Myeloid Leukemia Of Down Syndrome Research Articles

Improved outcomes for myeloid leukemia of Down syndrome: a report from the Children's Oncology Group AAML0431 trial

Patients with myeloid leukemia of Down syndrome (ML-DS) have favorable event-free survival (EFS), but experience significant treatment-related morbidity and mortality. ML-DS blast cells ex vivo have increased sensitivity to cytarabine (araC) and daunorubicin, suggesting that optimizing drug dosing may improve outcomes while reducing toxicity. The Children's Oncology Group (COG) AAML0431 trial consisted of 4 cycles of induction and 2 cycles of intensification therapy based on the treatment schema of the previous COG A2971 trial with several modifications. High-dose araC (HD-araC) was used in the second induction cycle instead of the intensification cycle, and 1 of 4 daunorubicin-containing induction cycles was eliminated. For 204 eligible patients, 5-year EFS was 89.9% and overall survival (OS) was 93.0%. The 5-year OS for 17 patients with refractory/relapsed leukemia was 34.3%. We determined the clinical significance of minimal residual disease (MRD) levels as measured by flow cytometry on day 28 of induction I. MRD measurements, available for 146 of the 204 patients, were highly predictive of treatment outcome; 5-year disease-free survival for MRD-negative patients (n = 125) was 92.7% vs 76.2% for MRD-positive patients (n = 21) (log-rank P = .011). Our results indicated that earlier use of HD-araC led to better EFS and OS in AAML0431 than in past COG studies. A 25% reduction in the cumulative daunorubicin dose did not impact outcome. MRD, identified as a new prognostic factor for ML-DS patients, can be used for risk stratification in future clinical trials. This trial was registered at www.clinicaltrials.gov as #NCT00369317.

CRISPR/Cas9-Directed Reassignment of the GATA1 Initiation Codon in K562 Cells to Recapitulate AML in Down Syndrome.

Using a CRISPR/Cas9 system, we have reengineered a translational start site in the GATA1 gene in K562 cells. This mutation accounts largely for the onset of myeloid leukemia in Down syndrome (ML-DS). For this reengineering, we utilized CRISPR/Cas9 to generate mammalian cell lines that express truncated versions of the Gata1s protein similar to that seen in ML-DS, as determined by analyzing specific genetic alterations resulting from CRISPR/Cas9 cleavage. During this work, 73 cell lines were clonally expanded, with allelic variance analyzed. Using Tracking of Indels by DEcomposition (TIDE) and Sanger sequencing, we defined the DNA sequence and variations within each allele. We found significant heterogeneity between alleles in the same clonally expanded cell, as well as among alleles from other clonal expansions. Our data demonstrate and highlight the importance of the randomness of resection promoted by non-homologous end joining after CRISPR/Cas9 cleavage in cells undergoing genetic reengineering. Such heterogeneity must be fully characterized to predict altered functionality inside target tissues and to accurately interpret the associated phenotype. Our data suggest that in cases where the objective is to rearrange specific nucleotides to redirect gene expression in human cells, it is imperative to analyze genetic composition at the individual allelic level.

Treatment results in children with myeloid leukemia of Down syndrome in Saudi Arabia: A multicenter SAPHOS leukemia group study

Despite the high incidence of Down syndrome (DS) in Arab countires, the incidence and outcomes of myeloid leukemia of DS (ML-DS) have not been studied. We evaluated 206 pediatric acute myeloid leukemia (AML) patients diagnosed between 2005 and 2012 and identified 31 (15%) ML-DS. The incidence of ML-DS was 48 per 100,000 compared to 0.6 per 100,000 for AML in non-DS children. Thus, patients with DS had 80-fold increased risk of ML-DS compared to AML in non-DS children. The median age at diagnosis was 1.8 years, male/female ratio was 1.2, majority (84%) of patients had FAB-M7 subtype, and the cytogenetic abnormalities were normal karyotype (constitutional trisomy 21) in 48%, additional trisomy in 23%, and other aberrations in 29%. Complete remission, cumulative incidences of relapse (CIR), toxic-death, and 5-year event-free survival (EFS) rates were 96.8%, 19.4%, 13.1%, and 67.7±8.4%; respectively. In the present study, multivariate analysis revealed favorable outcome (5-year EFS 86.7±8.8%) for patients with normal karyotype. The incidence and clinical characteristics of ML-DS in Saudi patients were comparable to other reports. However, there is a need to optimize risk stratification and treatment intensity to reduce CIR and toxic death rates to further improve outcomes of patients with ML-DS.

