Core Binding Factor Research Articles

Altered Transcriptome Uniquely Associated with AML Vs. Normal Hematopoiesis

Differential expression profiling has identified specific genes and pathways that are variably expressed in different subsets of acute myeloid leukemia. However, comprehensive evaluation of genes and pathways whose expressions are uniquely altered in AML compared to that in normal hematopoiesis is lacking. Previously we used whole transcriptome sequencing (RNA Seq) to investigate the expression profiles of t(8;21) and Inv(16) translocations, defining that down-regulation of homeobox (HOX) gene family plays an important role in CBF (core binding factors)-AML hematopoiesis compared to other AML variants. In this study, we used RNA seq data from diagnostic specimens obtained from 473 children and young adults with AML to compare their transcriptome to that in normal bone marrow (NBM) from healthy individuals (N=20).Initial comparison of AML vs. NBM transcriptome identified 2,151 genes to be differentially expressed with 2-fold change and FDR <0.01 using GLM (Generalized Linear Models) analysis; the most up/down-regulated genes are DUSP10 (1.06E-42), FCRL6 (5.11E-146) PLIN3 (2.19E-28) and TTC16 (22.46E-124); GSEA (Gene Set Enrichment Analysis) showed that these 2,151 genes were mostly involved in “p53 signaling pathway” (hsa04115; FDR=0.0013), “regulation of T cell mediated cytotoxicity” (GO:0001914; FDR=0) and “decreased circulating interleukin-17 level” (MP:0008615; FDR=0).In an additional analysis, in order to more appropriately capture transcripts whose expression might be limited to a small subset of patients, we used a customized filtering approach (RPKM value >5 in at least 10% AML and <5 in at least 80% normal samples) to maximize our ability to identify genes that are uniquely expressed or silenced in AML vs. NBM. This analysis identified 1,173 genes to be most AML-specific with the customized cutoffs. This analysis identified expression of a number of Cancer Testis Antigen (CTA) family of genes including PRAME, and CCNA1 that were highly expressed in a significant subset of the patients. GSEA top terms for these AML-specific genes were “mRNA surveillance pathway” (hsa03015; FDR=0.0024), “autophagosome assembly” (GO:0000045; FDR=0.0027) and “increased circulating interleukin-13 level” (MP:0008608; FDR=0).There were 261 genes identified by both methods, including MSLN (Mesothelin), PRAME (Melanoma antigen preferentially expressed in tumors) and CCNA1 (Cyclin-A1) (Figure 1A). GSEA of these 261 genes showed that the most significant GO pathway was “negative regulation of transcription from RNA polymerase II promoter” (GO:0000122; FDR=0.0025).We also applied the same pipeline for the comparisons between NBM with each AML cytogenetic subgroup (Inv(16), t(8:21), MLL and normal karyotype(NK)), each comparison resulted in 2,000~3,000 DE (differentially expressed) genes from GLM, with t(8;21) vs. NBM the largest number (2,318 genes). There were also 200~400 overlapped genes identified by both GLM and cutoff approaches; For GSEA results, “MicroRNAs in cancer” (hsa05206; FDR=0.0017) and “NF-kappa B signaling pathway” (hsa04064; FDR=0.0018) were shown in Inv(16) vs. NBMs; “regulation of transcription involved in G1/S transition of mitotic cell cycle” (GO:0000083; FDR= 0.0015) was shown in t(8;21) vs. NBM; “platelet degranulation” (GO:0002576; FDR= 0.0021) was shown in MLL vs. NBM; “NOD-like receptor signaling pathway” (hsa04621; FDR=0.0023) was shown in NK vs. NBM.MSLN and PRAME displayed distinct expression patterns among different cytogenetic groups: MSLN was highly expressed in most inv(16) samples; however, PRAME was highly expressed in the majority of t(8;21) samples. Moreover, we grouped all samples (N=493) based on the expression of MSLN (RPKM cutoff=5) or PRAME (RPKM cutoff=4); and performed the same analysis using GLM and GSEA (Figure 1B). 164 differentially expressed genes were identified, and the most significant GO pathway was “platelet degranulation” (GO:0002576; FDR=0.00057) in MSLN-based groups; 186 genes were identified, and “G-protein coupled receptor binding” (GO:0001664; FDR=0.0024) was the most significant one in PRAME-based groups.This analysis provides a comprehensive gene expression profile comparison between AML and normal hematopoiesis, incorporating current knowledge of pathways/interactions from databases, especially for those of clinical interests/significance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Significance of Monosomy 7 with and without Complex Karyotypes in Acute Myeloid Leukemia (AML): A Single Institutional Analysis

Background: Over the past few years, the presence of monosomies in AML has emerged as an important prognostic factor. The incidence of monosomal karyotype (MK) increases with age, from 4% in patients ≤ 30 years to 20% in those > 60 years. Studies show that autosomal monosomies such as monosomy 7 confer very poor prognosis with overall survival in the range of 0-3%. About 30% of patients with AML have unfavorable karyotype with a complete response rate of 50% and a 5-year overall survival of 10-20% when treated with conventional chemotherapy. Of these unfavorable subgroup, about half will present with a complex karyotype (CK), which is associated with a poor outcome, and 40% of those with CK will have a MK which carries an even worse prognosis. In a single institutional retrospective study, we analyzed the prognostic significance of monosomy 7 with/without complex karyotype.Method: Cytogenetic and overall survival were analyzed in 137 patients with median age 65 (21-91). The analysis encompasses patients at Beaumont Hospital with confirmed AML treated between years 2010-2015 who had monosomy 7 with/without complex karyotype. Statistical analysis was performed by an independent investigator and overall Survival (OS) assessed by Kaplan-Meier method.Results: Out of 137 patients studied, we identified 99 patients with cytogenetic abnormalities. Among the latter subgroup, 16 (16.2%) patients had monosomy 7 with one patient <30 years old. While 3 (18.8%) patients with monosomy 7 also had core binding factor (CBF), 10 (62.5%) patients had monosomy 7 in the presence of CK and 5 (31.2%) patients had monosomy 7 without CK. Twenty-two (22.2%) patients had CK without monosomy 7. Statistical analysis using the SAS System for Windows version 9.3 showed that monosomy 7 in the absence of CK carries a worse prognosis than in the presence of CK (5-year OS, 0% and 6.3% respectively, p<0.002) (Table 1). The 5-year OS observed in patients with CK alone was 22.3% (Table 1). Thus, the prognostic impact of structural chromosomal abnormalities is intensified when they occur in association with a MK such as monosomy 7. Based on our analysis, MK is more predictive of poor outcome than CK and possibly other individual unfavorable karyotypes associated with very poor outcome.Conclusion: Occurrence of MK in the presence of CK is fairly common among patients diagnosed with AML and carries a worse prognosis. Although, our study represents a single institutional analysis, it concurs with recent reports on the prognostic significance of MK either alone or in combination with CK. Currently, the best chance at cure for patients with unfavorable karyotype who achieve compete response with induction therapy is allogeneic transplant. Such reports in our opinion should necessitate a more aggressive approach in terms of treatment options as well as early discussion with patients and families on the prognostic implications of these unfavorable karyotypes inorder to make more informed decisions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Cancer Testis Antigens (CTAs) As Immuno-Therapeutic Targets in Pediatric AML

The Cancer Testis Antigen (CTA) superfamily of genes have long emerged as potential targets for immunotherapy and response monitoring due to their relative restrictive expression in testis and variable expression in some cancers. Much of the data on CTA expression in cancer have been in solid tumors with little data on CTA expression in leukemias. Identification of leukemia associated antigens (LAA) may provide attractive targets for immunotherapy or monitoring of residual disease.In order to discover AML associated CTAs, we performed transcriptome sequencing on 446 de novo pediatric AML samples and 20 normal bone marrows (NBM) from healthy donors and interrogated the expression of 245 candidate CTAs obtained from the CT database (http://www.cta.lncc.br). Differential expression using Limma (https://bioconductor.org/packages/release/bioc/html/limma.html) identified 32 CTA genes to be significantly differentially expressed with 7 being up-regulated compared to NBM and 15 genes with expression in NBM which are silenced in AML. The upregulated genes include: CCNA1, PRAME , CT45A5, IGF2BP3, SPAG6, TEX14, and ZNF165 and are collectively expressed in 330 of 446 patients (75%) with the highest contributors being PRAME and CCNA1 (Fig. 1A, Fig. 1B) . These 2 CTAs are expressed in every fusion group and constitute 65% of the total cohort, with a notable enrichment in core binding factor (CBF) patients (80%). High CCNA1 expression are present in 2/3 of patients with Inv(16), 71% of patients with a NUP98 fusion, 88% of DEK - NUP214 carriers, and 64% of cytogenetic normal cohort. PRAME is expressed in 79% of patients with a RUNX1 - RUNX1T1 fusion, 57% of CBFA2T3 - GLIS2 positives, and 20% of Inv(16) patients. While the oncoprint (Fig. 1A) shows that most patients express 1 or 2 CTAs, with some genes showing exclusivity, patients with a KMT2A fusion tend to express multiple CTAs. As a group, 77% of patients with KMT2A fusions express at least 1 CTA.To determine the underlying mechanism of dysregulated CTA expression in AML, we inquired whether promoter methylation status might be associated with CTA expression. We correlated the expression level of the 7 upregulated CTA genes with the methylation of their corresponding promoters. Overall there was a strong signal demonstrating anti-correlation of expression of the above mentioned genes with their promoter methylation. In specific, for PRAME, differences in methylation at the region chr22:22899991-22901267 explain over 87% of the variation in expression, and this locus is normally methylated in NBM (Fig. 1C). Likewise, for CT45A5, it's normally methylated in NBM, and demethylation of this locus explains about 61% of the variance in expression (Fig. 1C).Additionally, we evaluated the clinical implications of CTA expression, and demonstrated that CCNA1 expression was significantly associated with clinical outcome. The range of expression for CCNA1 in the AML samples was 0-561.4 TPM (mean = 31.8 TPM), whereas expression for NBMs ranged from 0.3-2.5 TPM (mean = 0.9) (p=8.45E-08). After binning expression values to determine the best cutpoint for high and low expressors of CCNA1, patients classified with high expression had statistically significant worse event-free (EFS, Fig.1D) and overall survival (OS) (p=<0.0001, p=0.0393 respectively, Log-Rank test). As a sub-group, patients with KMT2A rearrangements and high CCNA1 expression also show prognostic significance with correlation of poor outcome (EFS, p=0.0009, Fig. 1E). In addition, cytogenetically normal (CN-AML) patients with FLT3 ITD had a much higher overall expression of CCNA1 than did CN-AML without FLT3 ITD (p=0.009, Mann-Whitney test).In this analysis we identify candidate CTA genes that are cumulatively expressed in the majority of AML cases and can provide novel therapeutic options by targeting with modified T-cell receptor therapies. They can also be used as biomarkers for response monitoring. Further, as aberrant expression of these genes are regulated by altered methylation, use of hypomethylating agents might enhance therapeutic efficacy by increasing target expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of Distinct Combinations of Molecular Mutations in AML Patients. Results of Two-Center International Cooperation Study

