50 × 109/l. The age distributions varied between the DS and non-DS cases, with age peaks at 2 and 3 years, respectively; none of the DS patients had infant ALL (P = 0.029). The platelet counts were lower in the DS-ALL group (P = 0.005). Abnormal karyotypes were more common in non-DS-ALL (P < 0.0001), and there was a significant difference in the modal number distribution, with only 2% high hyperdiploid DS-ALL cases (P < 0.0001). The 5-year EFS and 5-year OS were significantly worse for DS-ALL (0.574 and 0.691, respectively) compared with non-DS-ALL (0.783 and 0.894, respectively) in the NOPHO ALL-1992/2000 protocols (P < 0.001).ConclusionsThe present study adds further support for genetic and clinical differences between DS-ALL and non-DS-ALL.

Spontaneous Remission of Transient Leukemia in Down Syndrome: Extrinsic or Intrinsic Mechanism?

Myeloid leukemias in childhood Down syndrome (DS) comprise a unique disease entity. Transient Leukemia (TL) in neonates with DS is a neoplastic disorder characterized by Acute Myeloid Leukemia (AML)-like hematological abnormalities, which spontaneously resolves in several weeks or months. On the other hand, AML in young children with DS (AML-DS), which occurs several years later, usually after spontaneous remission of TL, does not resolve spontaneously and is a lethal disorder unless treated. These two types of myeloid leukemia in DS are a spectrum of disorders with common GATA1 gene mutations and a background of trisomy 21, but arise in different organs at different developmental stages. TL is thought to arise in the fetal liver and is often accompanied by hepatic fibrosis in severe cases, whereas AML-DS arises in the postnatal BM and is often accompanied by myelofibrosis, with fibrosis of both organs being caused by a common mechanism through cytokines produced by leukemic blasts. The mechanism of the spontaneous remission of TL is unclear and two major hypotheses have been proposed: 1) a transition of major hematopoietic organs from the liver to the BM after birth might stop TL blast growth (extrinsic/ environmental theory); and 2) the genetic mechanism controlling the switch from fetal- to adult-type haematopoiesis might trigger the end of TL blast growth (intrinsic/genetic theory).

The Applicability Of The WHO Classification In Pediatric AML. A NOPHO-AML Study

IntroductionThe World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia was revised in 2008 and is based on genetic characteristics and morphology. The classification incorporates newly recognized entities and emphasizes, more than previously, the pivotal role of cytogenetic abnormalities aiming at identifying biological and clinical entities as basis for a stratified treatment. The classification of acute myeloid leukemia (AML) is primarily based on adult studies. However, the frequency of cytogenetic changes varies among children and adults and the relevance of the 2008 revised WHO classification in pediatric AML has not yet been elucidated. The aim of this study was to evaluate the usability of the WHO classification when applied to a large population-based pediatric AML cohort. MethodsWe included children 0-18 years of age diagnosed with de novo AML in the Nordic countries (Sweden, Norway, Finland, Iceland and Denmark) and Hong Kong treated according to the NOPHO-AML-1993 and 2004 protocols in the period January 1993 to December 2012. Clinical, morphologic, and genetic data were retrieved from the database. Patients with myeloid leukemia of Down syndrome, acute promyelocytic leukemia, and therapy-related AML were excluded. The karyotypes from the Nordic countries were centrally reviewed with annual evaluation of the karyograms by the NOPHO cytogenetic working group. Morphological and molecular analyses and immunophenotyping were carried out at regional centers. The patients were classified according to the revised 2008 WHO classification using genetic parameters and the French-American-British (FAB) classification. ResultsIn the NOPHO database we identified 609 children aged 0-18 years diagnosed with AML. In 13 cases, the karyotype analyses either failed or considered uninformative and were excluded. Among the remaining 596 informative cases (98%), 241 (40%) were categorized as AML with recurrent genetic abnormalities; t(8;21)(q22;q22);RUNX1-RUNX1T1 (n=69), inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 (n=47), t(9;11)(p21;q23);MLLT3-MLL (n=61), t(6;9)(p22;q34);DEK-NUP214 (n=5), inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVI1 (n=1), t(1;22)(p13;q13)/RBM15-MKL1 (n=4), NPM1 mutated (n=13), and CEBPA mutated (n=10). Furthermore, 23 children were classified in the provisional entity AML with FLT3-ITD. Based on the karyotype, 92 children (16%) were classified as AML with myelodysplasia-related cytogenetical changes (AML-MDS). This group was dominated by unbalanced abnormalities (9%) with -7/del(7q) being the most common (4% of the total cohort) and complex karyotypes (6% of the total cohort). AML with balanced myelodysplasia-related changes was found in 0.5%. The largest group was the highly heterogeneous AML not otherwise specified (AML-NOS) (n=263) (44%), and included some of the well-defined 11q23 abnormalities. In the 2001 version of the WHO classification, “AML with abnormalities of 11q23; MLL,” was listed separately, but in the 2008 edition only AML with t(9;11)(p22;q23);MLLT3-MLL was listed as an entity. The AML-NOS cases were sub-classified according to morphology, with FAB M5 being most common, found in 22% of NOS, followed by FAB M1, FAB M2, and FAB M4, all found in 18%.In conclusion, 40% of the children were classified in clinically relevant entities, but the WHO classification allocated 16% of the children to AML-MDS, the relevance is unknown in children, and 44% to the NOS group. This large and unspecified group limits the applicability of the WHO classification in children with AML suggesting that additional considerations are warranted for relevant classification of pediatric AML. Disclosures:No relevant conflicts of interest to declare.

