Syndrome Acute Megakaryoblastic Leukemia Research Articles

Genomic Landscape of Pediatric Non-Down's Syndrome Acute Megakaryoblastic Leukemia in China

Acute megakaryoblastic leukemia (AMKL) accounts for 4% to 15% of newly diagnosed pediatric acute myeloid leukemia (AML), and different from Down's syndrome AMKL (DS-AMKL) and non-AMKL, clinical outcomes of non-DS-AMKL are characterized by an inferior prognosis. Although comprehensive genomic studies have revealed key genomic aberrations of non-DS-AMKL in Western cohort, there is still no reports about Chinese patients. Here we report a comprehensive transcriptomic study on 100 Chinese pediatric non-DS-AMKL cases through 2009 to 2022 in Shanghai Children's Medical Center. A total of 133 rearrangements were detected involving 78 patients, including previously reported recurrent fusion events which defining subgroups of AMKL, namely CBFA2T3-GLIS2 (n=20, 20%), NUP98-KDM5A (n=16,16%), rearrangements of HOX cluster genes (HOXr, n=11, 11%), RBM15-MKL1 (n=5, 5%) and MLLr (n=3, 3%). Two new recurrent gene translocations, XPO1-TRNC18 and STAG2 rearrangements (STAG2r) were detected in 5 (5%) and 11 (11%) cases, respectively. Further unsupervised clustering analysis was done with the most variant coding genes defined by standard deviation, the results showed that cases carrying STAG2r and XPO1-TNRC18 fusion exhibited distinguishable gene expression pattern from other subgroups and were clustered together respectively. It suggested these two rearrangements as potential driver genomic alterations in AMKL. By introducing XPO1-TNRC18 fusion and three STAG2 aberrations (STAG2 knock-out, STAG2-GNA12 and STAG2-OCRL) into 32D cell line, we proved these gene alterations promoted cell proliferation and colonies formation, suggesting potential function in tumor transformation. Based on these evidences, we defined XPO1-TNRC18 and STAG2r as two new subgroups of AMKL. We identified 222 nonsynonymous sequency mutations affecting 137 genes totally, including 202 single-nucleotide variants and 20 insertion/deletion, with a median of two mutations for per case (range 0-6). Among driver abnormalities, 7 aberrations occurred in more than 3% of cases in current cohort, including JAK2 (n=15, 15%), MPL (n=13, 13%), PTPN11 (n=7, 7%), NRAS (n=5, 5%), GATA1 (n=4, 4%), CTCF (n=4, 4%) and JAK3 (n=3, 3%). All the mutations could be clustered into 8 pathways, and the most frequently mutated pathways were JAK signaling (30%), transcription regulation (29%) and RAS signaling (14%). Mutation frequency varied across different subgroups. For example, JAK/STAT lesions were enriched in STAG2r subgroup (p<0.05). Distinct genomic aberration profile was revealed while comparing results of this non-DS-AMKL cohort to the mutation profile of non-AMKL cohort which we have published before. CBFA2T3-GLIS2, NUP98-KDM5A, STAG2r, XPO1-TNRC18 and RBM15-MKL1 subgroups were merely detected in AMKL, while driver aberrations like FLT3-ITD, CBFB-MYH1 and so on were not detected in AMKL. For driver mutations, JAK2 (15% vs 3.7%, p<0.0001), MPL (13% vs 0%, p<0.0001 ) and CTCF (4% vs 0%, p=0.007) were detected more frequently in non-DS-AML, while NRAS (5% vs 18.8%, p<0.001), KRAS (2% vs 10.6%, p=0.008), KIT (1% vs 21.6%, p<0.001), CSF3R (1% vs 6.1%, p=0.047), FLT3 (0% vs 22.5%, p<0.001), CEBPA (0% vs 9.8%, p<0.001), AXSL2 (0% vs 8.6%, p<0.001), GATA2 (0% vs 6.1%, p=0.007) and DHX15 (0% vs 4.5%, p=0.038) occurred less frequently in non-DS-AMKL cohort. Survival analysis revealed association between key aberrations and clinical outcomes. Carrying CBFA2T3-GLIS2 (p=0.018), NUP98-KDM5A (p<0.001) and FUS-ERG (p=0.018) was associated with unfavorable prognosis. Driver mutations in JAK2 (p=0.03) and RAS pathway (p=0.01) were associated with shorter overall survival. Among the two new subgroups, the new XPO1-TNRC18 subgroup demonstrated similar clinical outcomes to MLLr cases, with 5-year EFS and OS of 50%, and STAG2r was characterized by a favorable clinical outcome, with 5-year EFS and OS over 75%. As the first comprehensive genomic landscape of Chinese pediatric non-DS-AMKL, our results defined two new subgroups with distinct expression patterns and prognosis characteristics. We further revealed a distinct mutation profile between AMKL and non-AMKL and shed light on association between driver aberrations and clinical outcomes. These findings revealed non-DS-AMKL as a heterogenous disease, and suggested the importance of exploration of risk-stratification and targeted therapy in different mutations driven cases.

CBFA2T3::GLIS2-positive acute leukemia with RAM and mixed T/megakaryocytic phenotype.

