non-DS Acute Megakaryoblastic Leukemia Research Articles

Survival Outcomes of Acute Megakaryoblastic Leukemia in the United States: A 10-Year SEER-Based Study

Background: Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), commonly observed in children with Down Syndrome (DS) but also seen in adults. The cytogenetic abnormality t(1;22)(p13.3;q13.1), has been reported in children with non-DS AMKL, which involves the fusion of RNA-binding motif protein-15 and megakaryoblastic leukemia-1 resulting in abnormal proliferation of megakaryocyte progenitors. Population-level data on outcomes of AMKL is scarce. Methods: We conducted a retrospective analysis using the Surveillance Epidemiology and End Results (SEER) database, a U.S. population-based registry maintained by the National Cancer Institute. We included all cases with histologically confirmed diagnoses of AMKL and AML with t(1;22)(p13.3;q13.1) between the years 2010 and 2019. Survival analysis was performed using the Kaplan-Meier method, and differences in overall survival (OS) were assessed using the log-rank test. Results: The analysis included a total of 217 cases, consisting of 54 patients with AML with t(1;22)(p13.3;q13.1) and 163 patients with AMKL. Among the AML cases with the cytogenetic abnormality, the median age was 58 years (ranging from 0 to 89 years), and 61% were female. 74% (n=40) of these patients received chemotherapy. The 1-year survival rate was 54.0% for the pediatric group (age < 18 years, n=10) and 44.6% for the adult group (age > 18 years, n=44) (Median OS 20 vs 8 months, p=0.69). Patients who received chemotherapy had a significantly higher 1-year survival rate of 57.9% compared to 14.3% in those who did not receive chemotherapy (p=0.0005). No significant difference in survival was observed when patients were grouped based on the year of diagnosis, with 1-year survival rates of 50.8% for the period 2010-2014 and 41.3% for 2015-2019 (p=0.66). Patients with AMKL were divided into two age groups: pediatric AMKL (age < 18 years, n=96) and adult AMKL (age > 18 years, n=67). The median age in this group was 3 years (ranging from 0 to 86 years), and 55% were male. 81% (n=132) of these patients were treated with chemotherapy. The 1-year survival rate for the pediatric group was 79.4%, whereas it was 17.6% for the adult group (p<0.0001). Chemotherapy significantly improved overall survival (OS) for both pediatric and adult groups, with 1-year survival rates of 82.5% vs. 35.7% in the pediatric group and 27.6% vs. 0.00% in the adult group in patients who did and did not receive chemotherapy, respectively (p=0.041 for pediatric group and p<0.0001 for adults). No significant differences in survival were observed among the groups based on the year of diagnosis, both in adults and the pediatric group. Conclusion: AMKL is commonly seen in infants and children, but it can affect individuals of all age groups. The prognosis is more favorable in pediatric patients. In contrast, outcomes for adults remain poor with current chemotherapeutic agents. Interestingly, AML with t(1;22)(p13.3;q13.1), which has been predominantly reported in children in the literature, was found to be more common in adults in this study. Regardless of age group, current treatments show poor outcomes. This study emphasizes on the urgent need for investigational agents.

A Multicenter Clinical Study with Reduced Intensive Chemotherapy for Induction Remission of Pediatric Acute Megakaryoblastic Leukemia