Development of acute megakaryoblastic leukemia in Down syndrome is associated with sequential epigenetic changes

AbstractAcute megakaryoblastic leukemia (AMKL) is more frequently observed in Down syndrome (DS) patients, in whom it is often preceded by a transient myeloproliferative disorder (TMD). The development of DS-TMD and DS-AMKL requires not only the presence of the trisomy 21 but also that of GATA1 mutations. Despite extensive studies into the genetics of DS-AMKL, the importance of epigenetic deregulation in this disease has been unexplored. We performed DNA methylation profiling at different stages of development of DS-AMKL and analyzed the dynamics of the epigenetic program. Early genome-wide DNA methylation changes can be detected in trisomy 21 fetal liver mononuclear cells, prior to the acquisition of GATA1 mutations. These early changes are characterized by marked loss of DNA methylation at genes associated with developmental disorders, including those affecting the cardiovascular, neurological, and endocrine systems. This is followed by a second wave of changes detected in DS-TMD and DS-AMKL, characterized by gains of methylation. This new wave of hypermethylation targets a distinct set of genes involved in hematopoiesis and regulation of cell growth and proliferation. These findings indicate that the final epigenetic landscape of DS-AMKL is the result of sequential and opposing changes in DNA methylation occurring at specific times in the disease development.

Normal karyotype is a poor prognostic factor in myeloid leukemia of Down syndrome: a retrospective, international study

Myeloid leukemia of Down syndrome has a better prognosis than sporadic pediatric acute myeloid leukemia. Most cases of myeloid leukemia of Down syndrome are characterized by additional cytogenetic changes besides the constitutional trisomy 21, but their potential prognostic impact is not known. We, therefore, conducted an international retrospective study of clinical characteristics, cytogenetics, treatment, and outcome of 451 children with myeloid leukemia of Down syndrome. All karyotypes were centrally reviewed before assigning patients to subgroups. The overall 7-year event-free survival for the entire cohort was 78% (± 2%), with the overall survival rate being 79% (± 2%), the cumulative incidence of relapse 12% (± 2%), and the cumulative incidence of toxic death 7% (± 1%). Outcome estimates showed large differences across the different cytogenetic subgroups. Based on the cumulative incidence of relapse, we could risk-stratify patients into two groups: cases with a normal karyotype (n=103) with a higher cumulative incidence of relapse (21%± 4%) than cases with an aberrant karyotype (n=255) with a cumulative incidence of relapse of 9% (± 2%) (P=0.004). Multivariate analyses revealed that white blood cell count ≥ 20 × 10(9)/L and age >3 years were independent predictors for poor event-free survival, while normal karyotype independently predicted inferior overall survival, event-free survival, and relapse-free survival. In conclusion, this study showed large differences in outcome within patients with myeloid leukemia of Down syndrome and identified novel prognostic groups that predicted clinical outcome and hence may be used for stratification in future treatment protocols.