To analyze the prognostic impact of distinct combinations of molecular aberrations, a retrospective analysis of 620 acute myeloid leukemia (AML) was performed. The mean patient's age was 57 (16-85) years. 47% of patients were characterized by abnormal karyotype: favorable (11%), intermediate (67%), and adverse (22%) risk. We analyzed NPM1, FLT3, DNMT3A, IDH1/2, and c-KIT mutations in diagnostic samples. Molecular genetics and cytogenetics considered adverse risk for 28% of patients, favorable for 26%, and intermediate in 46% of patients. More common mutations in whole cohort of patients and in patients with normal karyotype (NK) were NPM1 (22% and 36%), FLT3- ITD (23% and 31%). In addition, DNMT3A and IDH1 mutations were found in 18% and 17% of NK-AML patients, respectively. IDH2 mutations were associated with older patients age (p=.0001). DNMT3A and NPM1 mutations were more frequently detected in patients with monocyte/myelomonocytic differentiation of leukemic blasts (p=.0001). C-kit mutations were more common in Core Binding Factor AML.The presence of NPM1 mutations were associated with low relapse rate (p=.02) and significant better disease free (DFS) and overall survival (OS), particularly in cases of single mutation (p=.05). NPM1mut AML patients with additional FLT3 -ITD mutations were characterized by the inferior DFS as compared with others combination of mutations (p=.01). Patients with DNMT3Amut / FLT3mut / NPM1wt have demonstrated more inferior DFS than DNMT3mut / FLT3wt / NPM1mut (p=.01). Moreover, in patients after allogeneic stem cell transplantations, combinations of DNMT3Amut and FLT3 -ITDmut was a significant adverse prognostic factor (p=.01). Among FLTwt patients, the favorable DFS was seen in patients with NPM1 mutation and poor outcome was estimated in those with DNMT3A mutation (p=.03). Among FLT3mut / NPM1mut patients, more favorable outcome was found in cases with additional IDH1 and IDH2 mutations. The data suggest that favorable prognosis of NPM1 mutation dominates in cases of DNMT3A but not in FLT3 mutations, but poor prognosis of FLT3 mutation prevails all others. We conclude that mutations status is important factor for risk stratifications of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Next-Generation RNA Sequencing in Myeloid Sarcoma Tissue Sample: Benefit on Risk Stratification of Myeloid Sarcoma

Introduction: Myeloid sarcoma (MS) is a rare hematological neoplasm whose knowledge is largely based on case reports. Whether MS can be divided into different risk group as acute myeloid leukemia (AML) is an issue of concern. Molecular testing with high sensitivity may be of great help in diagnosis and outcome prediction. This study analyzed the clinical features, outcome, cytogenetical and molecular characteristics of MS patients in West China hospital of Sichuan University, China between 2006 and 2016.Method and result: Thirty-one cases of MS with clinical data available were analyzed in this study. Among them, 8 tumor samples (5 formalin-fix paraffin-embedded tissue and 3 bone marrow tissue) were appropriate for RNA-seq (NGS). The average age at diagnosis MS was 35.2±13.2 years (range 8-61). The mean follow-up period was 32 months (range 1-58). The most common molecular abnormality was FLT3-ITD (29, 4/14) and AML1-ETO fusion (21%). Eighteen patients (58%) presented as de novo MS, 9 patients had simultaneous AML, 3 patients had other hematological neoplasms. Within 18 de novo MS patients, 7 had progressed to AML in 2 to 42 months, the mean progression time was 18.7±17.7 months. The overall survival of this series was range from 2 to 58+ months, 2-year survival rates was 75.8%. Sixty-six fusion transcripts were identified by RNA-seq, including one recurrent transcript: CBFβ/MYH11. Involved genes included a number of hematologic or solid tumor associated genes. Functional annotation of fusion transcripts revealed these fusion genes might involve hematopoiesis, apoptosis, telomere stability, core binding factor, ubiquitination, transmethylation, etc.Conclusion and discussion: The prognosis of MS patients was heterogeneous and the prognostic genetic markers were similar to AML. Clinical application of NGS-based RNQ-seq profiling in tumor sample is reliable and feasible. FLT3-ITD and AML1-ETO were the most common molecular abnormalities in MS. RNA-seq could identify fusion genes and prognosis associated gene mutation in MS, thus could help to diagnose, predict prognosis and guide therapy, especially in isolated MS, or MS with trace bone marrow involvement. RNA-seq based molecular profiling of MS needs to be further elucidated in the pathogenesis of MS and in the risk stratification of MS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeted Therapy for AML Expressing RUNX1 Mutation

RUNX1 is the DNA-binding subunit of the core binding factor (CBF) complex and a transcription factor (TF) involved in normal and malignant hematopoiesis. Transcriptionally active full-length RUNX1b isoform is transcribed from 6 exons, encoding a DNA binding ‘Runt’ homology domain and a more C-terminal transcription activation domain. Within its CTCF-anchored loop or TAD (topologically-associating domain), RUNX1 gene has a highly conserved +24-kb enhancer (eR1 in mouse), which is part of a large super-enhancer (>170 kb) covering the entire intron 1 between proximal P2 and distal P1 promoter of RUNX1 and controlling the transcription of RUNX1. TFs bind to super-enhancers/enhancers that loop over to interact with promoters to transcriptionally regulate oncogenes, such as RUNX1 and its targets, as well as multiple other AML-relevant oncogenes involved in growth and survival of AML blast progenitor cells (BPCs). Heatmap of Hi-C interaction scores within the RUNX1 TAD showed triangle shaped regions of high interaction scores highlighting interactions between +24-kb enhancer and the P2 promoter of RUNX1. To functionally validate these findings, we cloned and transfected into HEK293 cells expressing RUNX1, a luciferase reporter construct containing the DNA of the P2 promoter with or without the +24 kb-enhancer of RUNX1. Our findings confirm that, as compared to the promoter or the enhancer alone, the +24 kb-enhancer-driven P2 promoter exhibited superior luciferase induction representing increased transcriptional activity of RUNX1. Somatic mutations in RUNX1 commonly occur in MDS, as well as in the secondary (s) or de novo AML (~10%). Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function mutations. Presence of mtRUNX1 confers relative therapy-resistance and poorer survival in patients with AML. Lack of specific targeted therapy coupled with resistance to standard chemotherapy may account for the poorer outcome in AML expressing somatic or germline mtRUNX1. Therefore, there is a strong rationale and an unmet need to develop novel targeted therapies for AML expressing somatic or germline mtRUNX1. In the present studies, we demonstrate for the first time that shRNA mediated knockdown of RUNX1 expression inhibits growth and induces differentiation and apoptosis of cultured cell line (OCI-AML5) and patient-derived (PD) AML BPCs expressing mtRUNX1. After engraftment, doxycycline-inducible shRNA-mediated in vivo knockdown of RUNX1 in OCI-AML5 cells, compared to controls, markedly reduced AML burden and improved survival of the immune-depleted (NSG) mice engrafted with luciferase transduced OCI-AML5 cells. Interaction of TFs with DNA and transcriptional co-factors such as the histone acetyltransferase p300 at enhancers and promoters leads to acetylation of histone proteins and TFs. BET (Bromodomain Extra-terminal) protein (BETP) BRD4 binds to acetylated histones and TFs at super-enhancers and recruits transcriptional co-factors to promote RNA pol II (RNAP2)-mediated transcription of oncogenes and their targets. ChIP-Seq analysis with anti-BRD4 and anti-H3K27Ac antibody showed that Runx1 super-enhancer and P2 promoter are occupied by BRD4. shRNA mediated knockdown of BRD4 attenuated expression of RUNX1 and its targets, MYC, PU.1 and myeloperoxidase (MPO), inhibited growth and induced apoptosis of OCI-AML5 cells. We also found that BETP-PROTACs (proteolysis targeted chimera) ARV-825 and its pharmacologically superior version ARV-771 (20 to 250 nM) caused degradation and depletion of BRD4, reduced BRD4 occupancy at the+24-kb-enhancer and P2 promoter of RUNX1, repressing RUNX1 and its targets, and potently induced apoptosis of OCI-AML5, Mono-Mac-1 and PD AML BPCs expressing mtRUNX1. Treatment with ARV-771 (30 mg/kg SQ daily for 3 weeks also inhibited AML growth and improved survival of NSG mice engrafted with luciferase-transduced OCI-AML5 cells. By utilizing the RNA-Seq mRNA signature from RUNX1-depleted AML cells we queried for expression mimickers (EMs) through LINCS1000-CMap (Connectivity Mapping) analyses. This uncovered several novel agents that inhibit RUNX1 expression and induce in vitro lethality and reduce in vivo leukemia burden in AML BPCs expressing mtRUNX1. These findings highlight RUNX1-targeted novel agents to be developed and tested as potential therapy for AML expressing mtRUNX1. DisclosuresDiNardo:AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding. Qian:4Arvinas, LLC. New Haven, CT: Employment. Raina:4Arvinas, LLC. New Haven, CT: Employment. Takahashi:Symbio Pharmaceuticals: Consultancy.