Transient Abnormal Myelopoiesis and AML in Down Syndrome: an Update.

Children with constitutional trisomy 21 (Down syndrome (DS)) have a unique predisposition to develop myeloid leukaemia of Down syndrome (ML-DS). This disorder is preceded by a transient neonatal preleukaemic syndrome, transient abnormal myelopoiesis (TAM). TAM and ML-DS are caused by co-operation between trisomy 21, which itself perturbs fetal haematopoiesis and acquired mutations in the key haematopoietic transcription factor gene GATA1. These mutations are found in almost one third of DS neonates and are frequently clinically and haematologcially ‘silent’. While the majority of cases of TAM undergo spontaneous remission, ∼10 % will progress to ML-DS by acquiring transforming mutations in additional oncogenes. Recent advances in the unique biological, cytogenetic and molecular characteristics of TAM and ML-DS are reviewed here.

Dysregulation of DNA Methylation Involves in Progression of Myeloid Leukemia in Down Syndrome

Transient abnormal myelopoiesis (TAM) is a clonal pre-leukemic disorder in neonates of Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting acute megakaryoblastic leukemia (DS-AMKL) after spontaneous remission. As such, the study of TAM/DS-AMKL can provide insight into the mechanisms of leukemic evolution. Our recent study has shown that DS-AMKL from TAM is shaped by acquisition of new mutations and clonal selection, and genomic mutations in epigenetic regulators frequently preceded other driver mutations. In this study, we address biological significance of the mutations in a progression from TAM to DS-AMKL using a newly established xenograft model of malignant conversion of the leukemia.We first analyzed global DNA methylation profile of clinical samples including 21 TAM and 10 DS-AMKL, using Infiniun 450K DNA methylation array (Illumina). Although averaged DNA methylation levels of both TAM and DS-AMKL were approximately the same, we observed two distinct clusters delineating TAM and DS-AMKL samples by both hierarchical clustering and principle component analysis (PCA). We identified differentially methylated cytosines on the promoters (TAM/AMKL-DMCpGs) between the two groups, and found that most of TAM/AMKL-DMCpGs were hyper-methylated in DS-AMKL samples compared to TAM, indicating that hyper-methylation of the subset of CpGs is a hallmark of DS-AMKL.To follow sequential alterations of DNA methylation profile, we established a xenograft model of the leukemia, which mimicked the progression of TAM to DS-AMKL. We transplanted TAM blast cells (1x106 cells) derived from the patient, who developed DS-AMKL later, into NOD/SCID/γc null (NOG) mice through tail vein. One series of the xenografts exhibited engraftment in the mouse and could passage by serial engraftment in vivo, whereas the other exhibited engraftment, but could not maintain in the mice by serial transplantation. The global DNA methylation profile of engrafted cells from the first generation mice was closed to TAM patients, whereas those from the forth, fifth and sixth generations approached to DS-AMKL patients. Moreover, hyper-methylation of TAM/AMKL-DMCpGs was also observed in engrafted TAM cells with higher numbers of passages, demonstrated that the xenograft model mimicked dysregulation of DNA methylation in disease progression of TAM. We further investigated the impact of dysregulation of DNA methylation on gene expression. As expected, RNA-seq and hierarchal clustering analyses of genes regulated by TAM/AMKL-DMCpGs displayed distinct two clusters, one with TAM samples and the first generation engraftments and another with DS-AMKL samples and 4th, 5th and 6th generation engraftments.We propose that epigenetic dysregulation is a crucial step for malignant conversion of TAM into DS-AMKL. DisclosuresNo relevant conflicts of interest to declare.