Clonal selection in xenografted TAM recapitulates the evolutionary process of myeloid leukemia in Down syndrome

AbstractTransient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγnull mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.

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.

High Frequency of GATA1 Mutations in Childhood Non-Down Syndrome Acute Megakaryoblastic Leukemia

AbstractAbstract ▪888▪This icon denotes a clinically relevant abstract Introduction:Pediatric acute megakaryoblastic leukemia (AMKL) occurred in 6.6% (84/1271) of the children enrolled to the AML-BFM98 and 2004 studies.Despite a similar phenotype in morphology and immunophenotype, AMKL shows a heterogenous cytogenetic distribution (normal karyotype 23%, complex karyotype 21%, t(1;22) 9%; MLL-rearrangement 8%; monosomy 7 5%, trisomy 8 5%; other aberrations 29%).Mutations of the hematopoietic transcription factor GATA1 have been identified in almost all children suffering myeloid leukemia of Down syndrome (ML-DS). In addition, GATA1 mutations (GATA1mut) could be identified in children with trisomy 21 mosaic.Here, AMKL without evidence of Down syndrome or Down syndrome mosaic were analyzed for mutations in exon 1, 2 or 3 of the transcription factor GATA1. Patients:Seventy-one children from the AML-BFM Study group (n=51; 2000–2011), the Netherlands (n=10), France (n=3) and Scandinavia (n=7) were included.Within the AML-BFM Group the 51 analyzed patients showed similar characteristics compared to the total cohort of 84 children with AMKL of the AML-BFM 98 and 2004 studies.AMKL was confirmed according to the WHO classification by genetics (t(1;22)); morphology and immunophenotyping. Table 1a) summarizes the patientxs characteristics and b) the cytogenetic results. Methods:For GATA1 mutation screening genomic DNA was amplified by PCR reaction for exon 1, 2, and 3. PCR amplicons were analyzed by direct sequencing or following denaturing high-performance liquid chromatography (WAVE). Results:Seven different GATA1 mutations were detected in 8 children (11.1%; table 2).In all GATA1mut leukemia, a trisomy 21 within the leukemic blasts could be detected.Seven out of these 8 children and all other 64 AMKL patients have been treated with intensive chemotherapy regimens according the study group protocols. The results are given in table 2. All achieved continuous complete remission (CCR; 0.4 to 4.2 years). [Display omitted] In one newborn with typical morphology and immunophenotype a GATA1mut associated transient leukemia was supposed. The child achieved CCR (follow-up 6 years).In total, allogeneic stem cell transplantation in 1st CR was performed in 6 children with AMKL (GATA1mut leukemia n=1). [Display omitted] Conclusions:GATA1 mutations occurred in 11% of children with AMKL without any symptoms or evidence of trisomy 21 or trisomy 21 mosaic. GATA1 mutations are associated with a trisomy 21 within the leukemic blasts. Although non-response occurred, prognosis was significant better compared to other AMKL. Therefore, analysis of GATA1 mutation in infant AMKL is strongly recommended. Whether treatment reduction similar to ML-DS Down syndrome is feasible needs to be confirmed. Disclosures:No relevant conflicts of interest to declare.