Bcakground: Pediatric acute megakaryoblastic leukemia (AMKL) without Down syndrome (DS) is a genetically heterogenous myeloid malignancy and with dismal prognosis. Refractory and relapse remains a major challenge. Given to the young age distribution of AMKL and high frequency of abandonment in middle-income countries, we conducted a multicenter clinical trial (ChiCTR1800015875) in aims to lower the intensity of induction while without compromise the remission rate and survival. Methods: From June 2018 to December 2022, there were consecutively 43 cases with de novo non-DS AMKL, which accounted for 7.6% cases of acute myeloid leukemia (AML) during the same period, enrolled in this trial. All the patients received at least one cycle of low-dose induction with HAG regimen (homoharringtonine 1 mg/m 2, intravenous daily, days 1-7; cytarabine 10 mg/m 2, subcutaneously every 12 hours, 20 doses, and G-CSF 5µ/kg subcutaneous, daily, 10 doses) followed by 3 to 4 courses of intensive chemotherapy or HSCT as post-remission consolidation. Besides conventional molecule testing, RNA-sequencing and whole exon sequencing (WES) were performed as well to reveal underlying genetic landscape of this type of disease. Results: The median age of the 43 patients at diagnosis was 18.5 (range 4-114) months. The median while blood cell (WBC) counts and platelet at diagnosis were 17.2×10 9/L (range 1.8-105.7 ×10 9/L) and 36.5×10 9/L (range 4-472 ×10 9/L), respectively. Among them, 39.5% of cases exhibited hepatosplenomegaly. A diversity of genetic fusions and mutations were uncovered. The recurrent fusions in this cohort include CBFA2T3-GLIS2 (16.3%), MLLr (11.6%), NUP98-(9.3%), RBM15-MKL1 (7.0%), HOX fusions (7.0%) and PICALM-MLLT10 (4.7%). Other non-recurrent fusions accounted for 18.6% (Figure 1A). The common mutations detected in our pediatric AMKL were JAK2, NRAS, MPL, BCOR, CTCF, KRAS and PTPN11. Regarding treatment response, 20 of 43 cases (46.5%) attained complete remission (CR) or complete remission with incomplete blood cell recovery (CRi) after one cycle of induction. Among the 20 cases, 9 of them got minimal residual disease (MRD) negative by flow cytometry. Ten patients (23.2%) achieved partial remission (PR) while 13 patients (30.2%) showed no response (NR) to treatment. Thirty-one patients continued with another cycle of the same regimen and 21 of the 28 cases who were available for evaluation (75.0%) attained CR/CRi. Disease relapse was observed in 16/43 (37.2%) cases, with 7/43 (16.2%) patients relapsed after transplantation. The 3-year overall survival (OS) and event-free survival (EFS) were 49.6±8.2% and 29.5±7.1%, respectively (Figure 1B). In total, more than half of patients (22/43, 51.5%) underwent HSCT. The 3-year OS of transplant group and chemotherapy group were 57.9±11.7% versus 39.5±11.2%, P=0.036. As genetic characteristics often predict treatment response and prognosis, we wonder which subgroup could benefit from the reduced intensive induction. We further compared the features of patients who got CR/CRi after induction I with those achieved PR/NR. Interestingly, we found more patients with adverse fusions in PR/NR group, such as CBFA2T3-GLIS2 (26.0% vs. 5.0%), NUP98- (17.4% vs. 0%), MLLs (13.0% vs. 10.0%), and RBM15-MKL1 (8.7% vs. 5.0%); while other non-recurrent fusions were more common in CR/CRi group (4.3% vs. 35.0%). Two thirds of patients (67.4%) experienced refractory or relapse and almost half of them harbored adverse fusions or mutations such as CBFA2T3-GLIS2, MLLr, NUP98- fusions, NRAS and PTPN11. On the other hand, 14 patients without refractory or relapse had genetic features with more JAK2 and MPL mutations. No treatment-related mortality (TRM) occurred during induction. Only one patient with KRAS mutation died of infection after consolidation III. Conclusion: Insight into the genetic and molecular landscape improved our understanding of pediatric AMKL. Distinct molecular features can serve as a tool to tailor the therapy. Though treatment still lags behind and novel therapy is urgent for the adverse subgroups, our low-dose induction with HAG regimen could benefit for a subgroup with non-recurrent fusions, which showed efficiency while is tolerable for the young age group. Based on our results, we provide an induction option for these AMKL patients, especially for those in the middle-income countries.