Herein, we present a rare case of acute myeloid leukemia (AML) with CBFA2T3-rearrangement and the expression of megakaryocytic and lymphoid markers, highlighting the need for a high suspicion index in differential diagnosis and applying adequate workup to avoid misdiagnosing this entity. CBFA2T3::GLIS2-positive AML is primarily found in infants with non-down syndrome acute megakaryoblastic leukemia (non-DSAMKL). Flow cytometry immunophenotyping plays an important role in recognizing the unique immunophenotype of bright CD56 expression with dim/negative expression of HLA-DR, CD38, and CD45 termed the RAM immunophenotype in this entity. Still, CBFA2T3::GLIS2-positive acute leukemia with T/megakaryocytic markers could be misdiagnosed as T-lymphoblastic leukemia/lymphoma, early T-cell precursor acute lymphoblasticleukemia/lymphoma, NK lymphoblastic leukemia, AML with minimal differentiation, or AML with myelodysplasia-related changes.

Pediatric non-Down's syndrome acute megakaryoblastic leukemia patients in China: A single center's real-world analysis.

Non-Down’s syndrome acute megakaryocytic leukemia (non-DS-AMKL) is a subtype of childhood acute myeloid leukemia (AML), whose prognosis, prognostic factors and treatment recommendations have not yet to be defined in children. We conducted a retrospective study with 65 newly diagnosed non-DS-AMKL children from August 2003 to June 2020 to investigate the clinical impact of factors and clinical outcome. Among all 65 patients, 47 of them were treated at our center who received three different regimens due to time point of admission (CAMS-another, CAMS-2009 and CAMS-2016 protocol), and the efficacy were compared. Patients with newly diagnosed non-DS-AMKL accounted for 7.4% of pediatric AML cases. The median age of the patients was 18 months at diagnosis, and over 90% of them were under three-years-old. The overall survival (OS) rates were 33.3% ± 1.7%, 66.7% ± 24.4% and 74.2% ± 4.0% for three groups (CAMS-another, CAMS-2009 and CAMS-2016 regimen), respectively. In CAMS-2016 group, the complete remission (CR) rate after induction was 67.7% (21/31), while the total CR rate after all phases of chemotherapy was 80.6% (25/31). The 2-year survival probability did not significantly improve in patients underwent HSCT when compared with non-HSCT group (75.0% ± 4.7% vs. 73.9% ± 4.6%, p=0.680). Those who had a “dry tap” during BM aspiration at admission had significantly worse OS than those without “dry tap” (33.3% ± 8.6% vs. 84.0% ± 3.6%, p=0.006). Moreover, the results also revealed that patients with CD34+ had significantly lower OS (50.0% ± 6.7% vs. 89.5% ± 3.5%, p=0.021), whereas patients with CD36+ had significantly higher OS than those who were negative (85.0% ± 4.0% vs. 54.5% ± 6.6%, p=0.048). In conclusion, intensive chemotherapy resulted in improved prognosis of non-DS-AMKL children and subclassification may base on “dry tap” and immunophenotypic. Although some progress has been made, outcomes of non-DS-AMKL children remain unsatisfactory, especially in HSCT group, when compared with other AML types.

Are Induced Pluripotent Stem Cells a Step towards Modeling Pediatric Leukemias?

Despite enormous improvements in pre-clinical and clinical research, acute leukemia still represents an open challenge for pediatric hematologists; both for a significant relapse rate and for long term therapy-related sequelae. In this context, the use of an innovative technology, such as induced pluripotent stem cells (iPSCs), allows to finely reproduce the primary features of the malignancy and can be exploited as a model to study the onset and development of leukemia in vitro. The aim of this review is to explore the recent literature describing iPSCs as a key tool to study different types of hematological malignancies, comprising acute myeloid leukemia, non-down syndrome acute megakaryoblastic leukemia, B cell acute lymphoblastic leukemia, and juvenile myelomonocytic leukemia. This model demonstrates a positive impact on pediatric hematological diseases, especially in those affecting infants whose onsets is found in fetal hematopoiesis. This evidence highlights the importance of achieving an in vitro representation of the human embryonic hematopoietic development and timing-specific modifications, either genetic or epigenetic. Moreover, further insights into clonal evolution studies shed light in the way of a new precision medicine era, where patient-oriented decisions and therapies could further improve the outcome of pediatric cases. Nonetheless, we will also discuss here the difficulties and limitations of this model.

Abstract B34: Cord blood CD34+ HSPCs: An in vitro model system for characterizing NUP98 fusions

Abstract Chromosomal translocations of the Nucleoporin 98 gene (NUP98) are associated with multiple hematopoietic malignancies. In particular, NUP98 rearrangements can be found in up to 6.6% of de novo cases of pediatric AML. Specific subpopulations of pediatric AML appear to be even more enriched for NUP98 fusions, with over 16% of cytogenetically normal pediatric AML patients and 15% of non-Down syndrome acute megakaryoblastic leukemia (AMKL) patients harboring these fusions. Clinically, the NUP98-KDM5A fusion is associated with erythroid and megakaryocytic leukemias while the NUP98-NSD1 fusion is associated with myelomonocytic leukemias. When NUP98 fusions are present in AML, they confer a poor prognosis. To better characterize the role of these fusions in human hematopoiesis, we used cord blood CD34+ (cb-CD34+) hematopoietic stem and progenitor cells (HSPCs) as an in vitro model system. We transduced cb-CD34+ HSPCs with NUP98-NSD1, NUP98-KDM5A, or empty vector control and studied the effects on cell proliferation and differentiation in liquid culture media containing cytokines (IL-6, FLT3-Ligand, SCF, TPO, and SR1 (an aryl hydrocarbon receptor antagonist)). NUP98-NSD1 and NUP98-KDM5A fusions increase the proliferation rate in liquid culture by three-fold and 19-fold, respectively (p<0.05). Consistent with observations from patients with these fusions, cells expressing NUP98-KDM5A have increased expression of CD235A and CD71, consistent with erythroid differentiation (p<0.05). In addition, cells expressing NUP98-NSD1 have increased expression of CD11b compared to NUP98-KDM5A (p<0.01) expressing cells. When placed in methylcellulose, the NUP98 fusions tested had no effect on total colony number, but we observed more erythroid differentiation for NUP98-KDM5A compared to vector control (p<0.05). Overall, the results of these in vitro studies demonstrate increased proliferation and inhibition of differentiation in HSPCs by NUP98 fusions, and cell differentiation patterns parallel the different AML subtypes observed in patients with NUP98-NSD1 and NUP98-KDM5A fusions. We propose cbCD34+ HSPCs as a promising system for in vitro modeling of NUP98 fusions. Citation Format: Ryan Hiltenbrand, Jonathan Miller, Jeffery Klco. Cord blood CD34+ HSPCs: An in vitro model system for characterizing NUP98 fusions [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B34.