Outcome of Transplantation for Acute Leukemia in Down Syndrome

AbstractAbstract 1991Children with Down syndrome (DS) have a 10-to 20-fold increased incidence of acute lymphoblastic (ALL) and acute myeloid leukemia (AML) compared to the overall pediatric population. Available data on HCT in children with DS are scarce, suggest unsatisfactory outcomes and are conflicting as far as causes of treatment failure are concerned. All but one case series identified treatment-related mortality (TRM) as the major barrier to success. To better understand factors associated with HCT-outcomes we studied 28 patients with DS-AML and 27 patients with DS-ALL, the largest cohort of DS patients with acute leukemia to date. All transplants occurred in 2000–2009. Transplantations occurred in second remission for 43% of patients with DS-AML and 52%, with DS-ALL. With a median follow-up of 3-years disease-free survival (DFS) was 14% for DS-AML and 24% for DS-ALL. Leukemia recurrence was the predominant cause of treatment failure in the current analysis, 61% for DS-AML and 54% for DS-ALL, both substantially higher than expected for pediatric non-DS AML or ALL. In the subset of patients with DS-AML, we conducted a matched pair analysis. Cases (DS-AML) were matched to non-DS AML controls for age, disease status, cytogenetic risk group, donor-source, donor-recipient HLA match and graft source. The results of multivariate analysis, adjusted for interval from diagnosis to transplantation are shown below. Relapse risk was significantly higher in DS-AML than non DS-AML (62% vs. 37%; p<0.001). TRM was also higher in patients with DS-AML compared to non DS-AML (24% vs. 15%, p=0.04). Consequently, 3-year DFS was significantly lower for DS-AML compared to non DS-AML (14% vs. 48%, p<0.001). The corresponding probabilities of overall survival (OS) were 21% and 52% (p<0.001). Interval from diagnosis to transplant was significantly associated with OS; transplantation that occurred within 12 months from diagnosis was associated with higher mortality (hazard ratio 1.90, p=0.03). Interval between diagnosis and transplantation was not significantly associated with relapse (hazard ratio 1.42, p=0.27) or TRM (hazard ratio 2.17, p=0.15). In conclusion, after adjusting for known risk factors, leukemic relapse and TRM contribute to treatment failure after HCT in the recent treatment era. Cooperative group trials appear warranted that improve the selection of HCT candidates, optimize transplant-conditioning regimen and explore novel therapeutic approaches to improve the depth of remission prior to HCT in these children.TableHazard Ratio 95% confidence intervalP-valueTransplant-related mortalityDS-AML vs. non DS-AML2.52 (1.06–6.00)0.04Leukemia recurrenceDS-AML vs. non DS-AML2.84 (1.75–4.59)<0.001Treatment failure (death or relapse; inverse of DFS)DS-AML vs. non DS-AML2.75 (1.75–4.31)<0.001Overall mortalityDS-AML vs. non DS-AML2.86 (1.77–4.64)<0.001 Disclosures:No relevant conflicts of interest to declare.

Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid-primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.

Clinical characteristics and outcome of refractory/relapsed myeloid leukemia in children with Down syndrome

AbstractMyeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy and favorable prognosis. Because little research has been focused on refractory/relapsed (R/R) cases, we conducted a retrospective analysis for R/R ML-DS. Among ML-DS patients diagnosed between 2000 and 2010 in Japan, 26 relapsed (25 in the BM and 1 in the skin), and 3 refractory patients were enrolled. The male/female ratio was 18/11. The median age at initial diagnosis of ML-DS was 2 years, and the median time to relapse was 8.6 months. Each patient initially had been treated with ML-DS–specific protocols. Thirteen of the 26 patients achieved complete remission with various kinds of reinduction chemotherapies; 2 of 8 survived without further recurrence after receiving allogeneic hematopoietic stem cell transplantation, and 4 of 5 maintained complete remissions with chemotherapy alone. Treatment failures mostly were associated with disease progression rather than treatment-related toxicities. The 3-year OS rate was 25.9% ± 8.5%. A longer duration from initial diagnosis to relapse was a significant favorable prognostic factor (P < .0001). We conclude that clinical outcome for patients with R/R ML-DS generally are unfavorable, even in those receiving hematopoietic stem cell transplantation. Novel methods to identify poor prognostic factors for ML-DS are necessary.

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

Transient Leukemia-Associated Pericardial Tamponade in a Neonate with Down Syndrome

Approximately 10% of neonates with Down syndrome may develop a form of megakaryoblastic leukemia that usually disappear spontaneously during the first months of the life. Although it seems to have a benign course, it may also be lethal and severe in some cases, especially in the form of hydrops and/or cardiopulmonary failure. Herein, we report a male infant with Down syndrome who was admitted with respiratory distress due to severe pericardial effusion leading to pericardial tamponade in the first 2 weeks of life. Pericardiosentesis and pericardial tube replacement in combination with steroid therapy was performed. He responded well to these therapies and his leukemia resolved on the fourth month of life. This case suggests that severe pericardial effusion and pericardial tamponade may be life-threatening complications of transient leukemia of Down syndrome and also it may be managed successfully with appropriate treatments.