The Oncogenic Fusion Protein CBFB-SMMHC Downregulates CD48 to Evade NK Cell Recognition

Chromosomal translocations are a common cytogenetic aberration in acute leukemia, which frequently generate fusion proteins with oncogenic properties. We have previously studied the immune evasion mechanisms of two common oncogenic fusion proteins in acute myeloid leukemia (AML), PML-RARA and AML1-ETO. We investigated, in particular, the activity of natural killer (NK) cells, which are part of the innate immune system and play a significant role in eliminating leukemic cells. We found that both oncogenic fusion proteins downregulate the expression of CD48, a ligand of the NK cell activating receptor 2B4, to escape NK cell-mediated recognition of the tumor cells.In the current study we tested the possibility that other oncogenic fusion proteins are also able to evade NK cell recognition. To this aim we cloned several leukemic fusion proteins into lentiviral vectors and expressed them in U937 cells, which are commonly used to express leukemic fusion proteins. The leukemic fusion proteins included MLL-AF4, NUP98-HOXA9, DEK-NUP98 and CBFB-SMMHC. By using flow cytometry, we examined the effect of these fusion proteins on the expression of several NK cell ligands. CBFB-SMMHC was the only fusion protein which downregulated the expression of CD48.Next, we tested the functional significance of the downregulation of CD48 by CBFB-SMMHC by performing cytotoxicity assays with NK cells. We first used the NK cell line YTS eco since the cytotoxicity of these cells is mainly dependent on the 2B4-CD48 interaction. We found that cells which express CBFB-SMMHC were killed significantly less than the control cells, and that this reduced killing was due to CD48 downregulation. We also tested the killing of CBFB-SMMHC expressing cells by primary bulk NK cells and found that cells which express this fusion protein were killed significantly less.Mechanistically we show that overexpression of CBFB-SMMHC reduces the mRNA levels of CD48. Additionally, we tested whether treatment with HDAC inhibitors (HDACi) can reverse the downregulation of CD48 by CBFB-SMMHC. We treated cells which express CBFB-SMMHC with various HDACi and examined the effect of this treatment on the expression of CD48. Two specific class I HDACi, mocetinostat and entinostat, upregulated the expression of CD48 in cells which express this fusion protein.Taken together we show that modulation of CD48 expression is an immune evasion mechanism, which is specific to core binding factor (CBF) proteins (AML1-ETO and CBFB-SMMHC) and PML-RARA. Although the three fusion proteins which we found to down-regulate the expression of CD48 are associated with better prognosis in AML, about 30% of CBF-AML patients eventually relapse. Based on our findings we suggest that down-regulation of CD48 by these fusion proteins, which leads to NK cell immune evasion, contributes to the persistence of residual disease in these subtypes of leukemia and eventually to a clinical relapse. Finally, we propose that NK cell based therapies (i.e., NK cell infusion) or class I HDACi could be potential adjunctive therapies for CBF-AML or APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CBFA2T3 Enhances Transcriptional Activity of RUNX1: Implication in Leukemia

Pre-B acute lymphoblastic leukemia (BCP-ALL) is the most common type of pediatric cancer. Many expression-profiling studies have uncovered genes deregulated in BCP-ALL compared to normal pre-B cells. Yet, the roles of the deregulation of those genes in the onset and maintenance of BCP-ALL are still unclear. We were interested in two genes, CBFA2T3 (Core-Binding Factor, Runt Domain, Alpha Subunit 2; Translocated To, 3) and RUNX1 (Runt Related Transcription Factor 1), which are specifically overexpressed in BCP-ALL characterized by the presence of ETV6-RUNX1 fusion gene. CBFA2T3 is a hematopoietic corepressor. Alteration of its function is implicated in leukemia. RUNX1 is a transcription factor, whose activation or repression activity depends on cofactors. RUNX1 deregulation is also involved in many hematological disorders. Our goal was to explore the role of these two genes in BCP-ALL. We demonstrated the major role of CBFA2T3 in the control of RUNX1 activity.Based on our patients' data and public database, we not only confirmed that CBFA2T3 and RUNX1 transcripts were overexpressed in ETV6-RUNX1 BCP-ALL, but also found that their expression levels were positively correlated. Expression of EVT6-RUNX1 in Nalm6 cells, a BCP-ALL cell line that normally does not express the ETV6-RUNX1 fusion protein, showed an overexpression of CBFA2T3 and RUNX1 proteins, confirming the role of the ETV6-RUNX1 fusion protein as a priming event in CBFA2T3 and RUNX1 expression.To further determine the role of RUNX1 in the control of transcription in the context of BCP-ALL, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) with anti-RUNX1 in Nalm6 cells and in REH cells, a ETV6-RUNX1 BCP-ALL cell line. Notably, we observed several significant enrichments of RUNX1 binding at CBFA2T3 and RUNX1 promoters in both cell lines. Our work on the promoters of these two genes was carried out with luciferase reporter assay. The luciferase results demonstrated that RUNX1 controlled the transcription of CBFA2T3 and RUNX1 and showed that CBFA2T3 enhanced the transcriptional activity of RUNX1.As CBFA2T3 is neither a transcription factor, nor a DNA binding protein, we suspected that CBFA2T3 could directly modulate RUNX1 activity. Chromatin immunoprecipitation of RUNX1 coupled to mass spectrometry identified a putative interaction between CBFA2T3 and RUNX1 in BCP-ALL lymphoblasts. Using Proximity Ligation Assay, we next confirmed the protein interaction between CBFA2T3 and RUNX1, as well as the interaction between CBFA2T3 and the fusion protein ETV6-RUNX1 in two BCP-ALL cell lines and, more importantly, in BCP-ALL patients' cells. Finally, we identified the CBFA2T3 protein domain responsible for the interaction with RUNX1, thanks to coimmunoprecipitation assay with truncated CBFA2T3. Using this CBFA2T3 domain, we next performed luciferase assay and were able to inhibit the effect of full length CBFA2T3 on the transcriptional activity of RUNX1.To conclude, this work highlights the interaction between CBFA2T3 with RUNX1 and ETV6-RUNX1. We identified a regulatory loop between CBFA2T3 and RUNX1 that could explain their deregulation in patients. Our finding reveals a novel molecular mechanism of control of RUNX1 involving CBFA2T3 and RUNX1 itself. It may also open new perspectives to understand the mechanisms implicated in acute myeloid leukemia with AML1-MTG16 (RUNX1-CBFA2T3) fusion protein.This work is supported by Ligue Régionale contre le cancer, Région Bretagne, Rennes Métropole, French Research Ministry, the société française de lutte contre les cancers et les leucémies de l'enfant et de l'adolescent, the Fédération Enfants et Santé, the Société Française de Biochimie et Biologie Moléculaire, the Société Française d'Hématologie, Mrs. M-Dominique Blanc-Bert and the Marie Curie Actions of the European Union FP7 under REA grant agreement n°291851. DisclosuresNo relevant conflicts of interest to declare.