Outcome and Genomic Characteristics of Non-Down Syndrome Acute Megakaryoblastic Leukemia in Children Treated with Low-Dose Chemotherapy for Induction Remission

Background: Acute megakaryocytic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) with a dismal outcome in children without Down syndrome. Refractory or relapsed disease accounts for treatment failure in most cases. Hematopoietic stem cell transplantation (HSCT) is recommended as post-remission therapy, although some patients have been cured with intensive chemotherapy alone. Genetic and genomic abnormalities are common in AMKL, but their prognostic significance is not known. In this study, we analyzed AMKL using high-throughput sequencing in addition to conventional morphology, immunophenotype, cytogenetic, and molecular (MICM) approaches. Methods: From February 2015 to April 2019, 19 cases of AMKL in non-Down syndrome patients were diagnosed according to conventional methods in our center. All patients, except one, were treated using a low-dose chemotherapy (LDC) regimen (cytarabine 10 mg/m2, subcutaneously every 12 h, 20 doses; mitoxantrone 5 mg/m2 days 1, 3 and 5, and G-CSF 5µ/kg subcutaneous, daily, 10 doses) for induction remission followed by four courses of intensive chemotherapy or hematopoietic stem cell transplantation (HSCT) as post-remission consolidation[1]. Patients were treated with consolidation chemotherapy if parents refused HSCT. High-throughput mutational sequencing of leukemia cells obtained at diagnosis was performed. The average sequencing depth was 500-1000× and analyses were performed using mGATK, SAMTools, SIFT, PolyPhen2, LRT, and MutatioinTaster methods. Results: The average age at diagnosis of AMKL of the nine boys and 10 girls was 19.7 months (7-53 months). Eighty-four percent of the cases were younger than 2 years of age. The 19 patients with AMKL represent 10% of cases diagnosed with AML during the study period. The median initial white blood cell (WBC) count was 13×109/L(2.38-126×109/L)and the average blasts at initial bone marrow (BM) was 54.5% (13-85%). In all cases of AMKL, more than 25% of cells were positive for CD41. Sixty-three percent of patients had recurrent fusion genes, including NUP98-KDM5A, HOXA11-AS/ANGPT1, CBFA2T3-GLIS2, KMT2A-MLLT3, RBM15-MKL1. Also, we identified PICALM-MLL10, TNIP1-CSF1R, NUP98-TAF3, and CBFA2T3-GLIS1 fusion genes which had not been previously reported in AMKL. EVI-1 expression was evaluated in all cases and was overexpressed in two cases. Forty-seven percent of patients showed at least single-gene mutations, including JAK2, MPL, KRAS, NRAS, TP53, NTRK3, MYO16, PDGFB, FLT3, and others. The response rate after remission induction I (IND-I) were 84.2% with 63.1% (12/19) attaining complete remission (CR) and 21.1% (4/19) partial remission (PR). After IND-II, the CR, PR, and no response (NR) rates was 68.4%, 10.5%, and 21.1%, respectively. Four patients (21.1%) were refractory to induction chemotherapy. Seven patients (36.8%) relapsed, three during consolidation chemotherapy courses, and four after transplantation (Table 1). Currently, 75% (6/8) patients who received chemotherapy only for consolidation were alive (median follow-up time 27 months). Eleven patients underwent allo-HSCT, and 8 patients are alive, including 6 cases living without evidence of disease. Four patients relapsed after allo-HSCT, and two of them have died. One additional patient died of transplantation related mortality. The 4-year OS, RFS, and EFS in this cohort were 62.4%±13.9%, 54.1±13.0% and 40.5±11.5%, respectively (Figure 1).Conclusion: Non-Down syndrome AMKL is genetically heterogeneous with recurrent and new fusion genes. Patients treated with LDC for induction followed by intensive chemotherapy or HSCT for consolidation have outcome comparable to those treated with intensive induction regimens. New treatment strategies are needed to improve the outcome of pediatric AMKL. Figure 1 Disclosures No relevant conflicts of interest to declare.