Genetic heterogeneity in transient abnormal myelopoiesis

Leukemia arises through an evolutionary process of somatic mutation and selection. Transient abnormal myelopoiesis (TAM) is a clonal pre-leukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism underlying leukemogenesis, a xenograft model of TAM was established using NOG mice. Serial engraftment after cell transplantation from a TAM patient who developed ML-DS a year later showed the self-renewal capacity of these cells. We detected a GATA1 mutation but no copy number alterations (CNAs) in the primary patient sample by conventional genomic sequencing and CNA profiling. However, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, both of which are clinically associated with ML-DS. Detailed genetic 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 various leukemia-initiating potentials already exist in the neonatal TAM phase, and that ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model could be a valuable tool for gaining insight into the processes underlying leukemogenesis.

Collaborative Efforts Driving Progress in Pediatric Acute Myeloid Leukemia.

Diagnosis, treatment, response monitoring, and outcome of pediatric acute myeloid leukemia (AML) have made enormous progress during the past decades. Because AML is a rare type of childhood cancer, with an incidence of approximately seven occurrences per 1 million children annually, national and international collaborative efforts have evolved. This overview describes these efforts and includes a summary of the history and contributions of each of the main collaborative pediatric AML groups worldwide. The focus is on translational and clinical research, which includes past, current, and future clinical trials. Separate sections concern acute promyelocytic leukemia, myeloid leukemia of Down syndrome, and relapsed AML. A plethora of novel antileukemic agents that have emerged, including new classes of drugs, are summarized as well. Finally, an important aspect of the treatment of pediatric AML--supportive care--and late effects are discussed. The future is bright, with a wide range of emerging innovative therapies and with more and more international collaboration that ultimately aim to cure all children with AML, with fewer adverse effects and without late effects.

GATA1 mutations in a cohort of Malaysian children with Down syndrome-associated myeloid disorder.

Children with Down syndrome (DS) are at increased risk of developing distinctive clonal myeloid disorders, including transient abnormal myelopoiesis (TAM) and myeloid leukaemia of DS (ML-DS). TAM connotes a spontaneously resolving congenital myeloproliferative state observed in 10%-20% of DS newborns. Following varying intervals of apparent remission, a proportion of children with TAM progress to develop ML-DS in early childhood. Therefore, TAM and ML-DS represent a biological continuum. Both disorders are characterised by recurring truncating somatic mutations of the GATA1 gene, which are considered key pathogenetic events. We herein report, to our knowledge, the first observation on the frequency and nature of GATA1 gene mutations in a cohort of Malaysian children with DS-associated TAM (n = 9) and ML-DS (n = 24) encountered successively over a period of five years at a national referral centre. Of the 29 patients who underwent GATA1 analysis, GATA1 mutations were observed in 15 (51.7%) patients, including 6 (75.0%) out of 8 patients with TAM, and 9 (42.9%) of 21 patients with ML-DS. All identified mutations were located in exon 2 and the majority were sequence-terminating insertions or deletions (66.7%), including several hitherto unreported mutations (12 out of 15). The low frequency of GATA1 mutations in ML-DS patients is unusual and potentially indicates distinctive genomic events in our patient cohort.

Down syndrome and leukemia: insights into leukemogenesis and translational targets.