Abstract 1488: Role of niche requirements in transient leukemia of Down syndrome

Abstract Background. Children with Down syndrome (DS; constitutional trisomy 21) display an estimated 150-fold increased incidence of acute myeloid leukemia (AML/AMkL). Leukemic blasts are detectable in the peripheral blood of approximately 10% of newborns with DS. This pre-leukemic disorder, known as Transient Leukemia (TL) or Transient Myeloproliferative Disorder (TMD), predominantly undergoes spontaneous resolution, although 20% of cases go on to develop AML later in life. Available data suggest that TL is a disorder of fetal hematopoiesis, triggered by somatic mutations of the hematopoietic transcription factor GATA1. We hypothesize that the transition from fetal to post-natal hematopoiesis results in the elimination of TL blasts in the majority of cases, and that progression events acquired during the first three years of life account for the transformation of Down syndrome TL to AML. Methods and Results. During embryonic development, the fetal liver (FL) constitutes the primary site of hematopoiesis. Data from our xenotransplant model suggest that the post-natal bone marrow (BM) environment provides insufficient or inappropriate cues for the survival, proliferation and dissemination of TL blasts. To identify the unique combination of signals provided by the FL but not the post-natal BM, we reconstructed both hematopoietic niches in vitro by culturing primary human TL cells on murine stromal cell lines derived from either FL (Aft024) or adult BM (MS-5). We found that TL cells underwent a striking &amp;gt;80-fold expansion in total cell number when cultured on FL-derived stromal cells for 2 weeks. In contrast, BM-derived MS-5 stromal cells supported only a moderate 8-fold expansion of TL cells, simultaneously allowing for progressive induction of apoptosis, and inducing increased myeloid differentiation. The superior ability of fetal-derived stromal cells compared to those derived from bone marrow to support TL cells was confirmed under hypoxic culture conditions, in keeping with their fetal origin. Comparative gene expression analysis successfully identified both soluble and membrane-bound components of the fetal hematopoietic niche (modeled by Aft024), such as BMPs and mediators of cell adhesion, as candidate regulators of TL cell growth. Conclusions. We have developed an ex vivo culture system which allows the expansion of primary DS-TL blast cells. We demonstrate that survival/proliferative signals are preferentially provided by the fetal liver vs. adult bone marrow environment, consistent with a fetal origin of TL blasts. Our current focus is on the identification and functional validation of signaling pathways that are essential for TL blast survival and whose interruption in this pre-leukemic disorder may lead to prevention of subsequent AML in children with Down syndrome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1488. doi:1538-7445.AM2012-1488

Low frequency of type-I and type-II aberrations in myeloid leukemia of Down syndrome, underscoring the unique entity of this disease

We recently published in this journal an overview of the currently known genetic events required for the development of pediatric acute myeloid leukemia (AML).[1][1] These aberrations can be subdivided into type-I aberrations that result in uncontrolled proliferation, and type-II aberrations that

GATA1 mutations Outside the Blast Cell Population in Transient Leukemia of Down Syndrome