NUP98-KDM5A Fusion Induces Hematopoietic Cell Proliferation and Alters Myelo-Erythropoietic Differentiation

Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98 gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novo cases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5A is further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5A on the growth and differentiation of human hematopoietic cells has not been systemically studied. In this study, we transduced cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) with NUP98-KDM5A or control lentiviral vectors and examined cell proliferation and differentiation. Exposure to cytokines (SCF, TPO, IL-6, FLT3L, SR-1) selected for CD34+ cell growth, and expression of NUP98-KDM5A increased proliferation rate in liquid culture by 19.4-fold (p<0.01). In addition to a growth advantage, expression of NUP98-KDM5A led to differences in differentiation. NUP98-KDM5A expressing cells showed a 4.9-fold decrease (p<0.05) in myeloid terminal differentiation (defined as the CD33+, CD11B+ population) and 2.8-fold increase (p<0.05) in early stem cell progenitors (defined as the CD117+ population). Interestingly, NUP98-KDM5A expressing cells showed a 3.3-fold increase (p<0.01) in the erythroid population (defined by the CD235a+, CD71+ population). The increase in erythroid differentiation was confirmed by RNA sequencing, which showed an enrichment in genes involved in heme metabolism (Hallmark Heme Metabolism, FDR q-value 7.33x10-8) after only 72 hours post-transduction. Furthermore, RNA sequencing demonstrated upregulation of HOXA genes, including HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10 and HOXA11 (p 3.36x10-2 - 1.16x10-7), which has been previously described in other high-risk leukemic fusions (including other NUP98 fusions) as a primary mechanism of stem-cell renewal. To further elucidate NUP98-KDM5A effects on differentiation, cytokine supplements known to drive myeloid differentiation (SCF, TPO, G-CSF, and GM-CSF) were added to culture media, which demonstrated the same disruption in myelo-erythropoiesis based on a 4.0-fold decrease (p<0.001) in the terminal myeloid population, a 3.2-fold increase in CD117+ (p<0.001), and 2.6-fold increase in erythroid markers (p<0.001). Furthermore, NUP98-KDM5A recapitulated this phenotype in methylcellulose colony forming assays, with increased BFU-E and reduced CFU-GM colonies compared to control (p<0.01), despite no difference in overall colony number. Taken together, these in vitro studies demonstrate increased proliferation and altered differentiation in HSPCs by NUP98-KDM5A, with the pattern of differentiation by NUP98-KDM5A paralleling the M6 (erythroleukemia) AML subtype observed in patient samples harboring this fusion, supporting the clinical observation that NUP98-KDM5A is often a monogenic driver. To evaluate the leukemic potential of NUP98-KDM5A, cells were injected into NRG-SGM3 mice with NUP98-KDM5A inducing at 12 weeks a rapidly lethal myeloid disease in vivo (97.8% human CD45+ cells vs. 2.3% in control, p<0.001). Furthermore, spleens of the NUP98-KDM5A mice were markedly enlarged, weighing on average 926.7g vs. 47.3g spleens seen in control, a 19.8-fold increase in weight (p<0.05). In conclusion, the expression of NUP98-KDM5A in HSPCs drives proliferation and alters differentiation to maintain stem cell markers while driving an erythroid phenotype in vitro. Furthermore, this fusion is sufficient to engraft in the bone marrow and spleen of NRG-SGM3 mice in vivo and cause marked splenomegaly. Taken together, these data suggest this model properly recapitulates the human disease phenotype seen in patients expressing NUP98-KDM5A and future functional assays and drug screens may provide important insights into understanding the pathophysiology and pharmacologic vulnerabilities of this fusion class. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.

Non-Coding HOX Fusions in Pediatric Non-Down Syndrome Acute Megakaryoblastic Leukemia

The homeobox (HOX) genes are a highly conserved family of transcription factors involved in embryonic patterning as well as adult hematopoiesis. Dysregulation of HOX genes, in particular upregulation of HOXA cluster genes, is a frequent event in Acute Myelogenous Leukemia (AML). Recently, we performed a detailed genomic analysis on pediatric non-Down Syndrome Acute Megakaryoblastic Leukemia (non-DS-AMKL) and identified novel fusions involving a HOX cluster gene in 14.9% of the cases. While most fusions were predicted to lead to an in-frame functional protein, several fusions included a non-coding HOX antisense gene (PLEK-HOXA11-AS, C8orf76-HOXA11-AS, HOXA10-AS-CD164) that were predicted to result in a loss of function of these regulatory transcripts. The functional consequence of these events, however, remain unknown. HOXA11-AS (human) and Hoxa11os (mouse) have been previously shown to have mutually exclusive expression with the Hoxa11 transcript throughout development. We therefore hypothesized that loss of function of non-coding HOX antisense genes as a result of these structural variations would cause upregulation of nearby coding HOXA genes that in turn promote leukemogenesis. To test this hypothesis, using CRISPR-Cas9 technology, we genome edited the human AMKL cell line CMK to carry the PLEK-HOXA11-AS translocation. qRT-PCR of HOXA11-AS and HOXA9-11 transcripts in this cell line recapitulated the pattern seen in patient specimens. Specifically, HOXA11-AS expression was significantly diminished while HOXA10 and HOXA11 transcripts were upregulated 1.8-2.5-fold when compared to parental CMK cells (p=0.0385 and p=0.006 respectively). To further investigate the loss of HOXA11-ASin vivo a CRISPR-Cas9 Hoxa11os knockout mouse model was established. qRT-PCR on bone marrow confirmed the loss of Hoxa11os transcripts in heterozygous (Hoxa11os1+/-) and homozygous (Hoxa11os-/-) mice of both genders (p=<0.0001-0.0012). Consistent with Hoxa11os knockdown, Hoxa11 transcripts were upregulated in male (1.8-fold p=0.0023 Hoxa11os+/-, and 2-fold p=0.0052 Hoxa11os-/-)and female (1.3-fold p=0.0074 Hoxa11os+/- and 2.2-fold p=0.0226 Hoxa11os-/-) bone marrow compared to wild type gender matched littermates. Interestingly, flow cytometry analysis of progenitor subsets revealed gender specific findings. We found a significant increase in the frequency of the lineage negative, Sca-1 and c-Kit positive (LSK) population in males (0.13% of total bone marrow Hoxa11os+/+, 0.19% p=0.0214 Hoxa11os+/-, and 0.25% p=0.0001 Hoxa11os-/-) compared to wild type male littermates but not in female mice at 8 weeks of age. In contrast an increase in the megakaryocyte-erythroid (MEP) population was seen only in the female setting (0.07% Hoxa11os+/+, 0.15% p=0.0055 Hoxa11os+/-, and 0.165% p<0.0001 Hoxa11os-/-). Limiting dilution colony forming assay confirmed the higher LSK frequency with a 2-fold increase in the number of colonies for male knockout marrow compared to wild type marrow in contrast to the female setting where no significant differences were seen. As hormonal signals have been shown to regulate expression of HOX genes and differences in clonogenicity of male and female stem cells has been previously demonstrated, we reasoned this phenomenon could be secondary to extrinsic stimuli in vivo. The relatively uniform Hoxa11 levels in male and female knockout mice, however, suggested that cell intrinsic factors may also play a role. We therefore overexpressed HOXA11 into male and female wild type bone marrow ex vivo for colony forming assays to determine if elevated levels of the HOXA11 protein led to functional differences. This assay demonstrated a clear enhancement of self-renewal in male but not female bone marrow in contrast to HOXA9 overexpression which serially replated in both genders. Combined these data demonstrate that loss of function alterations in Hoxa11os transcripts lead to upregulation of Hoxa11 and gender specific hematopoietic progenitor cell perturbations. Ongoing efforts include competitive transplant studies as well as RNA and ChIP sequencing to identify gender specific downstream targets of Hoxa11 in the hematopoietic compartment in order to understand the selective expansion of progenitor subsets and male specific self-renewal capacity of this protein. These data will contribute to our understanding on how HOXA11-AS translocations promote oncogenesis. Disclosures Zwaan: Daiichi Sankyo: Consultancy; Sanofi: Consultancy; Roche: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Incyte: Consultancy; Celgene: Consultancy, Research Funding; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Janssen: Consultancy. Gruber:Bristol-Myers Squibb: Consultancy.