Children with Down syndrome (DS) have a significantly increased risk of childhood leukemia, in particular acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (DS-ALL). A pre-leukemia, called transient myeloproliferative disorder (TMD), characterised by a GATA binding protein 1 (GATA1) mutation, affects up to 30% of newborns with DS. In most cases, the pre-leukemia regresses spontaneously, however one-quarter of these children will go on to develop AMKL or myelodysplastic syndrome (MDS) . AMKL and MDS occurring in young children with DS and a GATA1 somatic mutation are collectively termed myeloid leukemia of Down syndrome (ML-DS). This model represents an important multi-step process of leukemogenesis, and further study is required to identify therapeutic targets to potentially prevent development of leukemia. DS-ALL is a high-risk leukemia and mutations in the JAK-STAT pathway are frequently observed. JAK inhibitors may improve outcome for this type of leukemia. Genetic and epigenetic studies have revealed likely candidate drivers involved in development of ML-DS and DS-ALL. Overall this review aims to identify potential impacts of new research on how we manage children with DS, pre-leukemia and leukemia.

Risk-Oriented Therapy for Myeloid Leukemia of Down Syndrome: A Nationwide Prospective Study By the Japanese Pediatric Leukemia / Lymphoma Study Group (JPLSG)

Introduction: Asmyeloid leukemia in Down syndrome patients (ML-DS) is known to have higher sensitivity against cytotoxic agents, children with ML-DS are treated with less intensive ML-DS-oriented protocol in recent clinical studies. On the basis of results of previous Japanese trials for ML-DS, we have evaluated an efficacy of the risk-oriented therapy in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-D05 study.Patients and Methods: Between January 2008 and December 2010, seventy-four patients with newly diagnosed ML-DS from 122 hospitals in Japan were enrolled in this study. All patients received induction chemotherapy consisted of pirarubicin (25 mg/m2/d for 2 days), cytarabine (100 mg/m2/d for 7 days), and etoposide (150 mg/m2/d for 3 days). Patients who achieved complete remission (CR) after initial induction therapy were stratified to the Standard Risk (SR) and received four courses of reduced dose intensification therapy. Patients who did not achieve CR were stratified to the High Risk (HR) and received intensfied therapy consisted of continuous and high-dose cytarabine. Prophylaxis for CNS leukemia was not included throughout the therapy.Results: Out of 74 patients registered in this study, two patients were excluded (one because of uncontrolled cardiac failure, another of non-Down syndrome), and 72 were eligible and were evaluated. Male/Female ratio was 40/32. The median age at diagnosis was 19 months (range, 10 months and 17 years old). Median follow-up period was 3.64 years (range, 0.05 -5.96 years. One patient died of sepsis during initial induction therapy. Sixty-nine patients were stratified to SR and 2 patients to HR. Both of the two HR patients achieved CR but one relapsed. No therapy-related death was observed during intensification therapy. The 3-year event free and overall survival rate was 83.3% ± 4.4% and 87.5% ± 3.9 %, respectively. Age at diagnosis less than 2 years old was significant favorable prognostic factor for relapse (p=0.009). Sex, history of TAM, and chromosomal abnormalities (Normal karyotype or monosomy 7) did not influence the risk of relapse.Conclusion: This study succeeded the previous Japanese strategy with very low-intensive chemotherapy regimen for ML-DS, and despite the dose reduction of chemotherapeutic agents compared to the previous studies, the overall outcome was good and further dose reduction might be possible for specific subgroups. However, considering that most relapse occurred in the SR group defined by morphological treatment response and that relapsed cases are rarely salvageable, more accurate method for identification of the poor prognostic subgroup is needed.This trial is registered with UMIN Clinical Trials Registry (UMIN-CTR, URL: http://www.umin.ac.jp/ctr/index.htm), number UMIN000000989. DisclosuresNo relevant conflicts of interest to declare.