Advances in molecular characterization of pediatric acute megakaryoblastic leukemia not associated with Down syndrome; impact on therapy development.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) in which leukemic blasts have megakaryocytic features. AMKL makes up 4%-15% of newly diagnosed pediatric AML, typically affecting young children (less than 2years old). AMKL associated with Down syndrome (DS) shows GATA1 mutations and has a favorable prognosis. In contrast, AMKL in children without DS is often associated with recurrent and mutually exclusive chimeric fusion genes and has an unfavorable prognosis. This review mainly summarizes the unique features of pediatric non-DS AMKL and highlights the development of novel therapies for high-risk patients. Due to the rarity of pediatric AMKL, large-scale multi-center studies are needed to progress molecular characterization of this disease. Better disease models are also required to test leukemogenic mechanisms and emerging therapies.

Clinical impact of genomic characterization of 15 patients with acute megakaryoblastic leukemia-related malignancies.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia but is approximately 500 times more likely to develop in children with Down syndrome (DS) through transformation of transient abnormal myelopoiesis (TAM). This study investigates the clinical significance of genomic heterogeneity of AMKL in children with and without DS and in children with TAM. Genomic evaluation of nine patients with DS-related TAM or AMKL, and six patients with non-DS AMKL, included conventional cytogenetics and a comprehensive next-generation sequencing panel for single-nucleotide variants/indels and copy-number variants in 118 genes and fusions involving 110 genes. Recurrent gene fusions were found in all patients with non-DS, including two individuals with complex genomes and either a NUP98–KDM5A or a KMT2A–MLLT6 fusion, and the remaining harbored a CBFA2T3–GLIS2 fusion, which arose from both typical and atypical cytogenetic mechanisms. These fusions guided treatment protocols and resulted in a change in diagnosis in two patients. The nine patients with DS had constitutional trisomy 21 and somatic GATA1 mutations, and those with DS-AMKL had two to four additional clinically significant somatic mutations. Comprehensive genomic characterization provides critical information for diagnosis, risk stratification, and treatment decisions for patients with AMKL. Continued genetic and clinical characterization of these rare cancers will aid in improving patient management.

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.

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.

Acute Megakaryoblastic Leukemia in Childhood: Comparison Between Patients with and without Down Syndrome

Introduction: Acute Megakaryoblastic Leukemia (AMKL) is an unusual AML subtype in childhood, being the most frequent AML in Down syndrome (DS) patients. AMKL corresponds to 19.3% of AML in our Hospital and some special characteristics have been observed.Objective: To analyze characteristics of AMKL and to compare patients with or without DS.Patients and Methods:From January-1990 to June-2014 AMKL was diagnosed in 77 cases: 57 in non-DS and 20 in DS children. Fivecases were secondary leukemias and 3 cases had been previouslytreated with chemotherapy due to wrong diagnosis. One patient was on induction at the time of analysis. These 9 cases were not evaluable for follow-up. Forty-eight non-DS and 20 DS caseswere evaluable. Megakayocyte lineage commitment was confirmed in all cases by fluorescence microscopy. MPO was analyzedboth by flow cytometry and cytochemistry. G-banding and RT-PCR were performed according to standard techniques. Four successive BFM-based protocols were administeredwith schedule modification for DS patients.Results: Mean age was:2 (range: 2 mo-13 y) years of age and mean WBC count was: 27,500 (range: 1,600-250,000)/mm3. No differences were observed in age or hematological findings at diagnosis. Extramedullar compromise was detected in 10 (17.5%) of non-SD cases. Immunophenotype defined ambiguous lineage leukemia in 3 (5.0%) of the non-DS cases, with compromise of T-lineage. Interestingly, in 4 cases the megakaryoblasts also co-expressed MPO.Cytogenetic findings showed hyperdiploidy (n=7), t(1;22)(p13;q23) (n=3), t(16;21)/FUS-ERG (n=2), abnormalities in chromosome 7 (n=5) and complex karyotype (n=9). Complete remission (CR) was achieved in 51 (33 non-DS and 18 DS) (75.0%) of 68 evaluable cases;null-responses observed in7(only non-DS, 10.3%). Early deaths occurred in 10 (14.7%) during induction phase, 2 of them before treatment onset. From the 51 patients who achieved CR, 16 (14 non-DS and 2 DS) presented relapses(p: 0.04) and 9 (6 in non-DS and 3 in DS) died in CR. EFS-probability for the total population was 34 (6)%, for non-DS 21 (6)% and for DS 63 (11)%(p-value: 0.0026). LFS-probability was 45 (7)% for the total population, for non-DS 29 (8)% and for DS 70 (11)% (p-value: 0.0117). Median follow-up: 148 (range: 2-194) months.Conclusions:1- Extramedullar compromise and T/myeloid mixed lineage or co-expression of MPO were observed only in non-DS AMKL, in 17.5% and 12.0% respectively.2- Null-response was observed only in non-SD patients.3- The most frequent event of non-DS was relapse, and toxic deaths in DS.4- The significantly better outcome of patients with DS was confirmed in our setting. DisclosuresNo relevant conflicts of interest to declare.