The changing scenario of non-Down syndrome acute megakaryoblastic leukemia in children.

Pediatric non-Down-syndrome acute megakaryoblastic leukemia (non-DS-AMKL) is a heterogeneous subtype of leukemia that has historically been associated with poor prognosis. Until the advent of large-scale genomic sequencing, the management of patients with non-DS-AMKL was very difficult due to the absence of reliable biological prognostic markers. The sequencing of large cohort of pediatric non-DS-AMKL samples led to the discovery of novel genetic aberrations, including high-frequency fusions, such as CBFA2T3-GLIS2 and NUP98-KDM5 A, as well as less frequent aberrations, such as HOX rearrangements. These new insights into the genetic landscape of pediatric non-DS-AMKL has allowed refining the risk-group stratification, leading to important changes in the prognostic scenario of these patients. This review summarizes the most important molecular pathogenic mechanisms of pediatric non-DS-AMKL. A critical discussion on how novel genetic abnormalities have refined the risk profile assessment and changed the management of these patients in clinical practice is also provided.

MRTFA Augments Megakaryocyte Maturation By Enhancing the SRF Regulatory Axis

Pediatric non-Down Syndrome acute megakaryoblastic leukemia (AMKL), a disease where megakaryocyte (MK) maturation is blocked, has a very poor prognosis. In 13% of AMKL cases of this type, the transcriptional co-factor MRTFA is expressed as part of a fusion protein. Normally, MRTFA levels increase in hematopoietic cells during megakaryopoiesis, making it likely that expression of MRTFA as part of a fusion protein leads to aberrant gene regulation, which results in leukemia. However, the mechanism by which megakaryopoiesis is blocked in AMKL is unknown. Therefore, we sought to parse out the role of MRTFA in normal megakaryopoiesis, so that we could better understand the cause for maturation block in leukemia.MRTFA is a co-factor of serum response factor (SRF). Knockout of either SRF or MRTFA in mice decreases MK maturation causing thrombocytopenia; and MRTFA overexpression (MRTFAOE) promotes MK maturation of primary human bone marrow (BM) cells in vitro. Our novel study shows that genomic regulation by MRTFA promotes MK maturation. In the human erythroleukemia (HEL) cell line, MRTFAOE enhances phorbol ester (TPA)-induced megakaryopoiesis, mimicking the effects of MRTFA on primary MK maturation. TPA-induced HEL cells with MRTFAOE achieve significantly higher 8N and 16N ploidy (p < 0.001, N = 4), compared to those without MRTFAOE. To identify the mechanisms underlying megakaryocytic differentiation, we bioinformatically analyzed anti-SRF chromatin immunoprecipitation (ChIP)-sequencing [approx. 15 million reads/sample] and RNA-sequencing data [approx. 20 million reads/sample], from non-induced and TPA-induced HEL cells, with and without MRTFAOE (N = 2/condition). We identified SRF peaks that change during TPA-induction, with and without MRTFAOE, and analyzed the strength of their binding and motif status. MRTFOE not only increased SRF binding to the genome during MK maturation (p ≤ 10-8), but preferentially retained binding at genomic CArG (CC[W]6GG) motifs, where SRF binds to in association with either MRTFA or specific ETS proteins (ELK1, ELK4). We then analyzed upregulated and downregulated genes during TPA-induction, with and without MRTFAOE, and identified those that had associated SRF peaks. As expected, TPA-induction upregulated megakaryocytic and cytoskeletal genes (VWF, ACTN1, CORO1A) and downregulated erythroid genes (KLF1, GYPB, GYPE). Interestingly, TPA-induction with MRTFAOE increased the number of upregulated genes by 27% and the number of downregulated genes by 10%. In TPA-induced cells, MRTFAOE increased the percentage of differentially expressed genes that had SRF peaks (25% versus 11% for upregulated genes, and 9% versus 4% for downregulated genes), further highlighting the direct role that MRTFA plays in MK maturation. Also, genes upregulated by TPA-induction alone have both ETS and CArG motifs, whereas those upregulated by TPA-induction with MRTFAOE lack ETS binding motifs, suggesting that MRTFAOE skews SRF binding toward CArG motifs. With anti-MRTFA ChIP-PCR in HEL cells, we confirmed the novel finding that along with SRF, MRTFA binds to regions associated with megakaryocytic and cytoskeletal genes (XRK6, CORO1A). Therefore, SRF and MRTFA together regulate expression of genes that are important for normal megakaryopoiesis, which explains why lack of these proteins adversely affects megakaryopoiesis in mice.We asked whether our findings in HEL cells were applicable to the more clinically relevant primary human BM cells. Anti-SRF and anti-MRTFA ChIP-PCR on day 0 primary human cells (CD34+ cells) and day 8 differentiated cell subpopulations (CD41+CD42-, CD41+CD42+) confirmed that both SRF and MRTFA have increased binding during megakaryopoiesis at target sites associated with upregulated genes, such as CORO1A, TNS1, and XRK6 (p < 0.001). Therefore, we illustrated that transcriptional regulation by SRF/MRTFA function similarly in human BM cells undergoing megakaryopoiesis.We show for the first time that MRTFA increases both the genomic association and activity of SRF, and upregulates genes that enhance primary human megakaryopoiesis. These findings suggest that aberrant expression of MRTFA as a fusion protein in AMKL may disrupt essential transcriptional regulation via the SRF/MRTFA axis, resulting in blocked MK maturation. This forms a crucial reference point for future studies to understand the altered SRF/MRTFA function in AMKL. DisclosuresNo relevant conflicts of interest to declare.