Abstract 2562 Background.Transient leukemia (TL), a disorder of fetal hematopoiesis, occurs in approximately 10% of infants with Down syndrome and in one fifth of cases is followed by the development of acute myeloid leukemia. The diagnosis of TL is based on the detection of a spontaneously resolving population of blasts in the peripheral blood of newborns with DS and, more recently, the detection of clone-specific somatic mutations of the hematopoietic transcription factor GATA1. The alternative terms for this condition, transient myeloproliferative disorder (TMD) and transient abnormal myelopoiesis (TAM), reflect the observation that the peripheral blood of affected infants frequently shows an expansion of immature granulocytic precursor cells at various stages of development as well as nucleated erythroid precursors in addition to a population of blast cells. We hypothesized that the immature myeloid precursor cells observed in TL arise from the same hematopoietic cell clone that is defined by the specific GATA1 mutation found in TL blasts. Methods.Cryopreserved mononuclear cells from peripheral blood samples of three male infants with TL of Down syndrome were stained at a density of 106 cells/100ul with the following antibodies: CD45-Alexa Flour 700, CD61-PE, CD11b-PE-Cy5, CD235a-PE-Cy7, CD3-APC, CD34-APC-Cy7, CD15-Pacific Blue. In addition, aliquots of these cell populations were fixed, permeabilized and stained with for intracellular myeloperoxidase (MPO-FITC). Fractions of cells corresponding to different hematopoietic lineages were prepared by fluorescence activated cell sorting (FACSAriaII, BD Biosciences, San Jose, CA) both as bulk populations and single cells (sorted into 96-well plates). Morphology of sorted cell populations was determined by Giemsa staining. Lineage-specific hematopoietic colonies were cultured from primary mononuclear cell samples in a standard CFC assay. DNA of sorted bulk and single cell populations as well as individual lineage-specific hematopoietic colonies was used to detect GATA1 mutations in exon 2 by PCR, restriction enzyme analysis or sequencing of PCR products, or nested PCR. The location of the GATA1 gene on the X chromosome and the use of samples derived from males meant that wild type GATA1 alleles were not derived from TL cells. Results.Primary TL blood samples could be fractionated in a blast cell (CD45+CD34+and/or CD61+), erythroid (CD45-CD235a+), immature myeloid (CD45+CD15+MPO+) and lymphoid (CD45+CD3+) cell population. Cell morphology was consistent with this sorting strategy. The cell fraction designed to enrich TL blasts in all cases showed the presence of cells harboring only the mutant GATA1 allele indicating a high degree of purity of the blast fraction. As expected, the lymphoid cell populations contained in the CD3+ fractions exclusively harbored wild type alleles. Similarly, both sorted erythroid cell populations and individual erythroid colonies (Bfu-E) derived from unsorted input cells harbored only wild type GATA1 alleles. In contrast, CD15+ and/or MPO+ myeloid cell fractions consistently showed the presence of both mutant and wild type GATA1 alleles. Quantification by single cell PCR is in progress. Conclusion.Somatic GATA1 mutations which result in the translation of a N-terminally truncated mutant protein (GATA1s), are pathognomonic for TL and myeloid leukemia of Down syndrome. Published data do not determine if the cell clone harboring GATA1 mutations in TL extends beyond the population of blast cells and megakaryocytic lineage. We confirmed the presence of GATA1 mutations in TL blasts and their expected absence from lymphoid cell populations. The absence of mutations from cells of erythroid lineage suggests that the altered GATA1 function induced by mutations found in TL is incompatible with erythroid differentiation. The detection of clone-specific GATA1 mutations in myeloid cells expressing MPO was unexpected and raises the possibility that either the progenitor cells giving rise to TL blasts or the blasts themselves retain myeloid lineage differentiation potential. The finding lends support to definitions of TL and myeloid leukemia in Down syndrome that are based on the specific mutational and cellular mechanism and transcend a megakaryoblastic blast phenotype. Disclosures:No relevant conflicts of interest to declare.

Refractory / Relapsed Myeloid Leukemia of Down Syndrome Is Resistant to Second-Line Chemotherapy and Hardly Salvaged by Hematopoietic Stem Cell Transplantation: A Retrospective Study by the Japanese Pediatric Leukemia / Lymphoma Study Group (JPLSG)

Abstract 4276 Introduction:Myeloid leukemia in Down syndrome patients (ML-DS) is known to have good sensitivity against cytotoxic agents, especially to cytarabine, and outcomes in recent clinical trials are favorable with more than 80 % probability of long-term event-free survival (EFS). However, little have been focused on refractory / relapsed cases, since most of the treatment failures consist of toxicities rather than resistant or recurrent leukemia. We present here a retrospective analysis of the patients with refractory / relapsed ML-DS. Patients and Method:Among the ML-DS patients diagnosed between 2000 and 2011 at the 120 hospitals in Japan, twenty-nine refractory / relapsed patients were enrolled in this retrospective study. Result:Median follow-up period for all 29 patients was 10.9 months (range, 2.8 –76.7 months). Male/Female ratio was 18/11. The age at initial diagnosis for ML-DS was between 7 months and 16 years old (median, 2 years old). All the patients were initially treated with one of the protocols specifically designed for ML-DS; twenty patients with AML99 Down protocol, eight with JPLSG AML-D05 protocol, and one with JCCLSG AML9805 Down protocol. There were 3 induction failures and 26 relapsed cases. Among the 26 relapsed cases, duration from initial diagnosis to relapse was 2.4 month to 71.8 months (median 8.6 months); Twenty-eight relapsed at bone marrow and one patient relapsed at skin site. Twenty-six out of the 29 patients received various re-induction chemotherapies and 13 of them achieved complete remission (CR). Eight of the 13 patients with CR subsequently received allogeneic stem cell transplantation (SCT) and 4 survived. On the other hand, five of the 13 cases with CR were treated with chemotherapy alone, and 4 were alive with no evidence of leukemia. All the 12 patients who failed to achieve CR and the 2 patients who only received palliative therapy eventually died. The 3-year overall survival rate was 22.6 ± 8.8 %. Achievement of CR with second-line chemotherapy and longer duration from initial diagnosis to relapse were significant favorable prognostic factors. SCT did not influence the prognosis even if performed after achieving further remission. Conclusion:The Japanese strategy treating ML-DS have been successful with very low-intensive chemotherapy regimen, however, rescue of refractory / relapsed patients with ML-DS seems difficult even with SCT.No relevant conflicts of interest to declare. Disclosures:No relevant conflicts of interest to declare.