Acute megakaryoblastic leukemia associated with trisomy 21 demonstrates a distinct immunophenotype.

Acute megakaryoblastic leukemia (AMKL) is a rare form of acute myeloid leukemia that encompasses three distinct subgroups: children with Down syndrome (DS-AMKL), children without DS (non-DS-AMKL), and adults (adult-AMKL). We hypothesize that the biological differences in the subtypes of AMKL may be reflected in distinct immunophenotypic (IP) features. Our aims were to employ mutiparameter flow cytometry to establish a comprehensive IP spectrum of AMKL in multiple demographic groups and to determine whether the subtypes of AMKL demonstrate distinct immunophenotypes. Thirty-seven AMKL cases were retrieved and subdivided into these three groups. Multiparameter flow cytometry was used to determine and compare the immunophenotypes. Available clinical, cytogenetic, and morphologic data were reviewed to confirm diagnoses. While there is commonality in the expression pattern for a wide range of myelomonocytic markers in these three subtypes of AMKL, blasts in DS-AMKL were more likely to express CD7 and CD11b than those in non-DS-AMKL and adult-AMKL. Furthermore, blasts in DS-AMKL were more likely to express CD13, CD33, and CD36 than non-DS pediatric AMKL, and showed greater CD56 expression compared with adult AMKL. These results indicate that DS-AMKL is related to but immunophenotypically distinct from non-DS-AMKL and adult-AMKL. This distinct immunophenotypic pattern, co-expression of CD7 and CD11b, has practical implications for characterization of AMKL.

Acute Megakaryoblastic Leukemia Associated with Trisomy 21 Demonstrates a Distinct Immunophenotype.

Background: Acute megakaryoblastic leukemia (AMKL) is a rare form of acute myeloid leukemia that encompasses three distinct subgroups, children with Down syndrome (DS-AMKL), children without DS (non-DS-AMKL) and adults (adult-AMKL). We hypothesize that the biological differences in the subtypes of AMKL may be reflected in distinct immunophenotypic (IP) features. Our aims were to employ mutiparameter flow cytometry to establish a comprehensive IP spectrum of AMKL in multiple demographic groups and to determine whether the subtypes of AMKL demonstrate distinct immunophenotypes. Methods: Thirty-seven AMKL cases were retrieved and subdivided into these three groups. Multiparameter flow cytometry was used to determine and compare the immunophenotypes. Available clinical, cytogenetic, and morphologic data were reviewed to confirm diagnoses. Results: While there is commonality in the expression pattern for a wide range of myelomonocytic markers in these three subtypes of AMKL, blasts in DS-AMKL were more likely to express CD7 and CD11b than those in non-DS-AMKL and adult-AMKL. Furthermore, blasts in DS-AMKL were more likely to express CD13, CD33 and CD36 than non-DS pediatric AMKL, and showed greater CD56 expression compared to adult AMKL. Conclusions: These results indicate that DS-AMKL is related to but immunophenotypically distinct from non-DS-AMKL and adult-AMKL. This distinct immunophenotypic pattern, co-expression of CD7 and CD11b, has practical implications for characterization of AMKL. © 2014 Clinical Cytometry Society.