3085 - Generation of a Novel Mouse Model Recapitulating Features of Human Acute Megakaryoblastic Leukemia

Non-Down syndrome (non-DS) acute megakaryoblastic leukemia (AMKL) represents approximately 10% of pediatric acute myeloid leukemia (AML) cases and is associated with poor prognosis (<40% 3-year survival). Transcriptome, whole-genome sequencing, FISH, and RT-PCR analyses of pediatric non-DS AMKL patients recently revealed the presence of cryptic oncogenic fusions. While the majority of pediatric non-DS AMKLs harbor only one fusion, none are recovered in adult AMKL. Despite intensive studies of these oncogenic proteins, the molecular mechanism(s) underlying the role of specific oncogenic fusions in tumor development remains poorly characterized. This is due, in part, to the lack of mouse models that mimic the development of human AMKL. Thus, the generation of new AMKL mouse models harboring specific oncogenic fusions is a prerequisite to study the in vivo transformation process underlying AMKL formation and to identify new therapeutic avenues. To generate AMKL mouse models, we reconstituted lethally irradiated mice with fetal liver cells transduced with oncogenic fusion proteins. AMKL was generated with a penetrance of 50–60% and affected mice displayed striking splenomegaly. Flow cytometric analysis of fusion positive cells revealed increased proportions of cells co-expressing CD41 and CD61, and a reduced proportion of cells expressing B cell, T cell, macrophage and erythroid lineage markers, indicating a skewing towards megakaryocytic differentiation. Moreover, we observed an enrichment of hematopoietic stem and progenitor cells, lineage committed progenitors, and immature megakaryocyte progenitors in the bone marrow and spleens of sick mice. Bioluminescent imaging allowed detection of leukemia cells in bone marrow and spleen. Finally, histology analysis of bone marrow, spleen and peripheral blood revealed the presence of cells with morphology consistent with megakaryoblasts, megakaryocytes and megathrombocytes. In summary, we developed and characterized a novel AMKL murine model with high penetrance that recapitulates many clinical features of human AMKL and provides a novel tool to conduct mechanistic and preclinical studies of this disease.

Abstract 1482: SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL)

Abstract Individuals with Down syndrome (DS) are highly predisposed to develop hematopoietic malignancies, especially acute megakaryoblastic leukemia (DS-AMKL). Generally, genes located on chromosome 21 tend to be increased in Down syndrome patients, but how these individual genes contribute to development of DS-AMKL is still unclear. SON, a gene located on chromosome 21, is upregulated in Down syndrome patients, and it has been reported that DS-AMKL cells have higher level of SON expression. The SON protein functions as a splicing factor and a transcription regulator in leukemia but the role of SON upregulation in DS-AMKL pathogenesis is still not fully elucidated. In addition, runt-related transcription factor 1 (RUNX1) is also located on chromosome 21, but its expression level is downregulated in DS-AMKL patients despite the increased gene dosage. We show that SON represses RUNX1 expression and blocks terminal megakaryocytic differentiation in DS-AMKL. Knockdown of SON in DS-AMKL cell lines leads to morphologic changes and increased cell adhesion. Moreover, cell surface marker analyses demonstrate that the depletion of SON induces increased CD41+ CD61+ populations as well as polyploidy cells, indicating megakaryocytic differentiation. Consistent with these results, SON deletion causes upregulation of RUNX1, which is a critical factor in the megakaryocytic differentiation. Furthermore, our data show that knockdown of SON increases several megakaryocytic transcription factors and decreases erythropoietic transcription factors, facilitating terminal megakaryocytic differentiation of leukemic blasts. Mechanistically, SON binds to the proximal promoter region as well as two intronic enhancer regions upstream of the proximal promoter at the RUNX1 gene locus, and suppresses transcription of RUNX1 by lowering the level of H3K4me3. Taken together, we propose that the increased genomic dosage of SON in Down syndrome impairs terminal differentiation of megakaryo-erythroblasts into mature megakaryocytes through binding to the RUNX1 promoter/enhancer to repress its transcription, which contributes to the higher incidence of AMKL in Down syndrome patients. Our results reveal a novel connection of SON to DS-AMKL pathogenesis and provide clinical importance of SON upregulation in DS. These findings suggest that SON could be a promising therapeutic target for DS-AMKL patients. Citation Format: Lana Vukadin, Eun young Park, Jung Hyun Kim, Erin Eun Young Ahn. SON represses RUNX1 expression and impairs megakaryocytic differentiation in Down syndrome acute megakaryoblastic leukemia (DS-AMKL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1482.