Differences of Somatic Mutations and Gene Expression in Blasts of Transient Leukemia and Acute Myeloid Leukemia of Down Syndrome

Background: Transient leukemia (TL) occurs in 30% of newborns with Down syndrome (DS) and typically resolves spontaneously. Approximately 20% of infants with TL go on to develop acute myeloid leukemia of DS (DS-AML) within the first four years of life. The blasts of both TL and DS-AML harbor somatic mutations of GATA1 . The objective of this study was to identify additional genetic events, which associated with the progression of TL to DS-AML.Methods: Leukemic blasts of TL, DS-AML and normal T lymphocytes were sorted from blood and bone marrow samples of five patients who successively developed both disorders. In addition, blasts of one patient with subsequent relapse of DS-AML were analyzed. Mutational spectrum and gene expression and were determined by exome sequencing and RNASeq (Illumina HiSeq2000). The presence of mutations, which were identified with this approach in DS-AML blasts, was examined by droplet digital PCR in TL blasts (BioRad QX200).Results: Blasts of TL overall harbored fewer mutations than those of DS-AML. Mutations of cohesin and RAS pathway genes were identified in a subset of DS-AML but not TL. In the patient who developed a relapse, different cohesin gene mutations were detected at initial diagnosis of AML and relapse; a minor clone present at initial diagnosis of AML emerged as the predominant clone at relapse. Concordant somatic GATA1 mutations were present in both TL and DS-AML blasts derived from the same patient. In contrast, other genetic events, which were detected in DS-AML blasts by exome sequencing, were confirmed to be absent in TL (by droplet digital PCR). The majority of differentially expressed genes showed higher expression levels in blasts of TL compared to DS-AML. They included genes encoding chemokines and related to IL1 and TGFb signaling.Conclusions: The pathogenic sequence starting with TL and culminating in AML is uniquely initiated in children with DS by somatic mutation of GATA1. In contrast, the events associated with the transformation of TL to DS-AML resemble progression factors also found in non-DS AML. These progression events were not detectable even in minor subclones of TL suggesting they are acquired after the onset of TL.This research was supported by funding from the Canadian Cancer Society Research Institute and Ontario Institute for Cancer Research. DisclosuresNo relevant conflicts of interest to declare.