NUP98/JARID1A Is a New Recurrent Genetic Abnormality in Pediatric Acute Megakaryoblastic Leukemia with a Distinct HOX-Gene Expression Pattern

AbstractAbstract ▪537▪This icon denotes a clinically relevant abstract Introduction:Cure rates in pediatric AML are currently in the 60–70% range despite treatment with intensive chemotherapy. To improve prognosis new treatment targets need to be identified, hence there is a need to better understand the underlying biology. It is hypothesized that AML results from at least two types of mutations which non-randomly collaborate in leukemogenesis. The type-I aberrations confer a proliferative advantage, type-II mutations lead to impairment of hematopoietic differentiation (Kelly et al, 2002). We recently described NUP98/NSD1 as recurrent event in cytogenetically normal AML (Hollink et al, 2011). Patients with NUP98/NSD1 had dismal outcome, and a stem-cell phenotype characterized by overexpression of homeobox (HOX) A and –B genes. Using split-signal FISH on 122 pediatric AML cases without driving oncogenic mutation, 26 NUP98- rearranged cases were identified, including 1 patient with acute megakaryoblastic leukemia (AMKL). We previously reported a patient with fusion of JARID1A, located on chromosome 12p13, to NUP98, located on chromosome 11p15, in a non-Down Syndrome (DS) AMKL case (Van Zutven et al, 2006). Therefore, a large series of non-DS AMKL patients was screened for NUP98/JARID1A and for other abnormalities, including the novel CBFA2T3/GLIS2 translocation (Gruber et al, ASH2011; #757). Methods:Samples from 105 pediatric non-DS AMKL cases, diagnosed between 1998 and 2011, were obtained from the DCOG, the AML-BFM SG, the Saint-Louis Hospital in Paris, and the COG. AMKL is more common in DS patients, therefore we also screened a series of DS AMKL (n=16). Centrally reviewed clinical and cell-biological data were provided by these study groups. Translocation of NUP98/JARID1A, MLL-rearrangements, RBM15/MKL1, and CBFA2T3/GLIS2 were identified using RT-PCR, as well as molecular characterization including hospots for the following mutations: FLT3, KIT, RAS, PTPN11, NPM1, WT1, and CEBPA. HOXA and –B expression levels were analyzed using gene expression profiling (Affymetrix) in 274 pediatric AML patients (Balgobind et al, 2011) including 9 AMKL patients, and validated with quantitative real-time PCR (n=37). Results:NUP98/JARID1A translocations were identified in 11 patients (11%). Four other patients had a NUP98- aberration with unknown translocation partner based on split signal FISH. We identified 16/105 patients with RBM15/MKL1, 13/105 with CBFA2T3/GLIS2 translocation, and 13/96 harbouring an MLL-rearrangement. Hence, specific non-random abnormalities could be defined in 61% of pediatric AMKL cases. Only 3/45 cases harboured a type-I mutation, all localized in the RAS gene. Comparing NUP98/JARID1A positive cases with negative cases in pediatric AMKL, no significant differences in patient characteristics including sex, age, and white blood cell count (WBC) were found. Considering prognosis, 5-year pEFS (22±14% vs. 36±6%, p=0.50) did not differ significantly from all other AMKL patients, nor did the cumulative incidence of relapse (56±19% vs. 54±7% p=0.9). CBFA2T3/GLIS2 translocated patients also did not differ from other AMKL patients (pEFS 19±16% vs. 36±6%, p=0.63). However, 5-year pEFS for RBM15/MKL1 translocated patients was significantly better (73±13% vs. 28±6%, p=0.043), but not in multivariate analysis adjusted for age and WBC. Gene expression analysis showed significantly higher HOXA5/A9/A10 and HOXB2/B3/B4/B5/B6 expression in NUP98/JARID1A compared to other pediatric AML cases. We did not identify any NUP98/JARID1A cases in the 16 DS AMKL patients. Discussion and conclusion:NUP98/JARID1A is a recurrent cryptic translocation in approximately 11% of pediatric AMKL cases. In 61% of all AMKL cases a type-II mutation could now be identified. Similar to NUP98-NSD1 a stem-cell phenotype was detected with persistent HOXAB-gene expression. Although NUP98/JARID1A did not influence prognosis, outcome in pediatric AMKL is unsatisfactory. NUP98 is known to recruit CREBBP/p300 resulting in histone acetylation, and transcriptional activation of HOX genes (Wang et al, 2007), suggesting that histone acetyltransferase inhibitors may be active. Moreover, JARID1A is unable to demethylate H3K4me2/3, which also results in sustained up regulation of HOX genes. This may provide options for targeted therapy. Disclosures:No relevant conflicts of interest to declare.