Tetraploidy in Acute Myeloid Leukemia Is Associated with Poor Outcomes Regardless of the Presence of Favorable Risk Features, and Requires an Allogeneic Stem Cell Transplant in First Remission

Introduction: While tetraploidy (92 chromosomes) and near-tetraploidy (>80 chromosomes) confer favorable prognosis in B-cell lineage pediatric acute lymphoblastic leukemia, their prognostic significance in acute myelogenous leukemia (AML) remains ill-defined. Moreover, the mechanisms underlying their pathobiology and impact on known prognostic factors in AML are unknown. Combining data collected at our institution along with a review of the literature, we provide the most complete analysis of tetraploidy and near-tetraploidy in AML including data on biology, treatment outcomes, impact on other prognostic markers, and survival to assist in clinical management.Patients and methods: A systematic literature search was performed and included studies published from June 1st, 1994 to June 1st, 2017 in PubMed, Embase, and Web of Science. We used the key words tetraploidy, near-tetraploidy, acute myeloid leukemia, acute myelogenous leukemia, and AML. Data was tabulated regarding the following seven variables: Fluorescence in situ hybridization (FISH) analysis, molecular analysis, karyotype, risk status, overall survival (OS), history of allogeneic stem cell transplantation, and chemotherapy. Data was reported as percent and total number of cases with available data of each variable.Results and discussion: We identified 104 AML cases with confirmed tetraploidy or near-tetraploidy (102 cases in 24 articles, and 2 cases from our institution). Incidence was nearly equal between tetraploidy (55/104) and near-tetraploidy (49/104). The median age at diagnosis was 54. A notable male predominance was observed with a male-to-female ratio of 2.2:1 (71 males and 33 females). While insufficient molecular data were available to include in risk stratification, cytogenetics including FISH and karyotype were available in all cases. We stratified our cases irrespective of tetraploidy or near-tetraploidy into favorable risk (defined by the presence of the following genetic alterations in core-binding factors: inv(16) or t(16;16) or t(8;21), or t(15;17)), unfavorable risk (defined by complex karyotype [≥3 clonal chromosomal abnormalities], monosomal karyotype, 5q-, 7q-, -5,-7, t(6;9), inv(3), t(3;3), 11q23 - non t(9;11), t(9;22)) and intermediate risk (the rest). Consistent with the literature, cases with other cytogenetic abnormalities in addition to those defined in the favorable-risk group, were included within the favorable-risk category (Byrd Blood 2002). Interestingly, t(8;21) was the only favorable risk feature represented, seen in 13 patients (12.5%), thus grouped under favorable-risk AML. 51 patients (49.0%) were grouped under unfavorable-risk AML and included 24 patients with complex karyotype, 9 patients with -7, 7 patients with 5q-, 4 patients with -5, 1 patient with 7q-, 1 patient with t(9;22), 3 patients with both -5 and -7, 1 patient with both 5q- and -7, and 1 patient with both -5 and 7q-. Intermediate risk was observed in 40 patients (38.5%) who were found to have normal or non-defined cytogenetics. We used the Kaplan-Meier method to stratify survival data, and the log-rank test to compare the survival between subgroups. The overall outcomes were poor, the median OS (mOS) of the whole cohort was 6 months (M) (Figure 1A). The prognosis was equally poor for both tetraploidy (mOS 6.2M) and near-tetraploidy (mOS 6M) (P=0.81; Figure 1B). Surprisingly, risk stratification showed no impact on overall survival (P=0.57; Figure 1C), which suggest that the unfavorable risk imposed by tetraploidy or near-tetraploidy may overcome favorable-risk features, such as t(8;21), challenging current data on AML risk stratification. Twenty-one patients (20.2%) had an allogeneic stem cell transplantation. Interestingly, we noticed a significant improvement in outcomes (mOS 17M) in patients who received an allogeneic stem cell transplantation compared to those who did not (mOS 4M) (P=0.0085; Figure 1D).Conclusion: Regardless of the risk status, the prognosis of tetraploid or near-tetraploid AML is dismal and should be incorporated within the unfavorable risk group. Prompt evaluation for allogeneic stem cell transplant should be initiated at diagnosis. Indeed, longer survival can be achieved with chemotherapy followed by allogeneic stem cell transplantation at first complete remission. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Survival Among Acute Myeloid Leukemia Patients Conditional on Years Already Survived and Relative to the General Population in the United States

Background: Prognostic models for AML patients (pts) predict outcomes based on clinical and cytogenetic features at diagnosis. However, as a pt lives beyond the initial date of diagnosis following treatment, his/her estimated survival period improves and these models lose accuracy. Conditional survival (CS) probability estimations use the time a pt has survived since diagnosis to improve on prognostic accuracy and provide personalized survival information. Here we report population-level, 5-yr relative cancer survival probabilities of AML pts conditional on having already survived ≥1 yr(s) after diagnosis.Methods: A total of 13,933 adult AML cases diagnosed during 2000-2009, and followed through 2014 were assembled from all 18 registries of the Surveillance, Epidemiology and End Results (SEER). Pts who did not receive chemotherapy in the first 3 months after diagnosis were excluded. Survival probabilities were calculated using the SEER Survival System (SEER*Stat). We calculated relative survival (RS) probability estimates that compare survival in the pt cohort relative to the expected survival of the general population (matched for age, sex, race and calendar period over a standard follow-up time). Trends in 5-year RS estimates conditional on already having survived 0-5 years from diagnosis were modeled using regression modeling. Estimates were adjusted for age group [adolescents and young adults (AYA) (18-39 yrs); middle-aged (40-59 yrs) and elderly (> 60 yrs)], yr of diagnosis, years already survived (YAS), sex and race. We performed subset analyses for AML categories with adequate sample size for analysis in SEER - AML with myelodysplasia (MDS)-related changes or AML-MRC (n = 794), core-binding factor (CBF) AML (n = 462) and acute promyelocytic leukemia (APL) (n = 1499). Tests of significance were based on two-sided hypothesis at the .05 level. Analyses were performed using STATA.Results: Figure 1A shows survival of all AML pts relative to the expected survival of the general population by age groups. At 10 yrs after diagnosis, the RS probabilities of AML (all categories) was 50% for AYA, 35% for middle-aged and 10% for elderly cohort. When conditioned on YAS, the estimated 5-year RS probabilities of AML pts consistently improved with successive yrs as shown in Figure 1B. Figures 2A-2C show RS probability estimates for specific AML categories by age subgroups - 10-yr RS was highest for APL [80% - AYA, 70% - middle-aged, 50% - elderly] as expected but surprisingly lower than expected in CBF-AML [65% in AYA group, 50% in middle-aged and 30% in elderly]. Figures 2D-2F illustrate the influence of AML categories and advancing age on CS estimates. The estimated 5-yr RS probabilities when conditioned on successive YAS increased for all age groups and for each AML category. The 5-yr relative CS was highest in APL pts among all AML categories and almost reached 100% for all three age groups, thereby approximating survival of the general population. Pts with CBF-AML had high 5-yr relative CS probabilities of 90% in all age groups, consistent with their favorable prognosis. The 5-yr conditional RS probability for AML-MRC exceeded 90% and 80% for AYA and middle-aged groups, respectively but reached only 60% in the older cohort. AML-MRC is considered a biologically heterogeneous disease and this finding suggests a biologically aggressive course in older patients. In multivariable analyses, the 5-yr RS estimates were significantly lower in > 60 yrs cohort (vs. 18-39 yrs, 36.09% drop (95% CI, 35.81-36.09)] but better in females (vs. males, 5.07% improvement (95% CI, 4.89-5.25), with YAS [conditioned on 1 to 5 YAS, 5-yr RS improved from 19.92% (95% CI, 15.03-24.81) to 53.77% (95% CI, 49.55-57.98)] and in recent treatment period [2006-2009 vs, 2002-2005, 9.07% improvement (95% CI, 8.93-9.21).Conclusions: Following treatment completion, survival prognosis in AML can be further refined using a population-derived, 5-yr CS estimates based on YAS. While CBF-AML is often considered “favorable-risk” AML, long-term RS probabilities belied this, with unexpectedly lower survival probabilities (~30-65% at 10 yrs). This data suggests the need for a longer period of leukemia surveillance following treatment completion. RS and CS information can be applied clinically at the individual level and can be used in the design of clinical trials to determine appropriate length of follow up. [Display omitted] DisclosuresAdvani:Takeda/ Millenium: Research Funding; Pfizer: Consultancy. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Majhail:Anthem, Inc.: Consultancy; Sanofi: Honoraria. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Therapy-Related Myeloid Neoplasms Are Not Always Dismal If They Have Better Cytogenetic Profile and Longer Latency Period from Preceding Anti-Cancer Treatment

Currently, any myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) developed after previous exposure to chemotherapy and/or radiation (CT/RT) are defined as therapy-related myeloid neoplasms (t-MNs). However, this medical history-oriented definition has an inherent uncertainty considering possibilities of pathogenetically only minimally related or even irrelevant to previous exposure to CT/RT in a part of patients. We investigated whether there were any specific factors that could separate ‘by definition tMNs’ into better vs. poorer prognosis, thereby necessitate different therapeutic strategies.From July 2006 to May 2017, 82 adult patients were diagnosed as MDS or AML in Seoul National University Hospital, Seoul, Korea, with clear medical records of prior exposure to ≥ 1 of followings; alkylating agents, topoisomerase 2 inhibitors (Topo2I), and radiotherapy. After excluding 8 patients with acute promyelocytic leukemia, 74 patients (43 t-AML and 31 t-MDS) were analyzed (median age 58, range 19-82). The patients were treated by conventional therapeutic modalities for MDS/AML including intensive chemotherapy (IC; n = 30), hypomethylating agents (HMAs; n = 26), upfront allogeneic stem cell transplantation (allo-SCT; n = 2), low-dose cytarabine (n = 1), and best supportive care (BSC; n = 15). Lymphoid malignancies (31.1%) and breast cancer (21.7%) were the most common preceding cancers. More than half of the patients (54.1%; 40 patients) had received both alkylators and Topo2Is and previously known differences of characteristics between alkylator/radiation vs. Topo2I groups including median time of latency and initial presentation of t-MNs (either t-MDS or t-AML) were not observed. Median time from the last exposure of CT/RT to diagnosis of t-MN was 21.8 months (range, 0.8-134).During median follow-up period of 20.6 months (95% CI 5.7-35.5 months) from diagnosis of tMNs, median overall survival (OS) was 11.6 months (95% CI 7.8-15.3 months). There was no difference of OS between t-MDS and t-AML (p = 0.136) or treatment with IC vs. HMAs (p = 0.143). Patients who had ≥ 3 abnormalities and/or complete or partial deletion of chromosome 5 or 7 (n = 35) showed inferior OS compared to those without the abnormalities (median OS 6.9 vs. 19.5 months; p = 0.048). By contrast, patients who had normal karyotype (NK) or core-binding factor (CBF) cytogenetics [t(8;21), inv(16), or t(16;16)] (n = 24; 16 for NK and 8 for CBF) showed significantly superior OS compared to their counterpart (median OS 38.8 vs. 6.2 months and 3-year OS 56.4% vs. 10.6%; p < 0.001; Figure). In addition to the absence of NK/CBF cytogenetics, age ≥ 70 years at diagnosis of t-MN, development of t-MN < 12 months from the last exposure to CT/RT, and metastatic or active status of preceding cancer at the time of t-MN diagnosis, were associated with inferior OS in univariate analysis. Multivariate analysis showed that absence of NK/CBF cytogenetics in addition to development of t-MN < 12 months from the last exposure to CT/RT, a parameter indicative of more recent damage of body by exposure to CT/RT, were independent factors of poor prognosis (Table).Among 59 patients after excluding 15 patients who received BSC only for palliative aim, poor prognosis of lacking NK/CBF cytogenetics was overcome by allo-SCT (hazard ratio 0.44, 95% CI 0.20-0.95, p = 0.036). When analysis was limited to patients with t-AML who received either IC or HMAs, patients with NK/CBF cytogenetics had higher rate of achieving a complete remission (CR) compared to those without NK/CBF cytogenetics (75% vs. 25%). patients who achieved CR showed higher 3-year OS compared to those who had persistent disease (64.3% vs. 16.2%, p = 0.030).Those results show that t-MN patients who have NK/CBF cytogenetics and fit to intensive treatment including allo-SCT can achieve comparable outcomes to de novo MDS/AML and suggest that those patients may have a different pathogenesis from that of genetically heavily damaged t-MNs. Future studies serially comparing genetic pathways and mutations within the heterogeneous tMNs would contribute to establish more fine-tuned, a genetics-oriented definition of tMNs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiR-126 Promotes Leukemogenesis in Inv(16) Acute Myeloid Leukemia

MicroRNAs (miRNAs) are short non-coding RNA molecules that inhibit the expression of multiple target proteins. MiRNA expression profiles can predict prognosis and are associated with distinct cytogentic abnormalities in acute myeloid leukemia (AML). Core-binding factor (CBF) AML, including inv(16)(p13q22)/t(16;16)(p13.1;q22) and t(8;21) chromosomal rearrangements, express significantly higher levels of miRNA (miR)-126 than non-CBF AML. We and others have shown that miR-126 is highly expressed in hematopoietic stem cells and leukemic stem cell (LSC)-enriched subpopulations and functions to promote quiescence. We previously reported that miR-126 expression is associated with an AML LSC-gene expression signature and independently predicts poor prognosis in cytogenetically normal AML. However, the role of miR-126 and the mechanism of dysregulation in inv(16) AML remains unknown.Inv(16) chromosomal rearrangement generates a leukemogenic fusion oncogene CBFB - MYH11 (CM). We utilized a conditional Cbfb-MYH11 knock-in (Cbfb56M/+ / Mx1-Cre) mouse that can be induced to express CM from the endogenous Cbfb promoter, thus mimicking human inv(16) AML. Time sequential miRNA-seq analysis revealed that miR-126-3p and miR-126-5p expression was significantly up-regulated (log fold change =2.09 and 2.36 respectively; p< 0.0001) early (1 month) and increased progressively throughout leukemia progression (log fold change = 4.11 and 4.45; p< 0.0001). Increased expression of miR-126 was observed in multiple preleukemic progenitor subpopulations, including LSK (Lin-Sca1+ckit+) and pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-ckit+Sca1-CD16/32-/loCD105-CD150+) in CM mice.To determine the functional contribution of miR-126 to inv(16) leukemogenesis, we crossed our CM knock-in mice with a conditional miR-126 knockout. Preliminary survival results indicate that CM/miR-126△/+ (heterozygote) exhibit significantly prolonged survival compared to CM mice, with a median survival of 266 vs. 159 days, respectively (p<0.05). Survival monitoring is ongoing for a cohort of CM/miR-126△/△(homozygote) mice. To date, this cohort is already demonstrating significantly enhanced survival compared to CM mice (p<0.05).We hypothesize that high miR-126 expression might contribute to survival and quiescence of CM AML LSK, which is enriched for leukemia-initiating cells. Thus, we performed lentiviral-mediated miR-126 knockdown in CM AML LSK cells and observed increased apoptosis and reduced quiescence in vitro . To assess the role of miR-126 in leukemia progression in vivo, we designed a novel CpG-anti-miR-126 inhibitor. This compound comprises an inhibitory anti-miR-126 RNA component that is linked to a Class A CpG motif for targeted cell delivery. We showed that the CpG-anti-miR-126 is efficiently taken up (60-90%) by AML blasts and LSK cells and can achieve approximately 50-80% knock-down of miR-126 in vitro and in vivo. Mice transplanted with CMAML and treated with CpG-anti-miR-126 (5 mg/kg, 5 times/week for 5 weeks) had a significant delay in leukemia progression and enhanced survival (median survival 92 days vs 35 days; n=9-10, p=0.0339), supporting the notion that high miR-126 contributes to leukemia growth. We further evaluated the effects of combining CpG-anti-miR-126 treatment with chemotherapy, adopting a regimen consisted of Cytarabine (Ara-C; i.p. 100 mg/kg for 5 days) and daunorubicin (DNR; i.v. 3 mg/kg for 3 days). We observed significantly improved survival in mice treated with CpG-anti-miR-126 combined with Ara-C and DNR compared to CpG-scrambled combined with Ara-C and DNR (median survival 183 days vs. 64 days; n=9-10; p=0.0305). These studies demonstrate a key role for miR-126 in promoting leukemogenesis of inv(16) AML. Studies to elucidate the mechanism underlying miR-126 dysregulation and function in inv(16) AML are ongoing. DisclosuresNo relevant conflicts of interest to declare.

Genetic Heterogeneity of t(8;21)(q22;q22.1) Acute Myeloid Leukemia Revealed By High-Throughput Targeted Sequencing

Background: Recently, a comprehensive high-throughput sequencing study of pediatric and adult core-binding factor (CBF) AML [t(8;21), n=85; inv(16), n=80] provided novel aspects of the genetic heterogeneity and disease biology of t(8;21) AML (Faber et al. Nat Genet 2016). In addition to well-established variants in genes encoding for proteins in tyrosine kinase (TK)/RAS signaling, epigenetic regulation (ER), and in the cohesin complex (CC), novel candidate genes were identified.Aims: We aimed to validate and to further extend recent findings by comprehensive profiling of the mutational spectrum of t(8;21) AML using a high-throughput targeted sequencing (HTS) approach.Methods: HTS was performed of the entire coding region of 244 genes with evidence in hematological malignancies. Pretreatment peripheral blood or bone marrow specimens of 143 adult t(8;21) AML patients (pts) were analyzed. 114/143 pts were enrolled in seven prospective AMLSG treatment trials. Libraries (total probes size: 1.36 Mbp) were prepared using SureSelectXT custom solutions (Agilent). Paired-end sequencing was carried out on a HiSeq platform (Illumina).Results: A mean on-target sequencing depth of 900x was obtained for all pts. Coding variants without known SNP annotation and with a minimum variant allele fraction (VAF) of 5% were called. In sum 756 mutations (missense: 70%; indels: 19%; nonsense: 9%; other: 2%) were detected with an average of 5.3 per pt (SD: ±2.5). 99% of pts harbored at least 1 mutation and 90% 3 or more. In line with previous studies, activating mutations in TK/RAS signaling pathways represented recurrent events: KIT (34/143; 24%), NRAS (24/143; 17%), FLT3 (15/143; 10%; point mutations only), and KRAS (4/143; 3%).In addition to these mutations conferring proliferative clonal advantage, a striking enrichment of mutations was observed in genes involved in histone modification and DNA methylation, ASXL2 (22/143; 15%), ASXL1 (21/143; 15%), KDM6A (14/143; 10%), EZH2 (11/143; 8%), CREBBP (8/143; 6%), SRCAP (8/143; 6%), TET2 (19/143; 13%) and DNMT3A (9/143; 6%), emphasizing their contribution to impairing differentiation in this leukemia subtype. Further, frequent aberrations were found in the cohesin genes that are involved in sister chromatid segregation during mitosis and DNA repair: RAD21 (17/143; 12%), SMC1A (10/143; 7%), STAG2 (5/143; 3%), and SMC3 (5/143; 3%). In general, mutations in genes belonging to the same functional subgroups were rarely co-occurring suggesting functional redundancy without additional synergistic effect. We also identified recurrent variants in previously detected candidate genes outside these functional clusters. A remarkably high incidence of loss of function mutations was found in the ZBTB7A gene(27/143; 19%), encoding for a transcription factor guiding hematopoietic lineage fate. Recurrent mutations were also observed in CCND2 (14/143; 10%) , involved in hematopoietic cell proliferation, MGA (11/143; 8%), a negative regulator of MYC, as well as DHX15 (10/143; 7%), associated with ribosome biogenesis and spliceosome function. When focusing on the clonal hierarchy we found that the median VAF in genes belonging to ER and CC (0.31; range 0.3-0.91; 0.35, range 0.05-0.96; respectively) was higher than in genes associated with TK/RAS signaling (0.16, range 0.05-0.53) suggesting that mutations affecting the epigenetic state and differentiation occur earlier than those impairing proliferation during development of t(8;21) AML.Conclusion: Using a comprehensive sequencing approach we could further delineate the molecular pattern of t(8;21) AML that despite shared genetic lesions is remarkably different to inv(16) AML. Here, mutation clusters in genes involved in TK/RAS signaling, ER and CC were confirmed as well as novel t(8;21)-associated gene mutations such as in ZBTB7A and CCND2 that play an essential role in regulation of hematopoietic cell proliferation and differentiation. Further analyses including sample size extension as well as correlation of findings with clinical parameters are ongoing. DisclosuresDöhner:Astex Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Honoraria; Arog Pharmaceuticals: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Research Funding; Boehringer Ingelheim: Research Funding; Seattle Genetics: Honoraria; Agios: Honoraria; Amgen: Honoraria; Celator: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dohner:Novartis: Honoraria, Research Funding.

Functional Properties of Exon 17 KIT Mutations in Pediatric CBF AML Predict for Inferior Outcome and Enhanced Response to Tyrosine Kinase Inhibition: A Report from the Children's Oncology Group

Introduction: KIT mutations are detected in approximately 20-30% of core binding factor (CBF, t(8;21) and inv(16)/t(16;16)) AML and have been associated with outcome and response to tyrosine kinase inhibitors (TKI). Mutations tend to localize to 2 regions- the receptor's kinase activation loop in exon 17 (E17) and a region of the extracellular domain in exon 8 (E8). The prognostic significance of the 2 mutations has varied in previous series. In this analysis, the functional significance of E8) and E17 KIT mutations in vitro and their response to TKIs were evaluated. We hypothesized that activating variants might be associated with inferior clinical outcome and be more susceptible to KIT-targeted agents.Methods: COG AAML0531 enrolled 1,070 patients, of which 800 underwent targeted next generation sequencing of the entire KIT gene coding sequence to identify somatic KIT mutations. To evaluate the functional impact of the most common KIT mutations, E8 (D419G, D419indel) and E17 (D816V, N822K) mutations were engineered and transduced into HEK293 cells for evaluation of KIT phosphorylation (pKIT) by immunoblotting and into Ba/F3 cells for cytokine independent proliferation studies. Mutations that resulted in pKIT were evaluated for response to the TKIs dasatinib and crenolanib. We further correlated our mutational and functional analysis to clinical outcome data from COG AAML0531. We analyzed outcomes by treatment arms (no-gemtuzumab ozogamicin (no-GO) vs. GO), focusing on the GO arm given the potentially favorable impact of GO on CBF AML response.Results: A total of 247 CBF patients were enrolled, with 218 undergoing screening for somatic KIT mutations. Mutations were detected in 55 (25%) patients; 27 (49%) in E8, 26 (47%) in E17, and 2 (4%) involved both exons. Among the 109 patients with CBF AML treated on no-GO arm, KIT mutations were detected in 29/109 (27%); 14 (48%) in E8, 13 (45%) in E17, and 2 (7%) involved both exons. E8 mutations were a combination of missense mutations or 3-9 bp indels that maintained reading frame, while E17 variants were primarily missense mutations involving the D816 or N822 codons, while. Cells transduced with E8 mutations showed baseline receptor phosphorylation and no IL-3 independent proliferation. In contrast, E17 mutations showed autonomous receptor phosphorylation and IL-3 independent proliferation, with D816V showing more robust cellular proliferation and higher levels of pKIT (Fig1A).We further evaluated the cytotoxic effect of 2 TKIs in engineered cells with activating KIT mutations and KIT wild-type (WT). Neither parental line nor cells expressing KIT -WT had any response to the two TKIs with IC50s > 10,000μM, however cells transduced with the E17 variants D816V or N822K were exquisitely sensitive to the TKIs. Following exposure to dasatinib and crenolanib, N822K+ cells had an IC50 of 3.8nM and 28.3nM respectively, with D816V+ cells demonstrating similar IC50s of 13nM and 14.3nM respectively (Fig 1B).Given the differential activating potential of E8 vs. E17 variants, we evaluated the clinical implications of the mutations in the 2 regions among CBF patients. On the no-GO arm, E8 KIT mutations lacked clinical significance, with an overall survival (OS) of 81±20% vs. 80±9% (p=0.91) for patients with and without E8 mutations, with a corresponding disease-free survival (DFS) of 57±26% vs. 62±11% (p=0.75) and relapse risk (RR) of 43±28% vs. 36±11% (p=0.59) respectively. In contrast, activating E17 mutations were associated with adverse outcome with an OS of 56±27% vs. 85±7% for those with and without E17 mutations (p=0.02; Fig 1C), with a corresponding DFS 33±27% vs. 65±11% (p=0.05) and a RR of 67±30% vs. 32±11% (p=0.03) respectively. Interestingly, when we evaluated outcomes on the GO arm, patients with E17 mutations had an OS of 77±23% vs. 83±8% for KIT-WT (p=0.62), suggesting that GO may abrogate the adverse prognostic impact of E17 mutations.Conclusions: Somatic E17 KIT mutations lead to autonomous receptor phosphorylation and cellular proliferation and are associated with inferior clinical outcome when treated with conventional chemotherapy alone. This is in contrast to E8 mutations, which lack functional significance. KIT E17 mutations are uniquely sensitive to the cytotoxic effects of KIT inhibition. Consideration of TKI therapy for at least this subset of pediatric KIT + CBF AML is prudent given their inferior prognosis and this preclinical correlate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prospective Study of Acute Myeloid Leukemia (AML) Clinical Epidemiology and Cytogenetic Risk Group in Older Adults in the ECOG-ACRIN E2906 Clinical Trial: The Acute Leukemia Epidemiology, Survival and Endpoints (ALESE) Study

Prospective Study of Acute Myeloid Leukemia (AML) Clinical Epidemiology and Cytogenetic Risk Group in Older Adults in the ECOG-ACRIN E2906 Clinical Trial: The Acute Leukemia Epidemiology, Survival and Endpoints (ALESE) Study

Factors Predicting Response and Resistance to Midostaurin in FLT3 Positive and FLT3 Negative AML in 483 Primary AML Patient Samples

Introduction: Midostaurin is an FDA approved, multi-target tyrosine kinase(TK) inhibitor shown to inhibit the FLT3 kinase and have therapeutic benefit in FLT3 mutated AML especially FLT3-ITD mutations (FITD+). However, midostaurin has also shown therapeutic response in select FLT3-ITD negative (FITD-) AML patients likely due to inhibitory effects on other TKs .We analyzed primary AML patient samples from our ‘Beat AML’ database to look at disease related factors, aside from FLT3 status, that may influence midostaurin drug sensitivity and affect patient outcomes.Methods: We collected peripheral blood and bone marrow samples from 483 newly diagnosed AML patients (348 FITD+, 135 FITD-), extracted mononuclear cells and plated them on a Midostaurin dose curve, without growth media or feeder cells. Metabolic viability was assessed after 72-hours and drug response was summarized with an area under curve (AUC) metric on a 0-100 scale. We annotated these samples for known chromosomal translocations and somatic mutations in leukemia-related genes and performed association-based analyses to identify genomic predictors of midostaurin drug sensitivity.Results: FLT3-ITD was predictably the strongest association with midostaurin-response, so further analysis was subsetted into FITD+ and FITD- samples (Figure A). For our analysis, we included FLT3-TKD mutant samples in the FITD- cohort as we found no significant difference between FLT3-TKD status and non-FLT3 mutated samples.Within FITD- samples, we found inv(16) Core Binding Factor AMLs demonstrated increased sensitivity to midostaurin (p < 0.05) . We investigated the frequency of c-Kit mutations in inv(16) AMLs, given Midostaurin's activity against c-Kit, but did not observe significant differences in drug response between any combination of inv(16) and c-Kit mutational status due to a relatively small sample size.(Fig B) Further, among all FITD- samples evaluated, we found mutations in KRAS and TP53 to be predictive of midostaurin resistance (p < 0.05, Fig B). This relationship was also observed through analysis of publically available cell line drug screens within the Genomics of Drug Sensitivity in Cancer (GDSC), where mutations in NRAS and TP53 suggested Midostaurin resistance.In FITD+ AML samples, we found the FLT3-ITD mutant allele fraction (MAF) to strongly correlate with the extent of Midostaurin sensitivity. FLT3-ITD MAF was significantly higher in Midostaurin-sensitive samples compared to resistant samples (p < 0.001)(Fig C). Additionally, we observed an increase in Midostaurin efficacy (p < 0.05) in FLT3-ITD positive(15;17) APL,a subset of APL with relapse rates 15-times higher than FLT3 WT APL (Breccia et. al. Haematologica 2013) (Figure C). Further, we found that neither NPM1 or FLT3-TKD status conferred additional sensitivity to Midostaurin. There was no significant difference in AUC between FLT3-ITD and NPM1 double-mutant samples and FLT3-ITD alone, and similarly no significant difference between FLT3-TKD and FLT3 WT samples.Discussion: With the approval and clinical use of Midostaurin in FLT3 positive AML patients it is important to find and validate factors influencing Midostaurin efficacy. Data from the RATIFY clinical trial has shown Midostaurin improved overall survival in patients with both high and low FLT3-ITD allelic ratios (Stone et. al. N Eng J Med 2017). We show that the ex vivo Midostaurin response is correlated with FLT3-ITD status and its MAF but not significantly with the FLT3-TKD mutation. Our data suggests that FITD+ APLs are responsive to this FLT3-inhibitor thus offering the potential of combination therapy and improved outcomes with current APL regimens.We also demonstrate that Midostaurin shows efficacy in FLT3-ITD negative patient samples, illustrating the multi-targeted nature of the inhibitor. Within these samples,inv 16 predicted Midostaurin sensitivity regardless of c-Kit mutational status. Additionally, mutations in TP53 and the Ras-pathway predict Midostaurin resistance (these mutations rarely co-occurred with FLT3-ITD in our cohort, thus limiting their impact in FLT3-ITD positive cases). Additional in vitro and clinical data is necessary to determine whether these findings should influence treatment decisions.Newer FLT3 inhibitors in clinical trials need to be characterized in a similar manner so as to efficiently target subsets of FLT3 mutated AML. [Display omitted] DisclosuresTyner:Takeda Pharmaceutical Company: Research Funding; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Janssen Pharmaceutica: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; Syros: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Agios Pharmaceuticals: Research Funding; Seattle Genetics: Research Funding; Gilead: Research Funding; Leap Oncology: Consultancy. Borate:Novartis, Jazz: Consultancy, Speakers Bureau.

Rhob Overexpression Boosts Relapse of t(8;21) RUNX1-RUNX1T1 Rearranged Acute Myeloid Leukemia.

Introduction. The core binding factor rearrangements, inv(16)(p13;1q22), t(16;16)(p13;q22) and t(8;21)(q22;q22), are among the most frequent cytogenetic abnormalities found in children with acute myeloid leukemia (AML). They are considered mutations associated with good responsiveness to chemotherapy and favorable patient outcome. Despite this, in the AIEOP LAM2002 trial, we found that t(8;21) RUNX1-RUNX1T1- rearranged patients had an unexpectedly high cumulative incidence of relapse (30%), which was comparable to that of patients considered at higher risk of treatment failure in light of recurrent molecular/cytogenetic alterations or levels of minimal residual disease at the end of induction therapy. Thus, we aimed at re-evaluating at both genetic and epigenetic level samples of t(8;21) AML cases collected at diagnosis addressing the hypothesis whether the methylation process may influence response to treatment and contribute to further stratify patients carrying the same genetic lesion.Methods and results. We performed high throughput profiling of DNA methylation (Reduced Representation Bisulfite Sequencing), gene (HTA 2.0 Affymetrix) and protein expression (Reverse phase protein array) on samples collected at diagnosis of 34 t(8;21)(q22;q22)/RUNX1-RUNX1T1 children enrolled in the AIEOP-AML 2002/01 trial who either did or did not experience leukemia recurrence. We found that the DNA methylation profile of t(8;21)-rearranged patients who relapsed was peculiarly different from that of patients maintaining a state of complete remission. We identified a new epigenetic signature of 337 differentially methylated regions(DMRs) separating relapsed (n=6) from non-relapsed patients (n=6). We investigated DMRs association to preferential Transcription Factor Binding Sites (ENCODE) and found that the CCCTC-binding factor (CTCF) is the most represented, it demarcates genomic regions called Topologically Associating Domains, known to be sensitive to CpG methylation with main consequences over gene transcription. We identified 652 differentially methylated CTCF binding sites between patients who either did or did not experience relapse, and looked at gene expression correlation matrixes (transcripts distant within 500 kb from differentially methylated CTCF were considered as a module), revealing that patients who relapsed lost gene expression correlation with respect to non-relapsed children. These results support the role of CTCF in modulating gene expression in relapsed patients. We then performed gene expression profile (GEP) to investigate the expression levels of DMRs neighbor gene transcripts. We found 77 DMRs-controlling genes being targets of RUNX1-RUNX1T1 chimera, and clustering analysis using their expression showed a discrete ability in identifying t(8;21) rearranged patients with respect to their risk of relapse. Through GEP analysis, we identified 18 genes differentially expressed between the two groups (fold change >|2|; unpaired t-test, p<0.05, 87 genes fold change >|1.5|), and Ras Homolog Family Member (RHOB) being the most overexpressed in relapsed patients. We integrated DNA methylation and gene expression platforms with STRING database defining an active network of pathways of cell adhesion and cell-cell signaling, where RHOB emerged as the key player of this network, validated in patient's samples by RPPA. We hypothesized that RHOB is the candidate gene promoting relapse. We moved to in vitro validation and modeled the t(8;21)-rearranged cells to grow in suspension or in adhesion, this latter experimental condition mimicking the bone marrow niche. We showed that RHOB overexpression enhanced cell adhesion properties, by strongly enhancing stress fibers formation, and protected blasts from the cytotoxic action of etoposide. On the contrary, RHOB silencing abolished these features, thus reverting the cell chemotherapy resistance by interrupting RHOB-phospho-COFILIN axis.Conclusion. This study identifies a novel role of RHOB in influencing the risk of leukemia relapse, indicating that the RHOB-mediated interaction of leukemia cells with the BM niche may support leukemia growth and disease progression. To the best of our knowledge, this is the first study that establishes a dynamic interplay of epigenetic heterogeneity within the same genetic in pediatric AML. DisclosuresNo relevant conflicts of interest to declare.

Transcriptomic and Network Signatures of Cryptic Fusion CBFA2T3-GLIS2 in Pediatric AML Reveals Clinical Considerations and Implications

IntroductionA cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion was initially reported in childhood acute megakaryoblastic leukemia (AMKL) and was shown to be associated with unfavorable outcomes. Although this fusion onco-protein is more prevalent in FAB M7 AML, it is reported in younger patients with all subtypes of AML. We present a comprehensive transcriptomic signature of an unselected cohort of children with CBFA2T3-GLIS2 fusion treated on COG AML trials.MethodsCBFA2T3-GLIS2 fusions were identified in 26 of 1042 (2.5%) patients enrolled in Children's Oncology Group (COG) studies AAML03P1 and AAML0531, with a prevalence of 8% in patients < 3 years of age. Whole transcriptome sequence data was available from 466 patients and 20 normal bone marrows (NBM) for expression profiling. Differentially expressed genes were identified using the Limma v.3.30.12 R package. Unsupervised hierarchal clustering was performed using Euclidean distance with the WPGMA linkage algorithm. Gene-set enrichment analysis was completed using the Broad Institute software v.2-2.2.4 with MSigDB C2 curated gene sets.ResultsOf 26 patients with CBFA2T3-GLIS2 fusions, 21 (81%) were < 5 years of age (Fig. 1A). KMT2A fusions co-occurred in only two patients, and none of the positive tumors carried core binding factor fusions. Few other variants accompanied CBFA2T3-GLIS2 fusions and overall, this subclass of AML had been categorized as cytogenetically normal (42% CN-AML, Fig. 1B). CBFA2T3-GLIS2 AML fared worse than the AML population as a whole; more than 75% of fusion-positive cases relapsed within two years (Fig. 1C, p = 0.001).Gene-set enrichment analysis between CBFA2T3-GLIS2 fusion and fusion-negative tumors revealed the top three positively enriched gene sets as 1) basal cell carcinoma (FDR = 0.003), 2) ECM receptor interactions (FDR = 0.006), and 3) hedgehog signaling pathways (FDR = 0.029). Consistent with previous reports, genes associated with WNT, hedgehog (HH), and BMP signaling were significantly up-regulated in CBFA2T3-GLIS2 tumors (Fig. 1D). CBFA2T3-GLIS2 fusions have been associated with induction of BMP signaling through downstream HH targets, BMP2 and BMP4 in pediatric AMKL. Moreover, concurrent dysregulation of WNT, BMP and HH signaling is a feature of CBFA2T3-GLIS2 AMKL (Gruber et al., 2015). Unsupervised hierarchical clustering of differentially expressed genes effectively clustered the fusion-positive samples, suggesting CBFA2T3-GLIS2 fusions drive a distinct transcriptional profile from other AML subtypes (Fig. 1E). CBFA2T3-GLIS2 tumors compared to NBM had upregulated expression of 4 cancer testes antigens (CTAs): CCNA1, PRAME, IGF2BP3, and KDM5B (fold-change > 2, adj. p < 0.001). CTAs are promising candidates for targeted T-cell therapies due their low expression in NBM and surrounding tissues, which could limit treatment related toxicity ( Ochsenreither et al., 2012) .We further performed unsupervised hierarchical clustering in 437 patients for whom flow cytometry data were available (Fig. 1F). Most fusion-positive tumors (7/10) clustered together by mean fluorescence intensity (MFI), indicating that CBFA2T3-GLIS2 tumors have highly distinctive immune profiles. Of particular interest, fusion-positive tumors expressed CD56 at high levels (median CD56 in CBFA2T3-GLIS2 : 1,505.0 MFI [range: 6.4 - 4,290.0] versus median CD56 in fusion-negative AML: 9.0 MFI [range: 2.4 - 3,360.0], p = 0.001). A novel immunophenotype in pediatric AML, termed RAM, has been associated with high CD56 expression and much lower event-free survival. A preponderance of CBFA2T3-GLIS2 AML displayed the RAM phenotype, with 70% of fusion-positive tumors identified as RAM compared to 0.23% (1/427) in the fusion-negative population (p < 0.001).ConclusionCBFA2T3-GLIS2 AML tends to have an aggressive disease pathology in much younger patients, who had been primarily classified as CN-AML. This suggests screening for CBFA2T3-GLIS2 in younger patients could be valuable as standard practice, though not yet recommended by the World Health Organization. Our associations with CBFA2T3-GLIS2 fusions provides a potential link between genomic and immunologic characteristics of a high-risk group of pediatric AMLs, which could be employed in the future for clinical screening through molecular or flow-cytometric methods. [Display omitted] DisclosuresBrodersen:Hematologics Inc: Employment. Loken:Hematologics Inc: Employment, Equity Ownership.