Development of Acute Megakaryoblastic Leukemia in Down Syndrome Is Associated with Sequential Epigenetic Changes That Target the Down Syndrome Critical Region on Chromosome 21,

Abstract 3451Down syndrome (DS) is associated with an increased frequency of Acute Megakaryoblastic Leukemias (AMKL). In the context of DS, AMKL is usually preceded by the development of a transient myeloproliferative disorder (TMD). Up to 10% of DS children may develop this disorder, and approximately 20–30% of those with TMD go on to develop AMKL, which may or may not be preceded by a return to normal hematopoiesis. While trisomy 21 (tri21) alone is not sufficient to develop AMKL, it is clear that the acquisition of somatic mutations in GATA1 is a requirement for the development of DS-AMKL. Much has been studied in relation with this genetic mutation, however not much is known about the epigenetic changes accompanying the development of AMKL in DS patients. Given the step-wise nature of the development of DS-AMKL, we hypothesized that distinct and cumulative epigenetic changes would be associated with each of the phases of the disease. In order to address this hypothesis we performed DNA methylation profiling of a cohort of 7 primary DS-AMKL samples using the latest design of the HELP microarray assay, which covers ∼230,000 CpG sites at ∼22,000 RefSeq promoter regions. In order to better understand how aberrant epigenetic patterns become established during the development of this disease we compared these DNA methylation profiles to those of mononuclear cells from normal fetal liver (FL-MNC) (n=4), tri21 FL-MNC (n=4) and TMD blasts (n=6). We identified a set of 711 unique genes that displayed significant loss of methylation in Tri21 FL-MNC with respect to control FL-MNC (FDR < 5% and methylation delta ≥ 25%). However, when tri21 FL-MNC were compared to the GATA1s positive TMD blasts, we observed that TMD samples acquired significant hypermethylation of 346 unique genes at the same significance cutoff. A direct comparison of the genes affected by these two opposing waves of methylation changes demonstrated that different sets of genes were being targeted at each stage. Next we compared the DNA methylation profiles of TMD and DS-AMKL blasts, and found them to be virtually identical, indicating that an epigenetic imprint becomes established at the TMD phase, which is still present at the leukemic phase. Finally we compared DS-AMKL to a cohort of 8 non-DS AMKL, and observed that DS-AMKL samples display significant hypomethylation of 267 unique genes, indicating that these two diseases are not only genetically but also epigenetically distinct. In determining the location of the epigenetic changes acquired at the different stages of the disease we found that, similar to what had been previously reported for gene expression, DNA methylation changes did not localize solely to chr 21, but that these changes were distributed along all chromosomes. However, detailed analysis of chr 21 revealed that changes on this chromosome preferentially targeted a specific region on the long arm, the Down Syndrome Critical Region (DSCR). This region becomes significantly hypomethylated in Tri21 FL-MNC and remains hypomethylated through all the stages of the disease. Several groups have sought to identify candidate genes on the DSCR that may contribute to the pathogenesis of the disease. Two genes, BACH1 and CXADR, have been identified in common by two different studies as being overexpressed in DS-AMKL vs. Non-DS AMKL. Both of these genes displayed aberrant hypomethylation at their promoter regions in Tri21 FL MNC, TMD and DS-AMKL samples: such hypomethylation was absent from all the control samples. Finally, given that GATA1 mutations are a requirement for the development of DS-AMKL, we compared the different aberrant DNA methylation signatures to the normal GATA1 targets identified by ChIP sequencing experiments, and found 20–25% overlap. In summary, we have completed the first comprehensive DNA methylation study of the developmental phases of DS-AMKL and have demonstrated that a) specific epigenetic changes are associated with the different stages of the disease, b) these changes occur in ‘waves’ of sequential hypo and hypermethylation, c) aberrant DNA methylation on chr 21 preferentially targets the DSCR and results in aberrant overexpression of the associated genes, and d) epigenetic profiles of TMD and DS-AMKL are virtually identical. These observations lead us to believe that an epigenetic memory established at the TMD stage may be at least in part contributing to the development of full-blown AMKL at a later time point. Disclosures:No relevant conflicts of interest to declare.

GATA1 Mutants Lacking Rb-Binding Motif Observed in Transient Abnormal Myelopoiesis in Down Syndrome

Abstract 1491Five to ten percent of neonates with Down syndrome (DS) develop transient abnormal myelopoiesis (TAM), which is characterized by a rapid growth of abnormal blast cells with the erythroid/megakaryocytic phenotype. In most cases, it resolves spontaneously within three months. However, 20 to 30% of patients develop acute megakaryocytic leukemia referred to as myeloid leukemia of DS (ML-DS) within four years. Blast cells in most of TAM and ML-DS patients have mutations in exon 2 of the gene encoding transcription factor GATA1, which is essential for the normal development of erythroid and megakaryocytic cells. These mutations lead to expression of a truncated GATA1 protein lacking the N-terminal 83 amino acids (GATA1s). However, the molecular mechanism whereby GATA1s contributes to the genesis of TAM and ML-DS remains elusive. From 2003 to 2010, we screened GATA1 mutations in clinical samples obtained from 106 patients with TAM upon request from referring hospitals. We performed direct sequencing analysis using cDNA prepared form total RNA extracted from white blood cells in peripheral blood and genomic DNA. Acquired GATA1 mutations were detected in a total of 99 (93.4%) patients among them. Most of these mutations were coding GATA1s. However, the mutations coding GATA1 mutants with internal deletion of 43 and 15 amino acids were detected in five and one patients, and we referred to them as GATA-ID type-1 and GATA1-ID type-2, respectively. Sequences of exon 3 were discrepant with that expected from the corresponding cDNA sequence. Instead, a 2-nucleotide insertion, a 2-nucleotide deletion and a 7-nucleotide deletion with a single-nucleotide substitution were detected in exon 3 of the GATA1 gene in the GATA1-ID type-1 patients, and a 21-nucleotide deletion encompassing the boundary of intron 2 and exon 3 was found in the GATA-ID type-2 patient. These findings suggest that the mutations in exon 3 lead to expression of alternative splicing forms of mRNA encoding GATA1-ID. To investigate the role of GATA1-ID on the pathogenesis of TAM and ML-DS, we next expressed GATA1-ID by retrovirus in a ML-DS cell line, KPAM1, or primary GATA1-deficient fetal megakaryocytic progenitors. Expectedly, the cell growth was markedly reduced upon transduction with full-length GATA1-expression retrovirus. However, ectopic expression of GATA1-ID type-1 and type-2 failed to restrict the proliferation of cells as similar as expression of GATAs. Interestingly, all these patients had high white blood cell counts in the peripheral blood at diagnosis. Furthermore, three patients developed liver fibrosis and two had effusion. All these factors are significantly associated with early death. Three patients were treated with low dose of Ara-C. However, early death occurred in three patients. In this study we found that GATA1-ID proteins are created by somatic mutations in DS patients and contribute to the generation of TAM phenotypes. Our newly identified GATA1-ID mutants have highlighted a much narrower set of sequences responsible for the pathogenesis of than was previously suggested. The missing region identified by the GATA1-ID proteins contains a consensus motif (LxCxE, amino acids 81–85) essential for the interaction with pRB protein and this motif is also lost in GATA1s. Our results suggested the lack of Rb-GATA1 interaction as the most likely pathogenesis for onset of TAM. Disclosures:No relevant conflicts of interest to declare.