Rbm15 Affects Notch Signaling and Myelopoiesis.

Acute Megakaryoblastic Leukemia (AMKL) presents with one of two different genotypic abnormalities; either Down Syndrome (DS) accompanied with GATA-1 mutations, or a non-DS translocation t(1;22)(p13;q13). In AMKL associated with DS, there is trisomy 21 and various partial loss of function mutations in GATA-1. In non-DS AMKL, the translocation t(1;22)(p13;q13) encodes a fusion protein, RBM15-MKL, comprised of the transcriptional co-factors RBM15 (chromosome 1) and MKL (chromosome 22). In order to elucidate the role of the RBM15-MKL fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL. Herein we demonstrate a role for Rbm15 in myeloid differentiation.Previous work in the lab showed that Rbm15 is expressed at highest levels in hematopoietic stem cells, and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Therefore, we hypothesized that Rbm15 plays a role in myeloid differentiation. Indeed, we demonstrate that shRNA-mediated knockdown of Rbm15 enhanced the differentiation of 32DWT18 myeloid precursor cells. Recent studies have shown the Rbm15 homolog, Sharp, interacts with RBPJk, a critical transcription factor in the Notch signaling pathway; and since Notch signaling is essential for hematopoiesis, we hypothesized that Rbm15 may also modulate Notch signaling. We demonstrate that Rbm15 alters Notch-induced HES1 promoter activity in a cell-type specific manner. Rbm15 inhibits Notch-induced HES1 activity in non-hematopoietic cell lines, but stimulates this activity in hematopoietic cell lines including 32DWT18 and HEL (human erythroleukemia). In addition, we show that the N-terminus of Rbm15 co-immunoprecipitates with RBPJk and has a dominant negative effect by impairing the activation ability of HES1 promoter activity by full length-Rbm15.Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated, at least in part, by stimulation of Notch signaling via interaction with RBPJk. Consistent with a potential mechanistic role of Rbm15 and Notch in myelopoiesis, we have shown that shRNA-mediated knockdown of Rbm15 in 32DWT18 cells promotes myeloid differentiation, suggesting that the RBM15 component of the RBM15-MKL fusion protein may act by blocking differentiation. Currently, we are examining the effect of RBM15 overexpression and knockdown in primary mouse bone marrow cells using in vitro and in vivo assays.

Absence of JAK2 V617F Activating Mutations in Children with Acute Megakaryoblastic Leukemia with and without Down Syndrome.