Modeling the Multi-Step Pathogenesis of Acute Myeloid Leukemia of Down Syndrome

The multistep pathogenesis of Down Syndrome (DS)-associated pre-leukemia and subsequent progression to acute leukemia is one of the better characterized of all human blood malignancies. Children with DS have a 150 fold increased risk of developing acute megakaryoblastic leukemia (AMKL) and greater than 30 fold increased risk of developing B cell acute lymphoblastic leukemia (B-ALL). DS-AMKL is often preceded in late fetal development or soon after birth by a pre-leukemic syndrome termed transient myeloproliferative disorder (TMD), which is characterized by high numbers of abnormal megakaryocytes and megakaryoblasts in the circulation, spleen and liver. Previous work has demonstrated that constitutional trisomy 21 results in expansion of megakaryocyte-erythroid progenitors (MEP) in fetal liver (FL) with a concomitant reduction in fetal pre-pro-B cells. The expanded MEP population subsequently acquires an N-terminal truncating mutation in the transcription factor GATA1 (termed GATA1s), leading to selective expansion of a pre-leukemic erythromegakaryocytic blast population. While the majority of DS-TMD cases spontaneously resolve within 3 months, up to 15% of DS-TMD neonates can develop lethal progressive liver fibrosis. Progression to AMKL following spontaneous resolution of TMD is associated with acquisition of at least one additional germline mutation. While murine models implicate a role for trisomy 21 and GATA1s in the leukemogenic process, they do not faithfully recapitulate the pathology of the human disease. Previous attempts to model DS-associated TMD through xenotransplantation of DS-FL and DS-TMD cells have proven technically challenging. Therefore, there remains a need for a human model to investigate the genetic steps required for initiation of DS-TMD and progression to DS-AMKL.We previously identified a leukemia stem cell (LSC)-associated miRNA signature by sorting 13 adult AML patient samples into 4 sub-populations based on CD34/CD38 expression, followed by supervised analysis guided by the in vivo leukemia initiating capacity of each sub-population in an optimized xenotransplant model. Interestingly, the top three LSC-associated miRNA candidates are all located on chromosome 21. To determine the role of these miRNA in human leukemogenesis, we engineered a tri-cistronic lentivector for enforced expression. Compared to control vector-transduced cells, tri-cistronic vector-transduced Lin‒CD34+CD38‒ cord blood (CB) cells generated a myeloproliferative syndrome in xenotransplanted mice, with splenomegaly, enhanced CD45+ human bone marrow cellularity and blocked B cell development at the pro B cell stage. Human grafts were enriched for CD45+CD33+CD117+CD123+CD41lo/CD42lo cells in bone marrow, peripheral blood, spleen and liver. In the CD45‒ compartment, a distinct lineage switch was observed, with CD41+ megakaryocytic output supplanting normal CD235+ erythroid output. High numbers of CD41+CD42b+CD61+CD34lo human platelets were detected in peripheral blood and spleen. Blood films revealed large dysplastic platelets and megakaryoblast-like cells. Histology showed hCD45+ packed bone marrow cavities, with loss of normal architecture. Bone marrow, spleen and liver all showed extensive reticulin deposition. In the lineage negative (Lin-) fraction of BM, we observed an expansion in the proportion of human MEP and multi-lymphoid progenitors (MLP). To further model leukemic progression, we expressed GATA1s in combination with our tri-cistronic miRNA vector. Mice transplanted with double transduced cells showed intermediate levels of splenomegaly and bone marrow cellularity compared to mice transplanted with cells transduced with tri-cistronic vector alone. The addition of GATA1s induced a complete loss of B cell development while restoring erythroid development. In human Lin‒ cells isolated from the BM, addition of mutant GATA1s further augmented the proportion and total numbers of MEP while restoring the MLP compartment to normal levels.These data demonstrate that we have generated a human xenograft model of DS-TMD through enforced expression in normal CB cells of a tri-cistron comprising 3 LSC-associated miRNA in combination with mutant GATA1s. With this model in place, we plan to further interrogate the genetic lesions involved in progression from DS-TMD to DS-AMKL. DisclosuresNo relevant conflicts of interest to declare.

Standard Induction Followed by Low Dose Cytarabine for the Treatment of Acute Myeloid Leukemia of Down Syndrome

Background: Although acute myeloid leukemia occurring in patients with Down syndrome (AML-DS) is generally chemosensitive, these patients are more susceptible to regimen-related toxicities, and the optimal post-remission therapy for AML-DS is unknown. This study aimed to evaluate the outcome of post-remission chemotherapy using low dose cytarabine for AML-DS. Methods: We reviewed the medical records of 142 patients who were newly diagnosed as de novo AML between 1996 and 2011. Among them, 8 patients (5.6%) had Down syndrome. Seven patients received standard induction therapy composed of cytarabine (or behenoyl cytarabine) and anthracycline. Once complete remis sion (CR) was achieved, repetitive courses of low dose cytarabine were given. Results: Patients ’ median age at diagnosis was 1.3 years (range, 0.4-1.9). All but one showed French-American-British (FAB) M7 morphology. Six patients achieved CR (75%) after induction therapy and then received 9 to 20 courses (median, 14) of low dose cytarabine. One patient had 2 episodes of neutropenic fever, whereas the other 5 patients did not suffer from any complication. All six patients are alive event-free with a median follow-up of 118 months (range, 33-208). The estimated 5-year overall survival of all 8 AML-DS patients was 87.5%, while that of non-DS de novo AML patients was 58.6% (P=0.18). Conclusion: Low dose cytarabine was safe and effective as a post-remission therapy for AML-DS. Due to the rarity of AML-DS, a multicenter cooperative study is essential to identify the optimal duration of treatment and to further determine the feasibility of low dose cytarabine for these patients.

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.

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.

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