Pediatric non-Down syndrome acute megakaryoblastic leukemia is characterized by distinct genomic subsets with varying outcomes.

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts. While rare in adults, AMKL accounts for 4-15% of newly diagnosed childhood AML cases. AMKL in individuals without Down syndrome (non-DS-AMKL) is frequently associated with poor clinical outcomes. Previous efforts have identified chimeric oncogenes in a substantial number of non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements, and NUP98-KDM5A. However, the etiology of 30-40% of cases remains unknown. To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult). We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes. These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes. Overall, these data shed light on the etiology of AMKL and provide useful information for the tailoring of treatment.

Prognostic Relevance of Recurrent Genetic Aberrations in Pediatric Acute Megakaryoblastic Leukemia

Genetic abnormalities and early treatment response are the main prognostic factors in pediatric acute myeloid leukemia (AML). Non-Down Syndrome (non-DS) acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML, with a poor prognosis. These cases present with diverse cytogenetic aberrations, which might be important for risk-group stratification. A well-known genetic aberration in non-DS-AMKL is t(1;22)(p13;q13), resulting in expression of the RBM15/MKL1(OTT/MAL) fusion transcript. In recent years, targeted and genome-wide sequencing has identified novel, recurrent molecular abnormalities in pediatric AMKL. New fusions identified are inv(16)(p13.3q24.3) resulting in CBFA2T3/GLIS2 (Gruber et al, Cell, 2012; Masetti et al, Blood, 2013) and t(11;12)(p15;p13) resulting in NUP98/KDM5A (de Rooij et al, Leukemia, 2013) as specific aberrations in pediatric non-DS AMKL. Also KMT2A(=MLL)-rearrangements are recurrent in non-DS AMKL. The prognostic relevance of these novel abnormalities is not determined in a large cohort, since this disease is rare.To assess frequencies, clinical characteristics and outcome parameters of recurrent cytogenetic aberrations of pediatric non-DS AMKL, databases of the BFM-SG, DCOG, AIEOP, and COG were combined. In this study, we analyzed 151 newly diagnosed pediatric non-DS AMKL cases diagnosed between 1998 and 2014 of whom a sample was available. All patients included were screened for NUP98/KDM5A, CBFA2T3/GLIS2 and RBM15/MKL1 with reverse transcriptase(RT-) PCR,and for KMT2A-rearrangements using split signal FISH and RT-PCR. To assess outcome, probability of event-free survival (pEFS) and probability of overall survival (pOS) were estimated by the Kaplan-Meier method, and groups were compared with the log-rank test; the cumulative incidence of non-response or relapse (pCIR) was analysed by the Kalbfleisch and Prentice method, and groups were compared with the Gray's test. A Cox regression analysis was done for EFS and relapse incidence with as co-variables cytogenetic subgroup, age, sex, WBC and stem cell transplantation (time dependent variable).Patients were treated with different protocols; however, all protocols consisted of intensive chemotherapy using an anthracycline and cytarabine backbone for both induction and consolidation; 26% of patients received stem cell transplantation (SCT) in first remission. Median age was 1.6 years (range 0.1-17.1 years), with 62% of the patients younger than 2 years at diagnosis; 45% of patients were males, and median white blood cell count was 13.7x109/L (range 1.1-378.5x109/L). Translocation NUP98/KDM5A was identified in 9%; the CBFA2T3/GLIS2 translocation in 16%; RBM15/MKL1 in 12% and KMT2A-rearrangements in 9%, and hence in 54% none of these abnormalities were detected. All these aberrations were mutually exclusive. Comparing patients with NUP98/KDM5A, CBFA2T3/GLIS, RBM15/MKL1, or KMT2A-rearrangements with other pediatric AMKL patients, no significant differences in sex, age, and white blood cell count were found. Outcome between the included collaborative groups did not differ significantly. The 5-year pOS of the entire pediatric non-DS AMKL cohort was 56±4%, 5-year pEFS was 51±4%. NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearranged positive patients showed a poor outcome (pOS 36±13%, 38±10%, and 38±13% respectively), compared to RBM15/MKL1 positive cases and other pediatric non-DS AMKL (70±11% and 65±5% respectively, p=0.04, Figure 1). Harboring NUP98/KDM5A, CBFA2T3/GLIS2 or a KMT2A-rearrangement resulted in an increased risk of experiencing an event (EFS; HR1.65, 95%CI;1.03-2.66, p=0.039) or relapse (RFS; HR2.14, 95%CI;1.12-4.11, p=0.022). SCT was not an independent factor for event or relapse free survival (HR1.20, 95%CI;0.65-2.20, p=0.565 and HR 1.13, 95%CI;0.57-2.24, p=0.717, respectively), nor did sex, age at diagnosis or WBC.Our results indicate that non-DS AMKL is a heterogeneous group within pediatric AML. Although the overall survival is poor for non-DS AMKL in general with a 5-yr pOS of ~55%, the poor outcome is specifically determined by cases with NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearrangements. Other variants, including those with RBM15/MKL1, are associated with a better outcome. These data show that international collaboration allows the identification of prognostic subgroups, which may lead to new and more refined risk-group stratification. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Caracterización de mutaciones en el gen GATA-1 en pacientes con Síndrome de Down y diagnóstico de mielopoyesis anormal transitoria o leucemia megacarioblástica aguda

Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.