Activating mutations at codon 617 of the Janus-2 tyrosine kinase (JAK2 V617F) have recently been described in hematological malignancies. In adult acute myeloid leukemia (AML), the reported frequencies vary, and JAK2 V617F mutations have mainly been detected in secondary AML following a myeloproliferative disorder. In adult de novo AML, the mutation was less frequent, and detected in 2/11 (18%) acute megakaryoblastic leukemia (FAB M7) samples (Jelinek et al., Blood 2005), and occasionally in other FAB-types.This prompted us to analyze a cohort of pediatric AML FAB M7 samples for this particular mutation. In children, at least 3 different subsets of AML M7 can be identified: infants with AML M7 characterized by t(1;22)(p13;q13), older children with random cytogenetic aberrations, and myeloid leukemia of Down syndrome (DS ML). DS ML is often preceded by transient myeloproliferative disease (TMD), hence we also screened TMD samples to detect whether JAK2 V617F mutations would be involved in clonal evolution from TMD to DS ML. To exclude germ-line mutations in DS, we tested normal mononuclear bone marrow cells (NBMC) from children with DS. These NBMC were obtained from a sternal aspirate from children undergoing cardiac surgery, after informed consent was obtained. Genomic DNA was harvested from leukemic cells, and JAK2 exon 12, including the intron-flanking regions, was amplified and sequenced to screen for the JAK2 V617F mutation. As a positive control for the JAK2 V617F mutation, we used HEL 92.1.7 cells (an erythroleukemic cell line). In a dilution experiment we could still detect the mutation, using direct sequencing, if 10% HEL/JAK2 mutated cells were mixed with 90% wild-type control cells. We tested 49 samples, comprising of 9 NBMC, 11 TMD, 14 DS-ML M7, 11 non-DS AML M7 and 4 relapsed non-DS AML M7 samples (including 2 initial diagnosis-relapse pairs). The median age of the TMD cohort was 3 days, for DS-ML children 1.9 years (range 0.9–3.8 yrs), and for non-DS AML 1.5 years (range 1.2–13.7 yrs). The median white blood cell count for TMD was 25.8x109/l, for DS-ML 13.8x109/l, and for non-DS AML 12.4x109/l. Cytogenetic data were available in 5/11 non-DS AML cases only, which showed no cases with a t(1;22). No JAK2 V617F mutations were detected in any of the clinical samples. We conclude that the role of JAK2 V617F mutations in pediatric DS and non-DS acute megakaryoblastic leukemia is limited at best. However, we were not able to screen the subgroup of non-DS AML cases with t(1;22).

Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling

Individuals with Down syndrome (DS) are predisposed to develop acute megakaryoblastic leukemia (AMKL), characterized by expression of truncated GATA1 transcription factor protein (GATA1s) due to somatic mutation. The treatment outcome for DS-AMKL is more favorable than for AMKL in non-DS patients. To gain insight into gene expression differences in AMKL, we compared 24 DS and 39 non-DS AMKL samples. We found that non-DS-AMKL samples cluster in two groups, characterized by differences in expression of HOX/TALE family members. Both of these groups are distinct from DS-AMKL, independent of chromosome 21 gene expression. To explore alterations of the GATA1 transcriptome, we used cross-species comparison with genes regulated by GATA1 expression in murine erythroid precursors. Genes repressed after GATA1 induction in the murine system, most notably GATA-2, MYC, and KIT, show increased expression in DS-AMKL, suggesting that GATA1s fail to repress this class of genes. Only a subset of genes that are up-regulated upon GATA1 induction in the murine system show increased expression in DS-AMKL, including GATA1 and BACH1, a probable negative regulator of megakaryocytic differentiation located on chromosome 21. Surprisingly, expression of the chromosome 21 gene RUNX1, a known regulator of megakaryopoiesis, was not elevated in DS-AMKL. Our results identify relevant signatures for distinct AMKL entities and provide insight into gene expression changes associated with these related leukemias.

CD36 (thrombospondin receptor) expression in childhood acute megakaryoblastic leukemia: In vitro drug sensitivity and outcome

The outcome for children with acute megakaryoblastic leukemia (AMKL) remains poor, except for cases associated with Down syndrome (DS). This study compared immunophenotypic and drug sensitivity patterns of childhood AMKL cases with or without DS. All DS-AMKL cases showed high expression of CD36 and were very sensitive to cytarabine and daunorubicin in vitro. In children without DS, high expression of CD36 on AMKL blasts identified a sub-group with immunophenotypic and drug sensitivity patterns similar to that of DS AMKL. Among non-DS AMKL patients, CD36-high cases had a superior outcome compared with CD36-negative cases. CD36 expression in acute myeloid leukemia cases other than AMKL was not associated with increased in vitro drug sensitivity. CD36 expression in AMKL may be an indicator of megakaryoblast maturation and chemotherapy sensitivity.