Two Cycles of Consolidation Chemotherapy Are Associated with Similar Clinical Outcomes to Three Cycles in AML Patients with Favorable Risk Cytogenetics

Background: Patients with core-binding factor acute myeloid leukemia (CBF-AML) have favourable outcomes when treated with standard anthracycline based induction and repeated cycles of cytarabine based consolidation. CBF-AML exhibit high rates of remission, overall survival, and relapse-free survival even without allogenic stem cell transplant (alloSCT). The exact number of consolidation cycles necessary for optimal outcomes, however is unknown. While early studies suggested that 3 or 4 cycles of high dose cytarabine (HiDAC) are associated with better outcomes relative to 1 cycle (Byrd, 1999 & Byrd, 2004), the effects of 2 cycles, has not been studied in this patient group. In this study, we evaluated the impact of 2 cycles of consolidative chemotherapy on the clinical outcomes of patients with CBF-AML.Methods: Prior to 2012 patients with CBF-AML treated in Edmonton, Canada, were intended to receive 2 cycles of HiDAC after a complete remission (CR) following a single induction cycle ± intensification treatment (given if having >5% blasts on a day+14 marrow). Following a change in institutional policy, patients treated after 2012 were intended to receive 3 cycles of consolidation treatment. c-kit testing was not available prior to 2009 but thereafter all patients with c-kit mutations were offered alloSCT in first CR. In contrast, patients treated in Vancouver, Canada underwent similar induction but were intended to receive three consolidative cycles throughout the entire study period. Pooled data was retrospectively analyzed for all patients with CBF-AML treated in these two Canadian centres between 2003-2017 using an intention to treat approach.Results: Overall, 108 patients were identified with a median age of 48 (range 17-75). Sixty-four patients (59%) were male. Seventy-four patients (68.5%) were intended for 3 cycles of consolidation therapy and 34 patients (31.5%) for 2 cycles. There were no differences in baseline characteristics of the analyzed patients (Table 1). There was also no significant difference in the number of patients who received intensification chemotherapy due to day +14 residual disease, the number of induction deaths, or CR rate at end of induction. Six patients intended to receive 2 cycles of consolidation were transplanted in CR1 compared with 5 who were intended to receive 3 cycles (p=0.09). Rates of hospitalization, median length of hospital stay, bacteremic events, intensive care requirements, and deaths during consolidation therapy did not differ significantly between the two groups.Median follow-up time from CR was significantly longer in the 2 consolidation cycle group relative to the 3 consolidation cycle group (85 months vs. 30 months, p<0.0001). Of the 34 patients intended for 2 cycles of consolidation, there were 9 deaths and an additional 4 relapses, for a total event rate of 38.2%. Within the 74 patients intended to receive 3 cycles of consolidation there were 18 deaths and an additional 13 relapses for a total event rate of 41.9% (p=0.83)There was no significant difference in overall survival (p=0.96; Figure 1, 5-year OS 73% for the 2 cycle consolidation group v 71% for the 3 cycle consolidation group), relapse-free survival when censored for transplant (p=0.61; Figure 2, 5-year RFS 63% for the 2 cycle consolidation group v 57% for the 3 cycle consolidation group, or event-free survival (p=0.88; 5-year EFS 54% for the 2 cycle consolidation group v 52% for the 3 cycle consolidation group) within the two groups. Multivariate analysis revealed that patients age, cytogenetics [t(8;21), inv(16)/t(16;16)], or transplantation in CR1 did not influence rates of overall survival or relapse-free survival between the two cohorts.Conclusions: These data suggest that the use of 2 chemotherapy consolidation cycles compared with 3 does not diminish relapse-free survival or overall survival in patients with CBF-AML. Reduction in chemotherapy may provide both economic and quality of life benefits for patients. Larger prospective studies are necessary to confirm these findings. [Display omitted] DisclosuresHogge:Novartis, Roche, and Sanofi: Consultancy.

The LSC17 Leukemic Stem Cell Signature Predicts Outcome in Pediatric Acute Myeloid Leukemia

IntroductionThe model of the leukemic stem cell was proposed nearly 60 years ago as a mechanism of leukemic pathogenesis. Recently, a 17-gene leukemic stem cell (LSC) prognostic signature, termed the LSC17, was developed (Ng et al., 2016). The LSC17 score was shown to reliably differentiate adult acute myeloid leukemia (AML) patients into high and low risk groups. However, its applicability to pediatric AML remains unknown.Prognostic indicators are sensitive to differences in age. For example, NPM1 mutations, which are highly prognostic of favorable outcome, have no significance in patients older than 65 years (Ostronoff et al., 2015). Similarly, core-binding factor AML, also associated with favorable outcomes, loses prognostic value in older patients (Prébet et al., 2009).Given that age remained a significant prognostic factor in multivariate analyses with the LSC17, we evaluated the impact of the signature in older versus younger adults in the TCGA AML dataset and further, applied it to transcriptome sequence data from pediatric AML to assess the prognostic power in younger patients.Patients and MethodsPediatric AML biological samples were collected from patients enrolled in the Children's Oncology Group protocol AAML0531 and RNA sequencing was performed by the British Columbia Genome Sciences Center (Vancouver, BC). The Cancer Genome Atlas AML RNA sequencing data was downloaded from the Broad Institute Firehose (TCGA Network et al., 2013; Broad Institute TCGA Genome Data Analysis Center, 2016). LSC17 scores were calculated from log2 RPKM normalized gene expression as described by Ng et al .ResultsWe initially verified that the LSC17 score is effective for risk stratification in adult AML by applying the method to the TCGA AML cohort, excluding PML-RARA, BCR-ABL1 fusions, and those who received no treatment (n = 155, range: 18 - 88 yrs). Patients with low LSC17 scores had significantly better overall survival (OS) compared to the high scoring group after 5 years (low-LSC17 OS: 38.0%, high-LSC17 OS: 15.2%, hazard ratio (HR) = 2.08, p < 0.001). To investigate age effects on LSC17, we divided TCGA into two groups based on the median age of 58 yrs. The low (n = 81) and high age groups (n = 78) were reanalyzed. Younger patients with low LSC17 scores fared much better (low-LSC17 OS at 5yrs: 57.9%, high-LSC17 OS at 5yrs: 16.0% p < 0.001, Fig. 1A). Strikingly, for TCGA patients above 58 years, the LSC17 signature showed no prognostic significance (p = 0.714, Fig. 1B).We then evaluated the LSC17 signature to identify risk groups using RNA sequencing from our pediatric AML cohort (n = 446, range: 0.02 - 28 yrs). Univariate survival analysis revealed strong prognostic value for both overall survival (low-LSC17 OS at 5yrs: 72.8%, high-LSC17 OS at 5yrs: 51.4%, HR = 2.06, p < 0.001, Fig. 1C) and event-free survival (low-LSC17 EFS at 5yrs: 60.3%, high-LSC17 EFS at 5yrs: 36.4%, HR = 2.04, p < 0.001). Although dividing the cohort by LSC17 scores above and below the median (as proposed by Ng. et al.) demonstrated prognostic value in pediatric AML, the choice of median as a threshold may not be ideal. Dividing pediatric AML patients into four quartiles, we found no statistical significance between the survival curves for the middle two groups. In contrast, the top and bottom quartiles show very strong trends (LSC17-q1: 77.0% at 5 years, LCS17-q4: 41.1% OS at 5yrs, HR = 3.34, p < 0.001, Fig.1D). The high-LSC17 cohort carried a higher incidence of adverse cytogenetics and recurrent mutations, such as FLT3-ITD, and conversely, the low-scoring group was found to have a higher percentage of favorable cytogenetics (Fig.1E). To determine if the prognostic significance would remain, we performed a multivariate analysis; though WBC, age, and NMP1 mutations continued to maintain prognostic significance, we found the LSC17 score to be a strong independent prognostic indicator (HR = 2.031, p < 0.001, Fig. 1F).ConclusionIn this study, we validated that the LSC17 score can be used as an effective risk stratification tool in children and young adults. The LSC17 remains a strong independent prognostic factor after controlling for age, WBC, adverse FLT3-ITD mutations and favorable NPM1 mutations. While the LSC17 can be used as an informative indicator of long term survival, and event-free survival, we showed that this signature loses prognostic value in older AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.