Comprehensive Fusion Gene Analysis Of Pediatric Non-Down Syndrome Acute Megakaryoblasitc Leukemia

▪ BackgroundPediatric non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL) is a biologically and clinically unique subset of acute myeloid leukemia. Though non-DS-AMKL was thought to be a poor prognostic disease with a survival rate of 14-34%, recent improvements of diagnosis and intensive chemotherapy with effective supportive care have increased the rate to 50-70%. Little was known about the specific molecular marker in non-DS-AMKL other than t(1;22)(p13;q13)/OTT-MAL, however, new diagnostic and prognostic markers have been determined using new techniques such as high-resolution array CGH and next generation sequencing. Recent studies have identified new recurrent cryptic translocations, inv(16)(p13.3q24.3) and t(11;15)(p15;q35), which created CBFA2T3-GLIS2 and NUP98-JARID1A gene fusions, respectively. It has been reported that CBFA2T3-GLIS2 is a novel independent factor for dismal clinical outcome and CBFA2T3-GLIS2 positive cases should be classified as the high risk group, meanwhile, the significance of NUP98-JARID1Ais less clear at present. The aim of this study is to identify the more accurate frequency and clinical impact of such fusion genes in non-DS-AMKL, which will dissect the biology of this distinct subtype of leukemia. MethodsWe analyzed 44 newly diagnosed pediatric non-DS-AMKL cases enrolled in two Japan clinical trials, the AML-99 study and AML-05 study. The median age was 1 year of age, and median white blood cell count at diagnosis was 23 x 109/l. Morphologic classification and karyotyping of the cases enrolled in AML-05 study were centrally reviewed. Molecular characterization included the analyses of CBFA2T3-GLIS2, NUP98-JARID1A, NUP98-NSD1, OTT-MAL, MLL-AF9, MLL-AF10, MLL-PTD, FLT3-ITD, KIT, RAS, WT1, and NPM1. They were determined by RT-PCR and/or PCR followed by direct sequencing, though only MLL-PTD was determined by MLPA method. Some survival analyses were restricted to the cases enrolled in the AML-05 study. ResultsCBFA2T3-GLIS2 was identified in 12 cases (27.3%), NUP98-JARID1A in 4 cases (9.1%), and OTT-MAL in 11 cases (25%), MLL-AF9 in 2 cases (4.5%), and MLL-AF10 in 1 case (2.3%). Only CBFA2T3-GLIS2-positive cases had a high white blood cell count compared with other cases (median, 33x109/l for positive cases vs 20x109/l for negative cases, p=0.0807). Seven of the 12 CBFA2T3-GLIS2-positive cases died, while 4 of the 11 OTT-MAL-positive cases died. In the AML-05 study, 2-years Overall survival rates (OS) of non-DS-AMKL cases (n=34) were 36.4% for CBFA2T3-GLIS2-positive cases (n=11) vs 73.9% for negative cases (n=23) (p=0.0773), and 70.0% for OTT-MAL-positive cases ( n=10 ) vs 58.3% for negative cases ( n=24 ) (p=0.75). Regarding NUP98-JARID1A-positive cases, the number was too small to perform outcome analyses, although 2 cases relapsed and died. Among all cases, 4 of 5 cases with normal karyotype and 5 of 6 cases with acquired 21 trisomy had CBFA2T3-GLIS2, suggesting that those karyotypes may be useful features to suspect the presence of CBFA2T3-GLIS2 in the cases. Twenty-eight DS-AMKL samples did not harbor any CBFA2T3-GLIS2, NUP98-JARID1A, or OTT-MAL. ConclusionIn addition to well-known OTT-MAL, CBFA2T3-GLIS2 and NUP98-JARID1A are new recurrent fusion genes in pediatric non-DS-AMKL, which demonstrates that pediatric non-DS-AMKL is a distinct subtype of pediatric AML. CBFA2T3-GLIS2 is an independent poor prognostic factor and OTT-MAL may be a relatively good prognostic factor, while the impact of NUP98-JARID1A is still unknown and further investigation is needed. Therefore, routine screening for these fusion genes at diagnosis will be essential for proper identification and stratification of pediatric AML cases. Disclosures:No relevant conflicts of interest to declare.

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.

Core-binding factor runt domain α-subunit 2 translocated to 3 (CBFA2T3)–GLIS family zinc finger 2 (GLIS2) fusion protein as a biomarker for non–Down syndrome acute megakaryoblastic leukemia (non–DS-AMKL)

Core-binding factor runt domain α-subunit 2 translocated to 3 (CBFA2T3)–GLIS family zinc finger 2 (GLIS2) fusion protein as a biomarker for non–Down syndrome acute megakaryoblastic leukemia (non–DS-AMKL)

An Inv(16)(p13.3q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein Defines an Aggressive Subtype of Pediatric Acute Megakaryoblastic Leukemia

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors.